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The EFSA Journal (2006) [343, 1-45]

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Opinion of the Scientific Panel on Plant health, Plant protection products and their Residues on a request from EFSA related to the evaluation of dichlorvos in the context of Council Directive 91/414/EEC.

(Question N° EFSA-Q-2005-246)

adopted on 1 April 2006

Summary of Opinion Dichlorvos is an organophosphate insecticide that acts by inhibiting acetylcholinesterase (AChE), which results in a disturbance of nerve signal transmission and induces rapid respiratory failure in most insects. The same mechanism is responsible for the acute toxicity in mammals, including humans. The only use of dichlorvos supported by the applicant is against flower bulb pests during storage.

The Scientific Panel on Plant health, Plant protection products and their Residues (PPR Panel) of EFSA was asked if i) related to the increased incidence of tumours observed in various tissues in rats and mice following dichlorvos exposure, it is possible to identify a mode of action (for any of the tumours) and if so, is it possible to set a threshold for this effect; and ii) in considering any identified modes of action for the tumourigenic responses to dichlorvos, is any of them relevant for humans.

Dichlorvos has been evaluated for carcinogenicity in five long-term studies in mice and in six long-term studies in rats. The substance was administered orally via the diet in the drinking water or by gavage, or by inhalation (one study in rats). Most of the studies provided no evidence for the induction of neoplasia and only in two gavage studies, one in F344/N rats and the other in B6C3F1 mice, was there some evidence for neoplastic responses. In these studies, increases in the incidence of mononuclear cell leukaemia in male rats, mammary fibroadenomas combined with adenomas in female rats, pancreatic acinar adenomas in male rats and forestomach tumours in male and female mice were reported. After considering all of the available data the PPR Panel concluded that with the exception of tumours of the forestomach in mice, there was no convincing evidence for a compound-related increase in tumour incidence. The response on mouse forestomach was a consequence of local, rather than systemic, exposure.

The PPR Panel concluded that the available data clearly demonstrate that dichlorvos is an in vitro mutagen, and that there is some limited evidence that dichlorvos is a site-of-contact in vivo mutagen but that the mechanism of this effect is unclear; the evidence for alkylation of DNA in vivo, a possible mechanism, is very weak.

The Panel concluded that there was insufficient evidence to identify a mode of action for the forestomach tumours produced by dichlorvos in the mouse. However, the Panel concluded that irrespective of the mode of action, the response appeared to be a consequence of the high sustained local concentrations of dichlorvos that could be achieved in this specific exposure situation and was therefore limited to this site. The Panel further concluded that there was a threshold dose for this response. The Panel was of the opinion that the weight of evidence suggests that this would not occur at the levels of exposure that would be encountered by the proposed use of the compound. In addition severe systemic toxicity would occur before any concentration in tissues other than in the forestomach is reached that would induce the tumourigenic effect. This is because the forestomach is a unique structure that retains material appreciably longer than the glandular stomach and oesophagus.



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Key words: Dichlorvos, cancer, forestomach tumours, mutagenicity, site of contact, DNA-interaction, mode of action.



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Table of contents

Summary of Opinion ________________________________________________________ 1

Background _______________________________________________________________ 4

Terms of reference __________________________________________________________ 6

1 Assessment Question 1 ___________________________________________________ 6 1.1 Introduction ____________________________________________________________ 6 1.2 Dichlorvos: Identity and Physicochemical Properties __________________________ 6 1.3 Dichlorvos. ADME and Kinetics in Rodents and Man__________________________ 7

1.3.1 Absorption and distribution______________________________________________________ 7 1.3.2 Metabolism __________________________________________________________________ 8 1.3.3 Excretion ____________________________________________________________________ 8

1.4 Cancer studies __________________________________________________________ 9 1.4.1 Mice _______________________________________________________________________ 9 1.4.2 Rats _______________________________________________________________________ 10 1.4.3 Non-Genetic Effects of Dichlorvos on Forestomach Tissue ____________________________ 18

1.5 Chemical Reactivity of Dichlorvos _________________________________________ 18 1.5.1 Protein Phosphorylation _______________________________________________________ 18 1.5.2 Alkylating Activity of Dichlorvos: General Considerations ____________________________ 19

