Mutagenicity studies on organophosphorus insecticides

3/Iutation Research, 32 (1975) 133-15o © E l s e v i e r Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in The N e t h e r l a n d s

I33

MUTAGENICITY STUDIES ON ORGANOPHOSPHORUS INSECTICIDES

DIETER

WILD

Zentrallaboratorium figr i~Iutagenitiitspriifung der Deutschen Forschungsgemeinschaft, Freib~rg i. Br. (Germany) ( R e c e i v e d M a r c h i 2 t h , 1974) ( R e v i s i o n r e c e i v e d J u n e i 7 t h , 1975) ( Accep ted J u l y 22nd, (1975)

CONTENTS Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . General a s p e c t s of c h e m i s t r y , t o x i c i t y a n d m e t a b o l i s m of o r g a n o p h o s p h a t e s . . . Effects on D N A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mutagenicity tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M i c r o o r g a n i s m s in vitro . . . . . . . . . . . . . . . . . . . . . . . . . . M i c r o o r g a n i s m s in vivo ( h o s t - m e d i a t e d assay) . . . . . . . . . . . . . . . . Drosophila . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M a m m a l i a n cells in c u l t u r e . . . . . . . . . . . . . . . . . . . . . . . . M a m n l a l i a n cells in vivo . . . . . . . . . . . . . . . . . . . . . . . . . S u m m a r y and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Addendum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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133 135 137 138 138 141 141 142 142 144 t 46 147 147 147

INTRODUCTION

Almost 800.000 species of insects are known, and among them more than 68.000 are considered by an entomologist as "pests" (cited in ref. 36). Because existing insectivorous animals cannot easily be manipulated for the control of nocuous insects, chemical insecticides have been developed for this purpose, of which chlorinated hydrocarbons and organophosphorus compounds are the most broadly used and economically most important. However, with continued and increasing pesticide use, unintentional deleterious effects on the environment and on human health became apparent or were suspected. Among these, teratogenic, carcinogenic and mutagenic effects received special attention ~,41. The concern over potential mutagenicity of organophosphates was increased when trimethyl phosphate, a simple organophosphate which, however, is not used as insecticide, was found in 197o to be mutagenic in mice 15. Thereafter, potential genetic effects of selected organophosphorus insecticides were investigated with various methods and in several laboratories, and considerable information has now accumulated for a preliminary and tentative estimation of the potential risk of this group ot insecticides for the human genome. The present article reviews a part of the results of a pesticide test programmc conducted by the Zentrallaboratorium ftir Mutagenit/itsprtifung of the Deutsche

134

D. WILD

TABLE I ORGANOPHOSPHORUSINSECTICIDES AND METABOLITES DISCUSSEDIN THIS ARTICLE Common name, other names

Pesticide index ~umber (ref. 22)

Scientific ~zame (according to r£[. 22)

Organophosphorus insecticides Bidrin® dicrotophos

0618

3-(dimethoxyphosphinyloxy)N ,N-dimethyl-cis-crotonamide

Structure

cH3o\ //o / \ P O- CH30

C~

I CH3

CH - - CO - - N{CH3)

CH~O\p//S ct Bromophos

o I 68

0,0-dimethyl O-4-bromo,2, 5dichlorophenyl phosphorothioate

CH30 / \o ~ B ~ Ct C2H50

Diazinon®

0458

Dichlorvos DDVP dichlorovos vapona®

0528

Dimethoate

0617

0,O-diethyl 0-(2-isot)ropyl 4methyi-6-pyrimidyl) phosphorothioate

O,O-dimethyl 2,2-dichlorovinyl phosphate

CH3

,,~S

I

/ \

/

CH30X / P ~O--CH

CH3O /

~

CCL2

cH~o\//s O,0-dinlethyl S-(N-methylcarbamoyhnethyl) phosphorodithioate

P CH30/ ~S

CH2CONHCH3

c%o\//s Fenitrothion folithion® sumithion®

0667

0,O-dimethyl O-(3-methyl-4-nitrophenyl) phosphorothioate

c%o/ \ o

N% CH3

c%o\ ~s Malathion®

0968

S- [I,2-bis(ethoxycarbonyl)ethyl] 0,O-dimethyl phosphorodithioate

P

CH30/

~ ' S - - CH - - C00C2H5

I

CH 2 -

COOC2H5

cH~o\//s Methylparathion lO48 parathion-methyl

O ,O-dimeth yl-O- 4-nitrophen yl phosphorothioate

Oxydemeton113o methyl metasystox R® methyl demeton-O-sulfoxide

O,O-dimethyl S- [2-(ethylsulphinyl) ethyl] phosphorothioate

cHao/ ~o CH30\

/ P\

CH30 /

N% o

II SCH2CH2SC2H5

~35

MUTAGENICITY STUDIES ON ORGANOPHOSPHORUS INSECTICIDES T A B L E I (continued)

Common name,

Pesticide

Scientific name

other names

index number

(according to

Structure

re.[. 22)

(ref. 22) C2H50~ ~/S

Parathion

i 144

O,O-diethyl-O-4-nitrophenyl phosphorothioate

P C2H50

0

NO;

c.3o\//o Trichlorfon dipterex®

143 I

O,O-dilnethyl (i-hydroxy-2,z,2trichloroethyl) phosphonate

/P\ CH30

CH - - CC[3

I

OH

Metabolites dimethylphosphate, ammonium salt

( C1"-130)2PO2NH4

diethylphosphate, ammonium salt

(C2HsO)2PO2NH4

dimethylphosphothioate, a m m o n i u n l salt

(CH30)2~ONH4

diethylphosphothioate, ammonium salt

(C2H50)2~ONH4

methylphosphate, dianlmonium salt

S CH3OP03(NH4)2

ethylphosphate, diammonium salt

C2HsOP03( NH4)2

p-nitrophenol

HO ~

S

NO2

Forschungsgemeinschaft as well as results published by other groups. A summary of the organophosphorus insecticides and some metabolites covered in this article is presented in Table I. GENERAL ASPECTS OF CHEMISTRY, TOXICITY AND METABOLISM OF ORGANOPHOSPHATES

Because the chemistry of organophosphorus pesticides, their alkylating properties, mechanisms of toxicity and ways of metabolism have been treated thoroughly in several recent publications3,19,% these topics are only shortly outlined here. Relevant information on dichlorvos and an evaluation of its human health hazards, based on the information available in I972, will be found in articles by GILLETT

et al.23, ~4. For all phosphorus-derived insecticides the general term organophosphorus insecticides can be used. Most of these are esters or amide esters of phosphoric acid and, therefore, also correctly named organophosphates; only a few (e.g. trichlorfon) are derived from phosphonic acids. Phosphoric acid, as a tribasic acid, can form mono-, di-, and tri-esters with alcohols, thiols and phenols. Typical insecticidal organophosphates are mixed tries-

