Genetic toxicity of some important epoxides

Mutation Research, 86 (1981) 1--113 Elsevi'er/North-Holland Biomedical Press

GENETIC

TOXICITY

OF SOME IMPORTANT

EPOXIDES

*

L. E H R E N B E R G a n d S. H U S S A I N

Department of Radiobiology, Wallenberg Laboratory, University of Stockholm, 3-106 91 Stockholm (Sweden) (Received 24 S e p t e m b e r 1 9 8 0 ) ( A c c e p t e d 29 S e p t e m b e r 1 9 8 0 )

Contents 1. 2. 3. 4.

Summary ................................................. Introduction ............................................... Available biological a n d c h e m i c a l d a t a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G e n e t i c e f f e c t s : qualitative a s p e c t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. P o i n t m u t a t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.1. E p o x i d e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.2. A l k y l e n e h a l o h y d r i n s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. G e n e c o n v e r s i o n and crossing-over . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. C h r o m o s o m a l a b e r r a t i o n s and related e f f e c t s . . . . . . . . . . . . . . . . . . . . . . . 4.4. C a r c i n o g e n i c i t y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4,4,1, E p o x i d e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4.2. A l k y l e n e h a l o h y d r i n s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. P r o p h a g e - i n d u c i n g activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6. H a e m a t o l o g i c a l and r e l a t e d e f f e c t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7: G e n e t i c e f f e c t s o f certain c o m p o u n d s o f t e c h n i c a l i m p o r t a n c e o r occurring in n a t u r e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7.1. I n s e c t i c i d e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7.2. E p o x y resins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7.3. E p o x i d e s in f o o d s , p h a r m a c e u t i c a l s , etc. . . . . . . . . . . . . . . . . . . . . . 4.8. Special s t u d i e s p e r t a i n i n g t o m e c h a n i s m s . . . . . . . . . . . . . . . . . . . . . . . . . . 4.8.1. D o s e - - r e s p o n s e r e l a t i o n s h i p s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.8.1.1. M u t a t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.8.1.2. C h r o m o s o m a l a b e r r a t i o n s . . . . . . . . . . . . . . . . . . . . . . . . . 4.8.1.3. C a n c e r . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.8.1.4. General c o m m e n t s . . . . . . . . . . . . . . : .............. 4.8.2. M o d i f y i n g f a c t o r s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.9. A p p l i c a t i o n s o f g e n e t i c e f f e c t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.9.1. Plant b r e e d i n g . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 3 8 19 24 24 27 27 28 30 30 30 31 31 32 32 33 33 34 35 35 37 38 39 39 42 42

* This paper was originally prepared in 1978 and has not undergone extensive revision. Therefore, we want to state that no more recent data have been presented to invalidate the conclu~ons drawn.

Abbreviations: i.p., intrapezitoneal; P.o., pez oral; s.c., subcutaneous; D, dose; DL$0 , dose causing 50~ lethality; CL50 , eoncentrat/on causing 50~ lethality; b.w., body weight; GSH, glutathione. 0 1 6 5 - - 1 1 1 0 / 8 1 / 0 0 0 0 - - 0 0 0 0 / $ 0 2 . 5 0 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press

5.

6.

7. 8. 9.

4.9.2. O t h e r a p p l i c a t i o n s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.9.3. U n w a n t e d s i d e - e f f e c t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Quantitative aspects .......................................... 5.1. G e n e r a l . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. C h e m i c a l r e a c t i o n s o f e p o x i d e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.1. R e a c t i o n m e c h a n i s m s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.2. R e a c t i o n p r o d u c t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.3. Reaction-kinetic aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 . 2 . 3 . 1 . R e q u i r e m e n t s f o r risk e s t i m a t e s . . . . . . . . . . . . . . . . . . . . . 5.2.3.2. Reaction-kinetic data . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. F a t e o f e p o x i d e s in vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. S t r u c t u r e - - e f f e c t i v e n e s s r e l a t i o n s h i p s . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.1. M o n o e p o x i d e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4,2. A l k y l e n e h a l o h y d r i n s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.3. I s o m e r i s m . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.4. Di- a n d p o l y f u n c t i o n a l i t y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.4.1. Diepoxides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.4.2. V i e w p o i n t s o n m e c h a n i s m s . . . . . . . . . . . . . . . . . . . . . . . . 5.4.4.3. "Mixed difunctionality" . .. ...................... Risk estimates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. R a d - e q u i v a l e n c e o f g e n e t i c e f f e c t s o f e p o x i d e s in m a m m a l s . . . . . . . . . . . . . 6.1,1. C h r o m o s o m a l a b e r r a t i o n s a n d r e l a t e d e f f e c t s . . . . . . . . . . . . . . . . . . 6.1,2. T u m o u r s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. F a l s e - n e g a t i v e t e s t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2,1. C a n c e r t e s t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2.2. D o m i n a n t - l e t h a l t e s t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2.3. Size o f s a m p l e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. T h e q u e s t i o n o f a n o - e f f e c t level f o r g e n e t i c e f f e c t s o f e p o x i d e s . . . . . . . . . . . 6.4. G e n e t i c r i s k s a s s o c i a t e d w i t h t i s s u e d o s e o f e p o x i d e s in m a n . . . . . . . . . . . . . 6.4.1. E t h y l e n e o x i d e a n d its p r i m a r y r e a c t i o n p r o d u c t s . . . . . . . . . . . . . . . 6.4.2. O t h e r e p o x i d e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5. O t h e r a p p r o a c h e s t o r i s k e s t i m a t i o n f o r e p o x i d e s in m a n . . . . . . . . . . . . . . . 6.6. E p i d e m i o l o g i c a l a s p e c t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . General conclusions .......................................... Acknowledgement ........................................... References ................................................

43 43 44 44 46 46 49 50 50 52 57 60 60 63 65 66 66 71 73 77 77 77 79 80 80 85 87 87 88 88 89 90 91 94 97 97

1. Summary In various biological systems, 1,2-epoxides are able to cause biological effects with genetic mechanisms: point mutations, deletions, chromosomal aberrations, gene conversion, crossing-over, cancer and virus (prophage)induction. Dose--response curves are linear, or should be interpreted to have a linear comp o n e n t that predominates at low doses or concentrations. In forward mutation systems the effectiveness is related to the dose in the target cells and to the chemical reactivity. By applying experimentally determined correction factors for difunctionality (in the case o f diepoxides or monoepoxides with a reactive substituent such as a carbonyl group), genetic risks m a y be estimated b y a unitary approach, valid for epoxides in general, if n o t for all alkylating agents. The use of this approach, to calculate type-II errors in negative carcinogenicity tests, indicates that no data presently available support a conclusion that certain epoxides are non-mutagenic or non-carcinogenic, i.e. are less effective than expected from reactivity data. Determination o f the rad-equivalence of genetic

risks, i.e. the use of corresponding risks from a unit dose of ionizing radiation as a standard, indicates that the risks associated with Threshold Limit Values for epoxides in work environments in Western countries are 1--2 orders of magnitude higher than permissible risks for radiological workers.

2. Introduction This review deals with genetic and related effects of 1,2-epoxides, sometimes called a-epoxides. These epoxides may be considered to be derivatives of the simple three-membered ring of ethylene oxide (oxirane):

C%2o /

CH2

(la)

The formulae of some of the most important compounds dealt with are given in Fig. 1. Compounds with four- and five-membered rings, i.e. derivatives of trimethylene oxide and tetrahydrofuran, which are often called 1,3- and 1,4epoxides, resp., will not be discussed. In general, any of the four hydrogens of ethylene oxide (la) may be substituted. Hence, the general formula of epoxides may be written:

R I R 2 C ~ CR3R4

\o j

(lb)

Substituents R1 ... R4 may form parts of other rings, as exemplified in formulae XIV, XIXb, XXI, XXII, XXIVb,c and XXVb (Fig. 1). Nomenclature. The systems used for naming epoxides are somewhat confusing and have also undergone radical changes. The compounds may be referred to by using the name of the basic hydrocarbon with the prefix "epoxy", but they may also be considered to be derivatives of the heterocyclic oxirane ring. In addition, trivial names are used for many compounds in widespread use. Table 1 gives a few examples of names of epoxides according to different systems of nomenclature. In this review we have chosen to use the trivial names because they are in current use among biologists, and from among the other possible names we have chosen the one most commonly used in the biological literature. Biologists (and we are no exceptions) have become addicted to the use of acronyms such as EO, PO and ECHH for compounds I, II and IX in Fig. 1. Such abbreviations would only add to the confusion; they are chemically meaningless and should genemUy be avoided. In this paper, we shall refer to the compounds by their names or by the Roman numerals given in Fig. 1. As revealed in Rapoport's pioneer study [349], ethylene oxide (I) and derivatives thereof (VIII, IX; see Fig. 1) are effective mutagens. 1,2-Epoxides owe their reactivity; with genetic consequences, t o the strain in the three-membered ring. They easily participate in reactions, releasing this strain through opening of the ring. These reactions involve nucleophilic displacement on one of the ring c a r b o n s - Sect. 5.2., formulae (9), ( 1 0 ) - i.e. the epoxides are a class of alkylating agents.

4

Epoxides are of great concern in environmental genetics because man and other living organisms are exposed to these compounds in several situations. ~0, CH2-CH2

~0,

~0 CH3-CH- CH2

I. Ethylene oxide

CH3CH2-CH-CH 2

II. Propyleneoxide

III. 1,2- Epoxybutane

/O\ ,H /O\ CH3-CH-CH- CH3

/O\ CH3 (CH2)rl-CH- CH2

~V. 2, 3- Epoxybutane

V.n =13 : 1,2- Epoxy-n-hexadecane %2- Epoxy-n-dodecane

/O\ fl\ CH~-~-~-CH 2

CH2-HC -C~/C , H2 XVa, DL- Diepoxybutane

XVb, mesa- Diepoxybutane

vt.n =g:

/O\ /0\ CH2-CH-CH2-CH2-CH- CH2 XVl. 1,2,5,6- Diepoxyhexane

/0\ CH2=CH-CH-CH 2 V l l . 3,4-Epoxy-1 -butene

/O\ /0\

CH2-CH-CH20H Viii. Glycidol

~o\

CH2-CH-CH2X

XVll. Olglycidyl ether

IX- Xl, Epihalohydrlns IX. X=Cl; X. X=Br; Xl. X:[

.%

0

/0\

/0\

CHzCH-C~

CH2-CH -C-H Xll. Glycidaldehycle

CH2-CH-~=)_ '

Xlll. Chloroet hylene oxide XlV.Styrene oxide

H XlXo Benzo[a] pyrene

O

'

/0\

CH~-CH-C H2-0 -CH2- CH- CH2

/O\

"

/ ~ k CH3/-=X

CH2-'CH-CH2-O"~\ //)-CJ~

XVllf. Bisphenol A diglycidyl ether

~

OH

H H

X I Xb. 713,8a-dihydroxy-g~,l O~-epoxy7, 8,9,10- tetrahydrobenzo ra] pyrene

HO..,~_~i~,~OAc

~o,

/~--~c.2oH

CH3 (CH2)I" CH- CH'(CH2)?COOH XX. cis-g,lO-Epoxyoctadecenoic acid

CH3 O XXIL Fu~renon- X ( 12,13- Epoxytrtchothecene)

CH3 o ~ H

~o\

//~'O-CH2-CH-CH 2

0 /CH2OH O-C-C~

XXL Scopolamine

~ 0

XXlII. Epoxycyclohexene

Fig. I . F o r m u l a e o f s o m e e p o x i d e s and a f e w e p o x i d e precursors. ( E x c e p t for X V , c o m p o u n d s w e r e m o s t l y n o t r e s o l v e d i n t o o p t i c a l i s o m e r s . U n q u a l i f i e d n u m b e r s , II, III, e t c . , t h e r e f o r e r e f e r t o r a c e m a t e s o r unknown mixtures of isomers.)

C~ C I ~

CI C l i O

ci

c"~

XXIVa.Aldrln

XXIVb. Dieldrin

C, o C!

cl XXIVc. Endrin CI

Ct

C|C t ~ ~

Cl~O

CI

C l O t

XXVo. Heptochlor

XXVb. Heptachlorepoxide

CH2Ct

CH3

CH2Cl

CH20H CHC| CHOH XXVI.Ethy~ne CH20H CH3 chlorohl~lrin XXVlI.Propylene XXVIII.sec-Propylene chlorohyclrln chlorohydrln Fig. 1. C o n t i n u e d . TABLE 1 NOMENCLATURE O F E P O X I D E S , E X E M P L I F I E D BY T H E N A M E S U S E D F O R A F E W O F T H E COMPOUNDS DISCUSSED IN THE PRESENT REVIEW N u m b e r s r e f e r t o f o r m u l a e i n Fig. 1 a n d i n t h e t e x t . N a m e s u s e d in t h i s r e v i e w a r e i t a l i c i z e d . N a m e s a c c o r d i n g t o I U P A C n o m e n c l a t u r e a t e d e n o t e d b y asteriskm. Number

Trivial n a m e

N a m e u s e d in Chemical Abstracts

O t h e r n a m e s (cf. r e f . [ 1 9 0 ] )

I

Ethylene oxide

Oxirane

1,2-Epoxyethane *, e t h e n e o x i d e

II

Propylene o x i d e

Methyloxirane

1 , 2 - E p o x y p r o p a n e *, p r o p e n e o x i d e

III

--

1,2oEpoxybutane *

1,2-Butene oxide, ethyl oxirane

IV

--

2,3-Epoxybutane *

2,3-Dimethyloxirane, 2,3-butene oxide

V

Hexadeeene oxide

Tetradeeyloxirane

],2-Epoxyhexadecane *

VI

Dodeeene oxide

Deeyloxirane

1,2.Epoxydodecane *

VII

Butadiene monoxide

Ethenyloxirane

3,4.Epoxy.l-butene *

VIII

Glycidol

2,3-Epoxy-l-propanol

IX

Epichlorohydrln

(Chloromethyl)oxirane

XII

Glycidaldehyde

Oxirane eerboxaldehyde

2,3-Epoxypropanal * ~ J~-Epoxystyrene *

l-Chloro-2,3-epoxypropane

*

XIV

S t y r e n e oxide

Epoxyethylbenzene

XV

Butadiene dioxide

2,2'-Bioxirane

1,2,3,4-Diepoxybutane *

XVII

Diglycidyl ether

2,2'-[ Oxybis(methylene)]. bisoxirane

XXIII

Cyelohexene oxide

7-Oxabieyelo[4.1.0]heptane *

Bis(2,3-epoxypropyl) ether * 1,2,6,7-Diepoxy-4-oxaheptane * 1,2-Epoxycyclohexane 1,2-Oxidocyclohexane

Some epoxides are important industrial chemicals [10] and therefore present occupational health problems. This is especially true of the simple, volatile ethylene ( I ) a n d propylene (II)oxides, as well as epichlorohydrin ( I X ) a n d other intermediates in the production of e p o x y resins (e.g. XVIII). Where epoxides are used as pesticides or for the sterilization of foods, medical equipment and other materials, the hazards arising from the exposure of the public to residues of the epoxides and/or any reactive products formed from them have also to be considered. Epoxides are formed in the oxidation of alkenes with peroxides [172,232a, 319], especially peroxy acids [232a,319]. Reactions of general air pollutants leading to the formation of epoxides have been demonstrated experimentally and have been predicted from theoretical studies [232a,b,319], b u t practically nothing is known a b o u t their actual occurrence in, for example, urban air [319]. Some epoxides occur in living organisms [152] and might reach man via his food. F o o d (and animal f e e d ) m a y also become contaminated with epoxides due to the growth of moulds producing mycotoxins (e.g. those produced b y Fusarium, Fig. 1: XXII) or m a y be formed in food processing. Although data permitting a discussion of harmful consequences are still lacking, it should further be recognized that epoxidation occurs, too, in the metabolism of natural cellular constituents. One o f the main reasons for studying the genetic effects o f epoxides is the n o w well-established fact that, during detoxication in mammalian tissues, a number of unsaturated and aromatic c o m p o u n d s are metabolized via epoxides, which are proximal or ultimate carcinogens or mutagens. This was concluded b y Boyland [43] nearly three decades ago by inference from the structures o f excreted metabolites, b u t was not proved experimentally until 1968 [157,201, 202]. A wide range o f general pollutants, as well as industry-specific pollutants (e.g. alkenes [111,253,296,300], substituted alkenes such as vinyl chloride [154,331] and probably styrene [252,301,309], benzene and substituted benzenes [78] and polycyclic hydrocarbons [48,75,201]), are converted to epoxides in this w a y in vivo through the action o f mixed-function oxidases. Because c o m p o u n d s belonging to these classes are formed in many processes concerned with energy generation or food production, the clarification o f the genetic risks associated with epoxides is a problem o f very deep general concern. This review will include a discussion of a few alkylating agents having the general structure RCH--CH2--X

I

(X = leaving group; cf. 5.2.)

(2)

OH There are three reasons for broadening the review in this way: (a) these compounds introduce onto nucleophilic centres of cell components the same group, the 2-hydroxyalkyl, as do the epoxides; (b) c o m p o u n d s of this structure, such as ethylene chlorohydrin (XXVI), are often encountered as residues in products that have been exposed to epoxides, e.g. foods [346,444], and in some cases they are rapidly formed from epoxides in the stomach; (c) 2-hydroxyalkylating c o m p o u n d s may react via internally-formed epoxides: R--CH--CH2--X -~ R--CH

I OH

\,O j

CH2 + H + + X-

(3)

Several compounds, including halohydrins, 2-hydroxyalkyl methanesulphohates, 1,6
*

See f o o t n o t e ,

p. 88.

of the hundred or so epoxides which have so far been subjected to routine cancer tests. The rapidly developing research field of epoxidized metabolites of polyaromatic hydrocarbons (PAH)will only be touched upon for comparative purposes (cf. Sect. 3.). The reaction-kinetic model has been tentatively applied not only to quantitare the risks associated with compounds that have given clearly positive results in biological tests, but also, by estimating fl-errors [93], to investigate whether negative test results could be false. Despite the scarcity of quantitative data -- often limited to semi-quantitative comparisons of compounds in essentially qualitative experiments -- these analyses show a consistency that cannot be coincidental. This consistency is seen in comparisons between species, of importance for risk estimates in man, as well as in comparisons of individual compounds. The latter indicate that it is permissible to estimate risks on the basis of data for related compounds, complemented with reaction-kinetic parameters. This generalized a p p r o a c h - which has already been used for the formulation of counter-hypotheses in estimates of/3~errors -- thus differs completely from the classical approach, used by IARC and WHO for example, according to which the evaluation of a compound is almost entirely based on information on the biological effects of that particular compound. 3. Available biological and chemical data The various effects of oxiranes related to genetic (heritable) damage are summarized in Table 2. As in the case of other classes of "strong" mutagens, this list is long. Qualitatively speaking, most oxiranes are efficient mutagens. With the ultimate aim of quantifying genetic risks arising from exposure to oxiranes, we have classified the cited reports as follows: (a) Statements which, for one reason or another, were judged to be qualitative, i.e. restricted to demonstrating that the studied effect was induced, are indicated by "Qual.", followed by "Sign.?" if the significance, for statistical or experimental reasons, seemed doubtful. (b) Reported effects appearing to be of value in efforts to quantify risk are indicated by "Semiquant." or "Quant." followed by "(rel.)" if the data seem directly applicable to risk estimation or give information on effectiveness relative to that of other chemicals, resp. Many studies are of the latter kind because parallel experiments were carried out with other compounds. (c) Certain investigations aimed at studying practical applications are designated "Techn.". (d) A fourth class of data has been marked "Theor.", indicating significant theoretical contributions to the clarification of mechanisms of action of oxiranes (or alkylating agents in general). This applies, for example, to studies of dose--response curves, aftereffects and specificities. Here, the long series of investigations of reversions in Neurospora crassa by M. Westergaard, G. K¢Imark, C. Auerbach, B. Kilbey, and co-workers occupy a prominent position. Their data are of a high standard from the quantitative point of view -- as all data to be used for the clarification of mechanisms have to be -- but they have the drawback that induced back-mutation seems less useful in generalizable risk

TABLE 2

9

GENETIC TESTS WITH EPOXIDES AND WITH A FEW COMPOUNDS G EN ER A TIN G EPOXIDES References bearing asterisks are discussed in the text. The following abbreviations are used: x :>, x is more effective or more sensitive; x ~ , x is less effective or less sensitive; ~, a p p r o x i m a t e l y equal to; ~, less than or equal to. System

Agents studied

Observed effect

Comment

Ref.

Transforming DNA from Bacillus subtilis

XV, ~-butyrolaetone, peroxides

R e d u c t i o n of transforming activity, XV ~ ~-butyrolactone

Theor.

302*

DNA, calf t h y m u s

XXVI, Br- and Ianalogues

Buoyant density of DNA increased

DNAase in P-buffer

XV, ~-butyrolactone

Increased activity (~ 20% )

Sign.?

303

TMV virus and isolated RNA

I, II, d i m e t h y l sulphate, I-acetate, S-mustard

Inactivation by 2 + 1 alkylations/particle. RNA :> virus. Mutation only by d i m e t h y l sulphate (at pH 9?)

Theor.

142"

T2 phage, plaque-type mutants

I, ethylenlmine, XVII and other alkylating agents

Oxiranes non-mutagenlc, m u t a t i o n with e t h y l a t i n g agents. Post-treatment inactivation, pH 5 ~ pH 7

Theor.

262*

T7 phage

XV isomers, other alkylating agents

Inactivation. L ~ D ~ m e s o XV. Interstrand-linking of DNA by L- and D-, not by m e s o - X V

Theor.

426",427"

Mutation (forward) in rII region of phage T4 tested in E. coil B

XII, other carcinogenic and non-carcinogenic agents

Effectiveness of XII comparable Theor. to t h a t of ~-propiolacetone and propanesultone

69*

T2 phage, host-range m u t a n t s studied

I, II, e t h y l mcthanesulphonate and epoxides of PAH

No response to I, II, pos. to ethyl m e t h a n e s u l p h o n a t e and PAH epoxides

Theor.

67*

E s c h e r i c h i a coli mutat i o n to s t r e p t o m y c i n non-dependence

XV~ other agents, incl. X-rays

Mutation frequency influeneed by genetic background

Semiquant. (tel.)

150

E. coil Sd4, simplified plate test

XV and 430 other chemicals

22 c o m p o u n d s including XV mutagenlc

Qual.

404

E. coli Sd4, t r e a t m e n t in buffer

I, m e t h y l methanesulphonate

Mutation frequency per alkylation

Quant.

E. coli S d 4 , t r e a t m e n t in buffer

I, XV, TEM

Relative and absolute effeetiveness establithed

Quant.

186"

E. coli Sd4, treated in phosphate buffer

I, XIII, Cl-acetaldehyde

Dose--response per DNA a l k y l a t l o n d e t e rmi ne d, C~-acetaldehyde ~'~> XIII ~" I

Quant.

187'

E. coli B/r, her+, herstrains, m u t a t i o n to s t r e p t o m y c i n resistance

XV, UV, caffeine

Mutagenic a c t i on of X V and UV additive in rich m e d i u m , increased by caffeine. ~i]llng by UV enhanced by XV in WP-2 her-

Qual.

372 (abstr.)

(A) Acellular systems

355*

(B) Prolcaryotyes Forward mutation

1O6*,cf. 185"

10

TABLE

2 (continued)

System

Agents studied

Observed effect

Comment

Ref.

Klebsiella p n e u m o n i a e , mutation to streptomycin resistance

X X V I a n d F-, Brand I-analogues; III, VIII, XII, IX a n d Br- a n d Fanalogues, 1,2ep o x - 3 , 3 , 3 - t r i chloropropane

C o m p o u n d s m u t a g e n i c . Elect r o p h i l i c g r o u p in t h e v i c i n i t y of the oxirane ring enhances mutagenicity

Quant.

429",431"

I (12%) with CCI2F 2 (88%)

No resistant forms observed

Techn.

365*

E. coli a m i n o a c i d a u x o t r o p h s in d i f f e r e n t g e n e t i c background

XV, UV, X-rays, TEM, N-mustard

Mutation to prototrophy, influence of genetic background

Quai.

150

E. coli B / r T r y - WWP-2, t r e a t m e n t in saline suspension

XV, UV, methylnitrosourethane

No influence of amino-acid pool on revertant frequency d u e t o X V in l a t e l o g - p h a s e a n d s t a t i o n a r y - p h a s e cells; b u t w e a k a c t i o n if c a f f e i n e also p r e s e n t in e n r i c h e d medium

Theor. Chemicaily i n d u c e d e v e n t s rainc o d e d (?), n o t error-prone

E. coli B / r T r y - , t r e a t m e n t in b u f f e r

I X , diethyl sulphate, U V

Protein synthesis i m m e diately after chemical treatm e n t not required for mutation fixation, in difference from UV-induced mutation

Theor.

391

E. coli, g r o w t h o f p o l A ÷ and polA-

X X V I and Brand I-analogues

Preferential effect o n growth of PolA-, X X V I being least active

Qual.

355*

• Salmonella typhimurium H i s - T A 1 5 3 5 , t r e a t e d in phosphate buffer at 25°C

I, X I I L X X V I , Cl-acetaldehyde, C1-acetic a c i d

Linear dose response, no effect of Cl-acetic acid

Quai.

348*

S. t y p h i m u r i u m H l s TA1530, TA1535, TA1538 and G46, strains used w i t h a n d w i t h o u t $9 activation

Vinyl chloride, XIII, C l - a c e t a l d e hyde, XXVI, a n d Cl-acetic acid

Positive response in base subst, reversion strains with and without $9; phenobarbital i n d u c e d 1 5 - - 4 0 % enhancement; no significant r e v e r s i o n i n T A 1 5 3 8 ; activity of $9 fractions varied with sources

Qual.

278

S. t y p h i m u r i u m H i s TA1535, TA1537 and TA1538, spot test on agar

I, X X V I , e t h y l e n e g l y c o l , glyoxal, glycolaldehyde, glyoxalic acid, glycolic acid

Neg. r e s u l t in T A 1 5 3 7 and TA1538, I and XXVI pos. in TA1535. Other compounds neg.

Qual. (Sign.?)

119"

S. t y p h i m u r i u m HisG46 and TA1535, treatment in b u f f e r

I, X V , T E M

L a c k of response to functinnaiity observed

Q u a n t . (tel.)

186"

S. t ~ p h i r n u r i u m HisTA1530, TA1535 and TA1538, spot and plate tests

X X V I a n d Brand I-analogues and s o m e other compounds

Pos. response in TAI530, T A 1 5 3 5 , n o t in T A 1 5 3 8 . O r d e r o f m u t a g e n i c i t y in TA1530 and TA1535: bromohydrin > iodohydrin > XXVI

Qua.[.

355",356"

* Clostridium botulinum, gas e x p o s u r e o f s p o r e s at 4O°C Back-mutation

62*

11 TABLE 2 (continued) System

Agents studied

Observed effect

Comment

Ref.

S. t y p h i m u r i u m His-, TA1530 and TA1538, plate test

Mixture of lchloro-2~prop a n o l a n d 2chloro-l-propanol

M u t a g e n i c i t y in T A 1 5 3 0 b u t n o t in T A 1 5 3 6 d e m o n strated

Qual.

357*

S. t y p h i m u r i u m H i s TA100 and TA1535, plate test with microsomes

XII, III, X V , 1.2,7,8-diepoxyoctane (+294 carcinogens and non-carcinogens)

Pos. r e s p o n s e w i t h all e p o xides. Mutagenicity not annnlled by metabolism

Q u a n t . (tel.)

299*

S. t y p h i m u r i u m H i s TA1530, TA1535, TA1537, TA1538 and T A 1 0 0 , VII m i x e d w i t h top agar; for butadiene g a s e o u s e x p o s u r e s in desiccators

Butadiene, "butadiene m o n o x i d e " (VII)

Butadiene positive in TA1530 and TA1535 with and without $9, n e g . i n o t h e r s t r a i n s ; VII p o s . in T A 1 5 3 0 , T A 1 5 3 5 a n d TA100 without activation

Qual.

300*

S. t y p h i m u r i u m H i s TA1535, TA1537. TA1538, TA98 and TA100; spot test

XIV, other styrene metabolites

Only XIV mutagenic and o n l y in s t r a i n s T A 1 5 3 5 a n d TAI00

Qual.

309*

S. t y p h i m u r i u m H i s TA1535, TA1537, TA1538, TA98 and TA100

Styrene and XIV

S t y r e n e p o s . o n l y w i t h $9 a n d only in TA1535; XIV pos. o n l y in T A 1 5 3 5 a n d T A 1 0 0 with or without activating s y s t e m . In o n e s t u d y X I V possibly pos. with activat i o n in T A 1 5 3 8

Qual.

301",414"

Bacillus subtilis, "super suppressor" mutation from His- Leu-

XV, monofunctional agents

Mutation induced, with an i n f l u e n c e o f b r o t h o n frequency

71

Prophage induction and related effects E. coli K 1 2 (k); t w o stage test

XV and other agents

N-mustard ~ XV ~ TEM

Theor.

171,

E. coil K 1 2 (k). t r e a t m e n t in b u f f e r

I and other alkylating agents

Mutagenic and prophageinducing effectiveness compared; I highly effective inducer

Theor.

185"

B. megaterlum a n d Pseudomonas pyocyanea, t r e a t m e n t in c u l t u r e s

XV and other agents

Pos. r e s p o n s e w i t h X V , T E M , H 2 0 2 ; n e g . w i t h 2chloroethylamine

Qua]. (Sign.?)

271',272"

E. coli. s t r a i n s B, B / r and one N-mustardresistant strain, treatment in M9 m e d i u m

I. X V , X V I I . ethylenimine, N-mustard

P h a g e - s y n t h e t i c c a p a c i t y less a f f e c t e d t h a n cell survival b y alkylation, with a smaller influence of functionality a n d o f b a c t e r i a l s t r a i n . Cell killing b y e t h y l e n i m i n e o f m u l t i - h i t , b y X V o f singlehit type

Theor.

268*

I in e t h e r s o l u t i o n non-mutagenic

Qual.

198

(C) Eukaryo tic microorganisms Neurospora crassa Ade- reversion to Ad +

I, e t h y l e n i m i n e , other compounds

12 T A B L E 2 (continued) System

Agents studied

Observed effect

Comment

Ref.

N . crassa A d e - W 4 0

I, X V , X V I I , ethylenimine

All c o m p o u n d s a p p l i e d in water solution mutagenic, I > XV > XVII

Qual.

229*

XV° H 2 0 2 + HCHO, UV

Strains show different mutation frequencies; Ade+ > Inos+ obtained in double mutant

Qual.

2 2 4 * , see also 445*

Monofunctional epoxides , ~ R~----7~2 ,o~

R 2 = H; v a r i a t i o n o f R 1 indicates mutagenic effectiveness to vary in order: C2H 5 ~ CH3< CH2OH C H 2 B r ~ CH2CI; R1 = R 2 = C H 3 , (IV): l o w e f f e c t i v e n e s s ( T a b l e 8)

Semiquant. (rel.)

226*

Qual.

347*

"distinctus", reversion to Ade+ (mostly true backmutations) N . crassa, 5 " p s e u d o -

allelie" Inos- strains, 1 Metstrain, 1 Ade- strain; 1 double mutant AdeInosN . crassa A d e - W 4 0

"distinctus" (ad 38701), reversion to Ade+

N . crassa a d - 3 A ( 3 8 7 0 1 )

inos (37401), reversion to prototrophy

XV (pretreatment), Interaction mutations photoUV reactivable and have characteristics of UV-induced ones

N . cruasa a d - 3 A ( 3 8 7 0 1 ) and inos (37401), reversion to prototrophy

XV, sometimes I, o t h e r a g e n t s

Studies of mechanisms (see S e c t . 4 . 8 . 2 . )

Theor.

15",18"--22" 24",25"

N. crassa a d - 3 s y s t e m , reversion to prototruphy

1,2,3,4-diepoxycyclohexane 1,2,4,5-diepoxypentane, 1,2,7,8-diepoxyoctane

Diepoxycyclohexane: no mutations; other diepoxides approximately equal response

Qual.

325*

N . crassa a d - 3 s y s t e m ;

1,2,4,5-diepoxypentane, 1,2,7,8-diepoxyoctane

A b o u t 8% o f m u t a n t s c a u s e d by diepoxypentane or monofunctional agents are multilocus deletions; about 42% o f m u t a n t s c a u s e d b y diepoxyoctane have this structure

Theor.

326*

intragenic mutations and recessive multi-locus deletions

Penicillium

XV

Mutation demonstrated

Qual.

173"

treatment of conidia at conc. 0.01--0.1 M

XV isomers

Mutagenic effectiveness compared with that of X-rays

Semiquant.

316"

Saccharomyces

Fusarenon-X (XXII)

A t 7 • "10 --4 M 4 % r e s p i r a t i o n deficient mutants were obt a l n e d as a g a i n s t 1% in t h e control

Sign.? Size of Expt. not reported

410"

VIII, X I I

"Petite" mutations observed w i t h XII, v e r y f e w (sign.?) w i t h VIII; a c r o l e i n w e a k l y active; both epoxides gave back-mutation in swain $211

Qual.

192*,see also 1 9 3 "

XV and many other agents

C o r r e l a t i o n o b s e r v e d between the ability to induce gene conversion and mutation

Quant. (tel.)