1.6. Alkylation of Nucleic Acid Bases by Dichlorvos ______________________________ 20 1.6.1 In Vitro Alkylation ___________________________________________________________ 20 1.6.2 In Vivo Alkylation ____________________________________________________________ 21 1.6.3 Summary of Direct DNA-Interactions of Dichlorvos _________________________________ 23

1.7 Studies on Mutagenicity _________________________________________________ 24 1.7.1 In Vitro Mutagenicity _________________________________________________________ 24 1.7.2 In Vivo Mutagenicity__________________________________________________________ 24 1.7.3 Summary on In Vivo Mutagenicity of Dichlorvos____________________________________ 30 1.7.4 Possible Contribution of Metabolite Dichloroacetaldehyde on In Vivo Mutagenicity Of Dichlorvos _________________________________________________________________________ 31

Conclusions on Question 1 ______________________________________________________ 32 Conclusions on Mode of Action of Genotoxicity ___________________________________________ 32 Conclusions on Mode of Action of Carcinogenicity _________________________________________ 33

2 Assessment of Question 2________________________________________________ 33 2.1 The Relevance of the Finding of Forestomach Tumours to Man ________________ 33 Conclusions on Question 2 ______________________________________________________ 34

Documentation provided to the EFSA Panel by the PRAReR unit ___________________ 34

References________________________________________________________________ 36

Scientific Panel members____________________________________________________ 41

Appendix _________________________________________________________________ 42



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Background1 General

Dichlorvos (ISO proposed), i.e. phosphoric acid, 2,2-dichloroethenyl dimethyl ester is an organophosphate insecticide. It is to be used in greenhouses and post harvest applications in stores. Dichlorvos inhibits acetyl cholinesterase (AChE), which results in a disturbance of the nerve signal transmission and induces a rapid respiratory failure on most flying insects.

The only representative use supported by the applicant is against flower bulb pests during storage, with an application rate of 2.2 g/100 m3, a maximum frequency of 3 times with an interval of 2 days between treatments. Application is by automatic fogging and vaporizing equipment.

The rapporteur Member State (RMS) for the draft assessment report (DAR) of dichlorvos is Italy and it was finalized in August 2003. The notifier is Denka (NL). Generally, the studies submitted for the evaluation by the RMS are old and the quality not always up to the latest standards. Sometimes only publications from the open literature are summarised. Initially, the RMS stated that due the uncertainty about carcinogenic properties and that a new long term study was requested as well as due to the general limitations of the data it was not possible to conclude on the risk assessment on dichlorvos.

Dichlorvos was discussed in the EPCO2 experts’ meeting in June 2005 on issues relating to mammalian toxicology. Two major points remained open relating to the mutagenic and carcinogenic properties of dichlorvos. Due to these outstanding questions the reference values could not be allocated and thus it was not possible to come to a final conclusion on the overall human risk assessment. The basis for the discussion was the DAR and respective addenda as well as the opinion of the UK Committee on Mutagenicity.

The mutagenic property of dichlorvos has also been discussed in the UK Committee on Mutagenicity and a statement is available from January 2002. The basis for their discussion was more than 100 scientific articles relating to the toxicity of dichlorvos. The opinion from the UK Committee on Mutagenicity is not considered in the DAR since it was not available at the time of finalization of the DAR but it is briefly commented in the addendum (to the DAR).

Scientific summary

Dichlorvos is highly toxic by oral, dermal and inhalatory exposure (oral LD50 is 80 mg/kg b.w., dermal LD50 is 120 mg/kg b.w. and LC50 is 0.083 mg/l). It was also demonstrated to be a skin sensitizer. The critical effect is inhibition of AChE (brain and blood) seen in all species tested. Following the discussion at the experts’ meeting it was concluded that it was not possible to derive a NOAEL or to conclude on the reproductive toxicity of dichlorvos due to the fact that the studies were not up to current standards particularly as the upper dose levels were possibly too low to identify any inherent reproductive toxicity.

Genotoxicity

There are a substantial number of studies presented in the DAR but many of them are old, the purity often not stated, additionally many of them are reports from publications in the open literature containing limited information and only regarded as acceptable as supplemental studies (see DAR, Vol. 3, B.6.4). It was concluded that dichlorvos is mutagenic in vitro as demonstrated in a number of studies but not in vivo and defined as a weak mutagen.