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D. WILD

ters with two methvl- or two ethyl-ester groups and a different, electron-withdrawing ester group. The nmltiplicity of organophosphates is further increased by the introduction of derivatives of thiophosphoric acid. I

The common structural element of all organophosphates,

I

P - O - C - , with F

phosphorus and carbon as electrophilic sites, offers the key for the understanding of the reactions of organophosphates with nucleophiles. A nucleophile can preferentially attack the phosphorus atom with subsequent cleavage of the P-O bond and undergo pho@horvlatio~ or preferentially attack the carbon atom with subsequent cleavage of the C-O bond and undergo alk3,lation (methylation or ethylation). It can also attack either electrophilic site. The type and rate of reaction with a given nucleophile depends strongly on its nature, in the presence of several nucleophiles such as occur in competitive reactions in a living cell. Because of the correlation between alkylating and mutagenic, carcinogenic and other deleterious biological activities of chemicals:", a rapid standard assay, the NBP colour test, has been developed for the assessment of alkylating agents. In this test, NBP (4-(4-nitrobenzyl)pyridine) serve:~ as a model nucleophile. It reacts with alkylating agents to form coloured alkylation products which can be analysed colorimetrically. The test, in several modification:s, has been applied to many different classes of alkylating agents'~": most organophosphates studied exhibit alkylating properties in this test "~,~1,'~9.Though the NBP reaction can be quantitated, the results do not allow conclusions on the extent of alkylation of nucleophilic sites in DNA of a living cell which contains many different nucleophiles. Reaction between organophosphates and water as nucleophile, that is hydrolysis, leads to diesters of pho:sphoric acid and alcohols, thiols or phenols. The diesters, depending on their structure, can be further hydrolysed to monoesters and eventually to inorganic phosphate. Organophosphates are, therefore, not persistent in the environment and--unlike chlorinated hydrocarbon insecticides -do not po:se a serious residue problem. The acute toxicity of organophosphates for mammals and probably also for insects is mainly due to the blocking of cholinesterase enzymes which are in part essential for normal neural transmission. The mechanism of this blocking is pho,~phorylation of a serine hydroxyl group in the active ~;ite of the enzyme (see refs. z9, 2o). The main primary routes of metabolism of organophosphates are oxidation and hydrolysis (see refs. 3, I9, 2o, 28, 29). Mammalian liver and insects convert thiophosphates oxidatively to phosphates, a conversion that enhances the phosphorylating activity of the corresponding compounds. Enzymic hydrolysis, on the other hand, is a detoxication which is due to several enzymes pre:ent in lnammalian liver and other organs. Insect esterases are also known. Methyl esters such as dichlorvos can react with glutathione under transmethylation 24. Several studies on dichlorvos indicate that the rate of metabolic degradation is rapid in mammals (for review see ref. 24). In a recent study by t~LAIR et 6tl. on the distribution of dichlorvos in manmmlian tissues ", a half-life of dichlorvos in male rat kidney of 13. 5 rain is reported. Measurable amounts of dichlorvos in rat kidneys were found only when the animals were treated with atmospheres containing IO fig dichlorvos per litre. This is 250 times the practical household concentration of the insecticide. On the other hand, tissues of rats exposed for 14 days to atmospheres con-

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

INSECTICIDES

137

taining o.5 and o.o5/~g dichlorvos per 1 contained no detectable dichlorvos, the same being true for blood of two humans who were exposed to o.25 and o.7/~g/1. The technique allowed detection of o.oi/~g dichlorvos per g tissue (4.5" Io-Smol/kg) except for blood where only o.I/~g/g (4.5" Io-7mol/kg) was detected. E F F E C T S ON D N A

Dichlorvos is the only organophosphorus insecticide whose alkylation reaction with nucleic acids has been investigated. In i97o L6FROTH reported the occurrence of the alkylation product 7-methylguanine in hydrolysates from calf thymus DNA treated with dichlorvos in vitro a3. More detailed results from the same group and from L A W L E Y et al. were published recently32, 47. LAWLEY et al. a2 studied the reaction of methyl taC-labelled dichlorvos with isolated salmon sperm DNA (I 4 m M dichlorvos) and E. coli DNA (7.4 m M dichlorvos). The extents of DNA methylation for a 1-h treatment were similar for both DNAs. The corresponding value for MMS and salmon sperm DNA was about I5 times higher. Between 63 and 85% of the total DNA-methylation products was 7-methylguanine; in addition I-methyladenine, 3-methyladenine, 3-methylguanine, 6-O-methylguanine and 3-methylcytosine were identified. The pattern of products was similar to the pattern found with methylating agents such as MMS which react by a SN2 mechanism. After treatment of E. coli cells with radioactive dichlorvos (2. 9 mM) the extent of DNA methylation was of the same order as in isolated DNA, but the analysis of the methylation products revealed 7-methylguanine as the sole product (93% of total known DNA methylation products). 3-Methyladenine was practically absent, in contrast with the experiments with isolated DNA where it contributed between 9 and i 4 % to the alkylation products. This difference could originate from elimination of 3-methyladenine by repair enzymes. 7-Methylguanine as major alkylation product in E. coli DNA is confirmed by WENNERBERG AND LOFROTH47. It has also been detected in DNA obtained from cultured human H e L a cells treated with 0.97 m M dichlorvos a2. Dichlorvos can also react, in cells, with proteins, through methylation and phosphorylation. The resulting radioactivity in the protein from treated E. eoli cells was 20-3 ° times higher than in nucleic acids3~, aT. In comparison, MMS labelled DNA and protein to about the same extent '~2. These data make it clear that dichlorvos reacts in cells preferentially with proteins. This reaction is only partly due to methylalion of protein, protein phosphorylation probably contributing as well. The Ielation between these components is unknown. Experiments aiming at a detection of methylated nucleic acid components in dichlorvos-treated mice were described by WENNERBERG and LOFROTH4~. Mice that received methyl ~4C-labelled dichlorvos by i.p. injection (19-39 mg/kg) or by inhalation (5 mg/kg) excreted labelled 7-methylguanine in the urine. This was explained by a direct methylation of guanine (label in the methyl group), but it was unclear whether tiffs reaction occurred on guanine in nucleic acids or on free guanine. A possible different explanation with involvement of the I-carbon pool and labelling ot normally occurring 7-methylguanine in the purine ring could not be eliminated. In a study on rats that received about 62 mg dichlorvos per kg intraperitoneally, the same authors measured the rate of urinary excretion of labelled 7-methylguanine and 3methyladenine 3~. The preliminary results, because of the indirect experimental ap-

