Genetic toxicology testing of 41 industrial chemicals

Mutation Research, 153 (1985) 57-77 Elsevier

57

MTR07184

Genetic toxicology testing of 41 industrial chemicals B.J. Dean, T.M. Brooks, G. Hodson-Walker and D.H. Hutson Shell Research Ltd., Sittingbourne Research Centre, Sittingbourne, Kent ME9 8AG (Great Britain) (Received 3 August 1984) (Revision received 24 October 1984) (Accepted 30 October 1984)

Summary 41 c o m p o u n d s o r m i x t u r e s o f d i v e r s e s t r u c t u r e a n d a p p l i c a t i o n h a v e b e e n t e s t e d for g e n o t o x i c activity. T h e m a t e r i a l s w e r e t e s t e d in b a c t e r i a l m u t a t i o n assays, in Saccharomyces cerevisiae J D 1 for m i t o t i c g e n e c o n v e r s i o n a n d in a c u l t u r e d r a t - l i v e r cell l i n e for s t r u c t u r a l c h r o m o s o m e d a m a g e . 11 c o m p o u n d s w e r e b a c t e r i a l m u t a g e n s , 4 i n d u c e d m i t o t i c g e n e c o n v e r s i o n in y e a s t a n d 5 w e r e p o s i t i v e in t h e c h r o m o s o m e assay. 5 o f t h e m a t e r i a l s w e r e p o s i t i v e in b a c t e r i a o n l y a n d 2 c o m p o u n d s i n d u c e d c h r o m o s o m e d a m a g e in c u l t u r e d cells in t h e a b s e n c e o f m u t a t i o n in b a c t e r i a o r g e n e c o n v e r s i o n in yeast. T h e m a t e r i a l s w e r e t e s t e d o v e r a 5 - y e a r p e r i o d a n d t h e p e r f o r m a n c e a n d e v o l u t i o n o f t h e 3 assays d u r i n g this t i m e is e v a l u a t e d . T h e results a r e c o n s i d e r e d in r e l a t i o n to t h e s t r u c t u r e o f t h e c h e m i c a l s a n d t h e genotoxicity of related compounds.

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Micro-organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. Culture media (microbial) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Rat-liver chromosome assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.1. Cell lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.2. Culture media (cell culture) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Bacterial assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.1. Plate-incorporation assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.2. Pre-incubation assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.3. Spot test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.4. Treat-and-plate method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Saccharomyces gene conversion assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1. Treatment of stationary-phase cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2. Treatment of log-phase cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Rat-liver chromosome assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.1. Volatile compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.1.1. Cytotoxicity assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.1.2. Chromosome assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0165-1110/85/$03.00 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

58 58 58 62 62 62 62 62 63 63 63 63 63 63 63 63 63 66 66 66 66

58

4. 5.

6. 7.

3.3.2. Non-volatilecompounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.2.1. Cytotoxicityassay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.2.2. Chromosomeassay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Structure-activity considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Performancesand evolution of the assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.1. Bacterialmutation assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.2. Mitotic gene conversionassays using Saccharomyces cerevisiae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.3. The rat-liver chromosomeassay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Short-term assays for the detection of genotoxic chemicals have been applied in Shell Toxicology Laboratory since 1975. The following years have seen the evolution of short-term assays both in terms of technical modifications and in the way in which they are incorporated into the general strategy of toxicological evaluation. The purpose of this paper is to present the results of three short-term tests on 41 compounds used in or manufactured by the chemical industry. An essential part of the paper will describe the way in which the battery performed in general, how the techniques changed, how the strategy evolved over a 6-year period and how the final results were interpreted and applied in practice. Before any practical testing begins, a structure/ toxicity assessment is prepared on each compound by a multidisciplinary group. This assessment considers probable routes of biotransformation, the presence of electrophilic centres in the compound or its suspected metabolites, structural relationships to known genotoxic chemicals, and the influence of these factors on the design of the shortterm tests. Attempts are made to identify structurally-related model compounds for use as controls, and appropriate solvents or formulations are sought. The battery of short-term tests consists of bacterial mutation assays, a yeast assay for mitotic gene conversion, both tested in the presence and the absence of a rat-liver metabolising ($9) system, and an in vitro assay for the induction of chro-

66 66 66 66 68 68 69 73 73 74 74 75 75

mosome damage in cultured rat-liver epithelial-like cells. It is important to note that the chemicals were not chosen for testing at random, but in many cases, were selected where the structure of the chemical suggested the possibility of genotoxic reactivity. 2. Materials 2.1. C h e m i c a l s

Details of the compounds and mixtures tested are given in Table 1 and those of the solvents and formulations used, in Table 2. The positive control compounds were ethyl methanesulphonate (EMS), methyl methanesulphonate (MMS) and cyclophosphamide (CP) (Koch-Light Laboratories, Colnbrook, Bucks); benzo[a]pyrene (BP) (Koch-Light or Aldrich Chemicals, Gillingham, Dorset); neutral red (NR) (Hopkins and Williams, Chadwell Heath, Essex); sodium azide (SA) (Fisons Laboratory Equipment, Loughborough, Leics.); 7,12-dimethylbenzanthracene (DMBA) ( E a s t m a n - K o d a k Co., Kirby, Liverpool); 4-nitroquinoline N-oxide (NQO) was a gift from Dr. J. Ashby, ICI Ltd., CTL., Alderley Edge, Cheshire. Benzo[a]pyrene, 4-nitroquinoline N-oxide and dimethylbenzanthracene were dissolved in dimethyl sulphoxide (DMSO); the remaining positive control compounds were prepared as aqueous solutions. The stability of the formulation of all the test materials and positive control compounds was assessed by appropriate analytical methods and all

59 TABLE 1 D E T A I L S OF T H E 41 TEST C O M P O U N D S Abbreviation

C o m p o u n d name and structure

Date tested

CAS No.

Batch No.

Purity

PO

Propylene oxide

1976

[75-56-9]

-

-

1976

[156-57-0]

-

98%

1976

[3033-77-0]

-

91% (typical sample)

H3C "HC ---~H 2

MCE GMAC

O Mercaptoethylamine - HCI (cysteamine • HCI) H S C H 2 C H 2 N H ~ •C1Glycidyl trimethylammonium chloride (cH3)~ N+CH2HC - - CH2 . C I -

%/

CHT

1,3,5-Cycloheptatriene

1977

[544-25-2]

ID-9730

96% (typical sample)

GAAE

Glycerol-a-allyl-ether H2OCH2CH= CH2

1977

[123-34-2]

ID-9730

99%

1977

[2461-15-6]

ID-9730

99%

1977

[75-98-9]

ID-9627

99%

1977

[504-20-1]

ID-9614

96-98%

1,5-Cyclooctadiene

1978

[111-78-4]

000308

98% (typical sample)

Shell metal extractant 529

1978

-

5255

50% active component (in hydrocarbon diluent) 98.8%

~

CHOH

I

CH2OH

EHGE

2-Ethylhexyl glycidyl ether Et

I C H3(CFI2)3CCIM2OCH2HC x-~ CH2

PA PHOR

O Pivalic acid (trimethylacetic acid) (CH3)3CCOOH Phorone

~3 ~H3 CH3--C ~ C H - - C - - C H = C - - C H 3 li © COD

SME

(2-hydroxy-5-tert-nonyl acetophenone oxime)

D N 102

D N 102S

Dobane * 102 (linear alkyl benzene) 1-pheny1-C10-18% _ ~ C l l = 43% R C12 = 35% C13 = 3% 2-phenyl isomers = 22% Dobane * 102 sulphonate R~

D L 23

1978

17(1520)

1978

3032/77

1978

118(4611)

SO3H

(R as D N 102) Dobanol * 23 (primary synthetic fatty alcohol) R-C11 = 41% R C H 2 O H R-C12 = 58%

24.8% active component

60 T A B L E 1 (continued) Abbreviation

C o m p o u n d name and structure

Date tested

CAS No.