467*

chrysogenum,

t r e a t m e n t o f s p o r e s (of. B r y s o n , cir. b y H e n d r y e t al. [ 1 7 3 ] ) P. m u l t i c o l o r ,

cerevisiae,

t r e a t m e n t in c u l t u r e medium S. c e r e v i s i a e ,

t r e a t m e n t in s o l i d m e d i u m

S. c e r e v i s i a e ,

d i p l o i d s t r a i n D4 c a r r y ing a defective non-comp l e m e n t i n g allele, g e n e conversion

13 TABLE 2 (continued) System

Agents studied

Observed effect

Comment

Ref.

S. cerevisiae,

Styrene and XIV

Positive r e s p o n s e o n l y w i t h XIV

Qua/.

260

XV + UV, other chemicals + UV

Synergism, photoreactivation involved at low doses but not at high doses

Qua/.

212", 213" 345*

XV, methyl methanesulphonate, Nmethyl-N'-nitroN-nitrosoguanidine

Sensitivity to XV was correfated with sensitivity to UV radiation for inactivation; response to other agents more complex

Theor.

241"

Styrene and XIV

XIV mutagenic, styrene mutagenic only in host-mediated assay

Quant. (tel.)

260

XV

Some Met- isolates responded, others did not

Qua/.

II, VIII, I X , X V , XVI

Mutagerflcity in the decreasing o r d e r : X V ~ IX ~ X V I ~> VIII ~> II

Q u a n t . (rel.)

175"

X V , I, N - m u s t a r d , 2-chlorocthylamine

At mutagenica/ly equivalent doses difunctional agents produced higher frequency of crossing-over

Q u a n t . @el.)

311"

XV and some other chemicals, ~-rays, UV

Differences in distribution of mutations at studied loci noted; XV produced a higher percentage of non-revertible mutants and transioeations than N-methyl-N '-nitro-Nnitrosoguanidine, nitrous acid and ICR-170

Qua/. Comparison done at 2--3% surviva/; d i f f e r e n c e s between a/kylating agents not drastic

XV, nitrous acid, UV

X V a n d o t h e r a g e n t s gave somewhat, although not very different, relative ratios o f m u t a t i o n s in t h e 3 l o c i

Q u a n t . (rel.)

220

XV, other a/kylating agents

O n l y N-methyl-N'-nitro-Nnitrosoguanidine produced "bleaching", indicating plasmatic mutation

Qua/.

297

treatment for 24 h

Rhizoclonium hieroglyphicum, c h r o m o s o m a l

I, e t h y l m e t h a n e sulphonate

I produced more laggards than ethyl methanesulphonate

Quai.

381

D4 s t r a i n s , g e n e c o n v e r s i o n

S. cerevisiae, survival

S. cerevisiae, 4 haploid and 4 diploid s t r a i n s , survival

Schizosaccharomyces pombe, strain ade 6-60/ rad 10-198/h-, forward mutation in 5 adenine loci

Sch. p o m b e , d i f f e r e n t Met- isolates, reversion to Met +

Sch. p o m b e , 5-h t r e a t ment, reversion of Arg-1, Leu-3 and Urd-1

Aspergillus nidulans, s a g r e g a n t s l o o k e d f o r in a diploid strain; mutat i o n t o 8 - a z a g u a n i n e resist e n c e in a sensitive h a p l o i d strain

A. nidulans, r e s p o n s e o f 8 loci compared, forward mutation

A. nidulans, r e v e r s i o n s in Su m e t h l A , su m e t h l B and Su methlC loci

63

2

(D) Algae Eug|ena gracilis,

aberrations studied

14 TABLE 2 (continued) System

Agents studied

Observed effect

Comment

Ref.

H o r d e u m vulgare ( b a r l e y ) cv. B o n u s , s e e d s ( k a r y o p s e s ) s o a k e d in w a t e r s o l u t i o n s m o s t l y a t 2 0 ° C; g r e e n h o u s e a n d field t e s t s (cf. [ 8 9 ] )

I, X V , I X , VIII, other chemicals, radiations

Dose--response curves for chlorophyll mutation, toxicity, sterility. Viable mutations: mutation spectra of chemicals different from that of radiations

Quant.

87',99", 102",103",

Treatment of seeds with gas c o m p a r e d w i t h s o l u t i o n

I

G a s t r e a t m e n t less e f f e c t i v e

Qual.

101.

H. vulgare, treatment with water solutions (volume?) 24 h at 24°C or 72 h at 3 ° C. M u t a t i o n a n a l y s i s a t

I, II, IX, X V , X V I and some other alkylating agents

XV highly effective, but of low efficiency in inducing chlorophyll mutations For chlorophyll mutations: X V ~ 5 0 × I ~ II ~ IX; X V I t> I

Semiquant. (tel.)

176.

Semiquant. (rel.)

174"

Dose response of waxy mutants

Quant. T h e or.

257" 395"

(E) H i g h e r p l a n t s "Point" mutation

~DLs0 14. vulgate, treatment of plants during meiosis with contaminated air; m u t a t e d p o l l e n g r a i n s studied

101"--103'

H. vulgate, s e e d s t r e a t e d

Allylglycidyl ether, butylglyeidyl ether

Same toxicity, chlorophyll mutation and sterility: allyl ether ~ butyl ether

Q u a n t . (rel.)

Ehrenberg, unpubl.*

H. vulgare, c h r o m o s o m a l aberrations and chlorophyll m u t a t i o n , s e e d s t r e a t e d in buffer

fl-HO-alkylating a n d fl-MeOalkyiating agents

Hydroxyisopropyl methanesulphonate ~ methoxyisopropyl methanesulphonate

Quant.

258*

A r a b i d o p s i s fhaliana, s e e d s t r e a t e d f o r 3---4 h

XV isomers

Toxicity, sterility, mutation (mostly chlorophyll): L ~ D ~ meso

Q u a n t . (rel.)

295*

A . thaliana, s e e d s t r e a t e d for 4 h at 25°C

Threitol-bisMutation: L ~ D ~ meso methanesulphonate isomers

Q u a n t . (tel.)

294*

Triticum aestivum (bread wheat, 2n = 6x), seeds treated in water solutions. Viable and chlorophyll mutations

I, o t h e r c h e m i c a l s , radiations

I and other monofunctional c h e m i c a l s gave l o w m u t a t i o n frequencies compared with radiations

Theor.

277*

Zea m a y s ( m a i z e ) , tassels treated during microsporogenesis; endosperm colour mutations analysed

XV

M o s t l y m u l t i p l e losses o f chromosome-9 dominant m a r k e r s , i n t e r p r e t e d as deletions

Qual.

234*

Z e a m a y s , tassels treated during microsporogenesis; endosperm colour mutations analysed

XV isomers

"Mutation" in markers on c h r o m o s o m e s 4 a n d 9: DL~>L~D~meso

Quant.

Zea mays, treatment of p o l l e n w i t h gas

I

Loss of dominant endosperm-colour markers

Qual.

130.

O r y z a sativa (rice), treatment of seeds with water solution

I, e t h y l m e t h a n e sulphonate

Mutational effectiveness of I inversely proportional to dose

Qual.

196", 362* (see also 2 1 0 . 364*)

29*

TABLE 2 (continued)

15

System

Agents studied

Observed effect

Comment

Ref.

Pisum sativum ( p e a ) , treatment of seeds with water solution

I, o t h e r c h e m i cals

L e t h a l a n d viable m u t a t i o n s

Qual.

Pisum sativum, m u t a t e d sectors (leaf-spots) in treated individuals

I, r a d i a t i o n s

Dose response non-linear w i t h 7 a n d I, l i n e a r w i t h neutrons

Quant.

463"

Lycopersicum esculentum (tomato), treatment of Seeds w i t h w a t e r s o l u t i o n

XV

Relatively low toxicity; high frequencies of mutation, mostly affecting chloroplasts

Semiquant.

122'

Lactuca sativa ( l e t t u c e )

I, o t h e r c h e m i cals

Induced heritable variation

Qual.

174"

35*--37*

Chromosomal aberrations (and related phenomena) Vicia faba, r o o t t i p s

XV isomers

Breaks, sister-unions, interchanges: L > D

Qual.

316"

V. faba, r o o t t i p s

L-XVa

Chromosomal aberrations, mitotic delay

Qual.

293*

V. faba, r o o t t i p s

XVII

Chromosomal aberrations. Dose defined. Dose response, Hnear for breaks; translocations, no influence of dose rate. Localization to heterochromatin

Theor.

350*

V. faba, r o o t t i p s

X V ± ¢x,cz-dipyrtdyl

Aberration frequencies enhanced by the chelator

Sign.?

65*

V. faba, r o o t t i p s

XV ± caffeine

C h r o m a t i d e x c h a n g e s , especially, e n h a n c e d b y c a f f e i n e

Quid.

398"~

V. faba, r o o t t i p s

XV, X-rays, other chemicals

I n t e r a c t i o n , t o f o r m exchanges, of XV with monofunctional agents but not with X-rays

Qua/.

209*

V. faba, Nigella damascena, t r e a t m e n t o f r o o t s o r seeds

Threitol-l,4-bismethanesulphonate, isomers

Chromosomal aberrations: L ~ D > meso

Quant. (tel.)

313",314"

V. faba, r o o t t i p s

I, VII, X V , IX, many other compounds

Chromosomal aberrations: epoxides wealdy to moderately effective; diepoxides more effective

Qual.

261'

V. faba, r o o t

XV

Frequency of aberrations dependent on conc. and temperature; increases by EDTA and protein-synthesis irthibitors, decreases by streptomycin and ehloramphenicol

Q u a n t . (rel.)

403*'

H. uulgare, t r e a t m e n t o f seeds with water solution

I, e t h y l e n imine, X-rays, neutrons '

High frequencies of chromos o m a l a b e r r a t i o n s (less if compared with those due to radiations)

Q u a n t . (rel.)

102" 315"

H. vulgate, t r e a t m e n t o f Seeds a n d r o o t s

XV, N-mustard, X-rays

Equal yield of aberrations in wild and desynaptic mutants for each agent •

Quant.

400*

H. vulgate, s o l u t i o n injected before meiosis into spike area

XV

I n c r e a s e o f cross/rig-over. (Physiological effect because actinomycin D equally effective. )

Qua].

382*

tips

TABLE 2 (continued) System

Agents studied

Observed effect

Comment

Ref.

H. s a t i v u m , Jess. var. Pirolene, seeds treated for 3 h at 25°C

L-Threitol-l,4bismethanesulphonate

Chromosomal aberrations after conversion to L-XV. G 1 a n d S p e r i o d s sensitive

Q u a n t . (rel.)

291"

A l l i u m cepa, H. vulgate, treatment of seeds

Threitol-l,4-bismethanesulphonate isomers

Chromosomal aberrations: L > D > meso, during or a f t e r c o n v e r s i o n to d i e p o x y butanes

Quant. (rel.)

290",294"

A l i i u m cepa var. S t u t t g a r t , roots treated with 0.1 mM L - X V f o r 1 h: f o r m y l e r a n analogue either a fresh soln. (pH 5.5) or completely degraded soln. (pH 5.5--6)

L-XVa; L-threitol1,4-bismethanesulphonate

Comparable effect due to X V a and transformed Lthreitol-l,4-bismethanesulphonate; fresh soln. ineffective; initially aberrations localized to heterochromatin but not later; sensitive stages S and G 1; G 2 and mitosis insensitive

Quai.

292

Tradescantia p a l u d o s a , treatment of dry pollen w i t h gas

I, m e t h y l c h l o r i d e and o t h e r agents

Chromosomal aberrations o b s e r v e d in p o l l e n m i t o s i s

Qual.

384

I, VIII, IX

Rec. lethals induced; 15--20 times higher concentration of glycols required for same effect

Quai. (original finding)

349*

I, X V , T E M ,

Relative effectiveness established

Q u a n t . (rel.)

32*

33*

(F) I n s e c t s and Crustaceans Drosophila me|anogaster

Spraying food with solution

Injection intradorsaliy compared with other techniques; sperm stage studied

one N-mustard

Intradorsal injection

XV

Detailed analysis. Mosaics for visibles a n d t r a n s i o c a t i o n s ; XV > X-rays; brood pattern; m a x . d a y s 6 - - 9 ; earliest s t a g e s s e e m m o s t sensitive t o d o r a . lethal induction

Qual.

Intradorsal injection; sperm stage studied

I, X V , o t h e r alkylating agents, X-rays

Sex-linked rec. lethals; other e f f e c t s : X V > > I. X V gives s m a l l d e f i c i e n c i e s in h i g h e r r e l a t i v e f r e q u e n c y t h a n Xrays

Q u a n t . (tel.)

132",133"

Intxadorsal injection; spermatogonia -- sperms studied

I, X V

A l t h o u g h X V > > I, s a m e ratio translocations/lethals obtained

Qua1.

318"

Intxadorsal injection; spermatids -- sperm studied

I, XV, o t h e r carcinogens/ mutagens

R a n k c o r r e l a t i o n ( l o w sign.?) induction of minutes/carcinogenicity. No induction of viable X - c h r o m o s o m e f r a g men~ (hyperploid attachedX females) by XV

Q u a l . (rel.)

135"

Treatment of mature sperm (post-copulatory vaginal douches)

II, d i e t h y l sulphate

X-linked rec. lethals observed

Qual.

366 (abstr.)

Recombination-deficient strains (c3G) or c3G × wild-type hybrids, treatment of mature sperm

XV, ethyl methanesulphonate, X-rays (XV, injected; ethyl methanesuIphonate given in food)

C o m p a r e d with wild-type, X V induces less and ethyl methanesulphonate m o r e rec. lethais in c 3 G animals. This strain is m o r e sensitive to X-rays for both lethals and translocations

Qual. ( t h e o r . )

440, 441 *

TABLE 2 (continued) System

Agents studied

Observed effect

Comment

Ref.

Tr eatm en t of sperm

I, XV, ethylenimine, TEM

Greater efficiency of difunetional agents for producing transiocations developed during storage of t re a t e d sperm

Qual. (teL)

438,439* (ef. 3 8 7 . )

Tr eatment of sperm, injection; tests for rec. lethals

Mannitol m y l e r a n and other agents

Linear dose response

Qual. (rel.)

134"

Musca domestica, 1 l~l saline solution injected in prothoracic region of 3day-old male; XV, dose 1--20/~g

XV (+12 chemosterilants, 6 azlridines, 3 methanesulphonates, bempal N-mustard)

6--100% m o r t a l i t y in 24 h due to doses higher t ha n 2 #g; 3--4 #g produc e d almost sterile survivors; d o m i n a n t lethals produc e d

Qual.

240*

Aedes aegypti, developing 4th-stage larvae treated in water w ith acetone solution of substance dispersed; t o x i c i t y tested in 2 cell lines and in mouse or rat

17 naturally occurring 12,13epoxytrichothec9-enes

Trichodermin alone larvicidal, non-toxic t o m a m m a l s and mildly de rma t i t i c

Qual.

156

Artemia salina, h atching of viable dry eggs i n c u b a t e d in t h e r m o s t a t i c a l l y k e p t m e d i u m containing substance to be studied

XV, 1,2,3,4diepoxycyclohexane and other chemical agents

XV was inh i bi t ory, whereas 1,2,3,4-diepoxycyclohexane not

Qual.

58

Human l y m p h o c y t e s , tr eated for last 24 h of cultivation or for 1 h at GO, GI--S or G 2

IX (in d i m e t h y l sulphoxide), TEPA

24-h test: 10 -4 M toxic, 10-S--10 -6 M i nduc e d chrom o s o m a l aberrations; TEPA 4--5 X IX

Effect of IX Sign.? (action of d i m e t h y l sulphoxide )

235*

Human l y m p h o c y t e s , treated for last 24 h of cultivation or for 1 h at GO, GI --S or G 2

IX (in d i m e t h y l sulphoxide), TEPA, X-rays

G-Banding applied; chemicals i n d u c e d less r a n d o m effects than X-rays

Human fibroblasts from patients with genetic instability s y n d r o m e s

XV, other agents

XV gives increased effect in Fanconi's anaemia, n o t in xeroderma pigmentosum

Qual.

453*

Chinese hamster, V79 cells, 8-azaguanine resistance

Styrene and XIV

Only XIV mutagenic

Quat.

260

Sprague--Dawley rat em br yo flbroblasts, elastogenicity, tumorigenicity, ultrastructure and histochemistry

XV, 1,2,3,4-diepoxycyclohexane

Aneuploidy, tetxaploidy, chromosom e breakage, rearrangements and tumorigenic effects observed after XV, b u t n o t after diepoxyeyclohexane

Quai.

454

Chinese h amster V79 cells, c y t o t o x i c i t y and m u t a t i o n to 8-azagnanine: ouabaln and t e m p e r a t u r e resistance

XIII, XXVI, CIacetaldehyde, CIacetic acid; I 0 POlycycllc hydrocarbons with different degrees of careinogenicity

Mutagenic response increased as a functio n of conc. of XIII and Cl-acetaldehyde; XXVI and Cl-acetic acid ineffective below 2.5 raM; carcinogenic and mutagenic effectiveness related

Quant. (tel.)

182" , 183

XV, TEM, myleran, X-rays

XV gave bridges and fragments in anaphase I and II, m a x i m a l effect 1--2 days after injection, no 2nd maxim u m for bridges found with myleran

Quant. (tel.)

312'

(G) Mammalian systems Cells cultured in vitro

I ntact animals Hybrid mice, 101 X C3H, i.p. injection, study of meiosis after different tim es

236*

18 TABLE 2 (continued) System

Agents studied

Observed effect

Comment

Ref.

Mouse, dominant lethal test

XV (27 mg/kg), other chemicals

At highest possible sublethal dose administered; " a v e r y slight e f f e c t ...; o n l y in t h e first 2 w e e k s a f t e r t r e a t m e n t ... a h i g h e r incidence of deciduomata"

No experimental details

61"

M o u s e , m a l e Swiss C D - 1 , dominant lethal test (routine)

XV (17 mg/kg), XIXa

Doubling of deciduomata per total implants, not significant (scoring 3rd w e e k , o n l y 21 f e m a l e s ) ; pos. with XIX

Cf. [ 1 2 4 ] where lack of experimental c o n s i s t e n c y is noted by authors

126"

M o u s e , I C R / H a Swiss, routine dominant lethal test of 174 compounds

X V (i.p. 1 7 m g / k g ) , X X V I , IX (i.p. 1 5 0 m g / k g ) other halohy drins, XXVb

XV, pos., but non-signific a n t w i t h i n c o n t r o l limits, others neg.

Semi-quant.

124"

Rat, Long--Evans, inhalation of contaminated air

I

5 2 5 0 p p m • h (in 3 d a y s X 7 h/day): chromosomal aberr a t i o n s in b o n e m a r r o w ; 2 0 0 - - 4 0 0 0 p p m • h (in 4 h ) : micronuclei in reticulocytes; 4 0 0 0 p p m • h (in 4 h): p o s . dominant lethal test (pos. control, TEM 0.25 mg/kg)

Quant.

119.,121"

Rat, Long--Evans, s.c. i n j e c t i o n , i n v e s t i g a tion of peripheral lymphocytes

XV

Fall i n m i t o t i c i n d e x 4 8 h a f t e r 1 m g • k g-I ; stickiness, clumping of chromosomes

Sign.?

320*

Rat, p.o.; femur bone-marr o w cells i n v e s t i g a t e d

I (9 m g • kg -1)

Increased frequency of chromosomal aberrations after 2 4 h, m u c h less a f t e r 4 8 h

Significance stated but questionable because high f r e q u e n c i e s in controls

392*

Rat, exposed to contam i n a t e d air, s p i n a l c o r d cells s t u d i e d a f t e r 4 d a y s

I (2 a n d 6 0 P P m for 66 days)

Increased incidence of chromosomal aberrations

Significance discussed in text

393*

Accidentally exposed groups of persons

I

Chromosomal aberrations in peripheral lymphocytes

Semiquant. (exposure dose may be roughly reconstructed from extent of lung damage)

104",105"

O c c u p a t i o n a l c h r o n i c exposure

I

Lymphocytosis

Qual., dose not known

104"

O c c u p a t i o n a l c h r o n i c exp o s u r e , e x t e n s i v e investigation of health parameters

I (5---10 p p m )

Increased leucocyte number (effect not mentioned by author)

Exposure dose supposedly 200--400 ppm • h/week

2 0 7 * (cf. 106")

Man

19 TABLE 2 (continued) System

Agents studied

O b s e r v e d effect

Comment

Ref.

Occupational chronic exposurc

I X ( 0 . 5 - - 5 mg/ m 3)

C h r o m o s o m a l aberrations in peripheral lymphocytes

Semi-quant.

2 3 8 ; cf. 388*

O c c u p a t i o n a l c h r o n i c exposure

Vinyl chloride

C h r o m o s o m a l aberrations in peripheral lymphocytes

Dose roughly estimated

83", 145"

O c c u p a t i o n a l c h r o n i c exposure

Styrene

C h r o m o s o m a l aberrations in lymphocytes

Qual., dose not known

304*

estimations [186]. Papers discussed in the text are marked with an asterisk (*) in the tables. Owing to the close relationship between mutagenesis and carcinogenesis, data from carcinogenicity tests on epoxides are given in Table 3. This list is limited to the relatively simple mono- and di-epoxides of primary interest to the present review: for information on a large number of other epoxides tested for carcinogenicity see reviews by Weft et al. [442], Van Duuren [415], and Lawley [246]. Tables 2 and 3 do not contain data on the large number of genetic and cancer tests performed with dihydroepoxy- and tetrahydrodihydroxyepoxy derivatives of polycyclic hydrocarbons. This field, which is developing rapidly, was reviewed in 1976 by Brookes [48]. (See also some important papers on the chemistry and biochemistry [41,184,380], mutagenicity [9,279,280,452] and carcinogenicity of these compounds [256].) A word of warning about the investigated chemicals is justified. The validity of all the conclusions drawn depends ultimately on whether or not the identity of, and absence of active impurities in, the studied compounds has been established. Furthermore, for quantitative data it is imperative that chemicals have been protected from degradation, e.g. hydrolysis [114]. With a few exceptions [415--425,437 ], publications, especially from purely biological laboratories, do not mention whether such problems have been considered or how they have been tackled. Owing to the relative stability of most epoxides, hydrolysis has probably rarely been a problem, but it is nevertheless necessary to keep uncertainties about the quality of chemicals in mind in discussions of results which are surprising or contradictory. Some epoxides have a tendency to undergo polymerization, which is by nature a concentration
TABLE 3

2 mg in acetone, 3 times weekly

E p i c h l o r o h y d r i n (IX)

(xXIID

Epoxycyclohexane

Styrene oxide (XIV)

(V)

skin

Mouse, C3H

Mouse, C3H

skin, 2-stage i.p.

skin

Mouse, ICR/Ha

Mouse, ICR/Ha

s.c.

Mouse, ICR/Ha

Mouse, ICR/Ha

s.c.

s.c.

Rat, Sprague--Dawley

Mouse, ICR/Ha

skin

Mouse, C3H Mouse, Swiss-Millerton

"Total 5 #M"

1 "brushful" undiluted, 3 times weekly

1 mg in tricaprylin weekly

2 mg in acetone once

2 mg in acetone, 3 times weekly

1 mg in tricapry]in weekly

1 mg in tricaprylin weekly

100 mg in tricap~ylin

10 mg in benzene, 3 times weekly

"Total 20 ~M"

3 times weekly

10 mg in benzene,

Millerton

"Total 20/~M" skin

10 mg in acetone, 3 times weekly

"Total 25 #M"

M o u s e , Swiss-

skin

skin

Mouse, C3H

Mouse, ICR/Ha

Mouse, C3H

Mouse, ICR/Ha

1,2-Epoxyhexadecane

"Total 20 #M"

Mouse, C3H

~100 mg undiluted, 3 times weekly

1 0 2 - E p o x y d o d e c a n e (VI)

skin

M o u s e , Swiss- M i l l e r t o n

Total 1500 mg/k~ in water twice weekly 10 mg in acetone, 3 times weekly

3 , 4 - E p o x y - l - b u t e n e (VII)

skin

s.c.

Mouse, ICR/Ha

Rat, albino

T o t a l 1 5 0 0 m g / k g i n a r a c h i s oil, t w i c e weekly

10 mg in acetone, 3 times weekly

T o t a l 1 0 0 0 m g / k g i n a x a c h i s oil, twice weekly

Dose

1 , 2 - E p o x y b u t a n e (III)

s.c.

skin

Rat, albino

M o u s e , Swiss- M i l l e r t o n

P r o p y l e n e o x i d e (II)

s.c.

Rat, albino

E t h y l e n e o x i d e (I)

Route of application

Species, s t r a i n

Compound

Unless s p e c i f i e d all t e s t s w e r e o f e x t e n d e d d u r a t i o n , r o u t e s o f a p p l i c a t i o n m e n t i o n e d w h e r e k n o w n .

CARCINOGENIC ACTIVITY OF SOME EPOXIDES

5/30

0/40

0 (11)/30

1 (9)/30

0/50

7/50

2/50

0/20

0/30

1/22

1 (2)/30

3/19

1(2)/41

8/30

0/30

4/21

1 (2)/30

0/30

3/12

8/12

0•30

0/12

Turnouts malignant (benign)/ number of surviving animals

1 skin turnout 3 real. l y m p h o m a s 1 pulm. adenoma

11 l u n g p a p i l l o m a s

9 papillomas

1 pulm. adenoma

3 papillomas

1 squamous cancer

Mal. l y m p h o m a s

7 malignant lymphomas, 1 pulm. adenoma

Malignant lymphomas

No skin irritation

Comment

232a

442

417

418

420

424

232a

423

232a

232a 419

419

232a

423

419

436

424

436

Ref.

b~ O

(structure not specified)

6 (10)/30

1 0 m g in a c e t o n e , 3 t i m e s w e e k l y 3 mg in acetone, 3 times weekly

skin, 2-stage

Mouse, Swiss-Millerton

Weak initiator

1 (1)/30 0 (1)/30

1 0 m g in a c e t o n e , 3 t i m e s w e e k l y

skin skin skin

Mouse, Swiss-Millerton Mouse, Swiss-Millerton Mouse, Swiss-Millerton

1 (1)/20

424

Strongly toxic

15140

"Total 1 mM"

M o u s e , C57B1

1 mg once

423

12 skin turnouts, 3 mal. lymphomas, c o n t r o l ? (of. t e x t ) Strongly toxic

10/50

232a

421

298

0/50

5/30

I.I mg in tricaprylin weekly

Cancerogenic potency revealed i n all tests; c o n trois neg.; skin damage reported

173

5 m g in t r i c a p r y l l n w e e k l y

s.c. gastric feeding

Rat, Sprague--Dawley Rat, Sprague--Dawley

7/50

0.I mg in tricaprylin weekly

Not reported

H i g h t o x i c i t y in mice; skin test negative

173

419

420

421

419 424

1 m g in t r i c a p r y l i n w e e k l y

s.c. s.c.

Mouse, ICR/Ha

inhalation and Not reported skin

M o u s e , A a n d C57B1

Mouse, ICRIHa

s.c.

Rat, Long--Evans

None

(b) 2 d r o p s o f a 2 5 % s o l u t i o n i n a c e t o n e for 3 weeks + 2 drops of a 12.5% solution twice during the following week

skin

M o u s e , C57B1

None

(a) 1 d r o p o f a 1 0 % s o l u t i o n in a r a c h i s oil f o r 3 m o n t h s ( 6 6 d o s e s )

skin

1/14

Mouse, C57Bi

3 (6)/41

20 mglkg twice weekly, 6--7 weeks + 10 mg/kg twice weekly, 6 weeks

i.p.

Rat, albino

10 mg in acetone, 3 times weekly

skin

Mouse, ICR/Ha

D,L-Diepoxybutane (XVa)

1/50 5/20

1 mg in t r i c a p r y l i n weekly 33 mg in tricaprylin weekly

s.c. s.c.

Rat, Sprague--Dawley Rat, Sprague--Dawley

I/50 0/5

3 3 m g in t r i c a p r y l i n w e e k l y

intragastric

1 m g in t r i c a p r y l i n w e e k l y

Rat, Sprague--Dawley

7 (1)/30

3.3 mg in tricaprylin weekly

s.c.

Rat, Sprague--Dawley

3/50

0.1 mg in tricaprylin weekly

s.c. s.c.

8 (8)/30

Mouse, ICR/Ha Mouse, ICR/Ha

0/20

3 mg in benzene, 3 times weekly

skin

M o u s e , SwissMiilerton

G l y c i d a l d e h y d e (XII)

5 mg in acetone, 3 times weekly

skin

Mouse, ICR/Ha

Glycidol (VIII)

to

i.p.

Mouse, AIJ

skin

s.c. s.c. s.c. skin

Mouse, Swiss-Millerton

Mouse, ICR/Ha Mouse, ICR/Ha Rat, S p r a g u e - - D a w l e y Mouse, ICRJHa M o u s e , C57B1

1,2,5,6-Diepoxyhexane (XVI), a n d s o m e o t h e r diepoxides

skin skin skin skin,2-stage

Mouse, Mouse, Mouse, Mouse,

meso-Diepoxybutane (XVb)

Swiss-Millerton Swiss-Mfllerton Swiss-Millerton Swiss-Milierton

i.p.

Mouse, A / J

L-Diepoxybutane

skin

Mouse, C 3 H

(Structure not specified)

Route of application

Species, strain

Compound

Table 3 (continued)

0.1 mg in tricaprylin, weekly 1.1 m g in t r i c a p r y l i n , w e e k l y 1 mg in tricaprylin, weekly 2 m g in a c e t o n e , 3 t i m e s w e e k l y "Total 1 mM"

5 m g in a c e t o n e , 3 t i m e s w e e k l y

1 0 0 m g in a c e t o n e , 3 t i m e s w e e k l y 3 mg in acetone, 3 times weekly 10 mg in acetone, 3 times weekly 1 mg once

3--192 mg in tricaprylin 3 times X 4 weeks

1 . 7 - - 1 9 2 m g in w a t e r , 3 t i m e s X 4 w e e k s

"1 brushful" 10% acetone solution, 3 times weekly

Dose

(2)/30 (1)/30 (5)/30 (4)•20

2/50 3/30 1/50 10 (13)/30 2•20

0 (4)/30

4 0 4 0

Lung turnouts

Lung turnouts

1 (1)/3

Turnouts malignant (benign)/number of surviving animals

423 424

375

442

Ref.

control? (cf. t e x t )

232a

419

C a r c i n o m a in diepoxypentane, and diepoxyheptane, not in d i e p o x y c y c l o o c t a n e 421

Somewhat toxic Weak initiator

Toxic

I n c r e a s e significant, despite high v a l u e in c o n t r o l s

Weak initiator

Comment

t~

1/20 ?/? Lung turnouts

0•50

"Total 0.25 mM" not given 0.034--34 mmoles/kg, 3 times weekly, 4 weeks 2 mg in tricaprylin, 3 times weekly 1 mg weekly

Rat, Long-Evans

i.v. i.v.

i.v. i.v. i.v. i.v. s.c.

Mouse CF1, female Mouse CF1, female

Mouse CF1, female Mouse CF1, female

Mouse, A/He, female Mouse, A/He, female Rat, Fischer

s.c.

Mouse, ICR/Ha

Ethylene chlorohydrin ( X X V I ) (also ethylene glycol)

skin

Mouse, ICR/Ha

3-Chloro-l,2-propanediol

i.p.

Mouse, A/J

Mannitol myleran

Neg. control + vehicle control

5/18 2/18

1.2 rag, 7 times monthly 0.2 ml Ringer solution, 7 times monthly (control) 1 . 2 m g , single inj. 0.2 ml Ringer solution (control) 0.3--10 mg/kg, 2 times weekly for 52 weeks females: 21/100 males: 8/120; females: 18/120

3/100;

10/45 7/48 males:

5/46 7/48

1 . 2 r a g , single inj. 0.2 ml Ringer solution (control)

1/50

4/10

"Total 0.75 mM"

s.c.