Carcinogenicity

There are two long-term studies (2-year), one in the rat and one in the dog (a publication), both from 1967 and only considered as acceptable as additional studies. Furthermore, there are 1 Submitted by EFSA’s PRAPeR sector (coordination of the pesticide risk assessment peer review of active substances) 2 EPCO: EFSA Peer Review Co-ordination



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seven carcinogenicity studies as well as summaries of publications in rodents all considered as acceptable as additional studies (see DAR, Vol. 3, B.6.5). Tumours were evident in one of the rat studies. Dichlorvos induced tumours in two studies but was negative in other studies of poor quality. In the rat, adenomas in the pancreas in males and females as well as mammary gland fibroadenomas in females were observed (Chan, 1989 and Chan et al., 1991). In the mouse, forestomach tumours were noted in both sexes (Chan, 1989 and Chan et al., 1991).

Statement on mutagenicity from the UK Committee on Mutagenicity

Based on the available data (over 100 reports from the open literature mostly) the UK Committee on Mutagenicity concluded that dichlorvos is an in vitro mutagen as demonstrated by DNA damage, chromosomal breakage as well as mutation in mammalian cells. It has been shown to interact with DNA via methylation but other mechanisms are theoretically possible (see statement from UK Committee on Mutagenicity).

Furthermore, the UK committee concluded that dichlorvos should be regarded as an in vivo mutagen at the site of exposure but not for systemic effects. The UK Committee on Mutagenicity agreed that there was limited evidence for a carcinogenic effect of dichlorvos and until evidence proves the contrary and in the absence of appropriate mechanistic data, a precautionary approach should be adopted and no threshold could be assumed for the mutagenic activity or carcinogenic effects of dichlorvos.

Discussion at the experts’ meeting

The issue of mutagenic and carcinogenic properties was discussed at the EPCO experts’ meeting (see report from the meeting). It was noted that the studies evaluated by the UK Committee on Mutagenicity had methodological deficiencies. It was concluded that dichlorvos is indeed mutagenic in vitro (both in bacterial systems and in mammalian cells). Although it was not mutagenic in vivo in the studies evaluated, concerns were raised by the experts that the assays were not appropriate for detecting site of contact effects with highly reactive compounds such as dichlorvos. The results were considered as inconclusive.

The experts discussed the carcinogenicity potential, and agreed that a positive or equivocal result was obtained in two of the eleven carcinogenicity studies. Although eleven studies would normally have been sufficient to provide a weight of evidence on the carcinogenic potential, the quality of the studies was low; the experts considered that while there was no consistent evidence of carcinogenicity, the database was not sufficient to exclude carcinogenic activity.

Conclusion

In the discussion of dichlorvos in the experts’ meeting within the peer review under the Directive 91/414/EEC it was not possible to come to a final conclusion on the mutagenic or carcinogenic potential of dichlorvos.

The poor quality of the dossier submitted by the notifier had a significant impact on the ability to reach reliable conclusions. However, it was concluded that dichlorvos was an in vitro mutagen but uncertainties remained regarding the in vivo results. Furthermore, it was not possible to establish either mechanism or threshold for the observed tumours.

Due to this, the experts agreed that it was not possible to define the reference values or the possible additional safety factor needed and thus this affects the overall risk assessment of dichlorvos. Thus, the uncertainties remaining in a number of areas carried over to the risk assessment for operators as well as consumers which cannot be completed (see list of endpoints).

Therefore, the panel is asked whether it is possible to identify a mechanism for the tumours as well as a threshold and whether the carcinogenicity observed in rats and mice is relevant and a concern for human exposure.



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Terms of reference The Scientific Panel on Plant health, Plant protection products and their Residues (PPR Panel) of EFSA is asked for an opinion on the mutagenic and carcinogenic properties of dichlorvos in the context of the human risk assessment. The following questions have been identified during the discussion at the EPCO experts´ meeting.

Question 1: Related to the increased incidence of tumours observed in various tissues in rat and mouse following dichlorvos exposure, is it possible to identify a mode of action (for any of the tumours) and in that case is it possible to set a threshold?

Question 2: Considering these modes of action of the tumourigenic responses is any of them relevant for humans?

1 Assessment Question 1 Related to the increased incidence of tumours observed in various tissues in rat and mouse following dichlorvos exposure, is it possible to identify a mode of action (for any of the tumours) and in that case is it possible to set a threshold?