Batch No.

Purity

D L 25-3

Dobanol * 25-3 (primary alcohol ethoxylate)

1978

-

1573

100% active mass (nominal)

1978

-

103(1164)

> 26% active mass

1978

-

T N 1581/77

95%

1978

[128-39-2]

ID-9729

> 98%

1978

[107-05-1]

U.8212

98%

1979

[321-14-2]

F5

99.8%

R-C9 = 20% -C10 ~ 30% - C l l = 30% -C12 = 30% Dobanol * 25-3S/27 (sodium salt of sulphated Dobanol 25-3) R C H 2 O C H 2 C H 2 O S O ; Na ÷ (R as D L 25-3) Bis-(4-dimethylaminophenyl)methane RCH2OCH2CH2OH

D L 25-3S/27

BDAM

I H3C.~.~CH2 N H3C/

DTBP

~2

2,6-bi-tert-butylphenol (Ionox * 99) OH

(CH3)3C+C(CH3)3 AC

CSA

A]lyl chloride C H 2= C H - C H 2CI 5-Chlorosalicylic acid

C

COOH

DMPC

3-(N, N-Dimethylamino)-I -propyl chloride hydroehloride HC 3 ~.+ H3c/.N HCH2CH2CH2C[ • C I -

1979

[5407-04-5]

2OR

100.7%

SIX

Sodium isopropylxanthate s

1979

[140-93-2]

SEC. 1734

80-90%

EB

(CHa)2CHOCSNO+ Ethylbenzene ~ CH3

1979-80

[100-41-4]

6417160

> 99%

EBHP

Ethylbenzene hydroperoxide

HOO~CH3

1979-80

-

78/12484

33.9% in EB

TBB

tert.-Butylbenzene

1980

[98-06-6]

S.16923(A)

96.4%

1980

[98-19-1]

S.16923(B)

98.2%

Ir

TBMX

~

H3)3

tert.-Butyl m-xylene

C(CH3) 3

61 TABLE 1 (continued) Abbreviation

Compound name and structure

Date tested

CAS No.

Batch No.

Purity

XE

3,5-Xylenol (3,5-dimethylphenol)

1980

[108-68-9]

191

99%

AA

Allyl acetate ¢H3~OCH2CH=CH2 O 1,3-Dichloropropane

1980

[591-87-7]

ID-9765

96%

1980

[142-28-9]

X8113

H

DCP

CN3

(~H2CH2CH2CI 1980

[98-51-1]

$19815

sample) 96%

1980

[98-54-4]

ID-9423

> 95%

1980

[98-29-3]

ID-9581

> 95%

1980

[104-91-6]

792-79

> 98%

1980

-

13723

-

1980

[75-97-8]

ID-9928

> 95%

1980

[141-43-5]

17589

-

1980

[111-42-2]

17584

99.7%

1980

[102-71-6]

19319

88.2%

cl PTBT

p-tert.-Butyl toluene (1-methyl-4-tert.-butyl benzene)

99% (typical

6

C(CH3)3

PTBP

p-tert.-Butylphenol OH

C(CH3) 3

PTBC

p-tert.-Butylcatechoi OH

C (C H3)3

PNP

p-Nitrosophenoi

POL

Polyol E40 (reaction product of ethylenediamine and diphenylolpropane (molar ratio 2 : 1) with propylene oxide) Pinacolone (3,3-dimethyl-2-butanone)

N=O

PC

~H3 H~C--C--C--CH~

CH3 0 MEA DEA TEA

Monoethanolamine HOCH2CH2NH2 Diethanolamine (HOCH2CH2)2NH Triethanolamine (HOCH2CH2)3N

62

TABLE 1 (continued) Abbreviation

Compoundname and structure

Date tested

CAS No.

Batch No.

Purity

CE

18-Crown-6-ether (1,4,7,10,13,16-hexaoxacyclooctadecane)

1980

[17455-13-9] ID-9420

-

o.j DMBO

1,5-Dimethylbicyclo(3,2,1 )oetan-8-ol

-@

1980

[60329-20-61 ID-9421

> 95%

MB

2-Methyl-2-butene (2-methylbut-2-ene) ~H3

1980

{513-35-9]

85%

1980

[55107-14-7] ID-9065

ID-9579

CH3--CN= C--CH 3

MPA

Methyl pivaloylacetate CH~O

> 95%

o

| °11 H CH3--C--C--CH2--C --OCH 3 CH3

* Shell Registered Trade Mark. formulations were shown to be stable for at least one working day.

2.2. Micro-organisms Salmonella typhimurium TA1535, TA1537, TA1538, TA98 and TA100 were obtained from Professor B.N. Ames, University of California. Their genotypes have been described previously (Ames et al., 1975). Escherichia coli WP 2 and WP 2 uvrA, described by Professor B.A. Bridges (1972), were obtained from Dr. M.H.L. Green, University of Sussex. Saccharomyces cerevisiae JD1, heteroallelic at the histidine-4 and tryptophan-5 loci, was obtained from Dr. J.M. Parry, University College of Swansea (Davies, et al., 1975), and has the following genotype: a ade2-1 ser-7 his8 his4C trp 5-U9 a + + + his 4ABC trp 5-U6 Mitotic gene conversion may be scored by supplementing yeast minimal medium with histidine to score tryptophan prototrophs and with tryptophan to score histidine prototrophs.

2.2.1. Culture media (microbial) For detecting revertants of both the Salmonella and Escherichia tester strains, ready-poured petri plates containing 25 ml of a minimal agar medium based on Vogel and Bonner (1956), were obtained from either Difco Laboratories, West Molesey, Surrey or Gibco Europe Ltd., Paisley, Scotland. The yeast complete (YEPD) and yeast minimal (YM) media have been described by Davies et al. (1975). 2.3. Rat-liver chromosome assay 2.3.1. Cell lines The rat-liver cell lines, RL 1 and RL 4, are both epithelial-type cell lines derived in this laboratory following the procedure described by Williams et al. (1971). R L x was initiated in 1973 from a 10-day old Carworth F a r m E rat and RL 4 was derived from a 10-day old Wistar rat in 1978 (Dean and Hodson-Walker, 1979). 2.3. 2. Culture media (cell culture) Stock cultures were maintained and assays were performed using Minimal Essential Medium

63 (Wellcome Reagents Ltd., Beckenham, Kent) supplemented with 10% foetal calf serum (Flow Laboratories Ltd., Irvine, Scotland) and 1% nonessential amino acids (Flow Laboratories Ltd.). 3. Methods

3.1.3. Spot test The method used was that described by Ames et al. (1975). 20 #1 of test compound was added to a 1-cm diameter sterile filter disc before adding to the centre of the seeded plate. The plates were then incubated at 37°C in sealed gas jars before the revertant colonies were counted.