4 (7)/30

1 mg in acetone, 3 times weekly

skin

Mouse, ICR]Ha

Mouse, C57BI

1,2,7,8-Diepoxyoctane

Diglycidyl ether (XVII)

Lung turnouts No significant i n c r e a s e in c a s e of ethylene glycol

289

181

417

298 375

Significant increase

232a

419

No effect

Only skin tumours, no mal. lymphomas

t~

24

In addition to the above types of effect, growth inhibition and lethal action, at the level of organisms or specific tissues such as tumours or bone marrow, have at least partly a genetic mechanism. The latter effects, including variations in leucocyte numbers, will be discussed below insofar as t h e y can yield information of use in risk estimation. An understanding o f mechanisms is essential to the clarification of dose-response relationships, especially at very low doses. This is because samples studied in experiments or epidemiological investigations are by necessity of finite size and have statistically a limited resolving power [93]. Therefore, no response or no effect is found at and below some low dose or level. The question of whether this dose/level should be interpreted as a "threshold" or "noeffect l e v e l " - - a s has often been done in discussions o f carcinogenicity [90, 188] o f chemicals and radiations -- can only be answered by: (a) clarifying the mechanism at doses at which a response is observed, and (b) proving that the same mechanism is valid, or not valid, at doses below the resolving power of the system investigated. For these reasons, certain characteristics, especially the shape of dose-response curves and the influence o f modifying factors, will be discussed in this chapter (Sect. 4.8.). An assessment of the influence of di-(poly-)functionality on genetic risk, as well as a discussion o f basic mechanisms pertaining to functionality, depends on quantitative chemical and biological data, and will therefore be treated in Sect. 5. 4.1. Point mutation 4.1.1. Epoxides Genetic changes recorded as point mutations have been observed in almost the whole spectrum of experimental organisms, ranging from microorganisms to higher plants and animals. This is illustrated in Table 2, where the investigations are listed according to the test organism. Mono- and difunctional epoxides have been applied to forward and backward mutation systems in prokaryotes and eukaryotes. Cell culture systems in vitro have been used for detecting induced mutation to 8-azaguanine [182,260,279] or ouabain resistance [182, 279] mainly in hydrocarbon (PAH) mutagenesis. In aceUular systems (Table 2A), including tests for mutation in transforming principle [302], viruses [142, 267, 268] or virus nucleic acid [142], inactivation usually predominates over mutation *. This is consistent with the general idea, developed by Loveless [262,263], that point mutation in a virus is caused by a specific, relatively rare type of chemical event, favoured by a relatively low substrate constant, s, characteristic of ethyl methanesulphonate and N-alkyl-N-nitrosoureas but not o f m e t h y l methanesulphonate and simple epoxides, e.g., I, II (cf. Table 4). Among epoxides there are, however, two important exceptions -- mutation in viruses

* O f t h e c o m p o u n d s s t u d i e d in T M V b y F r a e n k e l - C o n r a t et al. [ 1 4 2 ] , o n l y d i m e t h y l s u l p h a t e w a s m u t a g e n l c . T h i s m i g h t have b e e n d u e t o t h e high p H u s e d in s t u d i e s w i t h this c o m p o u n d ; at h i g h e r p H , G u a - O 6 is m o r e d i s s o c i a t e d and a v a i l a b l e f o r a l k y l a t i o n . T h e e p o x i d e s , t h e s o f w h i c h are n o t m u c h h i g h e r t h a n t h a t o f d i m e t h y l s u l p h a t e , w o u l d p r o b a b l y have b e e n e f f e c t i v e if s t u d i e d u n d e r the same conditions.

25

being observed after treatment with (a) glycidaldehyde (XII) [69], and (b) certain epoxy derivatives of polyaromatic hydrocarbons (PAH) of the general type XIXb [67,69]. As will be discussed below, compounds of these types interact with DNA through specific functions on the residual molecule, namely the aldehyde group or the hydrocarbon moiety (see Sect. 5.4.4.). A number of monofunctional and difunctional epoxides have been tested on S. typhimurium His- strains responding to base-pair transition (G46,TA1530, 1535, 100) or frame-shift mutation (TA1537, 1538, 98). It is clear that ethylene oxide (I), simple monosubstituted oxiranes (III, IX, XIII, XIV) and diepoxides (diepoxybutane XV, 1,2,7,8~liepoxyoctane) induce mutation by the former mechanism. This is also true of the primary reaction product of I with chloride, ethylene chlorohydrin (XXVI). Few studies have been carried out, however, to permit the conclusion that a frame-shift mechanism would not operate. Such studies have been carried out with the unsubstituted compound I [119], with styrene oxide (XIV) [301,309] and with XXVI [119,356], but not with difunctional agents (e.g. XV). A decision on this point would be of interest because base-pair transition in the Salmonella strains cannot account for the high effectiveness of such compounds in forward-mutation systems (see Sect. 5.4.4.). The same applies for the aldehyde XII, which is of interest because a frame-shift mechanism was shown to operate in addition to base-pair transition (mainly AT to GC) in a viral system [69]. Epoxy derivatives of benzo[a]pyrene and other PAH induce mutation in Salmonella strains responding to either mechanism, in some cases with a preponderance of the one mechanism, in others of the other [279,280]. The implications of such specificities for the nature of the reactive intermediates in the carcinogenic action of PAH -- which often give frame-shift mutations in the Ames test in the presence of activating enzymes [8,9] -- is beyond the scope of the present review. It is, however, of interest that XIXa induces dominant lethal mutations in the mouse [124,125], indicating that reactive epoxides are formed in, or are long-lived enough to reach, the gonads. Much circumstantial evidence indicates that, as in the case of many other alkylating agents and ionizing radiation, the majority of point mutations induced by epoxides in higher organisms are deletions. The importance to the phenotypic response of elimination of cells with gross deletions is well illustrated by a comparison of diploid barley with hexaploid wheat. At corresponding survival rates, monofunctional alkylating agents such as I produce higher mutation frequencies than do radiations (i.e. they are more "efficient") [87, 88], whereas the reverse is found in wheat where, owing to the genetic buffering of polyploidy [277], chromosomal aberrations are not lethal. For similar reasons, difunctional alkylating agents, despite a high mutagenic effectiveness at very low doses, are never able to give rise to high mutation frequencies in the diploids, although their mutagenic efficiency may be considerable in wheat, as inferred from experiments with myleran [277]. Sterility in later generations after chemical treatment of barley kernels is mostly due to mutation, whereas most radiation-induced sterility is caused by translocation [115] (cf. Sect. 4.9.). The ability of epoxides to induce deletions of different sizes may be inferred from studies of loss of chromosome markers for endosperm colour after treat-

26 ment of maize pollen with d i e p o x y b u t a n e (XV) [28--30,234] or ethylene oxide (I) [ 130,385]. In contrast, practically no mutations were observed in the following diploid plant generation, probably as a consequence of the elimination of ceils with drastic changes in the chromosome material [234]. This difference m a y have a parallel in the high frequencies of w a x y ( w x ) mutations phenotypically manifested in the mature pollen grains after treatment during meiosis of barley plants with I [257,395] or ionizing radiation [97,321] as compared with a moderate mutagenic efficiency of these agents as judged from frequencies of lethal and viable mutations scorable in the following generation [102,161,446]. For 7-radiation it was found that only few pollen grains carrying a w x mutation were able to transfer the mutation to the following generation [129]. This indicates that these mutations are mainly deletions affecting the normal functioning of the pollen grain, i.e. they are subjected to haplontic or diplontic elimination. It is also of interest that treatment of maize pollen with a highly efficient "point mutagen" such as ethyl methanesulphonate is claimed to give rise to relatively few endosperm mutations [28]. However, in the experiment described, with w x mutations in barley, mutations induced b y ethyl methanesulphonate are more frequently transferred to the following generation than mutations induced by 7-radiation. This observation agrees with other indications that ethyl methanesulphonate is a relatively more efficient inducer of point mutation than ionizing radiation and, probably, many epoxides. Sex-linked recessive lethals were observed in Drosophila in tests in which either XV was fed to larvae or the agents I or XV were injected into the males [32]. In an extensive cytogenetic study of the action of XV and other carcinogens and t u m o u r inhibitors, dominant, recessive visible, lethal and semi-lethal mutations were observed in feeding and injection tests [33]; also in this study a certain sex-linked visible mutation was found to be associated with a " t u m o u r " mutation. An analysis of the sex-linked recessive lethals and reduction in fertility showed that the t w o endpoints followed the same course, except for the fourth brood (9--12 days) where the reduction in fertility (probably due to dominant lethals) became very marked and the percentage of sex-linked recessive lethals declined [33]. Further studies [133] aimed at comparing the cytogenetic effects of physico-chemical agents showed that 200 new sexdinked recessive visibles differing in p h e n o t y p e and genetic position from those due to radiations were induced by chemicals (including XV); the distribution of the recessive lethals was the same for the chemicals, b u t was different from that of X-rays. The sex-linked recessive lethals due to XV and other chemicals appeared to be associated with a higher frequency of small deficiencies and a lower frequency of chromosomal breaks than those due to X-rays [133]. In this stud); the ratio of sex4inked recessive visibles to lethals was also noted to be the same among XV and other chemicals. In another study [135], no rank correlation between the induction of point mutations and carcinogenicity in mammals could be discerned for alkylating agents (XV included), whereas a correlation was observed for the induction of minutes and carcinogenicity. For a general discussion of the influence of functionality on relative frequencies of point mutation and deletion, see Sect. 5.4.4.

27 4.1.2. Alkylene halohydrins Alkylene halohydrins appear, for example, as products of the reaction of epoxides with chloride in fumigated foods [444,448, cf. 283,284] or in the stomach or blood. Mutation tests with these compounds are usually positive. For example, according to data in Table 2 [124,348,356,431], and a recent review [139], ethylene chlorohydrin is mutagenic, and the corresponding bromo- and iodohydrins seem to be even more effective [355,429,431], which is consistent with their greater reactivity. Of the reaction products of propylene oxide with CI-, 1-chloro-2-propanol (XXVIII) was tested as a mixture (of unstated purity) with the isomeric 2-chloro-l-propanol (XXVII) [357]. These tests were positive. Since, on the basis of reactivity, the latter compound is expected to be more effective, both isomers should be considered to be mutagenic. Depending on the pH, the halohydrins may alkylate "directly", i.e., with C1- as leaving group (formula (9)), or via the formation of an epoxide (Sect. 2., formula (3), see also Sect. 5.4.2.). Epichlorohydrins gave the same antifertility effects as the alkylene halohydrins [166,194,204]. 3-Chloro-l,2-propanediol ("~-chlorohydrin"), which has been considered for use as an antifertility agent [26,54,55,68,72,86,127,128,149,194,219,233,306, 408], induced an increased mutation frequency in E. coli Sd4 (S. Hussain, unpublished), but a dominant lethal test on this compound in the mouse gave no significant increase in the number of dead implants [124,204]. As discussed in Sect. 6.2., this test cannot be considered conclusive. Application of a-chlorohydrin to mouse skin, or intraperitoneal injections in mice, caused no tumours, whereas subcutaneous injections induced a non-significant (p > 0.05) number of malignant tumours [417]. It is evident that a trihalogeno propane such as 1,2
28 and meiotic crossing-over differed, and a remarkable mutagen specificity for certain regions was reported. High frequencies of somatic crossing-over were observed after treatment of barley roots with 2-hydroxyethyl methanesulphonate (A.T. Natarajan, unpublished). This indicates that 2-hydroxyalkylation, the chemical change produced by epoxides, effectively induces somatic crossingover. This effect has been suggested to be one step in cancer induction [466]. 4.3. Chromosomal aberrations and related effects Observations of chromosomal aberrations, mainly in plant roots such as those of Vicia faba [265,350], induced by difunctional epoxides (XV--XVII) and mustards, as well as observations of tumour growth inhibition [354], were important in the development of early hypotheses on mechanisms, especially with regard to the "radiomimetic" activities of difunctional agents [151,163, 164,265]. As discussed below (Sect. 5.4.4.), it was soon found that monofunctional agents, including monoepoxides, produced similar effects [ 31,261 ]. In his early comparative study of chromosomal aberrations induced in Vicia faba by X-rays and diglycidyl ether (XVII), ReveU [350], thanks to his exact, quantitative way of working, arrived at conclusions which have played a central role in the clarification of mechanisms. His main findings were as follows: (a) X-Rays produce aberrations during the whole interphase ("resting stage"), although sensitivity was greatest in its later part (nowadays called G2), whereas XVII was active only during G1. (b) Whereas X-rays produce relatively more chromatid fragments in G2 than in G,, the stage has no influence on the spectrum of effects of XVII during its period of activity. (It was later clarified that, in G1, radiation gives rise to chromosomal aberrations and alkylating agents to chromatid aberrations, whereas in G2, where most chemicals are inactive, radiations induce mainly chromatid aberrations.) (c) Chemically induced aberrations show a stronger tendency than the corresponding radiation effects to cluster in heterochromatic segments of the chromosomes. Revell was also the first to demonstrate the importance of the dose; within a 60-fold range of concentrations of XVII, equal effect was obtained if the product of concentration and time was kept constant; cf. Eqn. (8b), p. 53. Diepoxybutane (XV) has been applied in several studies aimed at the clarification of mechanisms. The demonstration of the additivity of breaks, exchanges and isochromatid deletions produced by X-rays and XV may indicate that the primary changes produced by the two agents are basically different in nature, although the separation in time of initial events may have played a role [64,65]. IrL contrast, interactions of the effects of XV with those of other chemicals [64,65], including monofunctional alkylating agents [209], were found, indicating that the primary effects are identical. In experiments with Allium eepa and Vicia faba, Matagne [290,293] obtained evidence of maximal sensitivity in the early S-phase. The frequency of aberrations, especially interchanges, induced by XV was enhanced by caffeine, post-treatment being the most effective [398,399,401]. Other experimental results are consistent with a maximal effect of caffeine in the S-phase following treatment with XV [402], which agrees to a certain extent with Matagne's observations. In barley a

29 desynaptic mutant was as sensitive as the wild-type to the production of chromosomal aberrations by L-threitol-l,4-bismethanesulphonate [ 291 ], which is active after conversion to XV [77, cf. also 400] (see Sect. 2 formula (3); Sect. 5.4.3.). Along with their reports of chemically induced mutation in Drosophila, Fahmy and Bird [132,133,135] have given detailed descriptions of the chromosomal aberrations observed. Deficiencies were most common in the dark-band regions [132]. (For further details see Table 2 and Sects. 4.8.1. and 5.4.4 .) Compared with alkylating agents of several other classes, epoxides, irrespective of functionality, are relatively efficient inducers of chromosomal aberrations. In plant seeds, ethylene oxide (I) is highly effective in this respect [315], as is ethylenimine. In contrast, several non-substituted alkyl methanesulphonares (except for the methyl ester) are not efficient in this respect but are highly efficient inducers of point mutation. The chromosome-breaking ability may be a general feature of 2-hydroxyalkylating agents, as indicated by biological and chemical comparisons with corresponding 2-methoxyalkylating compounds (cf. Sect. 5.4.4.3.(d)). A high chromosome-breaking efficiency of epoxides has also been found in mammals. Treatment of mice with XV gave rise to aberrations observed in male meiosis [312]. After exposure of rats to I, aberrations were observed in the bone marrow [119], also at low doses [392,393]. These aberrations included micronuclei [119,120]. Aberrations in bone-marrow cells of the mouse were also induced by epichlorohydrin (IX) [388]. In rats chronically exposed to ethylene chlorohydrin an increased frequency of chromosomal aberrations in the bone marrow was observed [371]. Abnormalities, including stickiness and chromosome fragmentation, were claimed to have been observed in cultured l~mphocytes from rats treated with low doses of XV [320]. Certain quantitative aspects of the mammalian data are discussed in Sects. 4.8., 5. and 6. In a few cases, chromosomal aberrations have been found in workers exposed to epoxides. Considerable frequencies of breaks, chromosome translocations and chromatid exchanges were observed in a group of 8 persons 18 months after an accidental exposure to high concentrations of I [104,105]. In persons occupationally exposed to IX, Ku~erov~ and coworkers [238] observed a slight, but probably significant rise in various types of aberration. They applied techniques elaborated in vitro [235--237]. Similar effects have also been associated with exposure to compounds that are presumably metabolized to epoxides, such as vinyl chloride (XIII) [83,145] and styrene (XIV) [304]. Benzene, probably metabolized to an epoxide, produces chromosomal aberrations in mammals (see review by Dean [78]) and gives, like benzo[a]pyrene (XIX), a positive result in the micronucleus test [383 ]. Data on epoxide-caused dominant lethal mutations in insects are scanty. Study of the mutation frequency in different broods of Drosophila, carried out by injecting 0.1% XV solution into males, which were then mated to successive sets of females, has indicated that for dominant lethality the earliest germ-cell stage (fourth .brood) is the most sensitive stage, whereas the ceils of the second brood represent the least sensitive stage of the 4 broods [33]. Increases in the frequencies of dominant lethal mutations and translocations have been reported during storage for a week in the seminal receptacles of untreated females, of

30 spermatozoa treated with XV or TEM [438,439]. (For further comments on effects of storage, see Sect. 4.8.2.) Dominant lethals were also reported following treatments of mature sperms of Musca domestica b y injection of XV solution into the males [240]; the associated damage to male genitalia was considered to be repairable to a certain extent. Dominant lethal mutations, which should be seen as a consequence of chromosomal aberrations in germ-line cells, were observed after acute exposure of rats to I [119,121]. In the mouse they were induced by XV [61,124] and there were indications that they were induced by XIX [126]. These effects will be discussed in Sect. 6. Exposure of male rats to I at 61 ppm for 66 days, immediately followed b y mating, gave a highly significant increase in embryonal mortality, whereas the increase produced by exposure to 2 ppm was only weakly significant [ 393 ]. No observation of precocious abortions following exposure of human males have been reported. (An increased frequency of miscarriages may be the most important finding revealed b y a medical examination of w o m e n occupationally exposed to I [458]. This effect is of d o u b t f u l significance because of the scanty material. Further studies, including recording of exposure periods, are required to clarify the aetiology.)

4.4. Carcinogenicity 4.4.1. Epoxides Some 100 epoxides have been evaluated for carcinogenicity, especially in the extensive work of Van Duuren et al. [415--425], Weil et al. [442] and Shimkin et al. [375]. Carcinogenic activity was revealed in a large fraction of the difunctional, and also many of the monofunctional, epoxides tested. (See Table 3 for c o m p o u n d s discussed in this review.) In two-stage carcinogenesis studies with IX, XII and XV it was established that the c o m p o u n d s act as initiators [417, 419,425]. Because cancer transformation of a cell requires homozygosity of a recessive mutation, it is of interest to note that somatic crossing-over m a y be induced b y epoxides (cf. Sect. 4.2.). There is thus no d o u b t that, in general, epoxides should be considered to be carcinogenic. To this should be added the n o w well-established fact that 1,2epoxidation is a step in the metabolic detoxication of, for example, aromatic hydrocarbons and unsaturated c o m p o u n d s such as alkenes (ethene, vinyl chloride) and that the mutagenic and related activities of their epoxide intermediates make it likely that they are the proximal carcinogens. No transformation experiments in vitro seem to have been carried o u t with simple epoxides. Although the value of this system is still under debate, n o t least because of the low plating efficiency of cells [80], it appears to be a tool for use in the detection of carcinogenicity. Positive results have been reported for PAH (for review see [79]) and PAH epoxides [239].

4.4.2. Alkylene halohydrins Cancer tests on ethylene chlorohydrin (XXVI) in mice [181] and rats [289] were negative (see Table 3), as were parallel tests on the reaction p r o d u c t of I with water, and ethylene glycol, in the same studies. (For a c o m m e n t , see Sect.

31 6.2.1.) The former study [181] also included an experiment with injection site transfer, to shorten the latency period. Cancer tests with 3~hloro-l,2-propanediol, applied to skin or s.c. in the mouse, showed no significant increase in local tumours [417]. A small increase in the number of lung adenomas was found after i.p. injection of mannitol myleran [375]. This is a bifunctionai agent which, like chlorohydrins, introduces a 2-hydroxy-alkyl group, possibly via epoxide formation in vivo (eqn. (3)). 4.5. Prophage-inducing activity Prophage-inducing activity has been correlated with carcinogenic potency [125], tumoricidai activity [123,171] and mutagenic activity [171,185]. The test system is sensitive and available in many microorganisms. So far, a prophageinducing activity has been reported for ethylene oxide (I) in E. coli K12 [185] and for diepoxybutane (XV) in Bacillus megatherium, Pseudomonas pyocyanea [271,272] and E: coli K12 [171]. As with other hydroxyalkylating agents, I had a high inducing efficiency. Several alkyl methanesulphonates have exhibited a relatively low inducing/mutagenic effectiveness ratio in E. coli, whereas after treatment with I this ratio approaches the value found for ionizing radiations [185]. 4.6. Haematological and related effects Because of rapid cell division, damage to bone-marrow cells is manifested soon after exposure to radiation or alkylating agents, as characteristic changes in the relative and absolute numbers of various cell types in peripheral blood. In fact, this effect is the main cause of death ("bone-marrow death") occurring within a few weeks of acute whole-body irradiation. Alkylating agents show certain specificities towards cells of the myeloid and lymphoid series, a fact utilized in specific chemotherapy of certain types of leukemia. Elson [117] recognized two main patterns in the action of alkylating agents, the "myleran t y p e " and the "chlorambucil type". The former is characterized mainly by a suppression of polymorphonuclear leucocytes and the latter by a rapid supPression of lymphocytes and simultaneously some suppression followed by a large increase (overshoot) in the number of polymorphonuclears. The effect of X-rays is an average of both types, with suppression of both lymphocytes and polymorphonuclears at sublethal doses and increased numbers of both kinds of cell some time after exposure to low doses. Diepoxybutane (XV) has been reported [117] to induce changes of the myleran type. However, after injection of 50 mg/kg into rats a significant "overshoot" of lymphocytes followed a transient drop in the number of cells of this type [117]. In rats, rabbits and dogs, diglycidyl ether (XVII) caused a rapid reduction in the total leucocyte count and in the number of nucleated bone-marrow cells, followed by an increase in the relative number of polymorphonuclear leucocytes [179]. These effects were found irrespective of the route of administration: skin contact, inhalation or i.v. injection. Short-term studies in rats on ethylene oxide (I) showed a similar pattern, i.e. a drop in

32 l y m p h o c y t e count [104,105]. As with XVII, a depletion of lymphoid organs was concluded from the significant thymus involution [179]. After percutaneous application, e p o x y resin A, a technical diepoxide structurally related to XVIII, produced effects similar to those obtained with XVII, although they were weaker [179]. The haematological picture in rats after daily injections of epichlorohydrin (IX) for one m o n t h , or thrice weekly injections for 12 weeks, of doses amounting to 10 or 20% of the DLs0 (in the latter case also including doses of 50% of the DLs0), was characterized by an increased variation both upwards and downwards in the relative and absolute numbers of different types of leucocyte [251]. After acute exposure to epoxides, humans react in a similar way. In personnel occupationally exposed to I a significant lymphocytosis was observed [ 104], the l y m p h o c y t e levels returning to normal within a year after improvem e n t in the ventilation. Similar effects were observed among radiological workers in the twenties and thirties, when daily or weekly doses of the order of 1 rad were n o t u n c o m m o n . (For review, see ref. [106].) A significant decrease in the haemoglobin content of the blood of workers has also been observed [104], in agreement with findings in animal experiments [179,223,251]. In many respects the haematological changes produced by epoxides resemble those produced by radiations, and probably have, partly at least, a genetic mechanism. With the introduction of stringent safety regulations such changes are rarely found n o w in normal radiological activities, b u t they might give some guidance as to current dose levels of chemical exposure [106]. It is important to note that apart from a variation in individual sensitivity, partly determined b y infections, the time after acute exposure, or incidental high-level peaks in chronic exposure, plays a great role in determining the number of peripheral lymphocytes. The possible role in leukaemogenesis of bone-marrow disturbances of these kinds is discussed in Sect. 6.6.

4.7. Genetic effects of certain compounds of technical importance or occurring in nature 4.7.1. Insecticides Certain chlorinated cyclodiene-type insecticides are effectively oxidized by mixed-function oxidases into relatively stable epoxides, some of which are also used as insecticides. (See [322] for a review of the structure and toxicity of cyclodienes.) Aldrin (XXIVa) is converted to dieldrin (XXIVb), isodrin (formula n o t shown) to endrin (XXIVc), a structural isomer of XXIVb, and heptachlor (XXVa) to heptachlor epoxide (XXVb). These epoxidations can be carried o u t with variable efficiency by mammals, insects, plants and microorganisms [51]. The epoxides XXIVb and XXVb are so stable that they accumulate, at least to an equilibrium level, in mammalian (including human) adipose tissue [369], and they remain intact for years in the soft. This fact, together with positive results in cancer tests on some of the c o m p o u n d s (XXIVa, b [76], XXVa, b [369]) have led to severe restrictions, if n o t prohibition, on their use in many countries. Endrin (XXIVc) is not accumulated in tissues, b u t is more toxic than XXIVb, hence its use is subjected to similar restrictions. The fate of

33 the compounds in vivo will be discussed briefly in Sect. 5.3. The above-mentioned insecticides all produce increased frequencies of liver turnouts in the mouse. The validity of this end-point for risk prediction for man has been doubted [108,155] because the production of liver tumours by these compounds may be due to a promotive action at high doses, rather than to initiation through a chemical reaction with the gene material. At least for XXIVb and XXVb, the reactivity of the epoxy function is certainly very low owing to steric hindrance (cf. Sect. 5.2) and the carcinogenic activity, when observed, is probably not connected with epoxide reactivity. In general agreement herewith, the results of mutation tests with XXIVa [136,299] and XXIVb [136] in several microbial systems were negative. It is consistent with the low reactivity of XXIVc that a small (but perhaps not insignificant) increase in chromosomal aberrations was induced in Vicia faba root tips [457] and barley [455], and that no significant increase in the mutation frequency [455] or disturbances at meiosis were observed after the treatment of seed or spraying of plants with this compound [456]. In Crepis sprouts, XXIVb gave rise to Cmitosis, rather than aberrations [288]. No mutation test data seem to be available for XXVb. Administration of doses of from 4% of the DLs0 of XXIVa, XXIVb and XXVa produced an increase in chromosome rearrangement frequency in mouse bone-marrow cells [287]. Teratogenic effects of XXIVa, XXIVb and XXIVc have been studied in hamsters and mice after oral administration [334]. The administered doses caused similar anomalies in both species but, in contrast with hamsters, there were no foetal deaths in mice. XXIVa, XXIVb, XXIVc, XXVa and XXVb were tested for dominant lethal mutations in mice by Epstein et al. [124]. All were negative except for XXIVa and even in this case the effect was not significant. However, the resolving power of these tests was very limited (Sect. 6.2.2.).

4.7,2. Epoxy resins In the absence of values for reactivity under physiological conditions, the question of the carcinogenicity of difunctional epoxides such as "bisphenol A diglycidyl ether" (XVIII) used in epoxy-resin technology cannot be discussed in quantitative terms (cf. refs. [92,197]). One reported cancer test on XVIII gave essentially negative results [442]. However, positi~;e results with other epoxides of similar structure and of comparable molecular weight [178,442], and also with a poorly defined modified compound described as an "adduct" of XVIII [442], justify the suspicion that XVIII is carcinogenic and call for further tests. These suspicions are strengthened by the demonstration of mutagenicity in the Salmonella back-mutation test of not only XVIII but also a resin (Araldite CY 1400 KS; Ciba-Geigy), the mutagenicity of which was stronger than could be accounted for by the butyl- and cresyl-glyc~dyl ethers present (A. Hedenstedt, personal communication). For compounds of this type, the ability of the large molecules to penetrate cell nuclei has to be considered when risk is estimated. 4.7.3. Epoxides in foods, pharmaceuticals, etc. Epoxidized fatty acids of the general type illustrated by epoxystearic acid (XX) occur naturally in oils of certain species [190] and may also be formed

34 during storage of, for example, sunflower seeds [307]. Hence, there is a chance of such c o m p o u n d s reaching man via his food. Owing to steric hindrance, epoxides of this t y p e have low reactivity (cf. discussion of IV in Sect. 5.2.) and constitute a relatively small genetic risk. The available results from cancer tests may be considered negative, although s.c. injection of cis-9,10-epoxystearic acid into BALB/c mice led to a few local sarcomas (reviewed by IARC [190]). However, "epoxidized oil" is considered to be carcinogenic [232a]. In view of the general carcinogenicity of epoxides (Sect. 6.), epoxidized fatty acids should be considered to be weakly carcinogenic until evidence to the contrary is produced. Epoxides of sugars and sugar alcohols play an important role in the preparative chemistry of carbohydrates. Heating dry sugars is said to give rise to "anhydro sugars" sometimes containing an oxirane ring, as in ~-D-glucosan formed from s-D-glucose [148]. The reactivity of such anhydro sugars is illustrated by their tendency to polymerize. It is n o t known whether, or to what extent, such compounds, which are potentially mutagenic, are formed during food preparation. In ~-D-glucosan the ethylene oxide ring comprises carbons 1 and 2, i.e. is functionally a lactol ring, and hence is expected to be highly reactive. This raises the question of whether glycol aldehyde (HOCH2CHO), shown to be a n effective mutagen in the E. coli Sd4 test (S. Hussain, unpublished), is in equilibrium with the lactol (CH2--CH--OH) (cf. Karrer [208] ).

\/

O Other examples of metabolic products with an epoxide structure are the drug scopolamine (XXI), occurring in species of Solanaceae, and fusarenon-X (XXII), a 12,13~epoxytrichothecene occurring in Fusarium and other fungi imperfecti [169]. The mutagenic potential of XXI has not been studied except for a clastogenic effect which has been observed in human cells CHeLa cells) in vitro [432]. Because most things in this life and the next seem to be clastogenic in cultured mammalian cells [374], it is difficult to judge to what extent such a result reflects a genetic risk. XXII [410] was reported to have some mutagenic activity but this is of doubtful significance, partly because it was tested in a non-established system (Table 2B). Leucopenia and bone-marrow damage are characteristic of the toxic s y m p t o m s produced b y the trichothecenes [138, 410]. Therefore the c o m p o u n d s may be considered radiomimetic (cf. Sect. 4.6.). It is n o t known to what extent the oxirane ring in XXI and XXII plays a role in these effects.

4.8. Special studies pertaining to mechanisms The chemical reaction mechanisms and the fate of epoxides will be discussed in Sects. 5.2. and 5.3., resp., and this section will mainly treat certain aspects of biological end-effects which can shed light on action mechanisms. Dose-response relationships for survival and mutation, together with cytological studies and background information on the modification of effects under different conditions reveal relevant features of the behaviour of these agents in biological systems. The data on cancer will only be considered for agents of immediate concern in this review, and then only in so far as they add to the understanding of the genetic data.

35 4.8.1. Dose--response relationships

Generally, genetic effects of radiations and chemicals have dose (D)--response (R) curves of the general shape [53,413]: R = C + aD + fJD2

(4)

where C is the spontaneous (control) frequency of the observed effect and and ~ are empirical coefficients. In certain cases, terms with higher exponents of D have to be added. In curves of this shape the linear component, indicating a one-hit mechanism, predominates at low-to-intermediate doses. At higher doses, the square component becomes more important, showing a contribution from effects with a two-hit mechanism. Superimposed on (4) the following deviations are often observed: (a) a rise with an exponent > 2 above some dose [409], and (b) a decrease of the response, evidently due to killing of target cells at high doses [109,409]. (This effect, which is of limited interest in risk estimation, may be described by multiplying the D and D 2 terms of eqn. (4) by a negative exponent of D [53,94] .) At very low doses or dose rates a deviation from linearity may also be found. Owing to experimental difficulties, relatively little is known about this region. A supra-linear "hump", a deviation having the consequence that linear extrapolation to zero dose would underestimate risk at very low doses and/or dose rates, has been reported for various experimental systems exposed to radiation [97]. It has also been suggested to occur in the case of radiation-induced mutation in the mouse [254,274] and radiation-induced cancer in man [53,96]. The resultant dose--response curves, which have been discussed as regards genetic effects of ionizing radiations [96,254,413], have a complicated shape that is often misinterpreted in risk estimation. The relative importance of the recognized components of the dose--response curve varies strongly with compound, dose rate *, biological system and end-point studied. 4.8.1.1. Mutation

In various biological materials, such as barley (compounds I, VIII, IX, XV) [87,99], E. coli (S. Hussain, unpublished) and Sch. p o m b e (compounds II, VIII, IX, XV, XVI) [175], data concerning forward mutation induced by epoxides are consistent with a predominance of the linear portion of the dose-response curve over a wide range of doses from low up to and above DLs0. This is illustrated in Figs. 2 and 3 which show dose--response curves for I and rac. diepoxybutane (XVa) in E. coli Sd4 and for I in barley (unpublished data; cf. [99]). These data are consistent with a one-hit mechanism for induction of mutation, the square term of (4) being negligible. Back-mutation in S. t y p h i m u r i u m His- strains is repeatedly claimed to exhibit [299], and has sometimes been demonstrated (e.g. refs. [119,348])to exhibit, linear dose--response curves. To what extent this could be a conse-

* T h e chemical intensity factor equivalent to the radiobiological c o n c e p t o f d o s e rate (e.g. expressed in t a d " r a i n - 1 ) is c o n c e n t r a t i o n , e x p r e s s e d as m o l a z (M = m o l e . 1-1 ), t h e c h e m i c a l d o s e being expressed in c o n c e n t r a t i o n X t i m e (ef. refs. [ 1 0 6 , 2 4 2 ] a n d e q n . ( 8 ) ) .

36

/ s° , / s X'¢ ,s

f

~2 j

a.

• Ss f

J

o

/

A*" .-"

/ //"

•

""

./

•

//" ."