138

D. WILD

proach, do not prove methylation of nucleic acids in mammals. Nucleic acids per se were not investigated in this study. In summary, dichlorvos can methylate DNA i~ vitro as well as in living bacterial and mammalian cells. On the basis of the methylation product pattern dichlorvos is considered as an S~2 alkylating agent. Compared with MMS, the reactivity of dichlorvos towards nucleic acids is lower, towards proteins of the same order. The DNA and protein-alkylating ability of other organophosphates is certainly affected by the chemical nature of the ester groups, but in view of the lack of data for more compounds and of the general chemical similarities between organophosphates the pertinent dichlorvos results deserve careful consideration. In addition to alkylation, DNA-strand breakage by dichlorvos was studiedT, 2s. These studies show that dichlorvos (3 13 mM)--like MMS induces breaks in E. coli DNA which can be rapidly repaired by DNA polymerase I. Higher dichlorvos concentrations (13 26 raM) produced non-random disintegration of DNA molecules rather than random strand breakage 2~. This type of damage was also observed after treatment of E. coli cells with iodoacetamide, an alkylating agent which, however, does not measurably alkylate DNA. This lack of correlation between DNA-alkylating and strand-breaking effects of iodoaeetamide suggested the existence of indirect mechanisms for the production of the observed DNA damage, e.g, via protein alkylation. These indirect mechanisms could also operate with dichlorvos, and this suggestion agrees with the observation that the ability of dichlorvos to produce either strand breaks repairable with DNA polymerase I or that DNA disintegration did not correlate with the ability to produce gene mutations 2s. M U T A G E N I C I T Y TESTS

Tile results of mutagenicity tests are classified according to the test organism. For general descriptions of test organisms and systems the reader is referred to the literature 27,~. All publications to be discussed in the following section and that report mutagenicity tests with individual compounds are summarized in Table II. Microorganisms in vilro Dichlorvos. Because of the wide exposure of hmnans to atmospheres containing dichlorvos, this compound with regard to mutagenicity is the most thoroughly investigated of all compounds under discussion. The first indication of mutagenic effects of diehlorvos came from preliminary experiments on induction of streptomycin-independent mutant cells in the streptomycin-dependent Sd-4 strain of E. coli ~4. Subsequently, with the tryptophan-requiring WP2 strain of E. toll, a positive effect 2 and no effect 9 of dichlorvos on reversion frequency were found in qualitative agar plate tests. Variable results with the very same test in a third group 7 made the agar plate test procedure with this strain appear inappropriate for dichlorvos. The same is probably true for other organophosphate insecticides. When WP2 cells in suspension rather than on agar plates were treated with dichlorvos (13 m M , I 3 h) a mutagenic effect was clearly observed 7. In addition, several repair-deficient strains derived from WP2 were studied, from the low mutability of recA or exrA strains it was concluded that the dichlorvos-induced mutations resulted from misrepair of induced lesions by the error-prone recA +/exrA + repair system ~.

MUTAGENICITY STUDIES ON ORGANOPHOSPHORUS INSECTICIDES

139

TABLE II SURVEY OF LITERATURE ON NBPALKYLATION AND ON MUTATIONINDUCTION BY OROANOPHOSPHORUS INSECTICIDESAND SOME OF THEIR METABOLITES An asterisk* indicates unpublished results of the Zentrallaboratorium ft~r Mutagenit/itsprflfung Compound

NBP test

Tests for mutation induction in Microorganisms Drosophila in vitro in vivo

Bidrin@ Bromophos Diazinon@ Dichlorvos

3 21

9, 17, 18, 38, *

Dilnethoate Fenitrothion Malathion@ Methylparathion Oxydemetonmethyl Parathion Trichlorfon Dilnethylphosphate, ammonium salt Diethylphosphate, ammonium salt Dilnethylphosphothioate, ammonium salt Diethylphosphothioate, ammonium salt Methylphosphate, diammoniuln salt Ethylphosphate, diamlnoniuln salt p-Nitrophenol

21

3,21

Mammalian cells in vitro in vivo

4 2, 18, 38, * 2, 7, 9, 13, 14, 17, 34, 37, 38 , 46 , 48 2, 17, [8, 38, *

8,I3

IO,*

49 II, 12, 16

4 3,39 3, 21, 39 39 3, 21, 39 3

2, 9, 18, 38, * 18, 38, * 17, I 8, 3 8 , * 18, 38, *

43, 50 43 44 4

43, 50

* * * * * * 18, *

8

D e f i n i t e m u t a g e n i c i t y a n d clear r e l a t i o n s h i p s b e t w e e n d i c h l o r v o s c o n c e n t r a t i o n , e x p o s u r e t i m e a n d t h e m u t a g e n i c effect w e r e s h o w n in t w o o t h e r g e n e t i c t e s t s in E . coli: i n d u c t i o n of s t r e p t o m y c i n r e s i s t a n c e (5-25 m M d i c h l o r v o s , i - I O h) (48) a n d of 5 - m e t h y l t r y p t o p h a n r e s i s t a n c e ( 0 . 3 3 . 2 m M d i e h l o r v o s , 0 . 5 - 6 h) 38. T h e c o m p a r i s o n of t h e t w o s y s t e m s i l l u s t r a t e s t h a t a low dose no l o n g e r p r o d u c i n g a d e t e c t a b l e m u t a g e n i e effect in one s y s t e m m a y well p r o d u c e an effect in a d i f f e r e n t s y s t e m a n d t h a t " n o - e f f e c t d o s e s " for m u t a g e n i c i t y can b y no m e a n s be d e r i v e d f r o m l a c k of a d e t e c t a b l e m u t a g e n i c effect in a specific t e s t s y s t e m . O t h e r b a c t e r i a l species also r e s p o n d e d t o d i c h l o r v o s : r e v e r s i o n s w e r e f o u n d in Serratia marcescens w i t h t h e a g a r p l a t e t e s t (9) a n d in Salmonella t y p h i m u r i u m 14. I n s e v e r a l b a c t e r i a l species, i n c l u d i n g E . coli, S. t y p h i m u r i u m a n d Klebsiella p n e u m o n i a e , t h e m u t a t i o n r a t e s to s t r e p t o m y c i n r e s i s t a n c e w e r e i n c r e a s e d 46. I n t h e D 4 s t r a i n of t h e y e a s t Saccharomyces cerevisiae a d o s e - d e p e n d e n t i n c r e a s e of m i t o t i c g e n e c o n v e r s i o n b y 5 - 4 ° m M d i c h l o r v o s (5 h) was demonstrated13,1L I n v i e w of t h e d i c h l o r v o s doses r e q u i r e d in t h e m i c r o b i a l t e s t s m e n t i o n e d to d e t e c t m u t a g e n i c effects it is n o t s u r p r i s i n g t h a t n o ad- 3 m u t a n t s w e r e f o u n d in Neurospora crassa 37 a f t e r e x p o s u r e to an a t m o s p h e r e c o n t a i n i n g d i c h l o r v o s : t h e dose for t h e cells in t h e s e e x p e r i m e n t s was p r o b a b l y o r d e r s of m a g n i t u d e l o w e r t h a n t h e doses in t h e t e s t s m e n t i o n e d on b a c t e r i a a n d y e a s t . E v e n t h o u g h t h e q u a n t i t a t i v e r e l a t i o n b e t w e e n m u t a g e n e s i s a n d a l k y l a t i o n of