3.1. Bacterial assays 3.1.1. Plate-incorporation assay The method used was basically that described by Ames et al. (1975), using $9 microsomal fraction obtained from a rat liver homogenate from rats pre-treated with Aroclor 1254. From 1975 to 1980, a range of amounts of each compound was tested (0.2, 2, 20, 500 and 2000 # g / p l a t e ) both in the presence and in the absence of $9 mix. Since 1980, a preliminary cytotoxicity assay has been carried out to assess both the cytotoxicity of the test compound and its solubility in the top agar. The amounts to be used in the mutation assays were selected on this basis. 0.1 ml of a dilution (1 : 20 000) of an overnight bacterial culture was added to 2 ml top agar, together with 20 #1 test compound to give final amounts of 125, 250, 500, 1000, 2000 and 4000 #g per plate and 0.5 ml $9 mix ( + $ 9 ) or 0.5 ml p H 7.4 phosphate buffer ( - $9). The top agar was poured onto nutrient agar plates and an assessment of cytotoxicity was made after 24 h incubation at 37°C. In the mutation assays control plates were set up with the solvent alone and with a known positive control compounds. All tests were carded out in quadruplicate (until 1980) or triplicate. Two replicate assays were carried out on different days in order to confirm the reproducibility of the results. 3.1.2. Pre-incubation assay The method used was that described by Brooks and Dean (1981). Bacteria (0.5 ml) and $9 mix or p H 7.4 phosphate buffer (2.5 ml) were incubated at 37°C with the test solution (0.1 ml) or solvent for 30 rain before incorporation of 0.5 ml of this pre-incubation mixture into 2 ml of top agar. All assays were carried out at least in triplicate (i.e. 3 plates per data point).

3.1.4. Treat-and-plate method Overnight broth cultures were washed and resuspended in phosphate buffer p H 7.0. The suspension was then distributed in 2-ml volumes into universal containers and 20 #1 test compound solution was added ( - $9). For studies incorporation microsomal activation ( + $9), 0.5 ml $9 mix was added to each 2-ml bacterial suspension culture together with 25 #1 test compound solution. All cultures were then incubated at 37°C for 1 h before 0.1-ml volumes were seeded onto minimal agar plates with the appropriate amino acid supplement. Appropriate dilutions were plated onto nutrient agar to determine the numbers of survivors. The plates were then incubated at 37°C before the colonies were counted. 3.2. Saccharomyces gene conversion assay 3.2.1. Treatment of stationary-phase cells Yeast cells were grown to stationary phase in Y E P D broth, washed and suspended in pH 7.0 phosphate buffer solution at a concentration of 20 X 106 cells/ml. This suspension was then divided into 1.9-ml amounts in 30-ml universal containers and test compound was added ( - $ 9 ) . For the experiments with metabolic activation ( + $9) 0.5 ml of $9 mix was added. The cultures were incubated, with shaking, at 30°C for the required time. Aliquots were then spread onto YM plates supplemented with either histidine or tryptophan to determine the number of prototrophs at each locus, and dilutions were spread onto YEPD plates to determine cell viability. 3.2.2. Treatment of log-phase cells Yeast cells were grown to log-phase, washed, and resuspended in 2 / 5 strength YEPD broth at a concentration of 10 × 106 cells/ml. This suspension was then divided into 1.9-ml amounts in 30-ml universal containers and 0.1 ml of test corn-

-

.

-

-

DMSO

DMSO

DMSO

DMSO

Hex~e

DMSO

Hex~e

Water

DMSO

GAAE

EHGE

PA

PHOR

COD

SME

D N 102

D N 102S

DMSO

DMSO

DMSO

DMSO

DMSO

DMSO

Water

D L 25-3

DL25-3s/27

BDAM

DTBP

AC

CSA

DMPC

23

NT

DMSO

CHT

D L

NT

Wa~r

GMAC

NT

+

-

-

-

-

-

-

NT

NT

NT

NT

NT

NT

Wa~r

$9

Water

-

TA1535

.

+

-

m

_

-

NT

NT

NT

NT

NT

NT

NT

NT

NT

+ $9

.

+

NT

_

- -

NT

NT

NT

NT

NT

NT

NT

NT

NT

NT

- S9

.

TA1537

+

NT

_

m

NT

NT

NT

NT

NT

NT

NT

NT

NT

NT

+ $9

F O R T H E 41 C O M P O U N D S

S. typhimurium

MCE

lation

Formu-

RESULTS

PO

Compound

SUMMARISED

TABLE 2

-

-

-

-

-

-

-

-

-

-

- $9

TA1538

--

_

--

_

--

+

.

NT

NT

- $9

+

m

m

m

+ $9

.

TA98

_

--

_

+

+

.

NT

NT

+ $9

.

_

_

+

+

.

NT

NT

- $9

TA100

.

q , -

--

- [ -

+

+

NT

NT

.

+ $9

.

.

+

.

.

.

+

+

.

- $9

WP 2

.

E. coil

.

. .

+

.

+

-

.

.

+ $9

.

.

.

+

.

.

+

+

.

- $9

.

.

.

+

.

.

+

+

+ S9

WP 2 uvrA

.

-t-

+

NT

- $9

JD1

.

4-

+

+

NT

+ $9

S. cerevisiae

RL

--

--

q -

+

+

assay

some

chromo-

DMSO

DMSO

Hexane

AA

DCP

PTBT

DMSO

DMSO

PTBC

PNP

Water

Water

DMSO

DMSO

Ethanol

DMSO

DEA

TEA

CE

DMBO

MB

MPA

or

or

_

.

.

.

.

.

.

.

.

.

NT

.

.

.

.

-

+

.

.

.

.

-

.

negative;

Water

MEA

-,

DMSO

PC

Abbreviations:

DMSO

POL

Methanol

DMSO

PTBP

DMSO

Hexane

DMSO

TBB

DMSO

DMSO

EBHP

XE

DMSO

TBMX

Water

SIX

EB

+,

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

NT

+

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

. .

positive;

.

.

.

.

.

.

.

.

NT,

.

.

.

.

.

.

.

.

.

NT

.

.

.

.

.

.

.

not

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

. .

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

tested.

NT

.

.

.

.

.

NT

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

NT

+

NT

NT

+

NT

NT

--

NT

NT

_

NT

NT

_

-

-

+

-

-

+

+

-

--

66 pound solution was added ( - $ 9 ) . For the experiments with metabolic activation ( + $9) 0.1 ml of test compound was added to 1.6 ml of yeast cell suspension, together with 0.3 ml of $9 mix. The cultures were incubated, with shaking, at 30°C for 18 h. Aliquots were then plated as for stationaryphase cells. The $9 fraction used in these assays were prepared from the livers of Aroclor-induced rats according to Ames et al. (1975).