1.1 Introduction

Dichlorvos was considered to have a weak carcinogenic effect at the side of contact (forestomach) in mice and possibly systemic carcinogenic activity in rats in two of eleven long-term carcinogenicity studies, following gavage administration. Both of the studies in which significant increases in tumour incidences were observed were conducted by the US NTP (Chan, 1989; Chan et al., 1991). The PPR Panel first considered the available data on the tumourigenic effects of dichlorvos to determine which if any responses could be considered compound-related and of biological significance. Considering the documented in vitro mutagenicity of dichlorvos to bacteria, fungi, plants, insects, and mammalian cells in culture, the PPR Panel sought to identify a mode of action for the tumourigenic activity of dichlorvos in rodents. The PPR Panel in particular revisited the literature on the chemical reactivity of dichlorvos with DNA, its in vivo mutagenicity and its carcinogenicity and analysed whether the reported tumourigenic responses in rodents could be linked to

• DNA-interacting activity of dichlorvos

• non-DNA-interacting activity, e.g. irritation and/or cell proliferation

• local versus systemic tumourigenic activity.

An analysis was carried out also to assess whether these modes of action allow a threshold to be set for any possible risk of dichlorvos carcinogenicity to humans.

1.2 Dichlorvos: Identity and Physicochemical Properties

The active substance dichlorvos (dimethyl 2,2-dichlorovinyl phosphate syn. phosphoric acid, 2,2-dichloroethenyl dimethyl ester; syn. Denkavepon, Vapona, Nuvan, Nogos, DDVP) (Fig. 1) is a trialkoxyorganophosphate and is used as an insecticide and vermicide.

It is a colourless to amber liquid with 95% to 99% (950-990 g/kg) purity if marketed. In older studies (1970ies) its purity was up to 93%. Its molecular weight is 221.0. It is soluble in water up to 18 g/l at 25°C. In the pH range of 5 – 9 the active compound does not dissociate in water. It is miscible with ethanol and other organic solvents at concentrations ranging from 5 to 95 % v/v. The log PO/W is 1.9 at 25°C. The solubility in water at 20°C is 1% and in kerosene and mineral oils is 3%. In the various carcinogenicity studies dichlorvos was administered by gavage in drinking water, by gavage in corn oil, with diet or by inhalation (vaporization). Its vapour pressure is 2.1 Pa at 25°C. The high vapour pressure makes dichlorvos very effective in closed areas.

Dichlorvos decomposes in air at room temperature to dimethylhydrogen phosphate and dichloroacetaldehyde at room temperature. In the presence of water, pH 7.0, it decomposes



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with a half-life of about 8 hours to dichlorethanol, dichloroacetaldehyde, dichloroacetic acid, dimethylphosphate, dimethylphosphoric acid and further water soluble compounds. Alkaline pH accelerates, whilst acidic pH slows down decomposition rates.

Figure 1

1.3 Dichlorvos. ADME and Kinetics in Rodents and Man

Since the 1960s many studies have been performed on the ADME properties of dichlorvos. These utilised radioactively labelled dichlorvos (14C-vinyl, 14C-methyl, 32P, and 36Cl), deuterated (2H)-dichlorvos or unlabelled dichlorvos. Many of the studies have been classified not to be acceptable in the Draft Assessment Report (DAR) due to deviations from currently used guidelines (DAR, 2003; 2006).

1.3.1 Absorption and distribution

Due to its high lipophilicity (log PO/W is 1.9 at 25°C) complete absorption of dichlorvos is observed after oral administration. In rats dosed orally with [32P]-dichlorvos (single gavage dose 10 mg/kg b.w.), 60 – 70 % of administered radioactivity was recovered in the urine and about 10% in faeces on day 7 after dosing. Radioactive phosphate was incorporated into the bones where it still remained after 7 days (Casida et al,. 1962). This study is the only one which gives stomach tissue concentrations. The highest concentration of radioactivity derived from [32P]-dichlorvos compared with other tissues was in the stomach tissue, particularly of male rats, 1 hour after gavage (twice the liver and five times the kidney concentration) where it remained over 4 hours. In stomach, liver, and kidney the [32P]-derived radioactivity declined rapidly within 1 day, e.g. was 1 % after one day of the peak seen in the stomach after one hour (in liver after 1 hour the dichlorvos content was 50% of stomach and declined to ca. 15 % after one day) (Casida et al., 1962, DAR, 2003; 2005).