I40

D. WILD

specific sites in DNA is not yet known, it is instructive to note that the doses used in the microbial mutation studies are of the same order as those used in the DNA alkylation studiesa2,4L LAWLEY el al. computed from their data that a dose of the order of 3o m M dichlorvos for I h would produce about I4OO methylations in the E. coli W P i genome a'a. If one compares the dose-effect curves for dichlorvos in E. coli and yeast with those for MMS, another methylating nmtagen, one finds dichlorvos concentrations producing the same nmtagenic effect as MMS about 2o times higher for streptomycin resistance in E. eoli% Io times higher for gene conversion in yeast 17 and about ioo times higher for 5-methyltryptophan resistance in E. coli as. The first two of these values appear to correlate with the 15 times higher DNA-methylating activity of MMS versus dichlorvos '~. Technical dichlorvos contains up to 0.8% trimethylphosphate, which is mutagenic in bacteria% 1<46 and, at very high doses, in micel'a,15,16. On a quantitative molar basis the mutagenicity of trimethylphosphate in bacteria, according to available information, is roughly comparable with or lower than that observed for dichlorvos. Therefore a significant mutagenic contribution of the trimethylpho:@mte in technical dichlorvos is not likely in the microbial experiments. Other insecticides. The 3 insecticides dimetboate, bidrin and oxydemetonmethyl were mutagenic in tests on E. eoli (induction of 5-methyltryptophan resistance a~ and of streptomycin resistance (WILD unpubh)) and on Saceharomyces eerevisiae (induction of mitotic gene conversion~7,~8). Streptomycin resistance and gene conversion were induced at concentrations of 3o 3o0 m M (4 and 5 h respectively), and 5-methyltryptophan resistance was detected with concentrations about one order of magnitude lower. Methylparathion exhibited low mutagenic activity in the 5methyltryptophan-resistance system in E. coli in IO mM emulsion (4 h) 3~ and convertogenic activity in yeast in I I 4 mM emulsion (3o h) TM, whereas o.3 m M solutions were ineffective in the streptomycin-resistance system (WILD unpubh). In contrast with the results mentioned, dimethoate 'a and bidrin ~ appeared non-mutagenic in the agar plate test with E. coli WP2. This and another negative result with bidrin in plate tests on Serratia marcesce~zs 9 probably result from the low sensitivity of plate tests as already observed with dichlorvosL Diazinon, malathion and parathion, in all tests on bacteria and yeast, had no detectable genetic effects2,9,~8,a~ (Wild unpubl.). Looking for correlations between structure and mutagenicity of these seven organophosphates and dichlorw)s, one finds that all methyl esters tested, except malathion, were mutagenic for microbes at high concentrations. Among these, dichlorvos was the most effective, followed by the about equally effective bidrin, dimethoate and oxydemetonmethyl. The lowest activity was exhibited by methylparathion. Kinetic results of NBP tests with dichlorvosa,% bidrin a, dimethoate% methylparathiona, 21 and malathion a parallel the differential mutagenicity of these compounds. The relatively low observed mutagenicity of methylparathion compared with bidrin and dimethoate, according to the NBP data, could be an underestimate owing to the use of emulsions rather than solutions in the mutation tests. Neither of the ethyl esters tested (diazinon and parathion) was mutagenic in the microbial tests but parathion was a feeble alkylating agent in a NBP test ag. Metabolites. Several mono- and di-alkyl esters of phosphoric and thiophosphoric acid, products of the partial hydrolysis of organophosphate insecticides (see

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

14I

Table i), were tested in vitro in E. coli and yeast. They did not show genetic activity at concentrations up to IOO m M (FAHItlG, MOHN, WILD unpubl.), and this is consistent with the lack of alkylating activity of dimethylphosphate in the NBP test 3. p-Nitrophenol, a metabolite of parathion and methylparathion, was not genetically active in E. coli (MoHN, WILD unpubl.) but induced mitotic gene conversion in yeast (2I m M , 3 ° h) ~8. A weak effect of p-nitrophenol on Vicia faba root tips has been described earlier 1. Microorganisms in vivo (host-mediated assay) Dichlorvos has been subjected to traditional host-mediated assays with shortterm culture of the indicator microorganisms in the peritoneal cavity of treated ,nice. In the histidine-requiring Salmonella typhimurium strain G46 and with a subcutaneous dichlorvos injection of 25 mg/kg, no increased revertant frequency was found 8. However, the significance of this finding is questionable because the same strain was not reverted measurably by dichlorvos in a parallel plate test in vitro 8. Similarly, a negative result was obtained with the same dose and the Serratia marcescens strain a2r leu- (ref. 8). DEAN et al. 13 reported host-mediated assay studies with dichlorvos using the D4 strain of Saccharomvces cerevisiae. Mice received dichlorvos by inhalation in atmospheres containing 60 or 99/~g dichlorvos per litre or orally (50 or IOO mg/kg). None of these doses induced mitotic gene conversion in yeast cells kept for 5 h in the treated animals. The highest dose used in these experiments, IOO mg/kg, corresponds to 0.45 mM/kg. This dose is high in terms of mammalian toxicity and human exposure, but on the other hand it corresponds formally (under the assumption of uniform distribution in the mouse) to 1/4o of the lowest concentration that produced, in parallel experiments in vitro, a significant (P < o.oi) genetic effect. Therefore, even if dichlorvos were not metabolically altered and thereby inactivated in the mouse, at the doses used in these host-mediated assays, it had very likely not produced a measurable genetic effect in the yeast system. In other words, the negative result of the host-mediated assay is not in conflict with the positive results in vitro, and its explanation does not require the rapid metabolism of diehlorvos in the mamnlal. Certainly, rapid metabolism will greatly reduce a potential dichlorvos effect. p-Nitrophenol which, in vitro, had no effect in E. coli but induced gene conversion in yeast was without effect on the reversion in mice of Salmonella typhimurium G46 and of Serratia marcescens a2I at a dose of 75 mg/kg (ref. 8). Drosophila Obviously, an insect is not an ideal test organism for mutagenicity testing of insecticides. Consequently only few Drosophila tests have been performed. Injection of sub-lethal amounts of bromophos, fenitrothion and trichlorfon and application of the Muller-5 or Basc technique did not show an increase in the frequency of sex-linked recessive lethal mutations 4. In the same test a negative result was also obtained with oxydemetonmethyl, when normal insecticide-sensitive Drosophila males were fed with solutions of the compound 44. However, when males of the insecticide-resistant Hikone-R strain were used, a 35 times more concentrated solution could be given. In these experiments a small increase (P < o.o5) of sex-linked recessive lethal mutations was detected ~*. It therefore appears possible that the resis-