3.3. Rat-liver chromosome Hodson- Walker, 1979)

assay

(Dean and

3.3.1. Volatile compounds (Method A) 3.3.1.1. Cytotoxicity assay. Monolayer cultures of rat-liver cells were prepared in 100-ml glass prescription bottles. The cultures were incubated at 37°C for 24 h to commence active growth before a range of freshly prepared solutions of the compound were added. After a further 24-h incubation the cell monolayers were stained and growth inhibition effects were noted. The concentration selected for the chromosome assay were 0.5, 0.25 and 0.125 of the GI 5° (50% growth inhibition). 3.3.1.2. Chromosome assay. Monolayer cultures of rat-liver cells were prepared and treated as in the cytotoxicity assay in 200-ml glass prescription bottles. Positive control cultures were run in parallel. After 22-h exposure colcemid was added to each culture. 2 h later the cultures were harvested and a hypotonic solution was added to the cell suspension. After the hypotonic treatment the suspension was centrifuged, the solution was decanted and the the cells fixed in 3 changes of fixative solution (methanol: acetic acid 3 : 1 ) . Chromosome preparations were made on microscope slides and stained with Giemsa stain. The preparations were randomly coded and 100 cells from each cultured were analysed microscopically.

3.3.2. Non-volatile compounds (Method B) 3.3.2.1. Cytotoxicity assay. Monolayer cultures of rat-liver cells were prepared in multi-well tissue culture trays. The cultures were incubated at 37°C for 24 h to commence active growth before treatment with the test compounds. After 24 h exposure the cell monolayers were stained and the

growth inhibition effects noted. The concentrations selected for the chromosome assay were 0.5, 0.25 and 0.125 of the GI 5°. 3.3.2.2. Chromosome assay. Cultured rat-liver cells were grown on microscope slides contained in petri dishes. Treatment was again for a 24-h period and positive control slides were included. Colcemid was added 2 h before exposure was complete. The slides were then subject to hypotonic treatment followed by fixation and staining as in Method A. The chromosome preparations were randomly coded and 100 cells from each culture were analysed microscopically. 4. Results

The results of all the assays are summarised in Table 2. The following materials did not induce mutations in bacteria, gene conversion in yeast or chromosome damage in RL 4 cells: glycerol-a-allyl-ether (GAAE), pivalic acid (PA), phorone (PHOR), 1,5cyclooctadiene (COD), SME 529, Dobane 103, Dobane 103 sulphonate, Dobanol 23, Dobanol 25-3, Dobanol 25-3S/27, 2,6-di-tert-butylphenol (Ionox 99), 5-chlorosalicyclic acid (CSA), sodium isopropyl xanathate (SIX), ethylbenzene (EB), tert-butylbenzene (TBB), tert-butyl-m-xylene (TBMX), 3,5-xylenol (XE), p-tert-butyltoluene (PTBT), p-tert-butylphenol (PTBT), p-tertbutylcatechol (PTBC), Polyol E40 (POL), pinacolone (PC), m o n o e t h a n o l a m i n e (MEA), diethanolamine (DEA), triethanolamine (TEA), 18crown-6-ether (CE), 1,5-dimethylbicyclo(3,2,1)-octan-8-ol (DMBO), 2-methyl-2-butene (MB) and methyl pivaloylacetate (MPA). The results of the remaining compounds are considered in chronological order of testing. Propylene oxide (PO). This chemical was tested before the Saccharomyces assay was incorporated into the test battery and before a full range of Salmonella strains was available. Because of the volatility of PO, bacterial incubation was performed in liquid culture in sealed vials and chromosomes were anlysed from sealed flask monolayer cultures. Exposure of either strain of E. coli for 1 h to medium containing 20 # g / m l PO increased the number of revertants. Studies with S. typhimurium TA1538 were negative. Exposure

67 of R L 1 cells to PO resulted in a significant dose-related increase in chromatid aberrations at concentrations of PO ranging from 25 to 100/~g/ml. PO proved to be a potent clastogen and, remarkably, induced chromatid gaps in 56% of cells at the highest dose. Mercaptoethylamine (MCE) failed to induce mutation in bacteria or mitotic gene conversion in yeast. However, a variety of chromatid and chromosome aberrations were observed in slide cultures of RL 1 cells at concentrations of M C E ranging from 5 to 400 # g / m l in the culture medium. In a subsequent cytogenetic study in Chinese hamsters (data not shown here) two daily oral doses of 100 or 200 m g / k g M C E did not induce any demonstrable chromosome damage in bone marrow cells sampled at 6 or 24 h after dosing.

Glycidyl trimethyl ammonium chloride (GMAC). This compound induced an increased frequency of mutation in both strains of E. coli and mitotic gene conversion at the histidine and tryptophan loci in Saccharomyces cereoisiae JD1. The incorporation of rat-liver microsomal fraction did not influence the frequencies of mutation or gene conversion. G M A C induced high frequencies of chromatid aberrations in slide cultures of RL~ cells at concentrations ranging from 10 to 8 0 / t g / m l . Cycloheptatriene (CHT) did not influence the mutation frequency of the bacterial tester strains or the gene conversion frequency of the yeast strain JD1 in the presence or absence of rat-liver microsomal enzymes. At concentrations of 25 or 50 /~g/ml CHT, the incidence of chromatid or chromosome damage in RL 1 cells did not differ from that in solvent control cultures. In cultures containing 100 #g of C H T per ml, the frequencies of chromatid gaps, breaks and exchange figures were increased over the control values. Thus, although not a microbial mutagen, C H T was demonstrated to have clastogenic activity though only at a concentration at which some growth inhibition was evident. 2-Ethyl-hexyl glycidyl ether (EHGE). In bacterial assays, E H G E gave positive responses in Salmonella strains TA1538, TA98 and TA100 after microsomal activation and showed some mutagenic activity in TA100 in the absence of microsomal enzymes. The compound also induced mitotic gene conversion in yeast in the presence of

a microsomal enzyme preparation. There was no evidence of chromosome damage in RL 1 slide cultures exposed to E H G E up to 40/~g/ml, which was the growth-inhibiting concentration. The results of the studies with bis(4-dimethylaminophenyl) methane (BDAM) indicate that it is transformed by rat-liver microsomal enzymes to a reactive metabolite capable of inducing mutation in Salmonella strain TA100. No mutagenic activity was detected in other bacterial strains or in TA100 in the absence of microsomal enzymes. Assays for the induction of gene conversion in yeast or chromosome damage in RL 1 cells were negative. Because of the volatility of allyl chloride (AC) conventional plate-incorporated assays failed to demonstrate mutagenic activity. Even after a 20min pre-incubation of bacteria with AC in sealed containers, no mutagenic activity was observed. However, a series of spot tests with undiluted AC was successful in demonstating the mutagenicity of the compound to both strains of E. coli. No quantitative data were produced in bacteria. Positive results were obtained in tests for gene conversion in yeast and the incorporation at $9 microsomal fraction did not significantly influence the conversion frequency. Results were not strictly dose-related, and this may be due to the volatility of AC. Exposure of sealed monolayer cultures of RL 1 cells to concentration of AC up to 2 5 / ~ g / m l did not result in significant compound-related chromosome damage.

3-[N,N-dimethylamino]-n-propyl chloride hydrochloride (DMPC) was mutagenic to Salmonella strains TA1535, TA1537 and TA100 in the presence of $9 fraction and in strains TA1535 and TA1537 in its absence. Results of the Saccharomyces gene conversion assay and the rat-liver chromosome were negative. The sample of ethylbenzene hydroperoxide (EBHP) contained 33.9% EBHP in EB. Applications of the sample to E. coli WP 2 resulted in an increase in mutation frequency at concentrations up to 200/~g/plate in the absence of $9 fraction. There were no consistent increases in the mutation frequency of the other tester strains, or in E. coli WP z in the presence of $9 fraction. EBHP induced a significant increase in gene conversion at both loci in Saccharomyces cerevisiae JD1. Exposure of RL 1 cells to EBHP did not result in significant chromosome damage.