J

//

[

../

/,6f

f

f/

/

/

ss~

sJ

// ~S

/

///

f

ss

o

//"

/"

./"

A/'A •

I 1 log (raM-h)

'

0

I 2

I 3

Fig. 2. D o s e - - r e s p o n s e c u r v e s f o r m u t a t i o n i n E. coli S d 4 i n d u c e d b y e t h y l e n e butane (XVa). (S. Hussain, u n p u b l i s h e d d a t a . )

oxide

(I) a n d DL-diepoxy-

quence of deficient repair capacity (cf. [ 2 4 5 ] ) does not seem to have been discussed. For epoxides in barley the exponential increase in mutation frequency at high doses found for many other alkylating agents is small or absent. It should be remembered that such an exponential rise at high doses may be counteracted by the drop of the curve due to cell death, observed with most mutagenic

//' ss

sI

sS /s JsS /s S /s

ss

Ij

ssisssssI Is/s/ /s S ss//s/ s s s s t

I

50

I

rnM.h

I

100

,

I

150

Fig. 3. D o s e - - r e s p o n s e curve for chlorophyll mutation in barley (percentage by e t h y l e n e o x i d e . (Ref. [89] and L. E h r e n b e r g , u n p u b l i s h e d d a t a . )

p e r spike progeny) i n d u c e d

37 agents above some critical dose, giving a resultant curve that cannot be distinguished from a straight line [52,53,96,413]. In the highly sensitive test for waxy mutations in barley [89], the curve for ethylene oxide (I) exhibits a strong exponential component [257,395] at doses above 100 ppm for 24 h, indicating involvement of two-hit effects or an involvement of saturation of repair enzymes. Similar effects were found with v-radiation [97 ]. In careful kinetic studies of reversions in N . crassa ad-3A induced by I and XV, KSlmark and Kilbey [228] could correlate mutation frequencies (M) to exponential functions of dose ( c t = concentration × time = dose) (5)

M = a • (ct) b

where a is a mutagen-specific constant and the exponent b assumed values slightly greater than 2 for both I and XV. Experiments with microconidia gave similar results, with b rather close to 2. Although a two-hit mechanism might appear attractive, it seems to be excluded by the fact that a value of b > 2 was consistently and significantly obtained in macroconidia. Curves obtained at different concentrations (c) were superimposable. Exponential curves of a similar shape were also obtained for mutation induced by L- and D-XV, in P e n i c i l l i u m m u l t i c o l o r [316]. At low dose rates (i.e., low concentrations: see footnote, page 35) an almost linear curve was obtained for XV i n N . crassa, and the somewhat scattered data with 1.5--5 mM of I showed a similar tendency (at least, they do not differ significantly from a linear curve [228]). The mechanism of this effect is discussed in the following sub-section (4.8.2.), and some quantitative aspects in Sect. 5.4. Owing to experimental difficulties, it has not yet been possible to carry out an analysis of deviations from linearity at very low doses of chemicals, although such deviations have been demonstrated unambiguously and with good statistical significance in certain systems exposed to ionizing radiation [97,321]. Data on the mutagenic action of ethylene oxide (I) in barley (waxy mutations induced during meiosis) [257] and in E. c o i l Sd4 (S. Hussain, unpublished) indicate t h a t I may exhibit the same " h u m p " as is found with v-radiation in these systems. However, these effects have not yet been shown to be statistically significant. Dose--response curves of this kind might be obtained if certain repair enzymes are inducible [45,245]. It should be pointed out that the opposite effect, a dip in the curve, might be expected for other pathways of mutation induction [cf. 46,95]. 4.8.1.2. Chromosomal

aberrations

Reliable dose--response curves for this effect of epoxides have been determined only rarely. Revell's [350] data for digiycidyl ether (XVII) in V i c i a f a b a indicate an exponential curve at low doses, approaching saturation at higher doses. Several sets of data are in agreement with a linear dose response for breaks, as exemplified by the effects of XV in Vicia roots [316] or at anaphaseII in mouse spermatocytes [312]. On the other hand, exchange frequencies show an exponential or linear-exponential dependence on dose, as in the Vicia experiments of Moutschen-Dahmen et al. [316], t h a t is not distinguishable from eqn. (4). However, in a careful study over a narrow concentration range,

38 Swietlifiska et al. [403] found that breaks and sister-chromatid exchanges increased exponentially with dose, in contrast with chromatid exchanges which increase with a lower exponent of the dose. Over a wide range, concentration (i.e. dose rate) had little effect. Leaf spots, a kind of somatic mutation scorable especially in leguminous species, have been reported to follow the same exponential function of dose with ethylene oxide and X-rays [463]. The value of the exponent, a b o u t 1.6, is consistent with a contribution of both the linear and square components of (4). High-LET radiation produces this effect in one-hit kinetics. The data thus agree with chromosomal aberrations being a cause of the effect. Sterility in the mature plants obtained from exposed karyopses of barley and other grasses is a sensitive measure of genetic damage [89,330], although the specificity of this end-point has to be considered with care. The effect is mainly caused by translocations, although dominant lethal mutations (large deficiencies) and recessive lethal mutations may also be important. The dose--response relationship is different for monofunctional I, where the square c o m p o n e n t of (4) predominates, and for difunctional XV, where data, although scanty, are consistent with a linear response [99]. A similar difference between XV and I was found as regards the colony-forming ability of E. coli strains and the phagesynthesizing ability of the bacteria [268]. 4.8.1.3. Cancer Data permitting the analysis of dose--response curves for cancer induced b y epoxides are scanty, most tests having been carried out at one or t w o dose levels. The dose--response curve for lung tumours induced by L-diepoxybutane [375] is consistent with a one-hit initiation mechanism, or rather, in view of the uncertainty at lower doses because of the high background value, it should be described as n o t inconsistent with this mechanism (cf. Fig. 4).

75

50

t ..............ti....................I........

.<

~ 2s

~5

,~

~15

2!o

2!5

mmol kg"1

Fig. 4. P e r c e n t a g e o f a n i m a l s ( A / J m i c e ) w i t h p u l m o n a r y t u m o u r s 89 w e e k s a f t e r 12 t h r i c e - w e e k l y i.p. i n j e c t i o n s o f L - d i e p o x y b u t a n e , p r e s e n t e d as a f u n c t i o n o f t o t a l a m o u n t i n j e c t e d , e , e x p e r i m e n t a l p o i n t s , with 80% confidence intervals; ,, general control value; ...... , t h e o r e t i c a l o n e - h i t curve [ r e s p o n s e = ( 1 - - e --(aCO+b)) X 1 0 0 % ] t h r o u g h c o n t r o l and h i g h e s t - d o s e p o i n t s ( 3 5 a n d 7 8 % , r e s p . ) . CO, t o t a l injected dose (correctly: initial concentration); a a n d b , c o n s t a n t s . D a t a f r o m S h i m k i n e t al. [ 3 7 5 ] . P o i n t s f o r t h e s a m e k i n d o f t u r n o u t s i n d u c e d b y X - r a y s i n R F M m i c e [ 4 6 1 , 4 6 2 ] ( a , s i n g l e d o s e ; u , fract i o n a t e d d o s e ) are i n t r o d u c e d i n t h e scale o f t h e t a d - e q u i v a l e n c e o f d i e p o x y b u t a n e (cf. t e x t S e c t . 6 . 1 . 2 . ) .

39 Where the linear term in (4) predominates and the variation in sensitivity is small, the fraction of tumour-bearing animals would follow the expression N * / N o = 1 -- e -~D

(6)

where N* = number of affected animals, and No = the total number of animals. The ability of a one-hit mechanism to explain the dose--response relationship -- in agreement with linearity at low doses -- is then best analysed by the semilogarithmic representation of the fraction of unaffected animals, N / N o = 1 - N* /No,

In N / N o = ---~D

(6b)

which gives a linear function of dose with slope --~ [465]. Analysed in this way [205, cf. 107], Bryan and Shimkin's [56] now classical, much
Available dose--response data for the induction by epoxides of genetic effects -- including point mutation, cancer, prophage and maybe certain chromosomal aberrations -- suggest the involvement of a one-hit process. This was carefully investigated with ethylene oxide in E. coli Sd4 (Fig. 2) where doses causing an increase of the mutation frequency by some 10% above the spontaneous level are, if anything, more effective than would be expected from a linear relationship. The conclusion of linearity is fundamental in risk estimation, especially when collective doses and collective dose commitments are applied to risk estimation in populations where there are variations in the doses received by individuals [ 191]. 4.8.2. M o d i f y i n g factors

A couple of epoxides, notably diepoxybutane (XV) and ethylene oxide (I), played a role in the early demonstration by Westergaard, KSlmark and collaborators of specific sensitivities of an Ade- and an Inos- allele in N. crassa to back-mutation by different agents [224,445]. In an Ade- Inos- double mutant the ratio of Ade/Inos back-mutations was 3 orders of magnitude higher with XV than with UV radiation as mutagenic agents. This was considered to indicate that back-mutation frequency was dependent upon how the gene was orig-

40 inally damaged [281,445]. This principle was later beautifully clarified for Neurospora b y Malling and De Serres [282] and is n o w utilized for routine testing in Ames's His- mutants of S. t y p h i m u r i u m [ 8 , 9 ] . It was no surprise that Inos- strains were found with a mutation pattern similar to that of the Adestrain (cf. K¢lmark, 1953 [224]). However, a closer analysis of the Ade/Inos case revealed a number of peculiarities with regard to the induced reversions, especially in the allele at hand in ad-3A (38701). These data, indicating great specificities in mutagen--gene interaction [15,22,211], together with the finding that Ade reversions in N. crassa exhibit a non-linear response to the dose of epoxides [228] (cf. Sect. 4.8.1., formula (5)), have made certain geneticists take a pessimistic view of simplified or general methods for risk evaluation [16,216]. It should be pointed o u t here that these data concern back-mutation and therefore are of little relevance [186] to risk estimation. It should also be remembered that the exponential dose--response curves were obtained at doses and dose rates (i.e. concentrations) several orders of magnitude higher than those that are of practical interest in environmental mutagenesis and that at reasonably low dose rates the dose--response curves approach linearity [228] (Sect. 4.8.1.). However, the allele specificities in Neurospora contain several leads to the understanding of mutagenic mechanisms and of synergisms. This justifies a brief review of the" data here. Some data indicate that d i e p o x y b u t a n e (XV) sensitized the cells to Ade reversions induced b y the same c o m p o u n d , i.e. a kind of "self-synergism", which might be of general importance. A role of residual XV in treated conidia was indicated b y a post-treatment storage effect on mutation frequency [225], which could be eliminated by the use of conditions, such as stirring the solution, which keep the conidia in suspension [227]. A similar after-effect of residual I was found, perhaps with the difference that I was more easily washed o u t than XV [217]. A similar storage effect, b u t probably with a different mechanism, has been observed in Drosophila; exposure of spermatozoa to I and XV, followed by storage in the female seminal receptacle, produced an increased frequency of translocations in the case of XV, whereas there was no storage effect after treatment with I [439,441]. This seemed to indicate a qualitative difference between the action spectra of difunctional and monofunctional alkylating agents. It was later established, however, that a storage effect developed, although more slowly, after treatment with monofunctional agents [17,387]. This finding may be taken as evidence that the influence of functionality is restricted to quantitative differences, as discussed in Sect. 5.4.4. In support of a role of residual XV in the after-effect, Auerbach and Ramsay [21] found, in Neurospora, that a " b o o s t e r " dose of XV, so low that it had little effect alone, given before or after the mutagenic treatment with XV, could considerably enhance the effect of the latter. The same authors also found [20, 24] that the sensitization b y the booster dose, which also enhances frequencies of mutation induced by other agents such as UV and HNO2, was transient, being lost within 2--4 h at 25°C. Kilbey [214] then showed that this decrease of the booster effect could be prevented b y treatment of the cells with the protein-synthesis inhibitor cycloheximide. This allowed, for the first time, a separation of the sensitizing action of low doses of an epoxide and the effect of

41 residual concentrations of the agent. He also showed that cycloheximide could maintain the sensitization by XV to induction of mutation by UV, demonstrated earlier by Auerbach and Ramsay [15,20] and by Rannug [347]. This strongly indicates that the exponentiality of dose--response curves [228] and the booster effect of XV [20,21] are due to an impairment of the ability of cells to cope with mutagenic damage. The linear curves obtained at very low dose rates seem to indicate that sufficient time was available for the reactivation of inactivated repair functions. This is in agreement with the observation that linear dose--response curves at low dose rates were changed to exponential ones if cycloheximide was present during the treatment [215]. The system affected in the sensitization by XV is evidently not the photoreactivation [345, 347]. The mechanism is complex, however, as indicated by the synergistic action of UV and XV on the survival of yeast [211--213,345]. At low doses of XV, photoreactivation was disturbed, although at higher doses it remained unimpaired, the synergism acting at some other level. A similar study of Inos revertants shows the opposite behaviour, with a very slow increase in mutation frequency with increasing dose and a tendency of the curve to bend downwards at high doses [18]. Similarly, pretreatment with XV decreases the frequency of UV-induced mutations [20]. Here, XV evidently interferes with the expression of the primary mutation event [17]. The nasty situation of having two opposite effects of XV on different genes would be avoided if a common formula could be found, e.g. by demonstrating that the pre-mutational lesion is repaired in an error-free way in one case and via an error-prone mechanism in the other. One difficulty, however, is the observation that several Inos- mutants are resistant to reversion by XV. Maybe a clue to the explanation is to be found in the observation [25,336] that the low Inos*/Ade÷ mutant ratios are "normalized" in the slowly growing poky strain, with a defective respiration and in the perhaps related phenomenon that the specificity is not found in growing cultures, as opposed to conidia in vitro [19]. These phenomena are probably not specific effects of epoxides. For instance, the "Inos-specific" N-ethyl-N-nitrosourethane exhibits a similar storage effect on Ade- reversions (but not Inos- reversions) in the double mutants [23]. Pretreatment with formaldehyde or H202, like pretreatment with XV, increases the yield of Ade reversions and decreases the yield of Inos reversions induced by UV irradiation [281]. Pretreatment with nitrous acid or a mixture of formaldehyde and H202 had a similar effect on UV-induced reversions in the 2 genes, whereas ethylnitrosourethane enhanced UV-induced Inos reversions as well [22]. Several monofunctional agents act synergistically with UV in yeast [345]. It is possible that the exponential part of the dose--response curves observed with, for example, ethyl methanesulphonate [109,409] reflects a related mechanism. The role of the genetic background was studied in some detail by Allison [7], who compared the abilities of XV, ethyl methanesulphonate and UV to induce reversions to Ade ÷ and Inos ÷ in double mutants of a few ad-3A and Inos- alleles. Some influence of the genotype on the mutability of individual alleles was observed, especially with the chemicals, although certain regularities may be noted. Neurospora ad-3A (38701) exhibited the highest mutation frequencies to both ethyl methanesulphonate and XV. in all combinations, and

42 the allele (0077) with the lowest sensitivity to XV also showed the lowest mutation frequencies after treatment with ethyl methanesulphonate. This indicates that difunctionality plays little role in the allele specificities of the ad-3A locus. On the other hand, whereas Inos reversions appeared at very low frequencies in all d i e p o x y b u t a n e series, the response to ethyl methanesulphonate showed a great variation, although the same Inos- allele (2626) showed the lowest response to both XV and ethyl methanesulphonate. This allele also showed the lowest mutability in response to UV irradiation [211].

4.9. Applications of genetic effects Under this heading a few general remarks will be made, without any effort to cover the subject completely. For example, the vast field of comparative mutagenesis in plants of epoxides and other agents should be summarized in a different context.

4.9.1. Plant breeding Several studies of the mutagenic action of epoxides in higher plants (barley [87,99,101--103,174,176], wheat [277], rice [196,362,364], maize [29,130, 234], oats [317], t o m a t o [122], pea [35--37], lettuce [174]) have aimed at finding suitable methods for the creation of a heritable variation that could be applied in plant breeding. One reason for studying various mutagens in plant breeding w o r k has been the hope of finding useful specificities with regard to mutation spectra. However, no systematic study has been carried o u t that permits the evaluation of the yield of beneficial mutations at any given size of starting material and input of work. By and large, monofunctional epoxides -as judged from studies of ethylene oxide (I), glycidol (VIII) and epichlorohydrin (IX) in barley [103] and I in the pea [37] -- give a b o u t the same spectrum of mutant phenotypes as do other alkylating agents. This is indicated, for example, by the relative frequencies of different types of so-called chlorophyll mutations and the ratio of viable mutations to chlorophyll mutations [102, 103,160--162,446]. In the eceriferum [270] and erectoides [339] phenotypes, mutations have been assigned to great numbers of loci, which exhibit certain specificities with regard to LET of radiations, and chemicals compared with radiations. Too few epoxide-induced mutants have been assigned to particular loci in sufficient numbers to provide an adequate basis for the comparison of individual chemicals. If differences occur, methanesulphonic esters seem to give a narrower spectrum of eceriferum mutants, with a high proportion of mutants in loci c, q and u, whereas ethylenimine and I seem to give broader spectra than that obtained with radiations [270]. Some mutants not obtained with alkyl alkanesulphonates were induced b y I. The spectra of erectoides mutants are similar; it is of interest to note that I and ethylenimine produced mutation in the "neutron-specific" locus c [ 339]. I has a mutagenic efficiency (i.e. the highest mutation frequency that can be obtained at DLs0, for example [88] ) which is approximately equal to or somewhat higher than that of radiations and thus lower than that of certain other chemicals such as ethyl methanesulphonate. This is possibly a consequence of the chromosome-breaking and lethal effects of 2-hydroxy-alkylating agents

43 (see Sects. 4.8.1.2. and 5.4.4.3.). It is in fact indicated that the mutation spectrum of I is intermediate between that of X-rays and methanesulphonic esters [446]. Diepoxybutane (XV) is a highly effective mutagen, when mutation frequency per unit dose is considered, but owing to its high toxicity , it is rather inefficient in so far as high mutation frequencies cannot be reached [87,99]. Sterility in barley appears to be proportional to the square of the dose in the case of monofunctional I [102] and methanesulphonates [330], whereas it is linearly related to the dose in the case of XV [102]; hence a high ratio of X1 sterility to mutation frequency is obtained with the difunctional XV, which is consistent with the action of nitrogen mustard [275]. Owing to the genetic buffering in hexaploid wheat, I and other monofunctional alkylating agents (ethylenimine, ethyl methanesulphonate, diethyl sulphate) are relatively inefficient mutagens in this material [276,277]. Because myleran appears to be more efficient in wheat than in barley, studies on other difunctional agents are of interest. The action of XV has never been studied in wheat, however. For various reasons, chemicals other than epoxides have been preferred in practical breeding work. In our view, the combination of mutagenic and chromosome-breaking efficiency that is characteristic of epoxides would be interesting in some situations. In such work, giycidol (VIII) offers several advantages. It gives a good yield of induced genetic variation [87,103] and, owing to its low volatility, it is easier to handle than I and ethylenimine. Furthermore, it gives more reproducible results, and involves less risk to applicators (cf. 4.9.3. below). Preliminary studies on the usefulness of epoxides for the breeding of drugproducing microorganisms have been carried out [406]; but for this purpose other mutagenic agents have sometimes been preferred.

4.9. 2. Other applications A number of other biological applications of epoxides or other alkylating agents utilize effects which are essentially produced by genetic mechanisms. This seems to be true for sterilization of food, soft, medical equipment and other materials with simple epoxides, sterilization of mammals (with alkylene halohydrins possibly acting via the generation of epoxides) and tumour-growth inhibition, etc. 4.9.3. Unwanted side-effects When epoxides are used for sterilization, there is a risk of inducing "adaptive" forms of microorganisms. The results of preliminary investigations have been negative [365], and in all probability this risk is small, as shown in more extensive studies on radiation sterilization. Furthermore, in all practical applications of chemicals having genetic toxicity, occupational risks to personnel and risks to consumers (e.g. of sterilized products) or to the general public have to be acknowledged. In many countries and situations those who utilize or carry out research on applications of biological effects, represent, or should represent, the expertise in unwanted side-effects. This concerns not least plant breeders with their basic education in genetics. These persons therefore have the responsibility to furnish administrators with the information that is basic to the introduction of measures aimed at the protection of public health [113].

44 It will be shown below (Chapter 6) that the above-mentioned risks are considerable, or in any case non-negligible; consequently, scientists, technologists and industrialists must assume this responsibility. 5. Quantitative aspects 5.1. General

As specified in the Introduction, the quantitative evaluation of data has two main purposes, namely (a) to quantify the hazards to human health posed b y current exposure levels of c o m p o u n d s already shown to be mutagenic and/or carcinogenic, and (b) to investigate the probability that negative results in tests for mutation or cancer are false [93], especially with regard to the possibility that, owing to limited resolving p o w e r [90,93], these tests may n o t have succeeded in excluding an unacceptable risk. Data of these kinds are required for a sound assessment of threshold limit values (TLVs), for comparative risk-cost--benefit evaluations of alternative technical procedures, e.g. for food preservation, etc. Quantification of risk requires a c o m m o n unit, allowing a given exposure dose [e.g. expressed in p p m (in air) X h (exposure time) or in mg X (kg b o d y weight) -1 X h] to be related to the risk of heritable damage or cancer. The only environmental factor that has so far been evaluated with regard to risk coefficients for expected frequencies of cancer and mutation per unit exposure dose is ionizing radiation. Although there may be other solutions to this problem, it appeared interesting n o t only to use the general principles of radiological health evaluation as a model b u t also to investigate the possibilities of applying the risk coefficients for unit radiation dose as a standard in expressing chemical risk. Several comparisons between chemicals and radiation with regard to effectiveness in inducing genetic effects have been made in the past (see, e.g., refs. [66,133,312]). Bridges [44] and Latarjet [242], in particular, have contributed to the development of the concept of "rad-equivalence" of harmful exposures to chemicals. The tad-equivalence appears useful in risk estimation for mutation and related effects, although it should be borne in mind, as pointed o u t by F a h m y and Bird [132,133], that XV, compared with X-rays, gives somewhat less aberrations and more small deletions associated with a certain number of "point mutations". Restricting comparisons to the linear or nearly-linear low-dose portions of dose--response curves (Sect. 4.8.1.), which is a necessity for application of the rad-equivalence in estimates of detriment t o exposed populations [95,191], and expressing mutation frequencies per unit dose, D, of alkylating agents in terms o f the number of fads of acute 7-radiation giving the same mutation frequency, the following expression was found to be approximately valid [95, 110,186] Mutation frequency = 1 • 107 • D • kn=2[], fi rad-equivalents (rad-equ.)

(7)

In (7) the various constants have the following meanings: D = "tissue d o s e " [106] or "target d o s e " [405] of the proximal or ultimate mutagen/carcinogen, defined as

45 D = J C ( t ) dt

(8)

t

(dimension M • h, i.e. mole per 1 or per kg b.w. X h); k,= 2 = rate constant (dimension M -1 • h -1) for the bimolecular reaction of the

alkylating agent at nucleophilic strength = 2, probably a mean value of some groups in D N A with a relatively low reactivity; fi = a product of correction factors for steric effects, influence of charge, difunctionality, distribution in the b o d y , etc. [91,186]; i • 107 = the inverse value of the degree of alkylation of nucleophiles Y- at

~

n=2,

[RY.=2] IV;=2] that, for simple monofunctional alkylating agents (where I~ fi ~ 1), gives the same mutation frequency as 1 tad of v-radiation; this degree of alkylation is equal to the product D × kn= 2. Expression (7) was found to be valid within a factor of a b o u t 2 for bacteria and plants (barley) and, where the dose (D) could be estimated in mammals, to agree acceptably with available data for mutation frequencies [95,106,328]. (The tad-equivalent as defined b y (7) differs from the " r e c " of Committee 17 [66] primarily in the definition of dose.) This leads to the following two generalizations: (a) An alkylating agent, or c o m p o u n d metabolized to an alkylating agent, is potentially mutagenic, with an effectiveness determined by its reactivity at n = 2. Possible exceptions might be c o m p o u n d s with a complete steric hindrance of the reaction with the critical groups o f DNA, i.e., where the corresponding " f " of (7) is zero; so far no such case is known. In all probability this conclusion is valid for induced cancer as well. (b) The risk expression (7) might be applied, at least preliminarily, to obtain an estimate of the risk of heritable damage in man. D a t a for dose--response curves for malignant tumours induced b y radiation and chemicals are n o t yet available in the same strains of laboratory animals, and the validity of (7) for estimating cancer risk is therefore n o t known. However, certain data in mouse strains thought to be comparable indicate that the cancer risk at low doses might be quantified by using the risk expression (7) (c;f. refs. [91,95] and Sects. 6.1.3. and 6.2.1. below). ::It is recognized that eqn. (7) is based on empirical parameters, and that the ~antiness of data upon which it is based calls for prudence in its application. In particular, it has to be anticipated that unforeseen metabolites of high mutagenic effectiveness may involve a risk that deviates from the estimate on the basis of the electrophilic predecessor. When we nevertheless permit ourselves to apply tentatively the rad-equivalence as a basis of risk estimation, it is mainly for the reason that no other method is at present available -- besides the rare possibility of using epidemiological data from human populations. Eqn. (7) indicates, further, parameters of importance to risk in inter-comparisons of related c o m p o u n d s or of species, e.g. laboratory organisms and man. For a generalized risk estimate for epoxides in accordance with these considerations, certain qualitative and quantitative information on reactions in vitro ~ d in vivo is required. The reactions of an epoxide of the general formula

46 RtCH

CHR2

~0

/

are complicated by: (a) the possibility that nucleophilic attack may occur on either of the two carbons, resulting in different products, (b) the fact that the reaction pattern might be quite different under the neutral conditions in cells and at the low pH of the stomach, and (c) the fact that the compound may exist in different steric configurations. The following simplified review of the chemistry of epoxides will try to elucidate some matters with the following objectives: (a) to provide a background to the understanding of the fate of specific compounds in vivo, when absorbed by different pathways; (b) to serve as a background to the evaluation of possible correction factors, fi, in (7), for steric effects or secondary reactivity of substituents R1, R2; and (c) to provide a basis for risk estimation in man. For (b) and (c) reaction-kinetic data are required, both for the determination of doses according to (8) and for the estimation of k,=2 in expression (7). 5.2. Chemical reactions o f epoxides This Section will summarize briefly those features, both qualitative and quantitative, of the chemical reactions of epoxides that are important to the understanding of their biological effects and that are basic to risk estimation. For extensive reviews of reaction mechanisms, see refs. [335,358,428,443]. In many cases the reaction-kinetic data required for risk estimates are not available for compounds or reaction conditions, especially temperatures, of interest. However, in certain cases general rules for the influence of substituents and/or temperature on reaction rates permit the estimation of rate constants. 5.2.1. Reaction mechanisms Reactions of the epoxide ring can be described in terms of the general alkylation reaction (see textbooks of organic chemistry, e.g. ref. [172]) RX + Y-~ RY + X(alkylating (nucleo- (alkylated (leaving agent) phile) product) group)

(9)

except for the specific feature that, in contrast with reactions of alkyl halides, alkyl alkanesulphonates, etc., the leaving group X- is a covalently bound part of the alkylated product. The alkylation of Y- by a disubstituted oxirane may be described by the formula: Y I

RtC~H.~o//CHR2 + Y--* RtCH--CHR2 O-

(i0)

The negatively charged oxygen atom then takes up a proton from the solvent water Y Y I

i

RICH--CHR~ + H20 -> RICH--CHR2 + OHi

O-

i

OH

(11)

47 to give a 2-hydroxyalkylated ("~-hydroxyalkylated") product. In the reaction with water (Y- = H20) a glycol is formed; OH i R I C "~.--O H j C H R 2 + H 2 0 -~ RICH--CHR~[

(12)

OH In studies of the biological effects of alkylating agents, changes of p H in media or in cells m a y play a role that is usually neglected. It is of interest to note that whereas the hydrolysis of alkylatingagents often produces hydrogen ions (Y- = H 2 0 in eqn. (9)) R X + H20 -~ R O H + X- + H ÷

(9a)

the hydrolysis of epoxides occurs without a change of p H (12). Reactions of epoxides with non-protonated nucleophiles (11) may, on the other hand, lead to an increase in pH. In a neutral milieu, the predominant reactions of most epoxides discussed here m a y be described in terms of an SN2 mechanism *, i.e.they occur via an activated complex of the two reactants,the rate of reaction (10) depending on the concentrations of both the epoxide and the nucleophile (Y-). In addition, hydrogen ion (H+)-catalysed reactions of an SN1 type occur (13a, b, c). After protonation of the epoxide oxygen (13a), the rate-limitingstep is the ring opening (13b): RIHC~-~CHR2

R1CH

+ H ÷

\o

CHR2

(13a)

j,.

• --..).0÷/

@

R1CH--CHR2 I OH

(13b)

subsequent nucleophilic attack by Y- on the positively charged carbon atom will be very fast (13c): Y I RI~H--CHR2 + Y- -~ R1CH--CHR2 (13c) I I OH OH The

Reactions (13b and 13c) thus resemble the reactions of isopropyl methanesul* SN1 and SN2 symbolize "Substitution Nucleophilic Unimoleculas" and "Substitution Nucleophilic B i m o l e c u l a r " , r e s p e c t i v e l y , a n d i n d i c a t e w h e t h e r a r e a c t i o n o f t y p e (9) is first o r s e c o n d o r d e r [ 4 4 3 ] , i.e. w h e t h e r t h e r a t e o f r e a c t i o n o f R X is d e t e r m i n e d b y t h e c o n c e n t r a t i o n o f R X a l o n e o r b y t h e c o n c e n t r a t i o n s o f b o t h R X a n d Y - . A l t h o u g h t h e classical d e s c r i p t i o n o f r e a c t i o n m e c h a n i s m s in t e r m s o f S N 1 a n d S N 2 m a y b e s u f f i c i e n t f o r t h e p r e s e n t p u r p o s e s , r e a c t i o n s o f a n i n t e r m e d i a t e t y p e bre m o s t c o m m o n . F o r i n s t a n c e , m a n y a l k y l a t i n g a g e n t s g e a c t a c c o r d i n g t o S N 1 w i t h w e a k l y n u c l e o p h i l i c o x y g e n s b u t a c c o r d i n g t o S N 2 w i t h s t r o n g n u c l e o p h f l e s , s u c h as t h i o l s u l p h u r s . T h i s h a s l e d t o m o r e a d v a n c e d t h e o r i e s , s u c h as t h a t o f t h e i n n - p a i r m e c h a n i s m p u t f o r w a r d b y S n e e n a n d L a r s e n [ 3 8 6 ] ( f o r a r e v i e w , see r e f . [ 3 2 8 ] ) . F o r c e ~ a i n a r g u m e n t s a g a i n s t t h e s u i t a b i l i t y o f usdng t h e c o n c e p t s S N 1 a n d S N 2 t o d e s c r l b e t h e r e a c t i o n s o f e p o x i d e s , see Eliel [ 1 1 6 ] .

48 phonate and not the SN2-type reaction of methyl methanesulphonate [330]. Whereas hydrogen-ion-catalysed reactions of the type (13a--c) predominate for many ethylenimines (which are stronger bases and become protonated in neutral solution), they are relatively unimportant for most (but not all!) epoxides in neutral solution. This mode of reaction may, however, be decisive for the fate of epoxides absorbed via the stomach. The likelihood that epoxides will undergo H*-catalysed reactions having an SN1 mechanism is enhanced by electron-releasing substituents, such as CH3- or C2Hs- in propylene oxide (II) and epoxybutanes (III, IV), and is reduced by electron-attracting substituents such as C1CH2- and HOCH2- in epichlorohydrin (IX) and glycidol (VIII), resp. [344,358]. A quaternary ammonium nitrogen adjacent to an oxirane carbon caused a still stronger suppression of the H*-catalysed reactivity [358]. Thus, these types of substituents weaken or strenghten, resp., the acid strength of the protonated ring oxygen (reverse reaction, eqn. 13a). This is analogous to their influence of the pK a of carboxylic acids, which increases in the order HCOOH ~ CH3COOH ~ C2HsCOOH and decreases in the order CH3COOH > HOCH2COOH > CICH2COOH. Conversely, through their inductive effect, electron-donating substituents decrease and electron-attracting substituents increase, resp., the SN2 reactivity, although field effects and, above all, steric effects also affect the reaction rates (cf. Sect. 5.2.3. and Table 4). The relative availability of the two ring carbons for nucleophilic attack (SN2) is mainly determined by steric factors, and only when the steric configuration is approximately equal at the carbon atoms will inductive effects determine the way the reaction proceeds [137]. Hence, in monosubstituted epoxides of the general type RCH--CH2 (e.g. compounds II, III, VIII--XI in Fig. 1), substitu-

,,o/ tion mostly occurs exclusively on the unsubstituted (primary) carbon, giving the product RCHOHCH2Y [335]. In accord herewith, the SN2 reactivity of disubstituted oxiranes (IV, XX, XXIII) is, for steric reasons, very low, as is their mu~genic effectiveness (2,3-epoxybutane [226] is an example). Steric hindrance appears to be one reason for the low 8N2-type reactivity of certain molecules with epoxidized cycloalkane residues, such as epoxycyclohexane (XXIII). This is especially true for dieldrin (XXIVb) and endrin (XXIVc), where a methylene bridge across the cyclohexane ring further prevents nucleophiles from approaching the oxirane carbons, thus rendering these compounds extremely stable to, for example, alkali [189]. Whereas XXIVb and XXIVc are not stable to strong acids, heptachlor epoxide (XXVb), which has a chlorine close to the epoxy group, is stable even to concentrated sulphuric acid [189]. Accordingly, these compounds give no colour reaction with 4-(4-nitrobenzyl)pyridine, an agent sensitive enough to detect alkylating agents as weak as chloroacetic acid and trimethyl phosphate [343]. In contrast with SN2 reactions, where among monosubstituted oxiranes one reaction product, RCHOHCH2Y, predominates, the SN1 reaction (13a--c) often leads to the formation of a mixture of both isomers [335] (i.e. both RCHOHCH2Y and RCHYCH2OH). Products of SN2 and SN1 reactions differ further with regard to changes in the steric configuration. Because the nucleophfle attacks the alkylating carbon of the ring from the side opposite to the

49 C - O bond, the SN2 reaction occurs with inversion of the configuration, if the carbon is asymmetric (cf. Sect. 5.3.). Thus the D configuration changes to L and vice versa. In SN1 reactions, the ligands of the intermediate carbonium ion (13b) try to assume a planar configuration with the possibility of attack from either side, resulting in partial racemization [335]. When an aromatic group (as in styrene oxide, XIV), a C=C double bond (vinyl group, as in epoxybutene, VII), or an ether oxygen are directly attached to an epoxide carbon, the positive charge on this carbon is stabilized by conjugation [335]. Under certain conditions [1,144, cf. 13, 14,259] this favours substitution on the substituted oxirane carbon even in neutral-alkaline solution, despite the steric hindrance. (This has been called a "modified SN2" reaction [335].) Because the secondary carbons in substituted oxiranes are asymmetric, two products are expected to be formed through attack on either epoxide carbon (e.g. in the reaction of DL-XIV with the SH group of L-cysteine).