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tanee mechanism protects the Hikone-R flies from the lethal but not from the mutagenic effect of the test compound. M a m m a l i a n cells in cullure Human phytohaemagglutinin-stimulated lymphocyte cultures were treated with dichlorvos at concentrations up to 4 °/~g/ml (o.18 raM). The cultures were terminated at various times after treatment, and for each of 3 different blood donors and each individual dichlorvos-concentration 200 400 mitotic cells analysed for chromosomal abnormalities TM. Only toxic effects but no increase of ehromatid gaps and chromatid breaks were observed. Another study with the same test confirmed the lack of a chromosome-breaking effect of diehlorvos in cultured human lymphocytes (ScHoELLER unpubl.). No induction of 8-azaguanine-resistant mutations were detected in cultured V79 Chinese hamster cells that had been treated in serum-containing culture medium for 2 h with up to 1 mM dichlorvos (WILD unpubl.). Cultured mammalian cells are obviously more sensitive to the toxic action of dichlorvos than are microbes. The maximally tolerated doses are, therefore, low compared with the microbial tests and, in addition, the protein-rich culture media for mammalian cells enhance the degradation of dichlorvos 6. Nevertheless, in dichlorvos-treated HeLa cells, DNA alkylation has been demonstrated a2. Mammalian, cells in vivo Studies on experimental animals. Specifically dichlorvos was tested in several dominant lethal and cytogenetic tests on bone marrow and testes of rodents. No significant effect was noted in dominant lethal tests with mice that received diehlorvos orally (5" IO mg/kg) or by intraperitoneal injection (13 and 16.5 mg/kg) 16. In another study 12 male mice were exposed to dichlorvos atmospheres (30 and 55 /~g/l, 16 h or 2.1 and 5.8/~g/l, 23 h per day for 4 weeks) and, after treatment, mated weekly with untreated females for 8 weeks. (In comparison, the initial concentration of dichlorvos in rooms containing dichlorvos-impregnated strips was in the order of 0,04/~g/1, ref. II.) None of these doses produced a consistent increase of early foetal deaths over controls, whereas with trimethyl phosphate (IOOO mg/kg i.p.) and with ethyl methanesulphonate (200 mg/kg i.p.) significant effects were observed. In cytogenetic studies, bone-marrow and testis preparations of treated mice and Chinese hamsters were analysed n. Mice were exposed by inhalation (72 /~g/l, 16 h, or 5 fig/l, 23 h per day for 21 days), and Chinese hamsters were exposed either per os (I. 15 or 2. IO mg/kg) or also by inhalation (32/~g/1, 16 h). After exposure and an intraperitoneal injection of colcemid the animals were killed, and the testes and femurs used for chromosome preparations. A total of 4800 bone-marrow cells from all treated mice and 2400 cells from control mice were evaluated, the corresponding numbels for Ctfinese hamsters being 1743 and 12oo cells. In different groups of treated mice the frequencies of cells with ehromatid aberrations including gaps ranged between o.17 and 1.25% : these values were not significantly different from the spontaneous frequencies (0.67% in male, 0.58% in female mice). In the experiments with Chinese hamsters similar values were obtained and no effect of the dichlorvos treatment was observed. The analysis of meiotic (diakinesis/metaphase I) chromosomes from dichlorvos-

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treated mice did not reveal any effect of the treatment, namely cells with univalents or aberrations. This result, however, is of doubttul relevance in view of the equally negative result in the positive control group. This group consisted of endoxan-treated mice (200 mg/kg i.p.) killed 24 h after the endoxan injection. This short time between chemical treatment and preparation of testes may explain the absence of univaleuts and the apparent contradiction of findings by GCHLEIERMACHER42 with the same endoxan dose. (This author found increased univalent frequency 2 or more days after endoxan or trenimon treatment.) But the value of univalents as indicators of mutagenic effects is limited by the high variability of their spontaneous occurrence 42. Chromosomal aberrations can, in principle, be detected in diakinesis-metaphase I chromosomes, but "according to the available results with the powerful chemical mutagens trenimon and cytoxan we must assume that mutation experiments with alkylating agents will produce relatively weak cytogenetic effects in diakinesis and metaphase I" (SCHLEIERMACHER42).The data of DEAN AND THORPE agree with this statement: no aberrations were seen in cells from cytoxan- (endoxan-) or dichlorvos-treated and control animals. This result, therefore, does not allow a definite conclusion about the potential mutagenic effects of dichlorvos. Another finding--though not genetical--on a dichlorvos effect on mouse testis must be reported : after a single oral dichlorvos dose of 4 ° mg/kg and histological preparation of testes at intervals of 9 days up to the 63rd day from treatment, serious reduction of the germinal epithelium in many tubuli was noted 30. In view of the negative results in the dominant lethal tests, this result cannot be explained in terms of gross cytogenetic aberrations. An indirect and possibly unspecific mechanism should therefore be considered. Studies on exposed humans. Despite the additional difficulties of mutagenicity tests in humans as compared with experimental animals, results of two studies on persons with high occupational exposure 5° or acute intoxication 43 have been recently published. In both studies chromosomal aberrations in blood cultures from the test persons served as indicators for mutagenic effects. The first of these studies 5° comprised 16 male insecticide applicators with variable and mixed exposure, mainly to organophosphorus insecticides such as trichlorfon, malathion, diazinon and others. Two blood samples were taken from each person, a first during winter (no recent exposure) and a second in the spray season. Cultures were incubated for 48 h at 38 °. Only 25 metaphases were scored per culture. The investigators observed a mean chromatid-break frequency of 6.2% in the spray season, this value being 5-fold increased over the winter result and over the results with a control group not exposed (1.2%). This increase, however, without additional studies could not be linked causally with potentially mutagenic insecticides. As result of this study no specific insecticide can be suspected as mutagen. Confirmation and clarification of the effect by matching animal experiments is not feasible in the absence of information on the insecticides and doses to which the individuals were specifically exposed. The other study 43 was done on 31 persons with acute intoxication and 15 healthy control persons. All intoxications were caused by a single insecticide, mostly malathion (14 patients), methylparathion (5 patients) or trichlorfon (5 patients), a few by dimethoate, dichlorvos or diazinon. Doses were not known. The intoxications ranged between mild and lethal. Three blood samples were taken from most patients: a few days after exposure and I and 6 months later. After a 48-h culture period, IOO meta-