68

Allyl acetate (AA) proved to be a direct-acting mutagen in Salmonella strains TA1535 and TA100. Negative results were obtained in assays using the other bacterial strains and in all strains in assays incorporating $9 fractions. AA did not induce gene conversion in yeast or chromosome damage in RL 4 cells. The results of bacterial assays indicate that 1,3-dichloropropane (DCP) is transformed by ratliver microsomal enzymes into a reactive metabolite capable of inducing mutations in TA1535. Mutagenic activity was not detected in other bacterial strains or in strain TA1535 in the absence of rat-liver microsomal fraction. DCP was negative in the yeast assay and in the RL 4 chromosome assay. p-Nitrosophenol (PNP) was shown to be a direct-acting mutagen, inducing mutation in TA98 and, to a lesser degree, in TA100. Incorporation of $9 fraction eliminated the mutagenic activity and also reduced the cytotoxicity of the compound to bacteria. No evidence of the induction of gene conversion was observed in yeast. Chromosome damage was induced in RL 4 cells exposed to culture medium containing 0.75-3.0 #g PNP per ml.

5. Discussion

5.1. General Since these experiments were performed, results of mutagenicity tests carried out by other workers on some of the compounds have appeared in the literature. PO, for example, has been widely investigated and its mutagenic activity confirmed (e.g. Pfeiffer and Dunkelberg, 1980). MCE induced considerable chromosome damage in RL 1 cells yet there was no evidence of a mutagenic effect in bacteria or yeast. MCE has undergone extensive investigation as a chemical radioprotective agent in a variety of test systems ranging from bacteria and cultured mammalian cells to whole animals. Its mode of action is generally considered to be related to its radical-scavenging activity (Hart et al., 1975). At a concentration of 50 mM, MCE has been shown to protect DNA molecules of cultured Chinese hamster cells against X-irradiation-induced single-strand breaks, double-strand breaks and nucleotide damage and

against mutation induction and cell-killing (Pitra et al., 1977). The same concentration of MCE protected bacteria (E. coli BH) and cultured mouse lymphoma L5178Y cells against the lethal effects of radiation (Antokv, 1975). Lronard and Deknudt (1973) discovered that when MCE was dosed to mice in a mixture with reduced glutathione, 2-fl-aminoethylisothiuronium-Br-HBr, cysteine and 5-hydroxytryptamine, spermatogonial cells were protected against irradiation-induced chromosome rearrangements. Paradoxically, MCE is radioprotective at high doses (10-50 mM) yet had adverse biological effects at low concentration (0.1-2.0 mM) (Vos et al., 1962). Low concentrations of MCE have been shown to be cytotoxic to cultured human kidney cells (Vos et al., 1962), to inhibit DNA synthesis and to induce DNA single-strand breaks in cell cultures (Sawada and Okada, 1970) and chromosome damage in cultured Chinese hamster cells (Delrez and Firkett, 1968). Following the finding of its clastogenic activity in RL t cells, MCE was studied in an in vivo system. No increase in the frequency of chromosome aberrations was observed in bone-marrow cells of Chinese hamsters after two daily oral doses of 100 or 200 m g / k g MCE (unpublished results). None of the five detergents tested showed any evidence of mutagenic activity in bacteria, yeast or cultured cells. Hope (1977) tested two detergents based on Dobanol 25 sulphate in the rat bonemarrow system. No chromosome damage was observed in bone-marrow cells of rats fed these materials for 90 days. BDAM induced a small but significant increase in mutation frequency in Salmonella strain TA100. This was detected only in the presence of an $9 metabolising system, and the compound was inactive in the other bacterial strains and in the other two assays. The only published study on the mutagenicity of BDAM reported negative findings in bacteria, though the criteria on which positive and negative results were judged are not clearly defined (McCann et al., 1975). A National Cancer Institute bioassay of BDAM reported carcinogenic effects in both rats and mice (NCI, 1978). Allyl chloride (AC) was shown to be a directacting mutagen in both strains of E. coli and to induce mitotic gene conversion in yeast. These

69 results were in general agreement with those reported by McCoy et al. (1978). In both studies, probably because of the volatility of AC, the c o n ventional agar-incorporation technique was unsuccessful in detecting bacterial mutation. A 20-min preincubation stage was required to demonstrate mutagenic activity in the spot test and in McCoy's studies. The xanthate, SIX, was not genotoxic in any of the assays. However, it was considered that under certain in vitro experimental conditions the compound may inhibit mono-oxygenase enzyme activity. In order to ascertain whether SIX interferes with the enzyme activity of rat-liver $9 microsomal enzymes, it was incorporated in an agar overlay together with standard $9 mix, Salmonella strain TA98 and 5, 10 or 20 /~g/plate benzo[a]pyrene. The activity of $9 fraction was not affected by the incorporation of concentrations of SIX up to 200 /~g/plate. There was some reduction of benzo[a]pyrene-induced mutation in plates containing 2000/xg/plate SIX, suggesting that the compound did inhibit mono-oxygenase activity at this concentration. The EBHP sample was a plant-stream containing 33.9% EBHP in EB. The volatility of EB presented some technical problems with the assays, and may have contributed towards inconsistencies in the yeast data on the EBHP sample. It appears probable that the genotoxicity of EBHP was caused by the peroxide group and the variable yeast data may be related to variation in the ability of yeast cells at different stages of the cell cycle to degrade hydrogen peroxide (Thacker, 1975). The other components of the EBHP plant stream sample, acetophenone and phenyl ethyl alcohol, must also be considered in relation to the genotoxicity of the material. Phenyl ethyl alcohol is known to interfere with DNA and RNA replication and with protein synthesis in E. coli though did not induce DNA-strand breakage (Nair et al., 1975). Acetophenone was negative in a differential repair assay in bacteria, in which hydrogen peroxide gave positive results (Fluck et al., 1976) and also gave negative results in a Salmonella microsome assay (Ames et al., 1975). EB itself failed to induce recessive lethal mutations in Drosophila (Donner et al., 1980). 1,3-Dichloropropane (DCP) has been reported

to induce a substantial frequency of reverse mutations in Salmonella strain TA100 (Stolzenberg and Hine, 1980), and in TA1535 (Bignami et al., 1977). In both these studies metabolic activation was not a requirement for mutagenic activity. In our study, DCP was detected as a mutagen in strain TA1535 only in the presence of $9 fraction. No activity was detected in TA100. PNP was a direct mutagen in Salmonella strains TA98 and TA100. The incorporation of $9 fraction eliminated (TA98) or reduced (TA100) mutagenic activity. Cytotoxicity was evident in both strains at 400/~g/plate in the absence of $9 and at 1600/~g/plate in its presence. These results agree, in general, with those of Gilbert et al. (1980) who reported mutagenicity and cytotoxicity to strain TA1538 with no detectable effects in strains TA1530 and TA1535. These workers did not use strains TA98 and TA100. The three ethanolamines, MEA, DEA and TEA were reproducibly negative in the three assays, thus confirming the finding of Hedenstedt (1978) that MEA and DEA were not mutagens in Salmonella strains TA1535 and TA100. TEA has been tested in a series of Bacillus subtilis strains and failed to induce mutation. However, after reaction with sodium nitrite, a mutagenic product was detected in the reaction mixture (Hoshino and Tanooka, 1978). N-Nitrosodiethanolamine, a known carcinogen and mutagen, was identified in the reaction mixture, but because of its mutagenicity and stability characteristics, N-nitrosodiethanolamine was not thought to be the major mutagenic product. The same authors also reported the development of malignant tumours in mice fed a diet containing 0.3 or 0.03% TEA. 5.2. Structure-activity considerations The 41 compounds have been organized, for the purposes of a structure-activity discussion, into groups which emphasize important common structural features. The compounds are assessed for their potential to act as mutagens per se and from the point of view of their susceptibility to metabolic activation by the $9 fraction. The generation of electrophilic intermediates is particularly useful in the prediction or interpretation of positive mutagenicity data (Wright, 1980).