5.2.2, Reaction products Essentially, genetic effects of alkylating agents are caused by reaction with certain groups in the gene material, mainly in the DNA. Reactions with proteins are involved in other toxic effects and, if the proteins are concerned with the metabolism of DNA, the extent of genetic damage may be modified [cf. Sect. 4.8.2.]. As other alkylating agents, epoxides react with all nucleophilic centres, at relative rates determined by their nucleophilic strengths and by steric factors. Besides reactions with biological macromolecules and other specific constituents of cells, epoxides react generally in vivo and in the environment, especially with water and chloride, giving rise to glycols (eqn. (12)) and halohydrins (eqn. (3)) in the case of asymmetrically substituted oxiranes often in two isomeric structures. In principle, a toxicological evaluation o f an epoxide should include these products as well. No systematic study of reaction products of epoxides with biological macromolecules has been carried out. However, there is no reason to doubt that, depending on nucleophilicity, substrate constants and steric factors, the same centres in DNA will become alkylated as with methylating agents [243,245, 250,376,378]. Several investigations have shown that, as with other simple alkylating agents, guanine-N-7 is the most reactive centre in nucleic acids. Ethylene oxide (I) and other epoxides (II, IV, IX) [49,248,353,450] were thus shown to give 7-(2-hydroxyalkyl) products in reaction with guanosine, deoxyguanosine or guanylic acids. In the former study [49], reaction of diepoxybutane (XVa) with guanosine was also shown to give rise to the diguaninyl derivative, 1,4
50 5'-phosphate, which all gave rise to 3-(2-hydroxyethyl)uracil products [411]. The reactions were favoured by alkaline conditions, showing that N-3 reacts after dissociation (pKa of uridine about 9.5 [394] ). Alkylation of DNA phosphates was deduced early on from studies showing a decreased binding of a basic dye [3]. This conclusion has been doubted because quaternization of Gua-N-7 upon alkylation [243] might have the same effects, through neutralization of the negative charges on ester phosphates. Walles and Ehrenberg [433,435] suggested that 2-hydroxyalkylation of DNA phosphodiester groups, creating an "RNA-like" situation, was a cause of the DNA backbone breakage and the high frequency of chromosomal aberrations characteristic of this kind of alkylation (vide Sect. 5.4.4.3.(e)). Shooter [376] reported that 2-hydroxyalkylation of diester phosphates occurs when R17 phage RNA was treated with 2-hydroxyethyl methanesulphonate, although his experiments indicated that other changes in the nucleic acid were largely responsible for back-bone breakage. As has been shown for XIXb, PAH epoxides exhibit a different reaction pattern towards DNA, a predominating product involving alkylation of the extranuclear guanine-N~ [48,230]. In proteins, the sterically accessible cysteine SH groups, histidine-ring nitro-gens and methionine sulphurs are most easily alkylated by ethylene oxide (I) in neutral solution [328,329,389]. In similarity with reactions towards other alkylating and acylating agents, these residues vary in reactivity [98] and, as in the alkylation by XIV of certain essential cysteines of alcohol dehydrogenase, enzyme inactivation may occur even at moderate doses [222]. In neutral medium the amino terminal -NH2, e.g. the valine of the haemoglobins, is sufficiently unprotonated [328] to be easily alkylated by I (data to be published). Other amino groups such as lysine's e-NH2 mostly require a high pH to become available as free bases; similarly tyrosine OH and arginine NH: can be alkylated under these conditions [389]. In certain experiments, epoxides, but not other alkylating agents, gave high yields of alkylation of primary amino groups in proteins [4,5]. This is certainly a pH effect -- the solution becomes alkaline (eqn. (11)) during exhaustive treatment. Specific reactions. The following specific aspects of reactions of epoxides will be dealt with in connection with the discussion of certain biological effects (Sect. 5.4.4.): influence of isomerism (XVa, b), inter- and intramolecular crosslinking by difunctional epoxides (XV--XVIII) and reactivity of substituents ("mixed difunctionality", IX--XII). The consequences of intercalation are also related to mixed difunctionality. In this context the consequences of the reaction of epoxides, i.e. 2-hydroxyalkylation, with DNA on strand stability will be discussed.

5.2.3. Reaction-kinetic aspects 5.2.3.1. Requirements for risk estimates In risk evaluations, according to expression (7) (Sect. 5.1.), of substances, be they primary pollutants or metabolites, with electrophilic reactivity (RX in formula (9)), reaction-kinetic data are primarily needed on two levels. First, such data are required for the determination of dose, D, as defined in expres-

51

sion (8): D = f [RX] dt

(8a)

t

Owing to rapid diffusion it is generally possible, in the treatment of cell suspensions with relatively small, uncharged molecules, to consider intracellular and extracellular [RX] to be equal, i.e., the dose, in M • h, may be computed as [ 9 5 ] D = [RX] 0 × t D - [RXo] k' (1

D-

-

-

for a practically stable compound; cf. refs. [228,350]

(8b)

for a compound that decomposes partly during the experiment, or

(8c)

for a compound that breaks down completely during the experiment

(8d)

e-k't)

[RXo] k'

[RX]o = the (total,in the case of repeated administration) initial concentration of RX; k' = the sum of the pseudo first-order rate constants for reactions leading to elimination of the compound from the system. For instance, in a phosphate buffer, RX will be eliminated through reactions with H20, HPO~- (and H2PO~, but this is usually negligible), i.e. k' = k ~ 2 o + kHPO~-[HPO~-]

(14)

In experiments with more bulky materials, such as plant seeds, eqns. (8a--d) with a correction for diffusion are applicable [258]. In the more complex situation of intact mammals, physiological and biochemical processes of metabolism, transport and excretion usually predominate in the rate of elimination. The resultant elimination may, at least in simple cases, be described in terms of an exponential, i.e. first-order, process with rate constant h (time -I) [106, 329]. In mouse and man, the h of I is more than 100 times higher than k' in a phosphate buffer at the same temperature (Sect. 5.3.). In mammals, D may be monitored from the extent of alkylation, [RY]/[Y], of nucleophilic centres, Y-, in certain relatively stable macromolecules, such as histidine and cysteine residues of proteins or guanines of DNA [106,329]. In reactions of the general type (9) the extent of alkylation will be

[RY] _ - ky- • D [V-]

- -

(15)

from which the dose is calculated according to 1 . [RY] D = ky- [ Y - ]

(15a)

For reactive metabolites formed from unreactive compounds, such as I from ethene [111], XIII from vinyl chloride [331] or XIXb (etc.) [48] from benzo[a]pyrene (XIXa), dose monitoring should also be carried out in situations in

52 vitro. This can be done by applying cellular macromolecules or some suitable monitoring substance, such as 3,4
log(ky~/kH2o) = s" n

(16)

where k v ; is the second-order rate constant for reaction with a nucleophile Yof strength n (cf. formula 9), kn2o is the second-order rate constant for reaction with water, and s is a substrate co,lstant expressing the dependence of reaction rate on nucleophilic strength, n (cf. Table 4). The rate of hydrolysis is usually expressed as the pseudo first-order rate constant, k~2 o with dimension time -1. The second-order rate constant, kH2o, to be used in (16) is obtained b y division with the concentration of water; in dilute solution [H20] = 55.5 M. Hence: t

kH2o(M -1 " h -1) = kH2°(h

-1

)

55.5 (M)

(17)

In t h i s model the nucleophilic strength of water is defined as n = 0, and other nucleophiles are assigned values relative to certain standard compounds. From the values of k~2o and s the rate constants for alkylation of c o m p o u n d s with known nucleophilic strength, n, may be c o m p u t e d according to eqn. (16). Values of n for c o m p o u n d s of biological interest have been given, for example, by Ross [360], Osterman-Golkar [328,330] and Wells [443]. More precise estimates of rate constants, or of degrees of alkylation, require consideration of charge of the reactants [433], steric factors (especially with bulky or strongly lipophilic compounds), basicity, etc. [186,333]. Furthermore, it must be borne in mind that eqn. (16) is an empirical law and that, therefore, the values of s for the alkylating agents and n for the nucleophiles depend to some extent on the reference c o m p o u n d s used in specific investigations. The reaction kinetics of biologically important alkylating agents, including alternative models taking into account the basicity of the nucleophiles, have been reviewed b y Ross [359,360] and Osterman-Golkar [328]. For

53 the purpose of risk estimates, the Swain--Scott model appears at present to give sufficient information. The reactivity of important epoxides is illustrated in Table 4 by values of pseudo first-order rate constants for hydrolysis at 37°C and of second-order rate constants for reaction with ammonia (n = 4.16; calculated from ref. [11] at 20°C) and thiosulphate (n = 6.36) [335,358,416]. The substrate constants, s, are included in Table 4 where enough data are available for their calculation. Corresponding data are given for the reaction product of I with CI-, ethylene chlorohydrin (XXVI) and, for comparison, for a couple of alkylating agents often used in parallel experiments. The rate constants at n = 2 are given in the table, f o r p r a c t i c a l r e a s o n s in t h e d i m e n s i o n M -1 • h Inspection of Table 4 justifies the following general remarks. Except for certain drastic variations in the H÷-catalysed hydrolysis, the listed epoxides show relatively small variations in reactivity. Owing to experimental difficulties, especially the volatility of I, there are some doubts as to the relative reaction rates of I and II, but for practical reasons they may be considered equal. The rateenhancing effect of the negative substituent leads to 2--4 times higher rates for epichlorohydrin (IX) as compared with I and II. At n = 2, IX reacts about 3 times faster than the unsubstituted I and II. The reactivities of the corresponding epibromo- and epiiodohydrins (X, XI) do not differ much from that of IX, as judged from the rates of reaction with CNS-, which amount to 85 and 70%, resp., of that of IX [cf. 335]. In contrast, glycidol (VIII) reacts slightly more slowly than I and II. The diepoxides XV and XVII, which, like VIII, are substituted with an oxygen on the carbon atom adjacent to each epoxide group, are hydrolysed at the same rates as VIII. A very strong rate enhancement (about 6000-fold) is observed in XIII, where an oxirane carbon is chlorinated directly. As can be seen from the rates of reaction with ammonia, a prolongation of the hydrocarbon chain in the 1,2~poxyalkane series leads to a slight decrease in the reaction rate. Data for reactions of higher homologues in aqueous solution are not available, but the rates of reaction of V and VI with 4-(4-nitrobenzyl)pyridine in 2-methoxyethanol [343] and in methanol (K. Svensson, unpublished) are approximately equal to that of I. As expected, H÷~atalysed reactions are much more sensitive to the properties of the substituents, being enhanced by electron-releasing substituents and suppressed by those that are electron-attracting (Table 4, last column). The over-all rate of hydrolysis, including the H÷-catalysed reaction, may be written

d[RX] dt

- (kH2° + [I-l*] k~÷) [RX]

(18)

At pH 7, the second term will not be of importance for any of the epoxides listed in Table 4, but even at pH 6 it has to be taken into account for compounds with unsubstituted, i.e. electron-releasing,, methylene groups adjacent to the oxirane ring (II--IV, XVI; for a discussion of XVI, see [358]). In isobutylene oxide (2,2~limethyloxirane) with two methyls on the same oxirane carbon, more than 90% of the hydrolysis at pH 7.4 occurs by the H+~catalysed reaction [259]. In epoxycyclohexane (XXIII) [47], and, especially, epoxycyclohexene [447], the H÷-catalysed reaction is still more favoured, with kh÷

4

2.2 d

XIII, chloroethylene oxide

XII, g l y c i d a l d e h y d e

5.1 d

IX, e p i e h l o r o h y d r i n

14 5 0 0 k

( k c l - : 3.1 × I) e

1.9 d

( k c l - : 1.8 X I) e

low

V I I I , giyeidol

VII, 3,4-epoxy-l-butene

cf. V, VI, 1 , 2 - e p o x y - n - p e n t a n e a n d 1,2-epoxy-n-hexane

trans-IV

IV, 2 , 3 - e p o x y b u t a n e cis-lV

( k c l - : 0.7 X I) e

II, propylene oxide

III, 1 , 2 - e p o x y b u t a n e

2.5 b (2.0 c)

(see-l )

k ~ 2 0 X 105

I, e t h y l e n e o x i d e

Compound

30

6.98

4.32 4.47

0.75 f 0.43 f

5.45

7.25

6.87

(M-1 . s e e - l )

(20°C)

k N H 3 X 105 a

1.8 J

( 0 . 4 X I) g

( 0 . 5 X I) g

(~2)

(M -1 . sec -1)

k s 2 O2- × 102

/

4300

3.3

1.0

(2.5 X I)

(~I)

( 0 . 8 X I)

1.2

(1.1--)1.4

(M -1 . h - l )

kn=2 × 102

0.83 k

1.00 i

0.96 i

~0.96

0.96 b

s

1.9 d

12.5 d

fast e

(2 X If) h (4 X I I ) h

124 d

32 c

(M-1 . s e e - l ) P

k H + X 103

F r o m the r a t e s of u n c a t a l y s e d h y d r o l y s i s a n d t h e s u b s t r a t e c o n s t a n t , s, t h e r a t e c o n s t a n t s f o r r e a c t i o n a t n = 2 h a v e b e e n c o m p u t e d , a n d are g i v e n in M -1 • h -1 t o facilitate p r a c t i c a l use. W h e n n o t specified, d a t a are valid a t 37 ° C. F o r a f e w c o m p o u n d s , w h e r e n o d a t a f o r h y d r o l y s i s w e r e available, r a t e c o n s t a n t s f o r r e a c t i o n w i t h C1- (n = 3.0 [ 2 3 1 ] ) h a v e b e e n i n t r o d u c e d , r e l a t i v e l y t o t h e v a l u e f o r I. R e l a t i v e v a l u e s are e x p r e s s e d b y x A , w h e r e A is t h e n u m b e r o f t h e c o m p o u n d .

R A T E C O N S T A N T S ( T I M E U N I T : sec) F O R R E A C T I O N S O F S O M E E P O X I D E S A N D A F E W S T A N D A R D M U T A G E N S W I T H W A T E R A N D A F E W O T H E R NUCLEOPHILES

TABLE

0.0011 m

0.014 m

XXVI, ethylenechlorohydrln

a b c d e f g h i J k 1 m n P

(2/3 X I) d

3.5 0.195 0.033

7.8 2.6 0.14

0.0026 (0.003)

1.1

1.3

(~1.7)

0.89 0.69 0.68

~0.73 m

0.96 d

8300 d

6.4 d

177 d

1.9 d

Ref. [ 3 3 5 ] ( d a t a f r o m S. A n d e r s s o n [ 1 1 ] ) . Refs. [ 3 2 8 , 4 2 8 ] . D a t a f r o m ref. [ 2 3 1 ] w e r e u s e d t o c a l c u l a t e n v a l u e s . C a l c u l a t e d f r o m t h e v a l u e s a t 2 0 ° [ 4 7 ] a n d a t 25 ° [ 8 5 ] , b y u s i n g t h e a c t i v a t i o n e n e r g y E A = 1 9 . 0 k c a l • m o l e - I [ 8 5 ] . Refs. [ 3 5 8 , 3 5 9 ] , as f o r s, c a l c u l a t e d f r o m Ross [ 3 5 8 , 3 5 9 ] , el. [ 3 2 8 ] . R e l a t i v e values o f r a t e s o f a l k y l a t i o n o f (31- in 10% d i o x a n e a t 4 0 ° C . I n t h e case o f V I I , 86% o f r e a c t i o n s o c c u r o n t h e s e c o n d a r y o x i r ~ n e c a r b o n [ 1 ] . Ref. [ 1 1 ] . R e l a t i v e r a t e o f r e a c t i o n w i t h 0 . 2 M $ 2 0 2 - in b o i l i n g 50% a c e t o n e [ 3 5 8 ] . Ref. [ 3 4 4 ] . Refs. [ 4 7 , 1 6 7 , 4 4 3 ] . Ref. [416]. Refs. [ 1 8 7 , 3 2 8 ] . E s t i m a t e d f r o m 2.04 • 10 -3 M -1 • see- I a t 2 5 ° C ; ref. [ 3 3 5 ] . Osterman-Golkar, unpublished data. D a t a f o r m e t h a n e s u l p h o n a t e s f r o m refs. [ 3 2 8 , 3 3 0 ] . R a t e c o n s t a n t f o r H + - c a t a l y z e d hydrolymis; cf. e q n . ( 1 8 ) o n p. 53.

Triethylene melamine (TEM)

Methyl methanesulphonate Ethyl methanesulphonate 2oHydroxyethyl methanesulphonate

For comparison- n

20 1"1 0.92 (k~) H - = 1.1 M-1 • sec -1) 6.7 ( p H 7) d

(0.8 X XV) g

1.9 d

XVII, bi~(epoxypropyl) ether

2.25 J (0.5 X XV) g

6.4 d

XVb, meso-diepoxybutane

2.50 J

~0.8 1

X V I , 1,2, 5 , 6 - d i e p o x y h e x a n e

7.8

1.9 d

XVa, D L - d i e p o x y b u t a n e

XIV, styrene oxide

O1

56 150 and 20 000 times, resp., greater than those of I at the temperatures of investigation (20 and 25 ° C, resp.). Hydrolysis of epoxycyclohexene occurred by a general acid~catalysed mechanism, being enhanced by, for example, H2POZ [447]. In these cases, as with triethylenemelamine (TEM) (Table 4), H+-catalysed reactions will contribute already in neutral solution, and an accidental lowering of pH may lead to a rapid destruction of the compounds [114]. This is of further importance for the fate of epoxides reaching the stomach (cf. (Sect. 5.3. below and Table 7). In the H+-catalysed reactions (eqn. (13a--c)) the ring opening is the ratelimiting step, and the reactions are of the SN1 type. Hence, s values are more an expression of competition factors valid in certain circumstances where the reaction can be considered quasi-bimolecular [330]. In accordance with the relatively low s of SN1 reactants such as isopropyl methanesulphonate, the s values of H÷-catalysed reactions of epoxides are lower than s of the non-catalysed SN2 reactions of the same compound. From the rates of H÷-catalysed reactions of ethylene oxide with halide ions [85], s ~ 0.7 was estimated. This means that, in 0.1 M HCI, about 30% ethylene chlorohydrin and 70% ethylene glycol will be formed. As with reactions of isopropyl methanesulphonate (unpublished data), the rates of H÷-catalysed reactions of epoxides are enhanced by salts, i.e. by increasing ionic strength, as shown by BrSnsted et al. [47]. The influence of steric factors can be elucidated by comparing the rates of alkylation of ammonia and primary, secondary and tertiary amines by unsubstituted oxirane (I) and by the substituted oxiranes (II, VIII, IX) [167,168]. Taking due account of slight variations in s, the three substituted epoxides exhibit about the same relative reaction rates compared with I vis-a-vis ammonia, methylamine, dimethylamine and diethylamine, but their reactions with trimethylamine and triethylamine are about 3 times slower. This indicates steric hindrance due to an interaction between the oxirane substituent and the three nitrogen ligands of the tertiary amines. It is of interest, and possibly of importance to the elucidation of reactivities vis-a-vis DNA bases, that no such steric hindrance appears in reaction with the ring-bound nitrogen of pyridine [168] or of saturated cyclic amines [396]. Rate constants for reaction with biological macromolecules are so far available only for ethylene oxide, I (Table 5). The rates are in acceptable agreement with similar data for methyl methanesulphonate [370] which, in accordance with expectation (Table 4), reacts about 6 times faster than I with total mouse haemoglobin and total DNA. According to Walles [434], rate constants for the reactions of the higher homologues of I, propylene oxide (II) and 1,2-epoxybutane (III), with DNA in vitro deviate by a factor of 2 - 3 from those of I, recalculated per guanine: Compound

kDNA.Gu

I II

0.11 M -I " h -I 0.22 M -I • h -I

III

0.03

M -I

a (37 ° C)

• h -1

The titrimetric study of Lett et al. [255] of the reaction of II with DNA

57 TABLE 5 RATE CONSTANTS FOR REACTIONS OF ETHYLENE O X I D E (I) A N D SULPHONATE WITH SOME BIOLOGICAL MACROMOLECULES AT 37°C

METHANE-

Rate constant k (1 • g - I . h - 1 )

Nueleophile

Ethylene

Mouse hemoglobin

METHYL

oxide a

Methyl m e t h a n e sulphonate b

1.8 • 10 -4 0.5 • 10-4 0.2 • 10-4

1.2 • 10 -3 0.6 • 10 -3 0.12 • 10 -3

(in intact red cells)

Total Cysteine-S Histidine-NIm-3 Proteins in mouse spleen

4.3 • 10-4

cells (interphase fraction) DNA in mouse spleen cells Guanine-N-7

1.0 • 10 -4 c

0.6 • 1 0 -3

a Refs. [1061329] and unpublished data. b Ref. [370]. c C o r r e s p o n d s t o k G u a . N . 7 = 0 . 1 1 M -1 • h -1 .

indicates a lower rate of kDNA.Gua,about 0.07 M -~ • h - ' . Although it cannot be excluded that the reaction of III with DNA is sterically hindered, the deviation from expectation may be due partly at least to the unphysiologically high ionic strength of the solution (2.5 M sodium acetate, pH 5.8) [434]. 5.3. Fate o f epoxides in vivo This section will deal with a few general toxicological aspects of basic importance to the evaluation of genetic risk, i.e. primarily to the dose (eqn. (8), (15a)) in target organs, of epoxides and, in certain cases, of genetically toxic products of epoxides, such as alkylene chlorohydrins. The dose of an epoxide in the cells (cell nuclei) of an organ is a function of the exposure dose and is influenced by the route of administration, by the uptake and elimination, which occur through active transport and possibly through diffusion, and by destruction which occurs actively through metabolism with a contribution in some cases from chemical reaction. The resultant rate of elimination of the alkylating species from cells or tissues may be approximated by an exponential function, i.e. in principle by a first-order reaction, with the rate constant k (time-l), replacing the chemical rate constant k' in eqns. (8c), (Sd) (Sect. 5.2.3.1.). The tissue dose (cf. eqn. (8), Sect. 5.1) m a y then be expressed as D (concentration × time) =

Co (concentration) ~ (time_l)

(19)

In this expression, Co is a hypothetical concept representing the initial concentration, i.e. the concentration that would have been reached in the cells, cell nuclei, etc., if no processes of elimination were operat'.mg. It can be shown (un-

58 published) that this is valid for acute as well as repeated or chronic exposure; in the latter case, Co means the sum of all concentrations that would have been reached, again assuming no elimination. As illustrated by model experiments [106] with labelled ethylene oxide (I) absorbed by mice during 75-min exposure to contaminated air, the total radioactivity is initially highest in the liver and second highest in the kidneys, whereas in other organs, for example spleen and testis, the radioactivity is approximately equal to the average value in the body for about 1 h. The liver radioactivity drops more rapidly than the kidney value, the two becoming approximately equal after some 2--3 h, when other organ values have also started to drop considerably. This picture, confirmed by autoradiography [12], agrees with the general idea of a rapid active transport of I, which is recognized as a foreign compound, to the liver where it is subjected to enzymatic detoxication. The products of detoxication are then excreted via the kidneys. However, it is essential to note that, at least when the amounts absorbed are small, the dose of I, defined as the time integral of the concentration of free I (eqn. (8)), does not seem to be higher in the liver than in other organs of the body. This indicates that the transport of I to the liver and within this organ is carried out by a mechanism that protects the epoxide from chemical reaction. With larger absorbed amounts a tendency to somewhat higher relative doses in the liver is observed [ 106], possibly a consequence of damage to, and/ or saturation of, the transport/detoxication apparatus (v. infra). Two enzyme systems are known to participate in the further inactivation of epoxidized intermediates from aromatic and olefinic compounds (review: [157]). These are epoxide hydrases [323,324], which catalyse the hydrolysis to dihydrodiols and glycols, respectively (eqn. (12)), and epoxide-glutathione transferases [42,140], which in principle catalyse the alkylation of glutathione sulphur (Y- = GS- in eqns. (10), (11)). It is likely (and in agreement with the results of experiments in vitro) that the same enzymes participate in the detoxication of primary epoxides as well. There are indications that at least the epoxide hydrase system consists of more than one enzymic species with specificities for different epoxides. The accumulation in tissues of cyclodiene epoxides (XXIVb, XXVb) is partly counteracted by enzymes hydrolysing or transferring the epoxide function (which, despite its very high chemical stability, can be managed by enzymes to TABLE 6 RETENTION

T I M E S , IN T H E M O U S E , O F A F E W A L K Y L A T I N G

Ethylene oxide (I) Methyl methanesulphonate Ethyl methanesulphonate a b c d

AGENTS

k (h -~ )

Biol. half-life (h)

4.6 a 1.3 b 2.0 b 0.3 c 1.4 d

0.15 0.5, 0.35 2, 0 . 5

Ref. [106]. Ref. [370]. Estimated from ref. [ 7 4 ] , cf. [ 1 0 6 ] . Rate constant for clearance of ethyl methanesulphonate at administered amounts b e l o w 7 m g f k g b . w . (M.S.S. M u r t h y , personal communication).

59

some extent) and partly by solubilization initiated by enzymatic oxidation in other parts of the molecule [51]. The epoxide-metabolizing enzymes show a great variation in substrate specificity. Saturation at high concentrations of an epoxide or inhibition by other epoxides, effective in this respect [324], may tend to retard the elimination and, consequently, increase the tissue dose of the epoxide under study. (This is sometimes utilized in the Ames test [60].) Removal of giutathione, for example by diethylrnaleate, has a similar effect [414]. In comparison with methyl and ethyl methanesulphonates, which are more reactive alkylating agents (Table 4), I has a remarkably short retention time in vivo as shown by a comparison of the biological half-lives of the compounds (Table 6). This illustrates that, generally, the effectiveness of alkylating agents as substrates for deactivating enzymes need not be correlated to chemical reactivity and that chemical reactions have relatively little influence on the total rate of elimination from tissues. With highly reactive compounds, such as chloroethylene oxide [187,331], an intermediate in the detoxication of vinyl chloride, this is not necessarily so, unless there is a biochemical mechanism for transferring the epoxide from the site of origin (a mixed-function oxidase) to the hydrase or transferase enzyme. For ethylene oxide (I) the rate constant for elimination from tissues, assuming it to be a first-order process, was 4.6 h -l in the mouse [106,329]. Reasonably reliable measurements of tissue doses of I in persons exposed to known, or at least assessable, average concentrations during the 4-month lifespan of haemoglobin used for the monitoring dose, indicate that the rate constant for elimination of I from tissues, if it differs at all from the mouse value, could be somewhat faster [59]. In the estimation of risk shown below, the mouse value will therefore be applied to man. For other epoxides the rates of elimination are not known. Applying the value for I (~ = 4.6 h -I) to some other simple monofunctional and difunctional epoxides, in calculations of the rad-equivalence of the DLso, shows that the assumption that the rates of elimination of these compounds are equal cannot be far wrong (5.4.4.1.(a), Table 13). TABLE 7 A P P R O X I M A T E H A L F - L I V E S A T p H 1 (0.1 M HC1) A N D 3 7 ° C O F S O M E E P O X I D E S , T R I E T H Y L E N E MELAMINE AND METHYL METHANESULPHONATE F o r refs. s e e T a b l e 4 a n d t e x t .

N o . in Fig. 1

Compound

tI

I

Ethylene oxide

3.5 min

II

Propylene oxide

I min

IX

Epichlorohydrin

lh

XXII

Epoxyeyclohexane

1 . 5 sec

Epoxyeyelohexene

~ 1 0 -$ sec

Triethylene melamine Methyl methanesulphonate

0 . 8 sec 1 0 h (~---w a t e r s o l u t i o n )

60 These considerations are valid in situations where the epoxides are inhaled, injected i.p. or i.v., or absorbed via the skin. The latter process certainly occurs, as shown by the systemic toxicity observed in cancer tests with XV [173,423, 424]. The lethal action of IX applied on the skin of rabbits [251] or rats [143] is a b o u t one-fifth of that of the same a m o u n t given by injection. The importance of further investigations of this route is emphasized by the demonstration by Hine et al. [179, see also 180] of haematological disturbances following skin application of XVII in various species. When epoxides are absorbed via the stomach, H*-catalysed reactions may play a major role. As illustrated by half-lives in 0.1 M HC1 (Table 7), this is especially true for oxiranes substituted with electron
5.4. Structure--effectiveness relationships This section will discuss h o w variations in the mutagenic and related effectiveness [87,88], i.e. mutation frequency per unit dose, are correlated to the reactivity of the epoxide function and of substituents. The latter may contain a second epoxide function ("difunctionality") or some other chemical function ("mixed difunctionality").

5.4.1. Monoepoxides In 1955, KSlmark and Giles [226] studied 6 substituted monoepoxides (Fig. 1: II, III, IV, VIII, IX, X) with regard to reversion frequencies in N. crassa Ade- 3871. Direct comparisons of mutagenic effectiveness are complicated since, evidently because of differences in solubility, etc., the dose D -- see eqn. (8) -- was varied for some c o m p o u n d s b y a variation of time and for others by a variation of concentration. Furthermore, c o m p o u n d s were n o t always tested

61

at overlapping mutation frequencies. It was later demonstrated for ethylene oxide (I) and diepoxybutane (XV) that, above a certain concentration, the dose--response curves are of the general shape M

(eqn. (5), Sect. 4.8.1.)

= aD b

independently of concentration and time. Therefore, careful comparisons of effectiveness seem permissible in curves, or regions of curves, with equal values of b. With this restriction, relative effectivenesses would be proportional to doses giving the same response or, what amounts to the same thing, to a l / b . Although propylene oxide (II) and 1,2-epoxybutane (III) were not compared at overlapping concentrations, these considerations (Table 8) lead to the conclusion that the two compounds are about equally effective, which is consistent with their reaction rates being about the same (Table 4). The disubstituted oxirane (IV), is, however, much less effective, in accord with its lower reactivity. Epichlorohydrin (IX) and epibromohydrin (X) are decidedly more effective than III and II. IX was some 4--5 times more effective than I in the high concentration range, but because IX and X do not differ significantly in the

TABLE 8 R E L A T I V E M U T A G E N I C E F F E C T I V E N E S S OF SOME EPOXIDES [226] FOR A d e - R E V E R S I O N S I N N. crassa a ~and b are empirical c o n s t a n t s in E q n . (5); see S e c t i o n s 4 . 8 . 1 . and 5.4.1. Data for e t h y l e n e o x i d e and d i e p o x y b u t a n e are n o t directly c o m p a r a b l e w i t h the group o f 6 c o m p o u n d s at the top of the Table.

Compound

D o s e range, M • m i n (concentration)

a

b

Rel. effectiveness compared with III

II, p r o p y l e n e o x i d e

7.5--30 ( 0 . 5 M)

--

III, 1 , 2 - e p o x y b u t a n e -

0.15---6 (0.005---0.2 M)

0.28

1.6

IV, 2,3-epoxybutane

1.5--6 b ( 0 . 0 5 - - O . 2 M)

0.02

~1.6

v H I , giycidol

7.5--30 ( 0 . 5 M)

--

X, e p i b r o m o h y d r i n

0.15--2.4 (0.005---0.16 M)

0.8

iX, e p i e h l o r o h y d r i n

2.25--9 ( 0 . 1 5 M)

For comparison: c I, e t h y l e n e o x i d e XV, d i e p o x y butane

2.2 (1.6 at l o w e s t d o s e )

2.5 ( l o w e r at l o w e s t d o s e )

0.1--10 ( > 0 . 0 1 5 M)

2.61

2--8 (~>0.01 M)

0.17

(1) (I)

1 a

0.1--0.2 b

_

2.5 ( ~ 1 . 6 at l o w e s t d o s e ) 1.6

II

1.2 2

--

2.5--4

4--5

2.69

2.39

a Curves of II a n d III n o t significantly different in the region 6 - - 7 . 5 M • min0 where the d o s e ranges a p p r o a c h each other. b A t 12 M • rain there w a s a very l o w m u t a t i o n frequency; the tel. effectiveness therefore a p p r o x i m a t e . c From K~lmark and Kilbey, 1968 [226].