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phases were analysed per sample. Chromatid and isochromatid aberrations (gaps, breaks, and interchanges) and cells with unstable chromosome aberrations (acentric fragments, rings and dicentrics) were recorded. In addition, stable chromosome aberrations (deletions and translocations) were evaluated in IO randomly selected and karyotyped metaphases from each sample. Detailed consideration of the data with regard to sampling time, degree of intoxication, and type of insecticide led to the following results. (I) Immediately after intoxication, total chromatid breaks were increased 8fold compared with the healthy control (o.57 vs o.o7°/~,), unstable chromosome aberrations were increased 3-fold (I vs o.35%) and stable chromosome aberrations 5-fold (17.8 vs 3.3~/0)- After one month the increase of breaks was still 7-fold (o.48%) and of stable chromosome aberrations 6-fold (22.o°/~,). After 6 months all values had approached the controls. (2) A dose effect-like relationship between the severity of the intoxication and chromosomal damage was not observed. (3) Classification of the data according to individual insecticides indicated a highly significant increase of chromatid and isochromatid aberrations and unstable and stable chromosome aberrations in the malathion group immediately after exposure. In the methylparathion group no immediate change was found, but stable chromosome aberrations appeared one month later. A similar pattern was also obtained in the trichlorfon group. The dimethoate, dichlorvos and diazinon groups were to() small for an insecticide-specific analysis. The observed correlation suggests the possibility that chromosome mutations are induced in man by highly toxic doses of organophosphorus insecticides, specifically of malathion. However it is difficult in this kind of study on humans to exclude the influence of variables other than the agent studied. The high increase in chromarid-type aberrations persisting for a least one month after cessation of the exposure is difficult to explain for theoretical reasons and on the basis of experience with patients treated with ionizing radiations and clastogenic chemicals (ScHINZEL AND SCHMID, personal communication). Therefore, comparative studies on experimental animals appear desirable to confirm the effect. SUMMARY AND DISCUSSION

From a comparison of the data surveyed several points for a preliminary estimation of the potential genetic risk presented by organophosphorus insecticides can be derived. Organophosphorus insecticides are chemical alkylating agents. This property and its dependence on the individual chemical structure is revealed by the NBPtest 3,21,3~. DNA alkylation studies have been conducted with dichlorvos only; they prove alkylation of DNA bases in E . coli and HeLa cells at concentrations in the order of Io-3M (refs. 32, 33, 47). Despite attempts, DNA alkylation i n vivo has not been shown unequivocally35, 47. Induction of gene mutations in microorganisms i n vitro has been unequivocally detected with bidrini7,1s, aS, dichlorvos2,7,%13,1~,17.3%46, 48 dimethoate17,18, 38, methylparathioniS, a8 and oxydemetonmethyliT,18, 38 at IO-3 M or higher concentrations. The comparison of dose-effect relationships observed in several microbial systems sug-

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gests that the lack of detectable mutagenic effects at lower concentrations is due to a sensitivity threshold of the individual test systems, not to the existence of a principal no-effect concentration. The primary step in the production of microbial mutants is certainly alkylation of DNA bases. Misrepair of the primary lesions seems to be an additional requirement, at least for dichlorvos-induced mutants in E. coliL A different, indirect mechanism--possibly via protein alkylation--is probably involved in the production by dichlorvos of another DNA damage: DNA disintegration 25. In a relatively insecticide-resistant drosophila strain, oxydemetonmethyl seems to be a weak mutagen, whereas a lower dose in a normal strain was without effect 44. Mutagenieity experiments with mammalian cells and experimental mammals have been done exclusively with dichlorvos, although more compounds were covered in the studies on exposed humans. In mammalian cell cultures and mammalian organisms, dichlorvos doses as high as in the microbial tests are not compatible with survival of the organisms and are, therefore, not practicable in mutagenicity tests. Furthermore, the applicable doses, because of rapid degradation, do not lead to equivalent dichlorvos concentrations in tissues or cells ~. These factors, high toxicity and rapid metabolic degradation to non-alkylating compounds, are obviously the basis of the failure to detect mutagenic effects of dichlorvos in all tests using cultured mammalian cells or experimental mammals: in vitro, chromosome aberrations in human l y m p h o c y t e s 1° (ScHOELLER unpubl.) and mutations to 8-azaguanine resistance in Chinese hamster cells were studied (WILDunpubl.), the negative tests in vivo including tests on microbial indicator cells in the host mediated assayS, TM and on mammalian somatic ~x and germ cells ~2,~6. Several types of genetic effect are screened in these tests: gene mutations and gene conversion, structural chromosome mutations, dominant lethal mutations. A comparison of dichlorvos concentrations in the inhalation experiments with the maximal concentrations in normal household conditions to which humans are exposed, shows that the lowest experimental concentration (2.I/2g/l), used in the dominant lethal test for 4 weeks, exceeded the maximal household concentration by a factor of 52. For the highest concentration (99/,g/1), used in the host-mediated assay for 5 h, this factor was 2400. In similarly treated mice, dichlorvos concentrations in several tissues were determined and found roughly between IO-s mol/kg and 7" lO-6 mol/kg (ref. 6). However, these negative results in mammalian mutation tests with high doses and various routes of application should also be discussed with regard to the sensitivity of the test methods. The low sensitivity of the cytogenetic test on meiotic chromosomes 11 has already been mentioned in the section mammalian cells in vivo. The negative result with diehlorvos in one of the dominant lethal tests on mice 16 must be viewed together with an equally negative result with the potent mutagen dimethylnitrosamine in the same laboratory ~s (in contrast with positive results in another laboratory4°), and finally the use of positive controls in several other tests does not really help to clarify the sensitivity of the respective tests, because these positive controls were conducted with one high dose of a potent mutagen and thus do not indicate the type of dose-effect relation, in other words at what lower dose the mutagen would have remained undetected. An assessment of the potential safety of dichlorvos with regard to genetic hazard should therefore not be made exclusively on the basis of the negative mammalian