70

A ralkanes No simple alkanes were tested in this series but each of the 5 aromatic hydrocarbons tested possessed alkyl side-chains. These compounds, therefore, may be classified as aralkanes. There is no basis for supposing that any of these neutral, stable compounds are direct-acting mutagens. There are, however, numerous possible routes for their biotransformation. In principle, whenever a test compound contains a benzene ring, the possibility of oxygenation to an arene oxide must be considered. Whilst aromatic hydroxylation (via arene oxide formation) is a very common reaction in the metabolism of xenobiotics, only rarely does it seem to lead to genotoxic effects. It is likely that rearrangement or further metabolism of the arene oxides is usually too rapid for their action as arylating agents to be manifested. A problem in this context with in vitro test systems is that it is often surprisingly difficult to drive aromatic hydroxylation (cf. other xenobiotic oxygenations) in vitro. For example, the major in vivo reaction of phenobarbitone (4'-hydroxylation) has resisted several attempts to mimic it in vitro (Crayford and Hutson, 1980). The simplest member of the aralkanes, toluene, is not mutagenic in in vitro tests (Dean, 1978). Arene oxide formation and sidechain hydroxylation and sulphation (to form benzyl sulphate) could both afford arylating/alkylating species but this does not appear to happen either in vitro or in vivo. The linear alkylbenzene (Dobane 102) should undergo side-chain oxidation and degradation by r-oxidation. The terminal products of these reactions would be, depending on chain length, benzoic acid and phenylacetic acid. These metabolites would be expected to be non-mutagenic (Ames et al., 1975). The non-mutagenicity of the 5 aralkanes (Dobane 102, ethylbenzene, tert-butylbenzene, 4-tert-butyltoluene and 1-tert-butyl-3,5-dimethylbenzene) is therefore expected in the light of current experience.

Alkenes Alkenes per se would not be expected to be mutagenic (unless activated by a proximate carbonyl group) but may be so after oxygenation to their corresponding epoxides (alkene oxides). In in vitro test systems, the $9 liver fraction contains not only the alkene oxygenase (usually cytochrome

P450), but also epoxide hydrolase. These enzymes are located close together on the endoplasmic reticulum (present as microsomal vesicles in the $9 fraction). The success or failure to detect alkenes as mutagens, therefore, depends on at least 3 factors: (i) the efficiency of the oxygenation of the alkene to the alkene oxide, (ii) the mutagenic potency of the alkene oxide and (iii) the suitability of the latter as a substrate for epoxide hydrolase. Factors (i)-(iii) operate similarly in vivo, together with the added factor, glutathione epoxide transferase, which is not particularly effective in the $9 fraction due to dilution and oxidation of glutathione (Creedy et al., 1984). It is therefore possible that the in vitro systems are on the sensitive side for alkenes in comparison with those in vivo. Of the 7 alkenes tested (1,5-cyclooctadiene[COD], 1,3,5-cycloheptatriene [CHT], glyceryl a-allyl ether [GAAE], 2,6-dimethyl-2,5-hep tadiene-4-one [PHOR], 2-methyl-2-butene [MB], allyl chloride [AC] and allyl acetate [AA]) only the latter two were positive and apparently via direct alkylating action (see below). It is not possible to decide which of the 3 factors (i, ii and iii above) account for the nonmutagenicity of the other alkenes. It is of interest in this context that epoxide hydrolase is effective against lipophilic cis-epoxides (for example, as formed from COD) and against 1-substituted and 1,1-disubstituted epoxides, but is less effective against trans-disubstituted and tri- and tetra-substituted epoxides (Oesch, 1973). Thus the putative epoxides of MB and PHOR are likely to be poor substrates for the enzyme and therefore likely to persist longer; however, steric hinderance may limit their effectiveness as electrophiles. On balance it seems that alkenes tend not to be mutagenic in these in vitro test systems.

Epoxides Of the 3 epoxides studied, all were mutagenic, propylene oxide (PO) and glycidyl trimethylammonium chloride (GMAC) directly so, and 2-ethylhexyl glycidyl ether (EGE), after activation (apart from a very weak direct action on S. typhimurium TA100). $9 fraction slightly diminished the activity of PO and GMAC, possibly by epoxide hydrolase action or by the protective intervention of amino and thiol groups of $9 proteins. Glycidyl

71

trimethylammonium chloride is a particularly interesting molecule in that the effects of insertion of a cationic group into DNA have been very little studied. Similarly, the efficiency of charged compounds as substrates for epoxide hydrolase is unknown. The activity profile of EGE ($9 dependency) suggests that it is a glycidaldehyde generator. This aldehyde is a potent mutagen in these systems (Voogd et al., 1981). The result is in accord with a structure-activity study on a series of alkyl glycidyl ethers reported recently by Thompson et al. (1981). In this study, the lower members of the series (ethyl, butyl and hexyl GE) were all directly mutagenic, their action being enhanced only slightly by $9 fraction; higher members of the series (octyl, decyl, dodecyl GE) required activation. Another recent study of the mutagenic action of aliphatic epoxides, using Klebsiella pneumoniae as the test organism, revealed that 36 out of 45 epoxides were mutagenic (Voogd et al., 1981). Some useful structure-activity data emerged from this study: (i) the mutagenicity of 1,2-epoxides decreased with increasing chain length, (ii) terminal epoxy groups were more potent than non-terminal groups, (iii) electron-withdrawal from the epoxide ring increased the mutagenic activity and (iv) electrondonating groups decreased mutagenic activity. The relatively weak action of propylene oxide (PO) is in accord with this latter effect. Current evidence indicates, therefore, that epoxides must be assumed to be mutagenic until proved otherwise.

ione peroxidases (Sies et al., 1982), which protect aerobic life-forms from the damaging effects of a variety of endogeneous organic peroxides.