62

region of overlap (2.25--2.4 M • min), a value of about 3 seems more likely at lower concentrations, in accordance with what is expected from reaction rates. Although glycidol (VIII) reacts somewhat more slowly than II, it was slightly more effective than this c o m p o u n d . It would seem dangerous to make a direct comparison of these c o m p o u n d s on the basis of data determined many years later for ethylene oxide (I) and diepoxybutane (XV) [228] because changes in experimental conditions might well have changed the parameters determined. The exponential function of I would make this c o m p o u n d slightly more effective than IX at high doses, whereas XV, being some 3 times less effective than I, assumes a position between those of II and IX. At low dose rates, i.e. concentrations, where the exponent b decreases towards 1 [ 2 2 8 ] , XV would be some 40% more effective than III, and I, if anything, somewhat less effective than III. However, these differences are n o t significant. To our knowledge, the dose--response curves of I and other monofunctional epoxides have never been determined in simultaneous experiments permitting a direct comparison with regard to mutagenic effectiveness. In his study of induced chlorophyll mutation in barley, Heslot [174] included I, II and a few other epoxides, but his data refer to only one or two doses around the DLs0. However, because dose--response curves for epoxide-induced mutations in barley are mostly linear up to doses above the DLs0 (Sect. 4.8.1.), the data may be taken to show that I and II are about equally effective (Table 9), which is consistent with their practically equal reaction rates (Table 4). Refined risk estimates must take into consideration whether the steric hindrance due to a substituent on an oxirane carbon, which is observed, for example, in reaction with tertiary amines (Sect. 5.2.3.2.), influences mutagenic events as well. Therefore

TABLE

9

MUTAGENIC

EFFECTIVENESS

Compound

OF EPOXIDES

IN BARLEY

A N D Sch. p o m b e

C h l o r o p h y l l m u t a t i o n s in b a r l e y , % ( m M • h ) - 1 D ~ DL50 a

S l o p e o f linear Part o f d o s e - - r e s p o n s e curve b,c (22 ± 2°C)

Arg-1 reversions in Sch. p o m b e ; s l o p e o f linear part o f d o s e - - r e s p o n s e curve e X 107 (mM.h)

-1

3°C

24°C

I, e t h y l e n e o x i d e

0.004

0.028

(0.013 c) 0.025 ± 0.005 d

II, p r o p y l e n e o x i d e

0.0036

--

--

0 . 8 1 (1 h ) ; 0 . 8 5 ( 5 h )

VIII, glycidol

--

--

0.06

1.8

IX, e p i c h l o r o h y d r i n

0.0030

0.027

0.08

3±1

0.11

1.28

0.0035

0.10

XV, diepoxybutane XVI, diepoxyhexane a b c d e f

(rac.)

f

~5

Heslot [174]. Ref. [330]. R e f s . [ 8 7 , 9 9 ] and q u o t e d papers. L. E h r e n b e r g , u n p u b l i s h e d data. Ref. [175]. D i f f e r e n t rel. e f f e c t i v e n e s s in o t h e r strains o f Sch. p o m b e ; w a s n o t c o n s i d e r e d significant.

63 the important comparison of I and II requires reinvestigation in suitable systems. Heslot [174] found epichlorohydrin (IX) to have a b o u t the same effectiveness as I and II; his single points at 3 and 24°C were probably determined at doses b e y o n d the maximum of the dose--response curve. Other studies in barley [87] and in Sch. pombe [175] indicate that IX is 3--4 times more effective than I or II. In these studies, VIII was again more effective than I or II (Table 9). Data for mutagenic effectiveness can be related to values for methyl methanesulphonate, which has been included in many experiments. This c o m p o u n d was some 6--8 times more effective than I or II in the above-mentioned studies on barley, and the same ratio was also found in E. coli Sd4, as expected from reaction rates (Table 4). For u n k n o w n reasons, possibly related to peculiarities of repair systems, methyl methanesulphonate was relatively more effective in Sch. pombe [131]. In the standard plate test for back-mutation in S. typhimurium His- strains, slopes of the mostly linear dose--response curves seem to conform less well to reaction-kinetic data, possibly for technical reasons: in this test, cells are not exposed to a well
5.4.2. Alkylene halohydrins Ethylene chlorohydrin (XXVI) and other alkylene halohydrins react as alkyl halides introducing, like the corresponding epoxides, 2-hydroxyalkyl groups onto nucleophilic atoms. Strong field effects from the negative substituents exert a retarding influence on reaction rates. Thus an OH in the 2-position decreases reactivity some 40 times and an OH in the 3-position decreases it some 4 times, as compared with the unsubstituted c o m p o u n d s (Table 10). The corresponding effects of halogen atoms are even greater (cf. Table 10 and ref. [4O]). As a consequence o f its low reactivity, XXVI is expected to be a relatively weak mutagen. Data from studies in four systems permit a comparison of the mutagenic effectiveness per alkylation of DNA (at k,=2) with that of other c o m p o u n d s tested concurrently. In all cases, XXVI turned o u t to be more effective than expected, and in at least t w o tests the difference was significant (Table 11). At least t w o mechanisms for this enhancement of the mutagenic effectiveness of XXVI have to be considered. It may be due to oxidation of XXVI to

64 T A B L E 10 D E C R E A S E O F R E A C T I V I T Y D U E T O E L E C T R O N E G A T I V E S U B S T I T U E N T S ON C A R B O N S 2 OR 3 ( E L E C T R O P H I L I C C A R B O N N U M B E R E D AS 1) O F A L K Y L H A L I D E S A N D A L K Y L M E T H A N E SULPHONATES T h e c o m b i n e d a c t i o n o f field e f f e c t s a n d i n d u c t i v e e f f e c t s is g i v e n as t h e f a c t o r b y w h i c h t h e r e a c t i o n r a t e is c h a n g e d at 37 ° . Compound

Substituent and position

C h a n g e of kH20

Ethyl bromide

2-OH 2-Br

X1/40 X1/100

C h a n g e of other k

Ref.

352 40, 1 1 2

n-Propyl bromide

3-OH

X1/4

352

Ethyl chloride

2-OH 2-C1

X1/10 a X1/85

Osterman-Golkar (unpublished)

Ethyl methanesulphonate

2-OH

X1/18

k s 2 0 3 2 - = X1/6 kOH-

2-C1

X1/67

330

= X4.3 • 103 3 3 0

kHPO42-= X1/62

112

a A s s u m i n g r e a c t i o n r a t e s of m e t h y l c h l o r i d e a n d e t h y l c h l o r i d e t o b e a p p r o x i m a t e l y e q u a l [ 3 5 2 ] .

chloroacetaldehyde which, probably through a different mechanism (via Schiffbase formation [27]), is an extremely effective mutagen [187] or through a conversion to ethylene oxide (see Introduction, formula 3) which alkylates T A B L E 11 M U T A G E N I C E F F E C T I V E N E S S PER A L K Y L A T I O N AT n = 2 OF E T H Y L E N E C H L O R O H Y D R I N IN DIFFERENT BIOLOGICAL SYSTEMS " s t d " , c o m p o u n d c o n s i d e r e d as s t a n d a r d (tel. e f f e c t i v e n e s s = 1); ns, n o t significant. V a l u e s f o r o t h e r c o m p o u n d s give r a t i o s of m u t a t i o n f r e q u e n c i e s f o u n d a n d m u t a t i o n f r e q u e n c i e s e x p e c t e d u n d e r t h e a s s u m p t i o n o f p r o p o r t i o n a l i t y w i t h kn= 2.

Compound

Reactivity kn= 2 (37° C) M-1 • h -1

R e l a t i v e m u t a g e n i c e f f e c t i v e n e s s p e r r e a c t i o n a t kn= 2

E t h y l e n e o x i d e (I)

1.4 • 10 -2 a

1 (std)

Epicblorohydrin (IX)

5

Ba~ley e

E. coli Sd4 d

KlebsielIa pneum, f

1 (std)

. 10-2 a

S. t y p h i m u r i u m

g

1 (std) 0.7 (n.s.)

Trimethyl phosphate

2.8 • 10 -4 b

~1

Methyl methanesulphonate

7.5 • 10 -2 b

~1

Ethylene chlorohydrin (XXVI)

3

.10-S c

Chloroacetaldehyde

2

. 10-5 d

2 (1--5)

1

(std)

1 10 (4--25) 10 0 0 0

30

2.5 (sign?) 104

a T a b l e 4. b [112]. c C a l c u l a t e d f r o m k H 2 0 = 5 • 1 0 -5 h - I , k s 2 0 3 2 - = 4 • 1 0 -2 1 • m o l e - I • h - I (S. O s t e r m a n - G o l k a r , u n published data). d R e f . [ 1 8 7 ] . T h e r a t e c o n s t a n t f o r X X V I , k n = 2 = 1 " 1 0 - 5 , g i v e n in t h a t p a p e r c o n c e r n s 2 5 ° C , t h e t e m p e r a t u r e u s e d b y R a n n u g et al. [ 3 4 8 ] . e 22 ° C, 5-h t r e a t m e n t of k a r y o p s e s (L. E h r e n b e r g a n d S. Hussain, u n p u b l i s h e d d a t a ) . f Ref. [430]. g 2 5 ° C , ref. [ 3 4 8 ] .

65 some 500 times faster than XXVI (Table 4). The latter type of reaction is catalysed by alkali [57,390] and is expected to proceed at a rate amounting to 20-40% of that of the reaction with water (Table 4) at pH 7--7.4. Dibromomannitol [118] and 2-hydroxyethyl methanesulphonate [330] exhibit a similar intra-molecular epoxide formation. 5.4.3. Isomerism Diepoxybutane (XV) contains twQ asymmetric carbon atoms and may be resolved into the optical isomers (enantiomorphs) of XVa, with L o r D configuration, resp., at each of the two asymmetric carbon atoms. Because each of these carbon atoms is attached to the same four substituents, only one diastereomer of XVa exists, the meso-diepoxybutane (XVb). Except for the ability to rotate the plane of polarized light, optical isomers have identical physical and chemical properties in their interaction with optically inactive materials. However, they may be expected to differ in rates of reaction towards, or passage through membranes containing, optically active materials, ubiquitous components of living matter. For this reason, comparisons of optical isomers with regard to a biological effect may ultimately contribute to our understanding of the mechanism of action. On the other hand, diastereomers are different compounds, with different physico~chemical properties in all respects. For a number of genetic effects, explicitly presented in Table 2, L-XVa was more effective than D-XVa, and these compounds were again more effective than the diastereomer XVb. These effects are: killing of T7 phage [427], mutation in maize pollen (mainly deletions) [29], Penicillium multicolor [316], Arabidopsis thaliana [295] and Schizosaccharomyces pombe [175], and also chromosomal aberrations in Vicia faba [316]. Bianchi and Contin [29] found a significant, so far unexplained, synergistic action of L- and D-XVa in the induction of mutations in maize pollen. B o t h DL- and meso-XV are strong inhibitors of the growth of Walker carcinoma in rats [173]. However, they were not tested at comparable doses; possibly because meso-XV was considered more toxic to the rat, it was tested at 5 mg]100 g, giving 55% growth inhibition, whereas DL-XV gave 97% growth inhibition at 16 mg/100 g. In skin-painting tests with 10% acetone solution, DLXV appeared to be more toxic than meso-XV [190,423]. In such tests with Swiss--Millerton mice, the carcinogenic activity of DL-XV was greater than that of meso-XV (cf. Table 3). Similar results (L > D ~ meso) were obtained with isomers of threitol-l,4bismethanesulphonate with regard to chromosomal aberrations in Vicia faba, Nigella damascena [313,314], barley [290,294] and Allium cepa, and mutation in Arabidopsis thaliana [290,292,294]. The latter of these effects was observed only after treatment in alkaline solution. The threitol diesters H OH I I CH 3SO2--O--CH2--C-- C--CH2--O--SO2CH I I OH H are converted, probably into the corresponding isomer of XV, in an alkalicatalysed reaction [77,291], and the order of effectiveness L > D > meso was also found in treatment of Allium roots after such transformation [294].

66 Because the meso c o m p o u n d , i.e. the erythritol bismethanesulphonate, is converted more slowly than the L- and D-isomers, the biological consequences of treatment with this ester are determined by the rates of t w o processes, i.e., conversion into epoxide and reaction of the latter. On the chemical level, reactions of meso-XV were indicated to be slightly slower than those of DL-XV [416] (Table 4), although the difference is t o o small to account for the large differences in biological activity. The inability of meso-XV, in contrast with L- and D-XV, to inter-link the strands of the double helix of T7 phage DNA seems to be of special importance [427]. The implications of these findings will be discussed in the following section. It should be possible to resolve a number of monofunctional epoxides, such as propylene oxide (II), glycidol (VIII) and epichlorohydrin (IX), into optical isomers, and the diastereomers of a c o m p o u n d such as 2,3-epoxybutane should exhibit an interesting cis-trans isomerism of the methyl groups (see Fig. 1: IV, and cf. Table 4). It is n o t e w o r t h y that, except for the studies on XV reported above, this possibility has n o t been investigated in biological experiments with simple epoxides and their related halohydrins. Such studies might reveal specificities of epoxide hydrases and epoxide-glutathione transferases and might also be important as models to further our understanding of the situation in the case of reactive epoxy- and h y d r o x y - e p o x y derivatives of polycyclic hydrocarbons. These were early understood [39] to be metabolized in vivo in specific patterns o f isomers.

5.4.4. Di- and polyfunctionality Every student of biological effects of alkylating agents is aware of the high efficiency or effectiveness of di- and trifunctional agents. For comparative risk estimations, based on reaction kinetic data, it is essential to know whether two or more alkylating functions lead to an ability of the c o m p o u n d to do something that cannot be achieved b y monoalkylation, or whether the difference in effectiveness can be treated satisfactorily in quantitative terms. To shed light on this problem, in this Sub-section we shall discuss available quantitative data and current ideas on mechanisms. In addition, the reactivity due to substituents of monofunctional alkylating agents will have to be treated in a similar way ("mixed difunctionality"). This exercise leads to the general conclusion that, although difunctional and monofunctional agents m a y give rise to different spectra of effects owing to differences in the dose--response relationship for individual end-points (cf. Sect. 4.8.), a quantitative treatment of the data will estimate risks with sufficient accuracy. This is especially valid at low doses, where lethal action and physiological effects due to monofunctional alkylation of proteins are excluded. 5.4.4.1. Diepoxides A number of hypotheses has been p u t forward to explain the greater biological effectiveness of di- and polyfunctional alkylating agents, as compared with their monofunctional analogues. These hypotheses have been developed, or tested, in experiments with d i e p o x y b u t a n e (XV) and o t h e r epoxides, alongside studies of mustards and ethylenimines. The functionality of alkylating agents played an important role in the development of the concept of "radio-

67 mimetic" agents, originally used by Dustin (1947) and Boyland (1948) to signify chemicals that were able to produce the same end-points as ionizing radiations [265,359]. When interest was directed to the development of growthinhibitory agents of potential use in cancer therapy, the observation of the superior efficiency in this respect of difunctional agents led at one time to dior polyfunctionality being considered a prerequisite for "radiomimetic" activity [151,265]. Agents able effectively to inhibit tumour growth were also found to elicit chromosomal aberrations in root tips of suitable plants, and this kind of radiomimetic effect was assumed to play a role in the inhibition of growth. By inference from the use of di- or polyfunctional alkylating agents as cross-linkers in textile chemistry [163], their radiomimetic properties were suggested to be caused by a cross-linking of chromatids [151,265]. After the establishment of the double-helix structure of DNA the critical cross-linking was suggested to consist in chemical bridges between the DNA strands [247]. However, it was soon realized that, in agreement with Rapoport's demonstration of the mutagenic activity of monofunctional alkylating agents [349], chromosomal aberrations and other genetic effects could also be caused by related monofunctional agents, such as glycidol, ethylene oxide and monofunctional ethylenimines [31,266]. Furthermore, it was not a question of qualitative difference but rather a matter of differences in quantitative effectiveness between di- or polyfunctional and monofunctional alkylating agents, the latter being some 50 times less effective, for example, in inhibiting tumour growth [261,264: p. 26, 266,342]. Whether one considers the difference between di- and monofunctional agents to be qualitative or quantitative will then depend on the definition of the end-point. If the end-point is a 90--100% inhibition of turnout growth, many agents, especially those which are monofunctional, are too toxic, owing to their reactions causing enzyme inhibition etc. which is not enhanced by difunctionality, for this end-point to be reached at all [342,359]. The influence of functionality on the biological effectiveness is easily visualized in comparisons of ethylene oxide (I) and diepoxybutane (XV) because the reaction rates of these compounds are practically equal (Table 4). In the linear region of dose--response curves for mutation, XV was found to be some 30 times more effective, per primary alkylation, than I (Table 12, cf. ref. [186]). Within the limits of accuracy of the experiments (+ a factor 3), this conclusion is also valid for forward mutation in widely different systems, such as E. coli Sd4 [186], reversions of suppressor (i.e. forward mutation) character in Sch. pombe [175], chlorophyll mutations in barley [174], and recessive lethals in Drosophila [32]. The 1,2,5,6
T A B L E 12 R E L A T I V E E F F E C T I V E N E S S O F D I E P O X Y B U T A N E ( X V ) C O M P A R E D W I T H M O N O E P O X I D E S (=1) I N V A R I O U S S Y S T E M S R E C O R D I N G F O R W A R D AND BACK MUTATIONS To illustrate t h e role o f f u n c t i o n a l i t y , d a t a f o r t r i e t h y l e n e m e l a m i n e are i n c l u d e d . D a t a f o r t h e r e l a t i v e killing e f f e c t i v e n e s s o f X V are i n c l u d e d as well.

System

Ethylene oxide

Diepoxybutane Mutation

Killing

Triethylene melamine

Ref.

186; u n p u b l , d a t a

Forward mutation E. coli Sd4

1

16 ( 1 0 - - 2 5 )

14

2500

Drosophila

1

43

16

2500

32

Barley, 22 ° c h l o r o p h y l l m u t a t i o n s

1

~102

9300

99; unpubl, data

Barley, 3 a n d 24 ° c h l o r o p h y l l m u t a t i o n s

1

Sch. p o m b e

1 a

~102 c 25--50 L: ~38 (50) D: ~30 (40) meso: ~ 1 7 (20)

174 175 ~1

"

10

Back.mutation S. t y p h i r n u r i u m HisG46 TA1535 TA1535

1 1 1 b

1 1.3 ~ 0 . 5 (0.2 b)

1 1

1--2 ~0.3 (effectiveness) ~1 (efficiency [87,88])

30 350

17 60

186; u n p u b l , d a t a 186; u n p u b l , d a t a 299

N. crassa ad°3A

L o w doses High doses

~I

228

a P r o p y l e n e o x i d e ( I I ) , h a v i n g a b o u t 5 0 - - 1 0 0 % o f t h e e f f e c t i v e n e s s o f I in this s y s t e m . Figures in p a r e n t h e s e s r e f e r t o t h e r a t i o o f t h e e f f e c t i v e n e s s t o t h a t of I L b Glycidol ( V I I I ) is u s e d as a r e f e r e n c e (figure in p a r e n t h e s e s ) . T h e e f f e c t i v e n e s s r e l a t i v e to I a s s u m e d f r o m a r a t i o V I I I / I a b o u t 2.5; cf. t e x t . c Earlier e s t i m a t e r e f e r r e d t o m u t a t i o n s p e r e p o x y g r o u p [ 9 9 , 1 8 6 ] .

69

crassa ad-3A [228]) I and XV are about equally effective per initial alkylation

(Table 12). This indicates that mono-alkylation is required and is sufficient to produce base-pair transitions, whereas additional effects, such as those due to the second epoxide function, are not scored by the back-mutation systems. Litfie is known about the chemical background to the greater mutagenic potential of difunctional agents (cf. Sect. 5.4.4.2.). A comparison of mutation frequencies in strains responding to different changes in DNA might help to elucidate this, but such experiments are still lacking (except for glycidaldehyde; v. infra Sect. 5.4.4.3.). Approximately equal mutation frequencies are observed with XV and diepoxyoctane in the back-mutation system [299]. For Table 12 the relative killing effectiveness was calculated from the ratio of the DLs0 of I and XV interpolated or estimated from available data. It is noteworthy that this value is approximately equal to, or higher than, the relative mutagenic effectiveness. This is consistent with a genetic mechanism being involved in the lethal action of XV. The high lethal effectiveness of XV is also expressed in S. t y p h i m u r i u m , and more in the excision-repair-deficient strain TA1535 than in G46. This shows that the killing of these cells, where XV and I are equally effective in inducing back-mutation, is at least partly caused by a "forward" genetic change. In N. crassa the two compounds are about equally toxic. This should be interpreted as a predominance of a monofunctional change in DNA [228] through efficient repair of the lesion due to the second function of XV or a non~enetic mechanism, e.g. o f the kind expected if spores cannot tolerate alkylation of a reactive SH~nzyme [221,222]. A more equal killing effectiveness of the two compounds is also found in barley, if the end-point is defined as immediate inhibition of germination, in contrast with the delayed death characteristic of genetic damage to plant seeds [99]. The values given in Table 12 for bacteria and moulds as well as for barley were obtained under conditions where the concentrations of the agents during the treatment were approximately constant, i.e. the doses (concentration × time) were known. In Bird's [32] experiment with Drosophila the agents were injected into the abdomen; the values given assume equal rates of decomposition in vivo of the compounds compared, and they should therefore be considered uncertain. In Heslot's experiments with Sch. p o m b e [175], ethylene oxide (I) was not included. Comparison was therefore made with propylene oxide (II), which in available studies showed about the same mutagenic effectiveness as I, in accord with the fact that the reaction rates are equal (Tables 4 and 9). In the induction of reversions in N. crassa ad-3A, I is about 3 times more effective than XV at high doses where dose--response curves are exponential (cf. Sect. 4.8.1.). If compared at equal degree of killing, which in this material is somewhat more effectively caused by I, the two compounds are about equally efficient mutagens. At low doses/dose rates, where linearity is approached, XV is, if anything, about twice as effective as I. The difference between them was not significant, however. A few of the studies in Table 12 included the trifunctional ethylenimine derivative, 2,4,6-tris(ethylenimino)-l,3,5-triazine (triethylene melamine, TEM) as well. For this compound the back-mutation systems appear to be one order of magnitude more sensitive than expected from the computed extent of alkylation at n = 2. It should be remembered, however, that H÷-catalysed reactions

4.1

2.3 c

0.29

0.38

0.01

V I I I , glycidol

IX, e p i c h l o r o h y d r i n

XV, d i e p o x y b u t a n e

X V I I , diglycidyl e t h e r

For comparison: TEM

•

kg -1)

g

300 f

~250 f

~250

100--300

~ 250

(~150) a

(rad-equ.)

e

(~125) ~175

250

4000

6000

1.2

1.8

28

42

( m m o l e • kg -1)

(1000)

90 ( r a t ) d

1740 (mouse) b 4000 (rat) b

(800)

(~150)

835 (mouse) b 1460 (rat) b

(1150)

(rad-equ.)

CL50 (ppm)

CLs0 (ppm)

Uptake

Other data

I n h a l a t i o n d u r i n g 4 h, rat: d a t a f r o m Weil e t al. [ 4 4 2 ]

0.63

18 28

9 10

( m m o l e • kg - 1 )

Uptake

(500)

(500) (800)

(250) (280)

(rad-equ.)

a I n j e c t i o n , i.p., of saline s o l u t i o n [ 1 2 ] ; in o u r o w n e x p e r i m e n t s w i t h m i c e a n d r a t s ( [ 1 0 4 , 1 0 6 ] a n d E h r e n b e r g , u n p u b l i s h e d ) , t h e a c u t e t o x i c i t y , w i t h d e a t h o n t h e f o l l o w i n g d a y s o m e t i m e s a m o u n t e d t o l o w e r values, 2 . 5 - - 5 m M . b From [195]. c S o m e w h a t l o w e r values ( 1 . 0 - - 1 . 5 m m o l e • kg - 1 ) h a v e b e e n r e p o r t e d [ 2 5 1 ] . d From [180];see [190]. e C o r r e c t i o n f a c t o r 1 - - 3 , cf. S e c t i o n 5 . 4 . 4 . 3 . (d). f C o r r e c t i o n f a c t o r f o f e q n . (7) se~ e q u a l t o 3 0 ( f o r f u n c t i o n a l i t y ) . g C a n n o t b e e s t i m a t e d b e c a u s e o f c o m p l e t e l a c k o f k n o w l e d g e of l i f e - l e n g t h o f t h e c o m p o u n d in vivo.

8.6

(~5) a

(mmole

Mouse, i.p. in a r a c h i s oil; d a t a f r o m Ross [ 3 5 8 ] DL50

II, p r o p y l e n e o x i d e

I, e t h y l e n e o x i d e

Compound

U p t a k e f r o m air e s t i m a t e d a s s u m i n g t h a t all m a t e r i a l in t h e m i n u t e v o l u m e (1.0 1 • kg -1 in t h e m o u s e , 0 . 6 6 1 • kg -1 in t h e rat, [ 2 6 9 ] , cf. ref. [ 1 0 6 ] ) is a b s o r b e d . Tissue ~ioses e s t i m a t e d a s s u m i n g t h e r a t e o f e l i m i n a t i o n t o b e t h e s a m e in b o t h species a n d f o r all c o m p o u n d s as for I in t h e m o u s e , k = 4.6 h -1 [ 3 2 9 ] . T h e rade q u i v a l e n c e was d e t e r m i n e d f r o m E q n . (7) t o 1 2 5 r a d - e q u , p e r m M • h [ 1 0 6 ] .

L E T H A L DOSES OF SOME MONO- A N D D I F U N C T I O N A L EPOXIDES

T A B L E 13

o

71 characteristic of ethylenimines certainly contribute: even at pH 7 the hydrolysis of triethylene melamine is enhanced some 15% by this type of reaction ([358]; Table 4). For this reason, the exact extent of alkylation is strongly dependent on the local pH and should therefore be measured directly. However, the results in the forward mutation systems agree, indicating that, per initial alkylation, triethylene melamine is 1--2 orders of magnitude more effective than XV. The toxicities of epoxides to mammals exhibit a similar trend, in agreement with a genetic mechanism at least for the diepoxldes. DLs0 of monofunctional epoxides are some 5--40 times higher than those of comparable difunctional compounds (Table 13). Values from studies with administration by gavage cannot be used in such comparisons. As indicated by the rapid increase in DLs0 in the order I < II < I I I [442], and in agreement with the weak carcinogenicity of epoxides given by stomach tube [415], H÷-catalysed hydrolysis is expected to occur in the stomach [421]. When this happens, the glycols and chlorohydrins so formed will strongly influence the DLs0 (cf. Sect. 5.3. and Table 7). Examination of the available data from studies in which the toxicities of mono- and diepoxides have been compared, by using i.p. injection in the mouse [358] or inhalation experiments in the rat [442], shows that in both studies, XVa is about 30 times more toxic than I (Table 13). This, and the fact that estimated rad~equivalents of the DLs0 agree acceptably with the DLso of acute exposure to ionizing radiation (Sect. 6.), supports the idea that fatal events have a genetic background. These estimates are very rough, because they were made by assuming that the retention time in both species was the same as that determined for I in the mouse (Sect. 5.3.). The DLs0 value of 8000 ppm • h determined in the study of Rowe et al. [361] on the toxicity of II in rats is about one half of that reported by Weil et al. [442]. From reports on tests for dominant lethality and carcinogenicity of epoxides, it appears that especially the difunctional epoxides cause a delayed death, which is also seen with ionizing radiation. However, the symptoms and the inactivation mechanism of death have never been described in detail. After exposure to monofunctional agents, death is more immediate, with a number of symptoms that might be related to alkylation of proteins. For this reason, the tad-equivalence of lethal doses of monofunctional epoxides would be somewhat under-estimated. This appears to be especially true for I which, when injected i.p., had a DLs0 estimated to correspond to 80--150 rad-eq. The rad-equivalence of the DLs0 of I administered p.o. would amount to about 150 in the mouse and about 250 in the rat [361,442], assuming that the major part of this compound, which is relatively stable to H÷-catalysed hydrolysis, contributes to the tissue dose. No dose--response curves for the carcinogenic action of epoxides have been established except those of Shimkin et al. [375] for the pulmonary-tumour incidence in mice of strain A. These studies concerned L-diepoxybutane (XVa) and three other diepoxides but no monoepoxide. 5.4.4.2. Viewpoints on mechanisms Using the criterion of reversibility of heat denaturation of DNA, XVa was shown by Lawley and Brookes [247] to interlink the strands of native DNA, an observation consistent with the appealing cross-linking hypothesis. From the

72 earlier demonstration of a product that is probably a diguaninyl derivative linked by a four-carbon chain, obtained in the reaction of myleran and DNA, the authors [247] inferred that DNA is cross-linked by XVa through the formation of the analogous diguaninylbutanediol (cf. 5.2.2.). The latter is obtained in the reaction of XVa and guanosine [49]. This diguaninyl derivative is also claimed to be formed in vivo [337]. Because myleran is a very ineffective crosslinker of DNA strands [426], it seems less likely that the diguaninyl derivative of XVa is formed in inter-strand cross-linking. Two further facts should be considered. First, the four-carbon chain supplied by XVa is much shorter (about 4 A) than the distance (about 8 A) between the N-7 of the guanines of a GC to CG pair [247,415 ]. This would require an appreciable flexibility of this distance within the DNA molecule [415] or a distortion of the DNA as a result of the first alkylation reaction [249]. Second, according to investigations in T7 phage, XVb lacks the ability of XVa to interlink the DNA strands [427]. The biological effectiveness of XVb shows the enhancement characteristic of difunctional agents, although it is somewhat less pronounced than that of XVa (Sect. 5.3., Table 3), and the two diastereomers have practically equal reactivity (Table 4). (Comparisons of XVa and XVb on the biological and molecular levels in materials other than T7 phage might contribute to the urgently needed elucidation of mechanisms.) Even if the length of the middle portion of diepoxides is increased, the sixcarbon chain offered by XVI is still somewhat short, whereas the maximal interatomic distance covered by 1,2,7,8
73 sons, the mechanism behind the effect of functionality is no doubt more complicated than the inter-strand cross-linking hypothesis could account for. Besides intra~strand cross-linking (which might involve phosphate groups [ 3 ] as well as bases) suggested by the phage experiment just referred to, inter-linkage of DNA with other cellular constituents, e.g. proteins [ 5], might contribute to the high effectiveness of difunctional agents. Various possibilities have been discussed by Trams et al. [407]. If changes of these kinds within DNA strands are less effectively repaired than the same number of changes caused by monofunctional agents, enhanced biological effectiveness of difunctional epoxides could be seen as a mainly quantitative problem (see also 4.8.). Lawley et al. [249] have discussed the formal similarity of intra-strand cross-links to thymine dimers characteristically produced by UV radiation. Perhaps the high sensitivity of S. typhimurium TA1535, which is uvr-, to XV but not to I (Sect. 5.4.4.1.) reflects this similarity. Laskowski and Lehmann-Brauns [241] found a similar cross-sensitivity to XV and UV in yeast strains, and several observations of this kind have been made with other difunctional agents (quoted in refs. [241,246]). As has been indicated by Clarke [62] (Table 2B), XV but not methylnitrosourethane shows a synergism with caffeine on number of revertants and killing under certain conditions in experiments with E. coli. This effect resembles that obtained more generally with UV radiation and caffeine. N. crassa has a high resistance to UV; this might explain the non-expression of functionality versus the lethal action of XV (G. KSlmark, personal communication). 5.4.4.3. "Mixed difunctionality "

Van Duuren [415,419] has repeatedly drawn attention to the necessity of considering other chemical functions in substituents of monoepoxides in the interpretation of carcinogenicity. A few problems of this kind will be discussed briefly in this section. (a) Epihalohydrins. Alkylation products of epichlorohydrin (IX) and its Br and I analogues (formulae X and X I ) w i l l have a halohydrin structure. Thus they will have a certain alkylating ability that could be enhanced through alkali-catalysed formation of a new epoxide ring (cf. the discussion of halohydrins in Sect. 5.4.2., and eqn. (3)). The latter reaction, which plays an important role in epoxy-resin technology, e.g. the synthesis and polymerization of compounds of the type XVIII, may lead to secondary effects related to crosslinking by difunctional alkylating agents [92]. As a basis for risk estimations it is therefore important to clarify whether the epihalohydrins and structurally related compounds exhibit an enhanced effectiveness due to such secondary difunctionality. The results of available mutation experiments that permit a comparison of IX with other compounds show a mutagenic effectiveness in acceptable agreement with relative primary reactivities. This concerns forward mutation in barley and Sch. p o m b e (Sect. 5.1.1., Table 9) and reversions in N. crassa (Table 8), although there is uncertainty because of. non-linearity of the dose--response curves. Corresponding values were found for X in N. crassa (Table 8). Although the relatively high toxicity of IX (Table 13) remains to be clarified, it seems that a secondary reactivity of epihalohydrins could influence their genetic toxicity by a factor

74 2 at the most. A relatively low carcinogenic p o t e n c y of IX (Table 3) is consistent with this conclusion. (b) Carbonyl groups. Compared with other monoepoxides, glycidaldehyde (XII) is an effective carcinogen [420,421,424] and its mutagenic effectiveness is very high [299] considering the moderate reactivity of the epoxide function (Table 4). XII was also much more effective than glycidol (VIII) in inducing petite mutation in yeast [192]. The need for clarifying the toxicology of this c o m p o u n d is underlined b y the possibility that it is an important metabolite of the ubiquitous pollutant acrolein (for review see [193] ). The fact that A : T to G : C transitions, as well as frame-shift mutation, are induced [69] indicates a mechanism different from that of other monoepoxides. (However, so far frame-shift-type mutation has only been observed in the experiments of Corbett et al. [69] with the phage T4rII system. XII does n o t seem to have been tested in Salmonella strains responding to frame-shift-inducing agents; cf. Table 2. Such experiments are urgently needed, and due attention must be paid to the difficulties of avoiding side reactions such as polymerization.) Van Duuren has repeatedly pointed o u t the importance of the reactivity of the aldehyde group to the biological activity of XII [415]. He and his collaborators were recently able to demonstrate the formation of an additional imidazole ring on D N A guanine by the introduction of two carbons b e t w e e n Gua-N 2 and -N-1 [422]. The initial reaction is probably the formation of a Schiff base with Gua-N 2, as shown for certain ketoaldehydes [373], followed b y epoxide-ring opening through attack on carbon-2 of the residue b y Gua-N-1. As a parallel, chloroacetaldehyde had a mutagenic effectiveness several orders of magnitude greater than that expected from its rate of primary alkylation [187] (cf. Table 11). This is possibly a consequence of similar reactions with adenine and cytosine of the D N A [27]. Epoxide o f vinyl chloride. In accord with experiments in vitro [154], the main alkylating metabolite of vinyl chloride is thought to be chloroethylene oxide (XIII) [331], a highly reactive epoxide (Table 4). In experiments with E. coli Sd4 [187], this c o m p o u n d exhibited per alkylation at n = 2 a mutagenic effectiveness some 20 times greater than that of I and other monofunctional alkylating agents. XIII was also more effective than expected in the S. typhimurium back-mutation test [348]. The alkyl group introduced is rapidly hydrolysed to the aldehyde function, 2-oxoethyl (-CH2CHO). Possibly, a secondary reactivity of this aldehyde residue is a property of 2-oxoalkylation in general [187; cf. 409]. (c) Unsaturated and aromatic groups. The possible role of these groups in carcinogenesis has been discussed [415,419]. (See also Sect. 6.2.) As far as we know, additional e p o x y groups might be introduced metabolically into structurally complicated m o n o e p o x i d e s with double bonds or aromatic residues. A role of unsaturatedness in mutagenicity is indicated b y the comparison of butylglycidyl ether and allylglycidyl ether in barley seeds (Table 2; L. Ehrenberg, unpublished). In 24-h treatment at 20°C the c o m p o u n d s were equally toxic (CLs0 a b o u t 8 mM), but only the unsaturated allyl ether induced mutation (P of difference found < 0.01). However, its mutagenic effectiveness, per mM • h, is only a b o u t a third of that of I, despite approximately equal reaction rates [335]. This may be due either to steric hindrance or to a slower uptake of

75 the bigger molecule, suggesting a need for studies in a more suitable system. The metabolic fate of unsaturated compounds in plant seeds is unknown. The unsaturated analogue (VII) of 1,2¢poxybutane (III) is carcinogenic [423] and mutagenic [300]. Comparative mutation experiments with III and VII would give valuable information for the elucidation of mechanisms and for risk estimates in general (cf. Sect. 6.2.). (d) Hydroxy groups. In mutation experiments with plants, 2-hydroxyalkylating agents gave a spectrum of effects somewhat different from that obtained with analogous 2-methoxyalkylating agents, despite great similarity in reaction rates and patterns as determined by the substrate constant, s. In barley, 2-hydroxyethyl methanesulphonate is about twice as toxic as 2-methoxyethyl methanesulphonate and its mutagenic efficiency (mutation frequency at DLs0) is somewhat lower [330]. The spectrum of effects of hydroxyethyl methanesulphonate resembles that of ethylene oxide (I), which introduces the same group, HOCH2CH2--, onto nucleophilic centres. It was earlier indicated [147], and later corroborated in studies with the more reactive 2-hydroxy-and 2-methoxy-isopropyl methanesulphonates [258], that the hydroxyalkylating agents, including I [315], are highly effective inducers of chromosomal aberrations (Sect. 4.3.), whereas in this respect the 2-methoxyalkylating compounds resemble the analogous unsubstituted alkyl alkanesulphonates. Results indicating a higher mutagenic effectiveness of the hydroxy compounds have been obtained [258,330]. It is possible that the difference in effectiveness is about two-fold in barley (the results in Fig. 8 in the study of Lindgren et al. [258] are not comparable); it was not observed in Arabidopsis thaliana [147] and if there is a difference in E. coli [409] it is small. It appears theoretically possible that 2-hydroxyalkylation of DNA phosphates (Sect. 5.2.2.) could lead to scission of DNA strands via secondarily formed cyclic glycol diesters (cf. [218]): B.