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tests. It appears that for this assessment the available data on DNA alkylation and on mutagenesis in microbes as well as on mammalian metabolism should be adequately considered. This would obviously require extrapolation of alkylation and nmtagenesis from high experimental to extremely low mammalian concentrations i~z rivo. Such extrapolation over several orders of magnitude is at present affected by numerous uncertainties, but the potential genetic risk of dichlorvos would thus be estimated on the basis of all available relevant information and this would be superior to an estimate which is based only on absence of detectable effects in mammalian tests. Whereas the results with dichlorvos in all genetic tests are consistent with each other and with those obtained with the other insecticides tested, the results of the studies on exposed humans ~3,50 are comparatively unexpected. But one has to realize that the two sets of data cannot be compared strictly because only dichlorvos has been studied in mammals, whereas the persons studied were exposed exclusively or mainly to insecticides other than dichlorvos. The earlier mentioned difficulties in the interpretation of this kind of result should also be considered. With regard to the report on chromosome aberrations in malathion-intoxicated humans 4a it appears interesting to note that the toxicity of malathion for mammals is markedly low with an LDa0 in rats per os of 12oo mg/kg, compared with dichlorvos with a corresponding LDs0 of 62 mg/kg (refs. 19, 2o). It could therefore be that the malathion intoxicated patients had incorporated the highest doses of insecticide in the entire patient group. The relatively low toxicity of malathion may be connected with its metabolism: malathion in mammals is converted by hydrolysis of a carbethoxy group mainly to "malathion acid" which is only a weak cholinesterase inhibitor ~,'-'°. Malathi(m acid is still a triester of phosphoiic acid and as such a potential alkylating agent ; it has per se not been tested for mutagenicity. Mutation tests with malathion and malathion acid in nmmmals are, therefore, important for a future judgement of malathion. With the insecticides bidrin, bromophos, diazinon, dimethoate, fenitrothion, methylparathion, oxydemetonmethyl, parathion, and trichlorfon in addition to the microbial or Drosophila tests, no tests on experimental mammals have been performed. Therefore, the potential mutagenicity of these compounds for humans cannot be judged. But it is possible that by analogy with dichlorvos even those compounds which are nmtagenic in microorganisms would not produce detectable genetic effects in mammals. This should be investigated. The microbial mutation tests with all mono- and di-esters of phosphoric acid were negative. Since all these organophosphate metabolites have, by analogy with dimethylphosphate a, probably no alkylating potential, according to present knowledge they do not constitute a genetic hazard. The data on p-nitrophenol, another metabolite of some organophosphates, are not sufficient for a preliminary analysis. CONCLUSION

At the end of this review it appears convincing to the author that at least dichlorvos, under conditions of normal use as a insecticide, most likely can not exert significant mutagenic effects in mammals including man. (A different judgement is required for different applications of dichlorvos, for instance as an anthelminthic!) This view is based mainly on the mutagenicity for microbes and DNA alkylating property of

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dichlorvos at high c o n c e n t r a t i o n s a n d on the results from a n a l y t i c a l dichlorvos det e r m i n a t i o n s in tissues of t r e a t e d m a m m a l s . Tissue c o n c e n t r a t i o n s in m a m m a l s are lower t h a n i o - a - i o -5 times the lowest concentrations m u t a g e n i c for microbes i n v i t r o ! B u t it appears nevertheless desirable to define in a q u a n t i t a t i v e w a y the extremely low a n d perhaps negligible m u t a g e n i c risk from this compound. Such an estimate, in the ongoing public discussion on the health hazard from this insecticide such estimate could help to reconcile diverging opinions a n d to assess the safety of dichlorvos at least from the genetical viewpoint. ACKNOWLEDGEMENTS

The a u t h o r t h a n k s his coleagues Dr. R. Fahrig, Dr. G. Mohn a n d Dr. L. Schoeller for u n p u b l i s h e d d a t a a n d Professor W. Schmid a n d Professor G. Z b i n d e n for critical reading of the m a n u s c r i p t . The help of the E n v i r o n m e n t a l Mutagen I n f o r m a t i o n Center (EMIC) in the search of the literature is gratefully acknowledged. The work of the a u t h o r was supported b y the Deutsche Forschungsgemeinschaft. ADDENDUM

Quite recently several u n p u b l i s h e d papers a n d c o m m e n t s relating to the safety of dichlorvos were s u b m i t t e d to the U n i t e d States Protection Agency. A m o n g these, a paper s u b m i t t e d b y the Shell Chemical C o m p a n y is of special interest for this review, since it reports the a t t e m p t of a direct detection of 7 - m e t h y l a t e d g u a n i n e in nucleic acids of dichlorvos-treated rats. These animals inhaled m e t h y l 14C-labelled dichlorvos at a household use c o n c e n t r a t i o n of 0.064 /~g/1 for 12 h. S u b s e q u e n t isolation a n d analysis of nucleic acids revealed no labelled 7 - m e t h y l g u a n i n e in R N A or D N A isolated from several pooled organs (lung, heart, liver, kidney a n d others). Concomit a n t l y "little or n o " labelled u r i n a r y 7 - m e t h y l g u a n i n e was found. These results suggest that, at the applied n o r m a l use concentration, dichlorvos did not m e a s u r a b l y alkylate nucleic acids. Thus t h e y do n o t support the conclusions d r a w n b y LOEROTH AND WENNERUERG from their indirect studyaS, 47 using high doses of dichlorvos. However for a safety assessment it appears i m p o r t a n t to know whether similar results would also be o b t a i n e d after higher doses of dichlorvos. REFERENCES

i AMER,S. M. ANDJ~. M. ALl, Cytological effects of pesticides. IV. Mitotic effects of some phenols, Cytologia, 34 (1969) 533-54 o. 2 ASHWOOD-SMITH,M. J., J. TREVlNO AND R. RING, Mutagenicity of dichlorvos, Nature, 24o (1972) 418-42o. 3 BEDFORD,C. T. AND J. ROBINSOX,The alkylating properties of organophosphates, Xenobiotica, 2 (1972) 3o7 337. 4 BENRS, V. AND R. SRAM, Mutagenic activity of some pesticides in Drosophila melanogaster, Industrial 3,led., 38 (I969) 5o-53. 5 BENES, V., R. SRAM AND R. TUSCANY,Testing of mutagenicity of Fenitrothione, ~V[utation Res., 2i (I973) 23-24. 6 BLAIR, D., E. C. HOADLEY,AND D. H. HUTSON,The distribution of dichlorvos in the tissues of mammals after its inhalation or intravenous administration, Toxicol. Appl. Pharmacol., 31 (1975) 243-253. 7 BRIDGES,B. A., R. P. MOTTERSHEAD,M. H. L. GREEN AND W. J. H. GRAY, Mutagenicity ot dichlorvos and methyl methanesulphonate for Escherichia coli WP2 and some derivatives deficient in DNA repair, Mutation Res., 19 (1973) 295 3o3 .