A lkyl halides Allyl chloride is a direct-acting mutagen, probably acting as a direct alkylating agent. The effect of the alkene group is to increase the reactivity of the carbon-chlorine bond (due to charge delocalisation in the transition state of the SN2 reaction). This accounts for the difference in direct mutagenicity between allyl chloride and the (saturated) chloroalkane, 1,3-dichloropropane. The latter is not a direct-acting mutagen but requires bioactivation; these findings are in agreement with those of Stolzenberg and Hine (1980) who have reported on a series of 2- and 3-carbon halides. The nature of the bioactivation process can only be speculative in the absence of detailed metabolic studies. It is likely to be oxidative and could involve (i) C-oxygenation or (ii) C/-oxygenation (Guengerich et al., 1980) as indicated in Fig. 1. There are several possibilities for the generation of electrophilic centres from these reactions. Dimethylaminopropyl chloride (hydrochloride) (DMPC), unlike the other propyl chlorides, is a direct-acting mutagen. This reactivity is unlikely to be due to C-CI bond reactivity. However, the facile chemical reaction outlined in Fig. 2 may have a biological equivalent and this would gener-

(i) C - Oxygenation

Ethylbenzene hydroperoxide This compound was weakly mutagenic in one bacterial strain and in the yeast assay. The action was direct and probably derived from active oxygen species liberated during decomposition of the hydroperoxide. $9 fraction abolished the mutagenicity, either because the peroxide was reduced by protein thiol groups or because it was destroyed by the peroxidase activity associated with cytochrome P450 (O'Brien and Rahimtula, 1980). In the latter case, the reactive species are likely to be similar to those generated during the normal action of this enzyme. In vivo, ethylbenzene hydroperoxide is rapidly reduced to 1-phenylethanol which is further metabolised and excreted (Climie et al., 1983). The reduction is probably effected by the glutath-

OH

[o]

I

CH 2 - CH z - (~H2 I I CI CI

:>

-

2

H

CH z - CH-- CH 2 I I CI | CI

[

~

- HCI

O II (~H2-- C-- CH2CI

CH 2-- CH-- CH 2

CI

CI

I

\/

O

(ii) C I - Oxygenation RCH2CI

[0]

~

RCH2CI , ~

Fig. 1. Possible routes for metabolic activation of the chloroalkanes.

72

CH3~. N H - - CH:CH2CHzCl CH3~ +

el-

-H

I 1

NH--CH2--CH--CH2CI

CH3/"

+

(CH3)2NH + CH2=CH--CH2Cl +HCl Fig. 2. Generation of allyl chloride from dimethylaminopropyl chloride.

ate allyl chloride in situ and account for the reactivity of DMPC.

Allft acetate Allyl acetate (AA) was also a direct-acting mutagen. This was unexpected in that the ester carbonyl group is too far away to exert a strong polarizing effect on the alkene bond. It is likely that C-3 of the allyl group is activated for alkylation by loss of acetoxy from C-1. a-Acetoxysaffrole is thought to be mutagenic via such a mechanism (Wislocki et al., 1977). The analogous reaction may also occur with allyl chloride (see above) if an SN1 mechanism were to predominate. The destruction of the mutagenic activity of AA by $9 fraction is probably due to the action of microsomal carboxylesterase which would catalyse hydrolysis to allyl alcohol thereby nullifying the activating effect of the acetoxy leaving group.

Alkanols Dobanol 23 (a mixture of linear C l l and C12 primary alkanols), Dobanol 25-1-3 (a primary alcohol ethoxylate) and its sulphate ester, DMBO (a cyclic secondary alcohol) and the aminoalkanols (MEA, DEA an TEA) exhibited no mutagenic activity. This is in accord with their lack of electrophilic reactivity.

were non-mutagenic in the test systems. These possess anti-oxidant properties and would be expected to form quinones under oxidising conditions, e.g. DTBP to 2,6-di-tert-butylbenzoquinone: O

(CH3)3C'~C(C H3)3 0 Quinones contain electrophilic centres and may be mutagenic; however, 2-tert-butyl-p-benzoquinone, for example, has also been reported as nonmutagenic to five S. typhimurium strains (Natake et al., 1982). 5-Chlorosalicylic acid was non-mutagenic, as was the o-hydroxybenzophenone oxime (SME). These compounds are unlikely to afford electrophilic species. p-Nitrosophenol possesses a rather different reactivity from the phenols above and is cytotoxic and a direct-acting mutagen (an observation which confirms the report of Gilbert et al., 1980). This structure exists in tautomeric equilibrium with pbenzoquinone monooxime, the nitroso form predominating in aqueous media. It reacts with primary aliphatic and aromatic amines with the formation of p-nitroso-N-alkyl (or aryl) anilines (Belyaev et al., 1973). These reactions (Fig. 3) probably proceed via attack by the amines at the carbonyl atom of the quinonoid tautomer to form Schiff's bases which subsequently tautomerise. The carbonyl carbon atom of the benzoquinone oxime must therefore be regarded as a centre of electrophilic reactivity capable of causing damage by reacting with functional and informational macromolecules. Lack of mutagenicity in the presence of $9 fraction is probably due to the NADPH-dependent reduction of p-nitrosophenol to p-aminophenol (Bernheim, 1973) which is non-mutagenic (Garner and Nutman, 1977).

OH

O

N/R

NHR

N.~O

N ~'~OH

N ~'OH

N~O

Phenols The simple phenols, 4-tert-butylphenol (PTBP), (DTBP), 4-tert-butylcatechol (PTBC) and 3,5-dimethylphenol (XE)

2,6-di-tert-butylphenol

Fig. 3. Reaction of p-nitrosophenol with amines.

73 Ketones, carboxylic acids and ethers The 4 compounds grouped under this heading: pivalic acid (PA), pinacolone (PC), methyl pivaloylacetate (MPA) and 18-crown-6-ether were not mutagenic, either in the presence or absence of $9 fraction. None would be expected to be or to form electrophilic species under the conditions of the tests. Amino compounds The aminoalkanols (discussed above) were non-mutagenic. The thio analogue of MEA, mercaptoethylamine (hydrochloride) was also not a bacterial mutagen. It is not clear why the latter caused chromosome damage in vitro. Schiff's base or disulphide-bond formation with proteins may afford significant structural changes. The polyol E40 (formed by the reaction of ethylenediamine, diphenylolpropane and propylene oxide), provided that it was free of the latter, would not be expected to furnish electrophilic compounds. Bis-(4-dimethylaminophenyl)methane (BDAM) was mutagenic only to S. typhimurium TA100 and only after bioactivation. This structure contains some of the features of dimethylaminobiphenyl and dimethylaminoazobenzene,both mutagens and carcinogens. The bioactivation of these compounds is thought to proceed by N-dealkylation followed by N-hydroxylation and esterification of the N-hydroxy group to form reactive species, which decompose to nitrenium ions (Miller, 1978). The stabilisation of the nitrenium ion by charge delocalisation is thought to be important. This is facilitated in the biphenyl and azobenzene structures but is very weak in diphenylmethanes. Hydroxylation at the methylene group, followed by dehydrogenation, would afford Michler's ketone. This compound, and its imino derivative, auramine are carcinogens, and would be stabilised at the nitrenium ion stage. It is possible that BDAM effects its action via conversion to Michler's ketone. 5.3. Performance and evolution of the assays The screening of chemicals for mutagenic activity began in 1975. The following section addresses the performance and evaluation of the assays since that time.