Once in blood the parent compound leaves the systemic circulation rapidly (within 15 min). Thus, when administered via bolus application (e.g. by gavage) a very short period of maximum exposure due to a very short half-life time in blood has to be assumed, most likely because substantial hydrolysis of dichlorvos occurs already in blood and also in the cells. Only traces of radioactivity were found to reside in some rat tissues (e.g. 4 days after single oral exposure 4.4 - 5% of [14C]-radioactivity from dichlorvos was retained in the liver, 6.5 – 8.6 % in the skin and 12.3 – 16% in the other parts of the body (Hutson et al., 1971).

Dichlorvos is very rapidly absorbed and distributed after intraperitoneal administration of dichlorvos occurs. The peak concentration of deuterated [2H]dichlorvos in brain (0.75 µg dichlorvos/g mouse brain homogenate) after an i.p. injection of 10 mg/kg b.w. to mice was reached within 1 minute and declined significantly within 3 min to 0.2 µg/g mouse brain. The activity of brain acetylcholine esterase (AChE) was lowest after 15 min (about 23% of non-blocked enzyme) but the enzyme reactivated linearly to reach ca. 80% of initial activity within 2 hours, indicating a significant spontaneous reactivation (Nordgren et al., 1978).

Inhaled dichlorvos is absorbed and degraded rapidly. The distribution of dichlorvos after inhalation was very similar to that after intravenous injection (Blair et al., 1975). Dichlorvos was detected at low concentrations in the blood, liver, testes, lung, brain, kidney, and fat of rats exposed by inhalation at 90 mg/ m3 (90 g/l, corresponds to 60 % of full air saturation) for 4 hours with the highest concentration in kidney and fat. Dichlorvos was hardly detectable in the lungs, which can probably be ascribed to its rapid absorption into blood and metabolism. At

Dichlorvos

CH3O

CH3OP

O

O C CCl2



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lower exposure concentrations (10 mg/m3) the parent compound was detected only in the kidney (Blair et al., 1975). There, the dichlorvos concentration was 2 µg/g kidney tissues and its half-life after inhalation exposure (50 mg/ m3) was 13.5 min in rats (Blair et al., 1975).

Dichlorvos was not detected in the blood of two men immediately after inhalation exposure to dichlorvos at 0.25 mg/m3 for 10 hours or 0.7 mg/m3 for 20 hours (Blair et al., 1975). In professional sprayers, dichlorvos (concentration unknown) was detected in blood within 24 hours of exposure but not at 48 hours (Fournier et al., 1978).

1.3.2 Metabolism

Dichlorvos is rapidly metabolized in the liver, blood, and other tissues (Chan, 1989). Several metabolites have been described (see Fig. 3, Addendum) (Wright et al., 1979; Chan, 1989). Unchanged dichlorvos was not found in muscle or fat of rabbits administered dichlorvos orally at 5 mg/kg per day for 2 weeks and killed 48 hours later (Majewski et al., 1979). When the same dose was administered daily for 2 weeks or for 25 days to pregnant rabbits by the oral route, dichlorvos was not detectable in foetuses 24 hours after the last dose (Majewski et al., 1979).

Two main biotransformation pathways are known from metabolism studies in humans, mice, rats, Syrian hamsters, pigs, goats and cows: 1) ester hydrolysis following protein phosphorylation and 2) demethylation, yielding methylated macromolecules. The phosphorylation pathway (reaction 1) is important for acute toxicity in mammals including man when B-esterases are targets but also leads to dichlorvos inactivation in the reaction with A-esterases. In this case, dimethyl phosphate and dichlororacetaldehyde are formed. Dimethyl-phosphate is excreted unchanged in the urine. The metabolism of dichloroacetaldehyde proceeds also to the urinary metabolite dichlororethanol. The hydrolysis by protein phosphorylation is several orders of magnitude more rapid than the demethylation pathway with a ratio of 3 x 107 : 1 (Pletsa et al., 1999a). Dichlorvos hydrolysis by rat plasma A-esterases is 12 µmol/hour/ml at 370C (Reiner et al., 1980). Others have determined the half-lives of 5 µmol/l dichlorvos in vitro in whole blood of rat, rabbit, and man yielding 12 – 30 min, 2 min, and 10 min, respectively (Blair et al., 1975).