O Bo

B1

[O-I B2

\ Iz % \ a \ --iIR--O--P--O--dR-- ~- --dR--O--P--O--~R-0 / O--H 0/%0 I I CH2--CH2

i [ CH2--CH2

B~

O

B

+

(20)

0/%

1 I CH2--CH2

(dR, deoxyribose; B~, B2, bases.) The cyclic group at the phosphate end of the broken strand may be opened by hydrolysis. The triester group is in principle alkylating, and may become bound to the other DNA strand. Indirect support for the hypothetical reaction (20), Which bears a resemblance to the hydrolysis of RNA, was provided by the demonstration of a decreased reversibility of heat denaturation of 2-hydr0xyethylated DNA, but not of 2-methoxyethylated DNA, indicating, strand scission by the former treatment [433,435]. To get more insight into the mechanism, Shooter [376] demonstrated in model experiments with R17 phage RN:A that 2-hydroxyalkylation of diester phosphates of nucleic acids does occur. These reactions were carried out with II, which gives a relatively low yield, and with 2-hydroxyethyl methanesulpho-

76 nate in slightly acidic solution. In later studies of T7 phage DNA [377--379] where a special technique of alkaline hydrolysis was used to differentiate between phosphotriester hydrolysis and depurination as causes of backbone breakage, the former mechanism was hardly discernible, although not excluded within the resolving power of the experiment. The objection may be raised that, in these experiments, alkylation was carried out at pH 8.5, a pH at which a considerable fraction of the 2-hydroxyethyl methanesulphonate is converted into ethylene oxide (I) (eqn. (3)) within the incubation periods used. This leads to a loss of the advantage of the lower s of the model compound, with its relatively higher extent of alkylation of low-n nucleophiles (Sect. 5.2.3.2. and Table 4). In fact, the tendency towards phosphotriester hydrolysis was observed at shorter incubation periods. Bearing in mind the relatively slow rate of depurination (half-lives for dissociation of 7-(hydroxyethyl)guanine and 7-methylguanine from DNA being about 340 and 6 9 h , respectively; see [106]), we conclude that phosphotriester hydrolysis is most probably a characteristic result of treatment of DNA with I, XXVI and other 2-hydroxyalkylating agents. This has recently been indicated, too, for II and III, in contrast with fl-propiolactone [434]. Further experiments are required to clear up this question. The enhanced mutagenic effectiveness of glycidol (VIII) is not yet understood. Alkylation by VIII will introduce mainly 2,3-dihydroxypropyl groups onto nucleophilic atoms. No direct evidence of a secondary function of two hydroxyls is available. One important consequence of the hydroxyl in VIII is an increased hydrophilicity, a property that seems to favour reaction with DNA (current work, Ehrenberg et al.). Perhaps the relatively high tumour-inhibiting efficiency of dianhydro-hexitols, formed in vivo from, for example, dibromomannitol and mannitol myleran [118], can be explained in a similar way. 1,6Dibromo-l,6~lideoxy-D-mannitol is in principle a difunctional halohydrin, which is partly converted in vivo to a diepoxyhexane (XVI), hydroxylated on carbons 3 and 4. Mannitol myleran, which is converted into the same diepoxide, is carcinogenic [375]. Studies of the possibly enhanced genetic effectiveness of these hydroxylated compounds would be of interest because of their structural relationship to the highly effective tetrahydro-diol-epoxides of PAH (e.g. XIXb). The pattern of genetic effects characteristic of 2-hydroxyalkylation might be caused by products of hydrolysis, although this does not seem likely. Certain effects on blood clotting in mice exposed to wood shavings sterilized by ethylene oxide (I) were ascribed to the reaction product of I with water, namely ethylene glycol [6]. At high concentrations, ethylene glycol seems to have a weak mutagenic activity in Drosophila [349] and barley (L. Ehrenberg unpublished; cf. [160]), possibly related to the chromosome stickiness characteristically induced [327]. Too little is known of these effects to permit a risk estimate. Ethylene glycol is to some extent oxidized in vivo to glycolaldehyde, and the latter compound eould be responsible for genetic toxicity, either through Schiff-base formation, for example with DNA guanine-N2 [367,373], or through alkylation by the internal lactol (Sect. 4.7.3.). (In this context it should be pointed out that an oxidation in situ of hydroxyalkyl groups intro-

77 duced into DNA, which would be expected to increase the mutagenic effectiveness, has never been investigated.)

6. Risk estimates

6.1. Rad-equivalence o f genetic effects o f epoxides in mammals Practically no data are available t h a t permit an unambiguous test of the validity of eqn. (7) (Sect. 5.1.) for genetic effects of epoxides in mammals. Strictly speaking, such a test requires comparable data on the effects of radiation and of the chemical in the same animal species and, especially in the case of cancer, in the same animal strain. Furthermore, knowledge of the rate of elimination (given as the pseudo first-order rate constant k; eqn. (19)) of the c o m p o u n d from the organism is also needed. So far k has only been determined for a few compounds in the mouse (Sect. 5.3.). In the following calculations, the value 4.6 h -1, used for ethylene oxide in this organism, will tentatively be used also for other epoxides and applied to both the mouse and the rat. We have assumed t h a t this extrapolation will not be too erroneous. An extrapolation between species is to some extent justified by the fact that the k value for I in man cannot differ very much from k for the mouse [59] (Sect. 5.3.). Certain support for these assumptions can be derived from the fact that if we calculate the tad-equivalence of DLs0's of epoxides for the mouse and the rat in this way, values in acceptable agreement with the DLs0 of radiation in the same species are obtained (Table 13). In inhalation experiments it has to be assumed that the minute volume of the rat is about 60% of t h a t of the mouse [269].

6.1.1. Chromosomal aberrations and related effects Moutschen [312] has directly compared X-rays and a few alkylating agents, i.a. diepoxybutane (XV), with regard to frequencies of induced aberrations at meiosis in hybrid 101 × C3H mice. At anaphase II the available dose--response curves are approx, linear, showing that XV at 10 mg/kg gives the same response as about 150 tad. Calculating the rad-equivalence, using eqns. (7) and (19), assuming k = 4.6 h -1, Co = (10/86) mM = 0.116 • 10 -3 M, and f = 30 for difunctionality (Sect. 5.4.4.1.; compared with ethylene oxide, this f contains a small correction for difference in kn=2), we obtain: Risk = 1 • 107 •

0.116 • 10 -3 4.6

• 1.4 • 10 -2 • 30 -- 1 • 102 tad-equivalents,

in acceptable agreement with the directly determined radiation dose for the same effect. Tests for induction of dominant-lethal mutations b y XV in mice are negative or of doubtful significance and will be discussed in the following section. In the rat (Long--Evans), Embree et al. [119,121] studied the effect of ethylene oxide (I) at 1000 ppm for 4 h, i.e. an exposure dose of 4000 ppm • h. In the mouse, 1 ppm • h. leads to a tissue dose of 0.58 • 10 -6 M . h , and is approximately equal in different organs [106]. If the rat absorbs 40% less, owing to its lower respiration rate, the total dose would be estimated to be 1.4 • 10 -3 M • h. According to (7), this dose corresponds to about 200 tad-equivalents.

78 A significant effect, limited to matings during the first 5 weeks, a m o u n t e d to an average for the 5 weeks o f a b o u t 1 (0.9) dead foetus per female, and 1.5 dead implants per female in weeks 2--3. The fractional increase in post-implantation deaths according to these figures corresponds to 0.13 for the whole 5-week period and 0.24 for weeks 2--3. The increase in pre-implantation deaths was n o t significant. Data for radiation-induced dominant-lethal mutations in the rat have n o t been published *. However, L y o n [273] has shown that, in ot her r o d e n t species, the frequencies o f post-implantation death do n o t vary much, averages per 100 rad for the post-meiotic period being: mouse 0.08 (weeks 1--3, doses 100 and 500 tad), guinea-pig 0.08 (weeks 1--5, dose 500 rad), and hamster 0.12 (weeks 1--5, doses 100, 200, 300 rad). The rabbit was an exception, most o f the e m b r y o n a l deaths (in weeks 1 - 3 following 500 rad) occurring before implantation. F r o m a comparison of these data for t he dominant-lethal response of the three r o d e n t species, and with due regard to differences in the rate of sperm devel opm e nt and variations in the sensitivity of different stages and animal strains, the response to I observed by E m bree et al. is concluded to correspond to the response to (200 + 100) rad. This is consistent with what is e x p e c t e d from the rad-equivalence of the chemical dose. Because Appelgren et al. [12] judged a similar test for the ability of I t o induce dominant lethals in the mouse to be negative, this test will be discussed under 6.2.2. In the same experiments, Embree [119] also investigated the induction o f micronuclei in p o l y c h r o m a t i c bone-marrow e r y t h r o c y t e s . A doubling dose of 200 p p m • h o f I was found. By comparing this with a doubling dose o f a b o u t 20 tad for genetic effects in general, he arrived at a risk estimate of a b o u t 100 mrad-equ, per ppm • h, in fair agreement with our previous value for the mouse established on the basis of the e x t e n t of protein alkylation. Although this remains to be investigated in detail, we have a feeling t hat risks are bet t er evaluated on the basis of absolute responses than from a relative measure such as the doubling dose. Variations in the shape of the dose--response curves after t r e a t m e n t with various mutagens [ 199, 200 ] make micronuclei difficult t o q u a ntitate and perhaps less suitable for generalized risk estimation. Partly because o f a delay in the d e v e l o p m e n t o f damaged cells at high radiation doses, X-rays appear to be m o r e effective at lower doses t han at higher doses (2.8--4 • 10 -4 rad -1 at a b o u t 200 rad [ 170, 199, 200] and 8 • 10 -4 rad -1 at 10 rad [199, 2 0 0 ] ) . I gives a similar dose--response curve, 10 • 10 -6 (ppm • h) -1 at 100 ppm × 4 h and 4 • 10 -6 (ppm • h) -1 at 500 ppm × 4 h, although a lower effectiveness at very low doses c a nnot be excluded [ 119 ]. However, there are indications t h a t m e t h y l m e t h a n e s u l p h o n a t e induces this effect in two-hit kinetics or possibly has a no-effect level at a b o u t 0.14 m mol e (kg b.w.) -~, corresponding to a b o u t 65 rad-equ. (}~ = 1.3--2; cf. Table 6). It has been suggested t hat m e t h y l m e t h a n e s u l p h o n a t e induces this kind of effect by primary changes which are subjected to error-free repair at low doses [ 19 9]. The slopes o f the dose--response curves o f I, relative to the effectiveness of X-rays, a m o u n t to at low doses: 10 • 10 -6 (ppm • h) -I = 12 mrad (ppm • h) -1 8 • 10 -4 rad -1 at high doses: 4 • 10 -6 (ppm - h) -1 = 12 mrad (ppm • h) -~ 3.4 • 10 -4 rad -1 • N o t e a d d e d in p r o o f : B.A. T a y l o r a n d A.B. C h a p m a n ( G e n e t i c s , 6 3 ( 1 9 6 9 ) 4 5 5 - - 4 5 6 ) h a v e s h o w n t h a t t h e s e n s i t i v i t y of t h e r a t is c o m p a r a b l e t o t h a t o f t h e m o u s e f o r t h i s e f f e c t .

79 The expected value, according to the above calculation for dominant lethal mutations, is 12 mrad-equ. (ppm • h) -1. This is about one-quarter of the value, 50 mrad-equ. ( p p m . h) -~, expected from the above calculation for dominantlethal mutations. Until the ratio of tissue dose to exposure dose of ethylene oxide has been determined directly in the rat (cf. above), it cannot be decided whether these values are really different *. Rather, for this end-point, the agreement should be considered reasonable. The absorbed dose of I giving an increased frequency of chromosomal aberrations in the rat in Strekalova's experiments [392] is of the order of 5--10 radequ. A large transient effect of the kind described (see Table 2) cannot be excluded, but requires re-investigation. In the semi-chronic exposure [393] the total dose was of the order of 100 rad-equ., thus corresponding more realistically to the observed effect. For a comment on chromosomal aberrations induced by XXVI in vivo, see Sect. 6.4.1. below. 6.1.2. Tumours The only dose--response curves for epoxide-induced tumours that could possibly be compared with a corresponding response to radiation are those for lung adenomas in A/J mice induced by L
80 including cancer, seem to exhibit a rad-equivalence that could be expressed in terms of one and the same formula, eqn. (7). 6.2. False-negative tests

The question of whether certain negative test results are false, i.e. whether the true mutagenicity or carcinogenicity of a substance is n o t detected because of limited resolving p o w e r [93] of the laboratory experiment, has a bearing on the question of whether a subdivision of chemicals of a class such as epoxides (or precursors of epoxides) into mutagens and non-mutagens or carcinogens and nonCarcinogens is possible at all. This question may be seen from a merely qualitative viewpoint, by studying the laboratory experiments as such, or it could be qualified b y introducing a quantitative aspect, namely the probability that a non-acceptable risk to man remains " h i d d e n " within the confidence interval of the response zero. The possibility that a negative test is false can be analysed statistically by calculating the probability (fl) that, provided a certain contra-hypothesis (H1) is true, the null hypothesis (H0) of no effect would have been accepted at the chosen level of rejection (a) (cf. ref. [93 ]). With access to positive test data for related compounds, H~ for the calculation of the fi-error can be formulated as the expected frequency of tumours or mutations, assuming that the response is proportional to the applied dose and to the reaction rate (at n = 2), with corrections for difunctionality, etc., where appropriate (7). In certain cases, especially when the response of the test system to radiations is known, it might be practical to formulate H1 on the basis of the rad-equivalence of the applied dose. On the other hand, this is n o t always possible, e.g. with skin tests for carcinogenicity. In such cases, H~ could be based on a positive test with a related c o m p o u n d . 6.2.1. Cancer tests A few data from negative cancer tests cited in Table 3 were considered in this way (Table 14). By making the reasonable assumption that cancerinitiating and mutagenic effectivenesses are proportional, contra-hypotheses H, (i.e. expected frequencies) were calculated for the negative tests b y using skin application of ethylene oxide (I) and epichlorohydrin (IX) on the basis of local t u m o u r frequencies induced b y the relatively powerful carcinogen D L d i e p o x y b u t a n e (XVa). For the skin application tests in Swiss mice [423,424] (Table 3) the probability, fl, that no t u m o u r will be obtained is 30% for I and 70% for IX. A similar evaluation for the s.c. application of I in the rat [436] is ambiguous because it is n o t known h o w comparable the rat strains are. However, in a comparison with Van Duuren's [421] positive test with XVa, where 10 tumours were found in 50 animals, the probability of finding no t u m o u r in 12 animals in Walpole's [436] old test amounts to 40%. A critical evaluation of the only reported cancer test on propylene oxide (II) [436] that was concluded to be positive leads to the conclusion that a difference in carcinogenic effectiveness between I and II has n o t been demonstrated unambiguously (see end of this Section). Furthermore, consideration of the much faster disappearance of I than of

81 TABLE

14

~-ERROR OF SOME NEGATIVE CANCER TESTS ESTIMATED FROM POSITIVE TESTS WITH RELATED COMPOUNDS

ON THE BASIS OF EXPECTATION

I n these simple cases, where the c o n t r o l frequencies are e x p e c t e d to be zero, ~ = ( 1 _ p ) n w h e r e p is the e x p e c t e d f r e q u e n c y according t o H 1 and n is the sample s i z e [ 9 3 ] . I n the e s t i m a t i o n of p , X V a was

a s s u m e d t o be 3 0 t i m e s and IX to be 2 t i m e s m o r e effective than I per unit dose. Compound

T e s t (Table 3)

Dose

Cancer frequency

fl (%)

XVa, DL-diepoxybutane

Mouse, S w i s s - M i l i e r t o n skin application o f acetone solution

3 X 3 rag/week =

Found:

--

0.105 mmole/week

I, e t h y l e n e o x i d e

XI, epichlorohydrin

XVa

Mouse, Swiss-Millerton 3 × 1 0 r a g / w e e k = 0.68 mmole/week acetone solution

E x p e c t e d ( H 1 ): p = 0.043; Found: 0 / 3 0

30

s k i n application o f

Mouse, S w i s s - M f l l e r t o n 3 X 2 r a g / w e e k = skin application o f 0.065 mmole/week acetone solution

E x p e c t e d ( H l ): p = 0.008; Found: 0/50

70

1 r a g / w e e k , totally 2--3 mmole/kg

Found: 1 0 / 5 0 = 0 . 2

--

totally 23 m m o l e / k g during 9 4 d a y s

E x p e c t e d ( H 1 ): p = 0.07; Found: 0 / 1 2 Found: 1 ( 2 ) / 3 0 = 0.03

Rat, Sprague--

D a w l e y , s.c. R a t , albino,

I

6/30 = 0.2

s.c. VII, 3,4-epoxy-l-butene

Mouse, Swiss-Millerton 3 X 1 0 0 m g / w e e k skin application

III, 1,2-epoxybutane

Mouse, Swiss-Millerton 3 X 1 0 r a g / w e e k skin application

E x p e c t e d ( H 1 ): p = 0.0033; Found: 0 / 3 0

~40

90

XV from the site of application leads to the conclusion that the calculated ~errors are certainly under-estimates. According to this way of reasoning, it is not justified to consider I non~arcinogenic *. IARC's [190] conclusion ("although no carcinogenic effect was observed, the data do not allow an evaluation") was certainly justified. The episode, described by Reyniers et al. in 1964 [ 3 5 1 ] , in which an outbreak of tumours in germ-free mice could be causally connected with accidental introduction of ethylene oxide sterilization o f the corn bedding, was disregarded in this evaluation by IARC. This position was certainly correct because the precise mechanism and the eliciting agent are still debatable. In the event, all male mice died from haemorrhages of the kind earlier [6] ascribed to ethylene glycol; reproduction failure was observed (this may be related to t h e action of chlorohydrins, see Sect. 4.1.2.), and over 90% of the females developed tumours at different sites. Considering the technical importance of I and the widespread use of products containing residual quantities of I and its reaction products it is astonishing that n o b o d y has felt it necessary to clarify this remarkable event experimentally. In the case o f IX, carcinogenicity was demonstrated in other tests (Table 3) which, in addition, indicate that application to skin is not a suitable procedure to test the carcinogenicity of these compounds [417]. This may be related to skin damage which counteracts the manifestation of tumours [ 4 1 9 ] . Van Duuren [ 4 1 5 , 4 1 9 ] is certainly right in pointing out the importance of an aromatic ring, as in styrene oxide (XIV), or a double bond, as in 3,4-epoxy* Concerning

a recent positive test o f I and II, see f o o t n o t e , p . 8 5 .

M E T H A N E S U L P H O N A T E (Original d a t a f r o m E.C. a n d J . A . Miller)

71

75 75 77

24

31 25 38

E t h y l m e t h a n e s u l p h o n a t e (6 i n j e c t i o n s )

2-Hydroxyethyl methanesulphonate (3 injections)

2-Hydroxyethyl methanesulphonate (6 injections)

S o l v e n t only (6 i n j e c t i o n s )

0

0

0

8

3 b

0

1 l e i o m y o m a (vagina)

0

3 s q u a m o u s cell c a r c i n o m a s (Zymbal's gland) 1 l e i o m y o m a (vagina) 1 o d o n t o m a (jaw)

1 endometrial carcinoma 1 melanocarcinoma (eye)

18

17

16

0

2

N u m b e r of r a t s killed w i t h o u t tumours at 18 m o n t h s

b e n i n g m a m m a r y t u m o u r s w e r e e i t h e r f i b r o m a s o r f i b r o a d e n o m a s ; a p p r o x i m a t e l y e q u a l n u m b e r s of e a c h t y p e w e r e seen. k i d n e y t u m o u r s w e r e m a l i g n a n t s a r c o m a t o i d t u r n o u t s t h a t h a v e b e e n d e s i g n a t e d as s t r o m a l n e p h r o m a s a n d c o m p l e x s t r o m a l - e p i t h e l i a l n e p h r o m a s b y R i o p e l l e J a s m i n (J. Natl. C a n c e r I n s t . , 4 2 ( 1 9 6 9 ) 6 4 3 ) . n u m b e r o f t u r n o u t - b e a r i n g rats in t h e g r o u p given 6 i n j e c t i o n s of e t h y l m e t h a n e s u l p h o n a t e is 23 (as c o m p a r e d w i t h 20 r a t s in t h e g r o u p ) b e c a u s e s o m e rats

b o r e m o r e t h a n 1 t y p e of t u r n o u t .

a The b The and c The

0

2 (2 b e n i g n )

1 (1 b e n i g n )

10 (4 a d e n o e a r c i n o m a s ; 6 benign)

12 (6 a d e n o c a r c i n o m a s ; 6 benign) a

83

38

E t h y l m e t h a n e s u l p h o n a t e (3 i n j e c t i o n s )

O t h e r sites

M a m m a r y gland

4 months

1 month

Kidney

N u m b e r of rats with t u m o u r s by 18 m o n t h s

Compound

A v e r a g e w e i g h t gain (g)

G r o u p s of 20 f e m a l e F i s c h e r rats, 6 w e e k s old a n d a v e r a g i n ~ 1 1 0 g in w e i g h t a t t h e b e g i n n i n g of t h e e x p e r i m e n t ~ w e r e i n j e c t e d i n t r a p e r i t o n e a l l y 3 o r 6 t i m e s . F o r t h o s e receiving 3 i n j e c t i o n s , t h e doses w e r e given o n d a y s 1, 5 a n d 9; f o r t h o s e r e c e i v i n g 6 i n j e c t i o n s , t h e d o s e s w e r e g i v e n o n d a y s 1, 5, 9, 16, 23 a n d 29. E a c h dose c o n t a i n e d 0.27 m m o l e of c o m p o u n d in 1 m l o f sterile 0.9% s o d i u m c h l o r i d e . T h e r a t s w e r e m a i n t a i n e d o n W a y n e B r e e d e r Blox ( A l l i e d Mills, C h i c a g o , I L ) a n d w e r e h o u s e d individually in s c r e e n - b o t t o m e d cages. F o o d a n d w a t e r w e r e available a d l i b i t u m . T h e r a t s w e r e a u t o p s i e d a t d e a t h , w h e n killed b e c a u s e of o b v i o u s t u m o u r s or sickness, or o n t e r m i n a t i o n of the e x p e r i m e n t a t 16 m o n t h s . T u r n o u t s or grossly a b n o r m a l tissues w e r e f i x e d in n e u t r a l f o r m a l i n a n d s t a i n e d w i t h h a e m a t o x y l l n a n d eosin. T h e d i a g n o s e s w e r e m a d e b y Dr. H e n r y C. P i t o t of t h e McAxdle L a b o r a t o r y .

C A R C I N O G E N I C I T Y OF E T H Y L M E T H A N E S U L P H O N A T E A N D H Y D R O X Y E T H Y L

T A B L E 15

to

83 butene (VII), in the vicinity of the e p o x y group, or of a long hydrocarbon chain (V, VI), for the carcinogenicity of certain monoepoxides. However, noncarcinogenic analogues were not always tested under comparable conditions. This is exemplified in Table 3 b y the pair VII and III; the former was applied undiluted to the skin, whereas in the negative test with the latter the comp o u n d was applied in acetone solution at a 10 times lower dose. If cancer frequency is proportional to the dose and to the reaction rate (Table 4), the probability that not a single t u m o u r would have been found in the test of the saturated c o m p o u n d (Table 14) is as high as 90%. A similar result is obtained from an analysis of the negative test of the 12-carbon epoxide, VI, which in the experiments of Van Duuren et al. [419] was applied at a 5 times lower dose than the weakly active higher homologue with 16 carbons (V). The long~hain monoepoxides V and VI were both positive in the experiments of Kotin and Falk [232a,b] (Table 3). However, these authors present little information on the methods used (from the types of t u m o u r observed, administration was probably by s.c. injection) and no control frequencies of the observed tumours are presented. This may cast some d o u b t on the relevance of the predominant t u m o u r type, malignant lymphomas, to carcinogenicity. In Table 5 of their paper, Kotin and Falk [232a] show approximately equal carcinogenic effectiveness for 5 epoxides, whereas no tumours were found for epoxycyclohexane (XXIII). From its low 8N2 reactivity [358] (Table 4), partly due to steric hindrance, this c o m p o u n d is expected to be a very ineffective mutagen/carcinogen. The possibility that the test with XXIII could serve as a control may be investigated statistically by assuming that all 6 tests have equal (true) t u m o u r frequency and then calculating the probability that zero lymphomas would appear in any one of the 6 tests. This probability is as low as 2.2%, which may be taken as an indication that the data reported do reflect a carcinogenic potential of the other 5 epoxides, including epichlorohydrin (IX) and styrene oxide (XIV), whereas XXIII is a very weak carcinogen. The impression that I (and other monoepoxides) are not very effective carcinogens is corroborated by studies of other agents introducing the 2-hydroxyethyl group onto nucleophilic atoms. This is shown b y tests of 2-hydroxyethyl methanesulphonate and di(2-hydroxyethyl)-N-nitrosamine in the rat. The carcinogenic potential of the former c o m p o u n d , synthesized in our laboratory [332], was compared with that of ethyl methanesulphonate in a test carried o u t by E.C. and J.A. Miller, Madison, WI (personal communication). The design and results of the experiment can be seen in Table 15. H y d r o x y e t h y l methanesulphonate has the advantage of being more easily handled than the gaseous I, and it has a lower s (i.e. less toxicity due to protein alkylation). However, it has the drawback of a low reactivity; at n = 2 it is about 10 times less reactive than I and a b o u t 20 times less reactive than ethyl methanesulphonate (Table 4). From the rate constant for reaction with OH- [330] it is calculated that, at pH 7.4 and 37°C, its rate of conversion into I (cf. Sect. 5.4.2.) is a b o u t 10 -3 h - ' ; i.e. less than 1% of alkylations in vivo will be caused by I. The data obtained (Table 15) show that if we assume (a) equal retention times in vivo, i.e. equal doses of the t w o compounds, and (b) that the numbers of tumours obtained in the methanesulphonate series are on the linear part of the dose-response curve, we would expect about 0.7 and 1.4 tumours at the two dose

84 levels of 2-hydroxyethyl methanesulphonate. These values lie within the confidence intervals of the values found (1 and 3 tumours, resp.). Excluding benign m a m m a r y tumours, we would expect 0.5 and 1 tumour, resp., which is n o t significantly different from the values obtained {0 and 1 t u m o u r , resp.). The expected incidence would be over-estimated if cancer initiation by ethyl methanesulphonate exhibits the same strongly exponential dose--response relationship above some intermediate dose as has repeatedly been found for mutation induction by this c o m p o u n d [109,409, cf. 147]. Conversely, it may be under-estimated (or rather the over-estimation mentioned might be reduced) if 2-hydroxyalkylation is more effectively cancer-initiating than the corresponding ethylation, as found for the induction of chromosomal aberrations in certain materials (cf. Sect. 5.4.4.3.(d)). The Millers have c o m m e n t e d on the data in the following way: "We would hesitate to attach significance to it, but it is perhaps interesting that more tumours were observed in the rats given 6 injections than in those that received only 3 injections." It may be concluded that, because the low frequencies obtained agree with the expected frequencies, an experiment with larger samples would have been required to permit a decision to be made as to whether the h y d r o x y e t h y l a t i o n is more, or very much less, effective than indicated by the experiment. Considering the 0, 1 and 3 tumours found in the control series and at the two dose levels, the statistical significance of a linear dose--response relationship is not low: a test of H0 gives 0.05 > P 0.01. The ability of 2-hydroxyethylation to induce tumours in the rat was indicated in the experiments of Druckrey et al. [81,82] with N-nitroso-bis(2h y d r o x y e t h y l ) a m i n e . In this case nothing is known about the doses of the ultimate carcinogen, assumed to be 2-hydroxyethyldiazonium ion, HOCH2CH2N~, or, which is by no means excluded, ethylene oxide formed spontaneously from this compound. Again, comparison with N-nitrosodiethylamine indicates t h a t the h y d r o x y e t h y l c o m p o u n d is a much weaker carcinogen: Druckrey et al. [81, 82] judged this c o m p o u n d to be some 200 times less effective than its ethyl analogue. In view of possible differences in metabolic rates, etc., and the fact t h a t Druckrey et al., working at high doses, ultimately giving tumours in 100% of the rats, laid more stress on latency time than on t u m o u r frequency, this comparison is n o t even semi-quantitative. The negative tests with ethylene chlorohydrin (XXVI) and ethylene glycol (Sect. 4.4. and, for XXVI, Table 3) do n o t allow the calculation of fl-errors because of difficulties in formulating the contra-hypotheses. In the rat study [289] the highest injected dose of XXVI a m o u n t e d to 10 • 2 • 52 mg • kg -1 = 13 mmole • kg -1 ~ 13 mM. In the 20 Fischer rats, ethyl methanesulphonate gave rise to 17 and 23 tumours at a Co of 7.2 and 14.4 mM, resp. Assuming ~ of the compounds to be approximately equal (of the two compounds XXVI is probably eliminated faster), and realizing t h a t the reaction rate at n = 2 is 103 times slower for XXVI than for ethyl methanesulphonate (Table 4), we would expect some 10 -3 tumours per animal in the test of XXVI. Furthermore, if we consider a possible conversion of XXVI to more toxic compounds (Sect. 2. and 4.1.2., Table 11), increasing the risk 10 times, we would expect some 10 -2 tumours per animal, corresponding to a total increase of about 1 t u m o u r at the highest dose where 80 animals were exposed. This effect would not have been

85 detected in the experiment. Concerning the mouse experiments [181], where a still lower dose with Co ~ 0.8 mM was applied ,' suffice it to say that samples of about 250 animals in each of the control and the test groups would have been required to have a 95% chance (fl = 0.05) of detecting a doubling of the frequency when H0 is rejected at the 5% level. Even allowing for the formation of more toxic compounds in vivo (cf. above), the tad-equivalence of 1 mM of XXVI is o f the order of 1 tad. The test system is thus far too insensitive. Propylene oxide -- a false or, rather, exaggerated positive. From Walpole's [436] positive cancer test with propylene oxide (II), carcinogenicity of I would be expected. The results of the test with II appear doubtful, however, and it seems plausible that this test accidentally exaggerated the effectiveness of II. According to an earlier publication [437], the vehicle (arachis oil) gave, besides m a n y neoplasms in other organs, 14 local sarcomata in 91 animals treated for 33--300 days and 5 sarcomata in the 24 animals treated for 300 days. The frequency found, 8 local cancers in 12 animals, is significantly above any o f these control levels (×2 tests giving P <: 0.01 and 0.01 < P < 0.05, resp.). This significance is valid provided the control frequency did not vary during the course of the experiments, owing, for example, to variations between batches of arachis oil as regards the content of aflatoxins [421]; in fact the yield of local tumours was said to be variable [437]. As for water as vehicle, no control data are available, but Walpole appears to consider this frequency to be zero. Thus, although the data seem to indicate that II is carcinogenic, a renewed test of this important chemical is called for. A comparison of Walpole's [436] negative test of I (in which arachis oil was used as a solvent) with the more unambiguous test o f II in aqueous solution (cf. Table 3) does not permit the conclusion that I is less effective than II (P > 0.10, single-tail test) * General conclusion. It m a y be concluded that, in m a n y cases, qualitative statements about the non-carcinogenicity (or, for II, carcinogenicity) of epoxides, are based on weak experimental evidence. Maybe our quantitative approach, basing expectancy, with the possibility of calculating the required size of samples, on tissue doses and reaction-kinetic parameters, could help to solve such qualitative problems. 6.2.2. Dominant-lethal tests Negative tests for dominant-lethal mutations can be analysed in a similar way. In view of Moutschen's [312] demonstration of chromosomal aberrations at the male meiosis, diepoxybutane (XV) is expected to reach the gonads and to induce aberrations scored as dominant lethals. An increase in the frequency of dead implants was indeed observed in 3 tests with this compound, but it was n o t significant. Cattanach [61] found an increased incidence of deciduomata in the first 2 weeks after the application of 27 mg • kg -1, but found this dose too toxic for the mice. He gives no details of the test. In two reports on mass screening of chemicals, Epstein et al. [124,126] give data showing an increase in the frequency of dead implants following i.p. injection of 17 mg • kg -1 into

[ 8 3 a ] r e p o r t e d r e c e n t l y that b o t h I and II c a u s e d local t u r n o u t s a f t e r r e p e a t e d s . c . i n j e c t i o n s in t h e m o u s e . I w a s f o u n d t o be m o r e e f f e c t i v e t h a n II.