5.3.1. Bacterial mutation assays In the early seventies, some screening of agrochemicals was performed using Escherichia coli WP2 and the Serratia marcescens strains H Y / a l 3 and HY/a21 though, of course, without any attempt at microsomal activation (Dean, 1972). Subsequent to that work, the uorA strain of WP2 was introduced (Bridges, 1972) and then the frameshift-tester strain Salmonella typhimurium TA 1538 (Ames et al., 1973). At this time, the paper by Ames was published describing the incorporation of rat-liver microsomal enzymes in the bacterial assays (Ames et al., 1975). The first bacterial screening tests used at Sittingbourne consisted of E. coli strains WP2 and WP2 uvrA and Salmonella typhimurium TA1538 tested in the presence and in the absence of an $9 fraction prepared from the liver homogenate from Aroclor-induced rats. New strains were added as they were validated and the current tests now include, in addition to the above, Salmonella strains TA1535, TA1537, TA98 and TA100. Salmonella typhimurium TA92 was also used for a short period but appeared to have no advantages over the other strains for screening purposes (Brooks and Dean, 1981). Since the completion of this work, the two E. coli strains have been superceded by E. coli WP2 uorA pkm 101 (Venitt and Crofton-Sleigh, 1981). Although the agar-overlay technique originally described by Ames et al. is still in regular use, modifications, such as the use of 30-min preincubation period (Brooks and Dean, 1981), are used for specific compounds, and important changes in experimental design have been introduced. In early studies, a standard range of test chemical concentrations was used, i.e 0.2, 2, 20, 200 or 500, 2000 (4000) /tg per plate. Following the realisation that in rare cases a chemical may only be detectably mutagenic over a very narrow dose range, i.e. a 'mutagenic window', a range of doses at 2-fold intervals was calculated from an identified cytotoxic concentration and/or limit of solubility. In addition, a statistical study showed that acceptable data could be obtained from 3 replicate plates per dose level and this replaced the 4 replicates used initially. The practice of testing each compound in all tester strains was also modified to a sequential testing system. Compounds

74 are now tested in TA98 and TA100 initially (and more recently E. coli WP z uvrA pkm 101). If positive in either strain a confirmatory study is performed and the other strains are not used. Compounds negative in these strains are tested sequentially through the other strains. All assays are performed twice, on different days, to ensure the reproducibility of results. In most experiments, data are interpreted on the basis of a consistent doubling of the spontaneous reversion frequency confirmed by a doseresponse relationship. Where the number of induced revertants is less than twice the spontaneous rate, but a reproducible dose-related increase in revert'ants is detected, this is interpreted as a positive response. A variety of statistical techniques have been applied to bacterial data in an attempt to detect weak positive responses. Although the standard t-test, the Control Chart method and the Quadrant Sum method have been applied to selected data, and other techniques have been evaluated, none of them have proved suitable for general application to bacterial mutation assays on a routine basis. 11 of the 41 compounds induced mutations in one or more strains of bacteria. An analysis of the performance of the individual strains has shown that none can be regarded as totally redundant. For example, although TA100 was generally more sensitive than TA1535 from which it is derived, in one case, DCP, a positive response was detected in TA1535 in the absence of detectable activity in TA100. TA1538 did not detect mutagenic activity when TA98 was negative. However, unpublished observations suggest that TA1538 is superior for testing certain classes of compounds i.e. certain polycyclic hydrocarbons. E. coli WP 2 detected mutagenic activity with EBHP where the other strains failed. The incorporation of appropriate model mutagens for control purposes has also become much more standardised. In earlier studies a positive control was not included in every study with every strain. Current practice in this laboratory is to use an appropriate control with every experiment to monitor the system, and, when possible, to also use a positive mutagen structurally-related to the compound under test. The bacterial assays are under constant ap-

praisal and new modifications are assessed as they appear. 5.3.2. Mitotic gene conversion assays using Saccharomyces cerevisiae Our first experiments with mitotic gene conversion in yeast used the Saccharomyces cerevisiae strain D4 (Zimmermann, 1977). A subsequent evaluation of strain JD1 (Davies et al., 1975) showed this strain to have significant advantages over D4 for screening purposes. The treat-and-plate method using liquid-suspension cultures has altered little over the period of testing, though treatment times of 1, 4, 18 or 24 h have been favoured at different times. Treatment of exponential cultures (Sharp and Parry, 1981) is now used in preference to stationary-phase cultures, although any advantages are debatable (Brooks et al., in preparation). Initially a range of concentrations of test compound (0.01, 0.1, 0.5, 1.0 and 5.0 m g / m l ) are tested if solubility allows. A second experiment is then carried out based on these results taking into account the effect of the chemical on cell viability and any possible positive effect. A chemical is considered to increase the rate of mitotic gene conversion if there is a reproducible, dose-related increase in the number of prototrophs p e r 10 6 survivors together with an increase in the number of prototrophs per plate. Any increase in prototrophs per 106 survivors without an increase in plate counts demands tedious reconstruction experiments. The value of including a yeast assay for mitotic gene conversion in the basic test battery has been evaluated on a number of occasions. Although none of the 41 chemicals described here were exclusively positive in JD1, a positive yeast result with EBHP confirmed its genotoxicity after the observation of mutation in a single bacterial strain. This confirmatory role of Saccharomyces cerevisiae has been repeated with other compounds. 5.3.3. The rat-liver chromosome assay This in vitro assay has undergone few changes during the course of these experiments. Because of the depletion of suitable stock cultures of strain RL~, another strain of rat liver epithelial-type cells, RL4, was isolated, validated and introduced into

75 regular use in 1978. The technique has proved to be an easily standardised and reproducible means of screening chemicals for clastogenic activity. The residual microsomal enzyme activity in the cells is sufficient to allow the detection of many classes of activation-dependent mutagens without recourse to supplementary metabolising systems. It is recognised that the incorporation of a microsomal preparation from rat liver e.g. Aroclor-induced $9, may increase the sensitivity of the test system to certain classes of chemical mutagens. However, this would conflict with one of the initial objectives of the rat-liver cell assay, that is, to provide a different spectrum of enzyme activities to that represented by the conventional $9 preparation used in our bacterial and yeast procedures. 2 of the 41 compounds induced chromosome aberrations in RL cells in the absence of bacterial mutagenic activity, thus confirming the value of the assay in the test battery. The slide-culture technique remains virtually unmodified. Volatile materials were tested in monolayer cultures in sealed flasks, but recently, those have been replaced by Leyton Tubes large enough to accommodate a 3 " × 1" microscope slide. N o suitable statistical treatment for these types of data has been identified and results are judged on the reproducibility and dose-responsiveness of the aberration frequencies.

5.3.4. The test battery The battery of 3 assays has been applied on a routine basis to many other compounds and products not described in this paper, and has proved a valuable component in the toxicity-testing programme. In some cases, the B H K transformation assay (Styles, 1977) and the DNA-elution assay for single-strand damage has been incorporated to give a 5-test battery. This, of course, produces a higher degree of confidence in negative results, confirmatory evidence of positive findings and is an additional help in the interpretation of results in human hazard terms. There have been no occasions when these two supplementary assays have given positive results when the basic battery has failed to detect mutagenic activity.

6. Conclusion The performance of the battery of short-term tests used to detect genotoxic chemicals has been evaluated, using data derived from assays in 41 chemicals conducted over a 5-year period. The bacterial assay using Salmonella typhimurium and Escherichia coli tester strains detected the highest number (11) of the chemicals as mutagenic. A further two chemicals induced chromosome damage in RL cells and the test for mitotic gene conversion provided valuable supplementary evidence of genotoxicity in a eukaryote for 4 of the compounds. The results of these assays have provided information that has been used to formulate safe handling procedures for the chemicals and in the design of manufacturing processes.

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