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8 BUSELMAIER, W., 0. ROHRBORN UND P. PROPPING, M u t a g e n i t g t s u n t e r s u c h u n g e n mit Pestiziden im h o s t - m e d i a t e d assay u n d mit dem D o m i n a n t e n Letaltest an der Maus, Biologisches Zentralblatt, 91 (1972) 311 323 . 9 DEAN, B. J., The m u t a g e n i c effects of o r g a n o p h o s p h o r u s pesticides on nlicroorganisms, Arch. Toxikol., 3 ° (19721 67 74. io DEAN, B. J., The effect of Dichlorvos oi1 cultured h u n l a n lymphocytes, Arch. Toxikol., 3o (19721 75-85. I I DEAN, B.J. AND E. THORPE, Cytogenetic studies with dichlorvos in mice and Chinese hamsters, Arch. Toxihol., 3° (1972) 39 49. 12 I)I';AN, B. J. AND E. THORPE, Studies with dichlorvos v a p o u r in d o m i n a n t lethal m u t a t i o n tests on mice, Arch. Toxikol., 3 ° (19721 51 59. 13 DEAN, B. J., s. M. A. I)OAK AND J. FI'NNELL, Genetic studies with dichlorvos in the hostmediated assay and ii1 liquid m e d i u m using Saccharomyces cerevisiae, Arch. Toxikol., 3° (1972) 61 66. 14 DYER, I(. F. AND P. J. HANNA, C o m p a r a t i v e mutageuic activity and toxicity of triethylphosp h a t e and dichlorvos in bacteria and Drosophila, Mutation Res., 21 (1973) 175-I77. 15 EPSTEIN, S. S., Vv'. BASS, E. ARNOLD AND Y. BISHOP, Mutagenicity of t r i m e t h y l p h o s p h a t e in mice, Science, i68 (197 o) 584-586. 16 EPSTEIN, S. S., E. ARNOLD, J. ANDREA, ~vV.BASS AND Y. BISHOP, Detection of chemical m u t a g e n s b v the d o m i n a n t lethal assay in the mouse, Toxicol. dppl. Pharmacol., 23 (1972) 288-325. [7 F'AHRIC;, R., Nachweis einer genetischen W i r k u n g von O r g a n o p h o s p h o r Insektiziden, Nali*rwissenschafien, 60 (I973) 5 °. 5 I. I8 FAHRIG, JR., Mutagenicity studies with pesticides, in Che~nical Carcinogenesis Essays, I A R C Scientific Publications, No. IO, I974. I9 FEST, C. AND N. J. SCHMIDT, Insektizide PhosphorsXureester, Book c h a p t e r in R. WEGLER (Ed.) Chemie der Pflanzenschutz- und Schddlingsbekiimpfungsmittel, Springer, Berlin 197 o. 20 I"EST, C. AND l { . - J . SCHMIDT, The c h e m i s t r y of o r g a n o p h o s p h o r u s pesticides, Springer, Berlin 197321 [?ISCHER, G. \~. AND I(. LOHS, Zur Einsch/~tzung des Alkylicrungspotentials onkologisch b e d e u t s a m e r Phosphors~iureester mit Hilfe der N B P - R e a k t i o n , Arch. Geschwulstforschung, 42 (I973) 34-4 o. 22 FREAR, D. E. H., Pesticide Index, 3rd Ed., 1965, College Science Publ., State College, Pennsylvania. 23 (;ILLETT, J. V~T., J. l~. HARR, A. D. ST. CLAIR, AND L. J. YVEBER, C o m m e n t on the distinction between hazard and safety in evaluation of h u m a n health h a z a r d s on use of dichlorvos, especially in resin strips, Residue Reviews, 44 (1972) I 6 I - I 8 4 24 (;]LLETT, J. ~¥., J. R. HARR, F. T. LINDSTROM, D. A. MOUNT, A. D. ST CLAIR, AND [,. J. WEBER, E v a l u a t i o n of h u m a n health hazards on use of dichlorvos (DDVP), especially in resin strips, Residue Reviews, 44 (19721 I I 5 159. 25 GREEN, M. H. L., A. S. C. MEDCALF, C. F. ARLETT, S. A. HARCOURT AND A. R. I,EIIMANN, D N A s t r a n d breakage caused b y dichlorvos, m e t h y l m e t h a n e s u l p h o n a t e and Jodoacetamide in Escherichia coli and cultured Chinese h a m s t e r cells, Mutation Res., 24 (1974) 365 378. 26 Health Hazards of the Human Environment, W H O , Geneva 1972. 27 HOLLAENDER, A. (Ed.) Chemical Mutagens, Principles and JVlethods for their Detection, Plenum Press New York, Vols. I, 2, 1971 ; Vol. 3, 1973. 28 HUTSON, D. H. AND E. C. HOADLEY, The m e t a b o l i s m of 1~C methyl dichlorvos in the rat and the mouse, Xenobiotoca, 2 (1972 ) IO7-I16. 29 HUTSON, D. H., E. C. HOADLEY AND I~. A. PICKER1NG, The nletabolic fat(, of vinyl-II¢C dichlorvos in the rat after oral and inhalation exposure, Xenobiotica, i (1971) 593 611. 3 ° KRAUSE, W. AND S. HOMOLA, Beeinflussung der Spermiogenese durch D D V P (Dichlorvos), Arch. Dermatol. Forschung, 244 (1972) 439-44 I. 31 LAWLVY, P. I)., Effects of some chemical m u t a g e n s and carcinogens ou nucleic acids, Progress in Nucleic Acid Research and ll,lol. Biol., 5 (I966) 89 I 3 I . 32 LAWL~:Y, P. D., S. A. SHAH ANt) D. J. ORR, Methylation of nucleic acids by 2,2-dichlorovinyl dinlethyl p h o s p h a t e (Dichlorvos, DDVP), Chem.-Biol. Interactions, 8 (19741 171 i82. 33 Id~FROTH, G., Alkylation of DNA b y dichlorvos, Naturwissenschaften, 57 (I97 o) 393 394. 34 L(~FROTH, G., CHUL t(IM AND S. HUSSAIN, Alkylating p r o p e r t y of 2,2-dichlorovinyl dimethyl p h o s p h a t e ; A disregarded hazard, E M S Newsletter, 2 (19691 21-27. 35 L/SFROTH, G. AND R. \¥ENNERBERG, Methylation of purines and nicotinamide in the r a t by dichlorvos, Z. Naturforsch., 29c (19741 651. 36 MELNIKOV, N. N., Chemistry of Pesticides, Springer, New York 1971. 37 MICHALEK, S. M. AND H. E. BROCKMANN, A test for m u t a g e n i c i t y of Shell " N o - P e s t Strip Insecticide" in Neurospora crassa, Neurospora Newsl., 14 (19691 8. 38 MOHN, G., 5 - M e t h y l t r y p t o p h a n resistance m u t a t i o n s in Escherichia coil K i y . Mutagenic

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a c t i v i t y of m o n o f u n c t i o n a l a l k y l a t i n g 3/Iutation Res., 20 (1973) 7-15.

agents including

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o r g a n o p h o s p h o r u s insecticides,

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