* Dunkelberg

86 mice (ICR/Ha Swiss). One of the tests was limited to 1 week and a few females; the incidence of dead implants found (4 cases) was higher than that expected from control data (1.7 cases) [126]. In the second test [124], in which 7 males were mated for 8 weeks after receiving the same dose, a non-significant increase of the fraction of females with dead implants was reported. 17 mg • kg -1 would correspond to about 170 rad, i.e. close to the detection level of small-scale dominant-lethal tests (100 rad are expected to induce some 0.8 dead implants per female in post-meiotic stages [273,274,368]). The test series [124] included epichlorohydrin (IX) and ethylene chlorohydrin (XXVI) applied i.p. at doses of 150 mg • kg -1. According to eqn. (7) this dose of these substances corresponds to about 70 and less than 10 rad-equ. (adjusted value, Sect. 6.4.1.), resp., and thus there is very little chance of detecting an effect in tests of such limited size. The data are so presented that a c o m p u t a t i o n of ~-errors is not possible. However, a calculation of tissue doses in positive tests in the same series illustrates the low resolving power of the system: tests with methanesulphonates were positive at doses corresponding to about 500 rad-equ. (methyl methanesulphonate injected at 150 m g " kg -1) and about 1000 rad-equ. (ethyl methanesulphonate at 380 mg • kg -1 ), resp. Srfim et al. [388] have presented sufficiently detailed data on their negative dominant-lethal test of epichlorohydrin (IX) in the mouse (ICR, random-bred) to permit a meaningful estimate of the/3-error. The highest doses applied to the animals were 40 mg • kg -~ p.o. and 20 mg • kg -~ i.p. In view of the slow H ÷catalysed reactions of IX (Table 7), tissue doses of IX may be assumed to be independent of the route of administration. If we use ~ (of I) = 4.6 h -~ and k,=2 = 3.3" 10 -2 M -1" h -~ (Table 4), 40 m g - k g -~ will correspond to about 30 rad-equ. (eqn. (7)). This would be expected to lead to an increase in the frequency of dead implants of about 0.3 above the control level (0.6 per female), i.e. an increase of about 50%. Each dose was tested in about 30 pregnant females per week, and thus the expected increase would be from 18 to 27 dead implants (A = 9; cf. ref. [93]) in a 1-week comparison. In a comparison restricted to 1 week, the fl-error of accepting H0 when the true effect is A = 9, amounts to 88%. Assuming the same increase during a 3-week post-meiotic period, the relative error would be reduced by a factor x/3, and fl to 25%. Hence, it is n o t surprising t h a t no effect was found. It has to be stressed t h a t these estimates o f fl are very rough because we lack precise knowledge of ~ of the particular c o m p o u n d and about the response to radiation of the animal strain used [412]. Appelgren et al. [12] tested I for its ability to induce d o m i n a n t lethals in NMRI mice, applying doses in the range of 0.025--0.1 g • kg -~, corresponding to 12--50 rad-equ. During weeks 1--3 after the t r e a t m e n t of males, the number of dead embryos per implant increased by about 3 • 10 -2, but this was considered n o t significant. The expectation from radiation experiments is 8" 10 -4 rad -1 in the mouse (Sect. 6.1.}, i.e. (1--4) • 10 -2 at the applied doses. Although the ~-error could certainly be calculated, we find this meaningless in a case where the expected increase was actually found. The lack of statistical significance then simply indicates that the experimental groups were too small. In this study [12], the non-significance claimed was based on a t test of proportions of dead implants found. Assuming the dead implants to be independent

87 events, a ×2 test of the whole material indicates a significant increase in weeks 1--3 ( 0 . 0 5 > P > 0.02). However, whether it is permissible to make this assumption is not apparent from the data presented (cf. [93] ).

6.2.3. Size of samples The last-mentioned experiment [12] illustrates the importance of calculating the sample size required to detect an effect considered to demonstrate risk. Because the rad~equivalence permits the calculation of a hypothetical response (,,A,,), the sample size required for the detection of this change may be estimated [93]. This applies to experimental as well as epidemiological data [153].

6.3. The question of a no-effect level for genetic effects of epoxides Risk estimates for man are primarily concerned with low doses at low concentrations (i.e. low dose rates). Under such conditions even the otherwise exponential dose--response relationship for epoxide-induced reversions in N. crassa ad-3A (Sect. 4.8.1.) approaches linearity. In fact it is expected that, as long as the dose and/or dose rate are low so as not to change any promoting or other co~arcinogenic factor (where modifying effects are mostly expected to follow multi-hit kinetics), both mutation and cancer initiation are expected to be linear functions of dose [46,107]. Deviation from linearity could be obtained if, above some dose, the agent investigated affects the induction status of activating, deactivating or repair enzymes, leads to stimulation or retardation of growth, or gives rise to an inhibition of DNA repair. No experimental indication o f a no-effect level for genetic effects of epoxides has been obtained. In fact, in a situation like that of man, in which there is permanent exposure to cancer-initiating and promoting agents, the exposure of a population to a chemical initiating cancer in a two-hit process (e.g. one mutation and one somatic crossing~ver in the same cell), or a chemical promoting t u m o u r development, can be shown to increase cancer incidence linearly from dose zero upwards [73]. Partly because of the argument that risk estimations at low doses by linear extrapolation would place health authorities in an impossible situation, alternative methods that are assumed to be less conservative have been proposed. The probit/log dose model o f Mantel and Bryan [285,286] is one example. A realization of the incompatibility of this model with biological theory, which is no d o u b t in favour of the mutation hypothesis and consequently with a linear dose response, has led to other kinetic models being suggested. For instance, Cornfield [70] recently suggested a thresholded one-hit curve obtained by the introduction o f an "irreversible deactivation" process, leading to tissue dose zero at low exposure doses. Although a situation approaching, but not completely agreeing with, this model, is possible in special cases [46], any threshold dose of this kind for an epoxide administered to a mammal must be very low. This is illustrated by the finding that the tissue dose of ethylene oxide in various organs of mice exposed to less than 1/1000 of that received by a person spending a working week (40 h) at the TLV of this c o m p o u n d (50 ppm in most Western countries) shows no deviation from the linear curve from higher doses to the origin [106]. There are at present no data to show whether the

88 active principle(s) of PAH, presumably epoxides, used to exemplify Cornfield's model, would behave otherwise. Dose--response curves for t u m o u r initiation by these compounds are consistent with linearity down to the lowest doses applied [107,205].

6.4. Genetic risks associated with tissue dose o f epoxides in man Knowledge of retention time (1/)~, Sect. 5.3.) in human tissues is of critical importance for a correct estimate of the genetic risk associated with unit exposure dose. For volatile compounds this is expressed, for example, in ppm • h or m g . m -3 . h , and for injected or ingested compounds in mg (kg b.w.) -1. Furthermore, a knowledge of the pulmonary ventilation is required, as well as of how it varies between individuals and its dependence on the intensity of physical activity.

6.4.1. Ethylene oxide and its primary reaction products The only epoxide so far investigated with regard to retention time is ethylene oxide (I). In the mouse I had a retention time corresponding to ~ = 4.6 h -1 (cf. 5.3 above), i.e. 1/)~ = 0.22 h = 13 min, or tl/2 (biol) ~ 9 min. Measurements of tissue doses in persons exposed to I in situations where the exposure doses could be reconstructed indicate that retention times are equal to or shorter than those in the mouse ([59], Sect. 5.3.). Until this point has been clarified, we find it most appropriate to retain the mouse value for )~ in estimates of risk to man [59]. Application of eqn. (7) would then give a risk estimate during light physical activity of about 11 mrad-equ, per ppm • h *, a value that we, because of its uncertainty, round off to 1 • 10 ~ mrad-equ. (ppm • h) -1. The earlier estimate [106], 20 mrad-equ. ( p p m - h ) -1, may apply during somewhat more intense physical activity in man ("intermediate physical activity"). The difference between these two estimates results partly from different assumptions of ratios of alveolar to pulmonary ventilation, and of the absorbed fraction of the air pollutant [ 59]. Furthermore, the coefficients of eqn. (7), 1 • 107 and kn=2, are still under evaluation, and the given values are present averages for a number of compounds and biological systems. We judge that the risk estimate of I is correct to within a factor about 2, and that the true value might be found ultimately to be somewhat lower. A working week of 40 h at TLVs [451] of 50 ppm (valid in the U.S.A. and * (a) C a l c u l a t i o n of tissue dose ( M " h) p e r unit e x p o s u r e dose ( p p m • h) for ethylene oxide. A s s u m e d alveolar ventilation d u r i n g light w o r k w i t h s o m e rebt: 0.I 5 1 air (kg b.w.) -1 • rain -I [59]. V o l u m e of 1 m o l e : 2 4 . 6 l~ k = 4.6 h -1.

dD

1 • 10-6

• 0.15

1 • rain -1

• kg -1

- 60

• 4.6

h -1

rain

• h -1

= 0 . 8 0 • 1 0 -6 M dDex

p

24.6

1 • mole

-1

C o m p a r e with observations [59]: ( 0 . 4 4 -+ 0 . 2 6 ) • 1 0 -6 M - h ( p p m

- h(ppm

• h) -I

• h) -I

(b) R a d - e q u i v a l e n c e of 1 p p m • h (eqn. (7)). k n = 2 = 1.4 • 1 0 -2 M -I • h -1 ( T a b l e 4 ) ~ II fi = 1 R i s k = 0.8 • 1 0 - 6 M

• h(ppm

- h) -I • 1.4 • 1 0 -2 M -I • h -I - 1 . 1 0 7 = 0 . 0 1 1 rad-equ. ( p p m

• h) -I

89 some other Western countries) and 10 ppm (valid in Sweden) would correspond to risks of 20 and 4 rad~equ. (week) -1, resp. These values are about 2 orders of magnitude higher than the risk associated with the maximal permissible dose to radiological workers (in most countries 0.1 tad low-LET radiation per week). The maximal allowable concentration of I in the U.S.S.R., 0.5 ppm [451], is more consistent with current radiation safety standards. Risks estimated from the doses determined in workers at ethylene oxide sterilization plants amount to a few rad-equ, per week [59]. These risk estimates are sufficiently accurate (cf. 6.5.) to call for urgent measures to decrease the exposure levels. It appears that the dose commitments to the personnel and to the public from sterilization activities with ethylene oxide could be decreased by an order of magnitude or more through relatively simple and cheap measures. Although dose measurements have so far been limited to no more than a b o u t a dozen persons, a clear trend is observed in that tissue doses are lower in operators of ethylene oxide ovens than in personnel handling sterilized goods on storage premises. This may partly be a psychological phenomenon: the oven operators are more conscious of the risks, and therefore participate more actively in protective measures. The finding is in agreement with the observation that very high exposure levels are sometimes found on storage premises [84,180a]. This is a consequence of the ability of certain materials to retain residual I for periods of time longer than the "quarantine periods" practised for sterilized goods. These materials include rubber and plastic materials for medical use and for use b y the general public [158,159,206,464], as well as foods [283~284,340,341] and drugs. (For a review, see ref. [448].) Burns due to residual I in sterilized rubber shoes (see ref. [ 1 9 5 ] ) a n d hospital garments [34] have been reported, indicating that occasionally high concentrations may occur. Dermatitis from contact with rubber gloves or screens have been observed [363] among persons working with I. For this reason, work with bare (dry!) hands m a y be recommended in many situations [113]. The risk associated with residual ethylene chlorohydrin (XXVI) occurring, for example, in foods fumigated with I, can at present be estimated only very roughly. Assuming k = 2 (as a mean value for some other alkylating agents) [370] and kn=2 = 1.4 • 10 -2 M -1 • h -~ (Table 11), the absorption of 1 mg of XXVI (kg b.w.) -1 would be associated with a risk of some 2 mrad-equ. However, in view o f the possible conversion of XXVI to more reactive c o m p o u n d s (Sects. 2. and 5.4.2.), it would be safer at present to assume a 10 times higher risk (Table 16). The observed increase in the frequency of chromosomal aberrations in rats exposed to XXVI at 13 ppm • h per day for 60--120 days [371] would correspond to a b o u t 30 rad-equ, at 60 days if this adjusted estimate be used, which is in better agreement with observation than is the uncorrected dose. The possibility of genetic toxicity of the reaction product of I with water, namely ethylene glycol, was discussed in Sect. 5.4.4.3.(d). (See also Sect. 6.2.1 .)

6.4.2. Other epoxides For other epoxides, ~ is not known, b u t because there is a reasonable agree-

90 TABLE 16 ESTIMATES OF RISK TO MAN FROM EXPOSURE TO UNIT DOSE OF SOME EPOXIDES Compound

m r a d - e q u . ( p p m • h ) -1

I, e t h y l e n e o x i d e II, p r o p y l e n e o x i d e VIII, g l y c i d o l IX, e p i c h l o r o h y d r i n XV, DL-diepoxybutane XVII, diglycidyl ether XXVI, ethylene ehlorohydrin

10 ~(5--10) ~7 ~25 ~300 ~300 ~20

ment of rad-equivalences with respect to DLs0, it would n o t introduce a large error if the value for I were applied. This would give risk estimates, for the exposure o f humans to a few important epoxides, shown in Table 16. For c o m p o u n d s of higher molecular weight (e.g. V, VI, XVIII, in Fig. 1) or with high reactivity of the alkyl residue (e.g. XII), correction factors, fi in eqn. (7), for distribution in the b o d y and reactivity etc. are required. Such factors have to be calculated from experiments, in the case of distribution in the b o d y , with suitable mammals. Risk evaluation for c o m p o u n d s converted metabolically into epoxides will have to be carried o u t in the same way. As demonstrated for ethene, which is converted into I, and vinyl chloride, which is converted into XIII, the activating and deactivating enzymes are of great importance [111,331]. This is the greatest obstacle in estimating risk in man from experimental data [107], and calls for dose determinations directly in exposed persons (cf. Sect. 6.6.). Compounds giving rise to short-lived metabolites, such as the vinyl chloride metabolite XIII (biological half-life a b o u t 2 sec [331]) and probably certain epoxides from PAH, are expected to give a dose that varies between organs. In such cases, relative organ doses might be estimated from animal experiments. 6.5. Other approaches to risk estimation for epoxides in man

Using a completely different approach, Sr~m et al. [388] have arrived at an estimate of genetic risk from exposure to epichlorohydrin (IX) which, with due regard to the scantiness of available data, seems to show a good agreement between the U.S.S.R. standard of acceptable annual uptake of this chemical and the limit of radiation risk to radiological workers. The risk from a yearly dose was estimated on the basis of the dose--response curve for chromosomal aberrations in the mouse bone-marrow after acute or fractionated exposure, which do n o t seem to give significantly different results. Administration p.o. gives a somewhat smaller effect, indicating a slower absorption by this route with a possible contribution of H÷-catalysed hydrolysis of the epoxide in the stomach (cf. Sect. 5.3. above). According to the approach of Bochkov et al. [38], an increase by a factor 2--3 in the spontaneous frequency of chromosomal aberrations is considered as the maximal admissible risk for an individual prognosis per year. After i.p. injection of IX the linear dose--response curve in the range of 1--20 mg • kg -1 corresponds to 1.4% aberrant cells per

91 mg • kg -1. Because the control level of aberrant cells is 4%, some 10% (i.e. 2.5 times increase) of induced aberrations is taken to correspond to the maximal admissible risk. During normal work an exposure level of 0.5 m g - m -s had been determined, i.e. one-half of the maximal allowable concentration in the U.S.S.R. [451]. Assuming the lung ventilation during light w o r k to be 0.25 1 • kg -1 • min -1 and resorption to be 66% (i.e. a b o u t the same values as assumed for I [59]; see also footnote, p. 88), the total uptake per year (48 weeks, 5 6-h shifts per week) is estimated to be 7.1 m g . kg -1. This is the amount required to produce 10% aberrant cells in the mouse, and is hence judged to be acceptable. With the use of radiation safety standards, this conclusion appears reasonable. If we assume the elimination rate to be the same as that of I (cf. 6.4.), an uptake of 7.1 m g . kg -~ would give a tissue dose of 0.017 mM • h, corresponding to an annual risk of about 5 rad-equ., equal to the acceptable limit (5 rad) of exposure for radiological workers [191]. Possibly, chromosomal aberrations are specifically useful for monitoring the risk of epoxides because they induce this kind of effect with a relatively high effectiveness (cf. Sect. 4.2. and r e f . [235]). Ku~erov~ et al. observed significant increases in the frequencies of chromosomal aberrations in lymphocytes from persons occupationally exposed to IX at 0.5--5 m g . m -3, a value that exceeds the SovietCzechoslovak maximal admissible concentration [238]. Fomin [141] arrives at a similar conclusion from experiments with rats. Using the somewhat d o u b t f u l criterion of nucleic acid content in blood, he found a decrease at a chronic exposure of IX, 2 mg • m -3, b u t no effect from 0.2 mg • m -3 air.

6.6. Epidemiological aspects As discussed above, ethylene oxide (i) absorbed has been indicated to give a b o u t the same dose in different organs [106], and this is expected to be true for unsubstituted and substituted homologues of low molecular weight that are soluble in both water and lipids. To the extent that the mutation hypothesis for cancer initiation is valid, these compounds are not expected a priori to induce cancer with a high specificity to organs. Until other data become available to show the contrary (e.g. a demonstration of organ-specific deficiency or inhibition of DNA repair) we would therefore assume these compounds, like whole-body irradiation [412], to induce cancer in various organs of the b o d y . It might seem that such a picture would come into conflict with experimental and epidemiological data for tumours with a chemical aetiology. We believe, however, that for different reasons both sources of information are misleading with regard to the true picture at low levels. Studies of experimentally induced tumours, or carcinogenicity tests, are mostly conducted at doses/levels so high that tissue damage and ensuing reparative growth, w i t h an organ specificity that may depend on route and method of administration, chemical structure and animal strain, leads to a promotive situation in particular organs. Since epidemiological observations of aetiological relationships are greatly facilitated b y shifts of the incidence in specific types of tumours, many differences between populations and exposure situations may reflect variations in the occurrence of promotors rather than of initiators -- agents with the kind of

92 activity we normally mean by the term carcinogenic. The carcinogenicity of vinyl chloride was observed because of a rise in the incidence of a very rare t u m o u r *. Application of the two-stage hypothesis of cancer induction makes this seem to be due, in addition to the formation of a mutagen (probably XIII), to a promoting action of vinyl chloride itself or some of its metabolites. In h u m a n populations one would expect somatic m u t a t i o n (or some related process causing initiation) due to electrophiles that have a general distribution in the b o d y , to induce cancer in patterns of sites, which reflect promoting processes and other co~arcinogenic events characteristic of the populations. Apart from investigations of populations exposed to ionizing radiation, epidemiological studies have rarely been conducted in a way that facilitates the detection of factors t h a t enhance cancer incidence in general. Leukaemias, with a median latency time about 10 years after exposure to ionizing radiation [412], are expected to appear before solid tumours, which probably have some 2--3 times longer median latency times. Radiation is k n o w n to induce myeloid leukaemias and acute lymphatic leukaemia, sex and age having an influence on frequencies and relative abundance of types [412]. Chronic lymphatic leukaemia, one case of which was encountered in a group of some 30 ethylene oxide operators [104], does n o t seem to be induced by (acute doses of) irradiation, as judged from the incidence in Japanese A-bomb survivors [412]. (However, the possibility t h a t development of this type of leukaemia could be p r o m o t e d by repeated low doses, should n o t be entirely excluded. Some indications t h a t this is so have indeed been f o u n d in patients subjected to great numbers of diagnostic X-ray investigations [146]. This effect, and its association with bone-marrow stimulation observed in radiologists as well as in epoxide operators [106] (cf. Sect. 4.6.), requires further investigation. In animal experiments a certain association between leukaemia and increased l y m p h o c y t e numbers has been indicated [305] .) In Joyner's study [207], 37 ethylene oxide (I) operators exposed to I at 5--10 ppm for about 10 years were observed, w i t h o u t any increased t u m o u r incidence (4 cases) compared with a control group (6 cases in 41 persons) being found. (The tumours reported were partly at least of a benign character. It does n o t appear from Joyner's paper whether he had considered cancer mortality during the period of study. We have been unable to find reports on the planned [207] follow-up of the health records of the exposed personnel.) Assuming the average level of I in the work-place studied by J o y n e r to a m o u n t to 7 ppm for 40 h × 40 weeks per year, the annual dose would a m o u n t to some 11 000 ppm • h, corresponding to about 110 rad-equ. The collective dose rate in 37 persons would thus a m o u n t to about 4 • 103 man-rad-equ, per year. 10 annual collective doses would a m o u n t to 4 • 104 man-rad-equ., with an expectancy of 2eukaemia incidence of about 4 • 104 X 3 • 10 -s = 1.2 cases. This shows t h a t an increase in incidence of leukaemia might well have escaped detection in a population of the size studied. However, the high annual doses actually received mean that there is an urgent need for epidemiological studies.

* Haemangiosarcoma [180c].

93 The expected incidence of leukaemia was in fact encountered among exposed staff at a Swedish ethylene oxide sterilization plant [ 59 * ]. In a population of 78 persons (77 regularly + 1 intermittently exposed), working at the enterprise for about 8 years during the period 1968--78, at an average level estimated to be 20 + 10 ppm, the risk was estimated to be as follows: Collective dose = 78 persons × 20 ppm × 40 h / w k × 40 w k / y X 8 y = 2 • 107 person • ppm • h. Collective risk = 2 • 107 person • ppm • h X 0.01 rad-equ. (ppm 10 -6 cases (man-tad) -1 = 6 cases.

•

h) -1 X 30 "

With a median latency time of 10 years somewhat less than half of the cases, i.e. ~ 3 , would have appeared before 1978. In the reported study, 3 cases were found, significantly above expectation (0.07 cases) based on the age-adjusted Swedish average. The cases are less likely to represent an occasional cluster because it was predicted a priori. (2 of the cases are of types demonstrated to be induced by radiation [59,412]. The 3rd case is a rarer type n o t studied in this respect. We feel that this case should be included as well in the evaluation of the data.) In 1978 an epidemiological follow-up study was carried o u t ** on those epoxide operators in a chemical industry manufacturing and using I, who had exhibited deviating blood-cell pictures in 1960--61 (Sect. 4.3. and 4.6. [105, '106]). The group of personnel exposed during the years 1942--1962 comprised 89 persons, with 8 years average e m p l o y m e n t time. A significant increase of the cancer incidence (to about 250% of the value expected from the Swedish average incidence) comprising leukaemias as well as tumours at other sites, was found. Control groups of unexposed persons (in all, 152 men) within the fact o r y exhibited a cancer incidence close to expectation *** In this study, exposure levels of I were n o t known, but the increase found agrees with the reasonable guess that it might have been in the range 3--30 ppm. In this case, and in contrast with the cold-sterilization plant discussed above, the clarification of the aetiological relationship is obscured, however, by some exposure to other chemicals as well, i.a. 1,2
* Original d a t a f o r t h e c a s e - c o n t r o l s t u d y have b e e n p r e s e n t e d b y H o g s t e d t et at. [ 1 8 0 a ] . ** F o r details, see ref. [ 1 8 0 b ] . *** Added n o t e . T h e s t u d y also i n d i c a t e s an increase in m o r t a l i t y f r o m cardiovascular disease a m o n g exposed personnel [180b].

94 should be treated as carcinogenic *, with a risk per unit exposure dose as given in Table 16. Above all, continued epidemiological studies, combined with improved estimates of exposure levels, are called for. The conclusion that I should be handled as a carcinogen should, in our view, be extended to comprise all epoxides * (including compounds metabolized to epoxides) with related reaction patterns. Heritable damage, embryological effects. Epidemiological data for heritable damage caused by any environmental factor are, if anything, too scanty to permit risk quantitation. In the absence of epidemiological data for mutations induced by radiations, UNSCEAR [412] and other organizations [191] have based risk coefficients on animal data. Estimates of risks of heritable damage are, therefore, much less reliable than cancer risks, where fairly consistent estimates of risk coefficients could be developed from epidemiological data. Considering the equal distribution within the body of the tissue dose of a low-molecular-weight epoxide [106], the fact that predictions of cancer risk could be confirmed, at least to the order of magnitude, in epidemiological studies, may be taken to sustain the validity of the tad-equivalence also for the risk of heritable damage due to such epoxides. The lack of experimental data for the response of mammals to exposure of premeiotic stages to epoxides, however, imposes an additional uncertainty on risk estimates. According to our estimates presented above, an epoxide operator might accumulate a 10-year risk of the order of 103 rad-equ., i.e. some 10 times higher than the risk associated with the average radiation dose experienced by surviving A-bomb victims in Hiroshima and Nagasaki [412]. These very high collective doses already at hand in relatively small populations, i.e. populations with a small background "noise" due to spontaneous incidence, may have created conditions suitable for the determination directly in man of risk coefficients for heritable damage (and, possibly, embryological damage) due to an environmental factor. Studies with such aims should comprise not only precocious abortions (as indicators of heritable damage or, especially after postconceptional exposure of females, embryological damage) and foetal malformations but, especially, traits with dominant or sex-linked inheritance. (The observed increase in abortion frequency in females exposed to ethylene oxide [458], so far the only available study of second-generation effects in man, was based on a limited material and requires confirmation; cf. p. 30.) In the estimation of risk coefficients, cold-sterilization plants using ethylene oxide present a model situation where the identification of aetiological relationships is mostly not confused by the simultaneous presence of other (potentially) electrophilic pollutants. 7. General conclusions

Epoxides are able to induce all kinds of genetic effects, including cancer. Dose--response curves for mutation induced by epoxides show no indications of deviation from linearity at low doses or low concentrations, and there is no * This conclusion is f u r t h e r sustained by Dunkelberg's [ S 3 a ] p o s i t i v e a n i m a l t e s t o f I a n d I I in m i c e . See f o o t n o t e , p. 8 5 .

95 reason to assume a "safe threshold" at some low dose. Partly by inference from data for other mutagens, this is probably valid for cancer too. Data are consistent with the assumption that the effectiveness of epoxides -- and hence the risk -- is proportional to the dose in target cells and to the reactivity towards certain groups of low nucleophilic strength, presumably in DNA. Difunctional agents, i.e. diepoxides or monofunctional epoxides with a reactive substituent such as a carbonyl group, have a greater effectiveness (with about the same enhancement factor in different biological systems) than would be expected from the dose and the rate of primary alkylation. Such quantitative interrelationships indicate that the problem of genetic risks of e p o x i d e s - if not of alkylating agents in general -- may to a large extent be treated b y using a Common approach, i.a., permitting the evaluation of risks of a c o m p o u n d on the basis of data on related chemicals. For a few relatively simple epoxides, enough chemical -- especially reactionkinetic -- and biological data are available to permit an exploratory application of the model for evaluating the tad-equivalence of genetic risk on the basis of the degree of alkylation. For a number of end-points in mouse and/or rat, comprising dominant-lethal mutation (ethylene oxide), chromosomal aberrations (ethylene oxide, diepoxybutane) and turnouts (diepoxybutane), the agreement with the expected results was so good that it is unlikely to be a coincidence. Using similar data to formulate the contra-hypotheses in estimates of ~errors of negative tests for cancer or dominant lethals, we have calculated high probabilities that the tests are all false negatives. This means that for no epoxide do the results of the biological tests permit the conclusion that it is non~arcinogenic. In a statistical sense, non-mutagenicity or non~carcinogenicity of a particular c o m p o u n d should be taken to mean that it is significantly less mutagenic or carcinogenic than expected from reactivity data and the tissue dose administered. If it is considered necessary to decrease the upper limit of the confidence interval of a found mutation or turnout frequency equal to zero, tests on a larger scale in suitable systems would be required. We must stress the preliminary character of these conclusions, including the risk estimates for a few industrially important epoxides (Table 16). To improve the accuracy of risk estimates, research along the following lines is required: (a) Refined evaluation of the parameters of the generalized risk equation, relating tissue dose to tad-equivalents (eqn. (7)) and basic research aimed at the clarification of the theoretical background of the presently empirical radequivalence of doses of chemicals. (b) Determination of retention times (l/X) of individual compounds, a necessity for the determination of the dose (cf. eqn. (19)). In particular knowledge of retention times, and variations therein, in man is required for proper risk estimates. For this, data on the extent of alkylation of, for example, haemoglobin at typical e x p o s u r e doses are needed. (c) To establish the validity of eqn. (7) for cancer, comparative studies o f dose--response curves of chemically and radiation-induced cancer in the same animal strains are called for. (d) Appropriate cancer tests, especially with ethylene oxide, propylene oxide and epichlorohydrin, are required. These tests should be carried out, combined t,

96 with tissue dosimetry, in animal strains having known response to radiation and/or related chemicals, and on a large enough scale to permit the detection, with acceptable probability, of the hypothetical frequency (cf. Sect. 6.5. and ref. [93]). Tests for cancer-initiating ability should be carried o u t at defined levels of activating and deactivating enzymes and in defined cancer-promotive situations. Compared with other alkylating agents, such as alkyl methanesulphonates, often applied in studies of induced mutation, ethylene oxide and related monofunctional epoxides are relatively ineffective [88] (i.e. they give a relatively low frequency of genetic effects per unit dose applied), and relatively inefficient [88] (i.e. they give a relatively low frequency of genetic effects at a given degree of some somatic effect such as DLs0). This is due to their relatively low reaction rates and relatively high substrate constants, s (eqn. (16)). In mammals the low effectiveness is exaggerated by the rapid elimination of epoxides from tissues (so far demonstrated for ethylene oxide only). This is probably connected with the existence of effective enzymic systems for hydrolysis or transfer to glutathione, also operating on epoxides formed as intermediates in the detoxication of unsaturated and aromatic compounds. Although many epoxides are thus weak mutagens/carcinogens, risk estimates in terms of rad-equivalence of some industrially important epoxides (Table 16) show that the TLVs adopted in most Western countries correspond to a much higher genetic risk than the maximal permissible doses for radiological workers. Maximal admissible concentrations in the U.S.S.R. seem t o be more consistent with recommended risk levels in radiological work. Case-control studies of cancer incidence in a couple of populations occupationally exposed to ethylene oxide indicate a considerable risk at current exposure concentrations. Furthermore, these findings are consistent with the risk estimate in Table 16, thereby confirming its order of magnitude. The groups studied so far have been small; therefore further epidemiological studies are called for. In certain w o r k environments -- such as premises where ethylene oxide is used for sterilization -- an epoxide is the sole or predominating mutagen/carcinogen. The model character of such exposure situations should be recognized, and the epidemiological investigations should be directed towards quantitation of risk per unit exposure dose or tissue dose. I t seems possible that, through international collaboration, sufficiently large collective doses could be obtained to permit meaningful studies of mutation in man, n o t only with regard to abortion frequencies b u t with consideration of the consequences to the live-born, e.g. of dominant or X-linked mutation. (The risk coefficients for radiation-induced heritable damage in man are exclusively based on animal experiments and therefore much less reliable than the corresponding coefficients for cancer.) In epidemiological studies, tissue doses -- or relationships between tissue dose and exposure dose -- are suitably determined b y means of haemoglobin alkylation. It should be stressed, however, that this m e t h o d is also useful for qualitative monitoring of the hygienic standard in different environments polluted b y mutagens/carcinogens.

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