The demethylation pathway (reaction 2) leads enzymatically to demethyldichlorvos which is further degraded to dichloroacetaldehyde and monomethylphosphate. Dichlorvos can react with glutathione (Dicowsky and Morello, 1971) to yield S-methylglutathione or with macromolecules such as DNA at nucleophilic centres. The methylation of glutathione (see Fig. 3, Addendum), and thereby demethylation of dichlorvos, is thought to generate carbon dioxide and also may prevent the incorporation of the methyl group into nucleic bases due to the competing reaction (Wright et al., 1979). However, it was noted that GSH-dependent demethylation of dichlorvos may play only a minor role in terms of DNA-protection, even at high dichlorvos concentrations (Wright et al., 1979).

With the exception of dichloroacetaldehyde, which is a main metabolite of both pathways (hydrolysis and demethylation-pathway), but with lesser acute toxicity than dichlorvos, all other metabolites are of very low chemical reactivity and are not further considered. Dichloroacetaldehyde has mutagenic activity in the Salmonella/microsome test (Löfroth, 1978) and in Aspergillus nidulans (Crebelli et al., 1984) as well as in the dominant lethal test in mice (see 1.7.4). However, it is only a very weak alkylating agent, being negative in the 4-(4-nitro-benzyl) pyridine (NBP)-test (Bedford and Robinson, 1972). Dichloracetaldehyde can be reduced to dichlorethanol which can eventually be glucuronidated (Fig. 3, Addendum).

1.3.3 Excretion

The main excretion pathways of the metabolites of dichlorvos are urine and expired air. 60-70 % of radioactivity from [32P] dichlorvos was recovered in urine and 10 % in faeces of rats within 7 days (Casida et al., 1962). In other animals (mice, goats, cows) this ratio was similar. The vinyl moiety is degraded to yield carbon dioxide and when this residue was radiolabelled 27% in



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man (within 8 hours) and 40 % in rats (within 4 days) of orally administered radioactivity was found as [14C]CO2 in expired air (Hutson et al., 1971; Chan, 1989).

1.4 Cancer studies

Summaries of the data were taken from: JMPR (1993), IPCS (1989), Chan (1989), Chan et al., (1991), and DAR 2003; 2006). Table 1 contains a list of all studies.

1.4.1 Mice

Gavage study 1:

Groups of 100 male and 100 female C57Bl/6/Bln mice received by gavage freshly prepared 0.2 mg dichlorvos (97% pure in 0.2 ml water) per mouse (equivalent to 10 mg/kg b.w.), either twice or three times per week for 50 weeks. Control groups received by gavage either 0.2 ml water, three times per week (about 50 males and 50 females) or no treatment (35 males and 35 females). Surviving animals were terminated after 110 weeks. The C57Bl/6/Bln strain of mouse is known for a high spontaneous incidence of mixed lymphomas (reticulocell sarcoma type B). From the age of 12 months onwards, some animals in all groups developed interstitial pneumonia. The incidence of mixed lymphomas was lower in both treated groups compared to controls (26-60% in control groups, 23-30% in treated groups). An increased incidence of focal hyperplasia (transitional cell hyperplasia) of the urinary bladder was found in both dichlorvos treated groups (0-8% in control groups, 5-10% in treated groups). The authors concluded that no neoplastic lesions were found which could be attributed to the treatment of the mice with dichlorvos (Horn et al., 1987).

Gavage study 2:

Groups of 50 B6C3F1 mice were given dichlorvos (99% pure) dissolved in corn oil orally by gavage at doses of 0, 10 or 20 (males) or 0, 20 or 40 (females) mg/kg b.w. per day, five days/week for 103 weeks. Dose selection was on the basis of toxicity in a 13-week pilot study. The dose volume of corn oil dosed was 10 ml/kg b.w. per day. Body weight gain and survival did not differ significantly between treated and control groups. Increased incidences of forestomach squamous cell papillomas were observed at high doses compared to controls (1/50, 1/50 and 5/50 in control, low- and high-dose males, respectively and 5/49, 6/49 and 18/50 in control, low- and high-dose females, respectively). The positive trend was statistically significant in both sexes while by pairwise comparison only the incidence in high-dose females was significantly higher than in controls. The mean and range for the overall historical incidence of papillomas at the study laboratory was 1% ± 2% (0% to 6 % in males) and the overall mean and range of historical incidence in NTP studies with this mouse strain was 0.9% ± 2% (0% to 8% in females).

Two forestomach squamous cell carcinomas were seen in high-dose females and none in the other groups. There was no increases in the incidences of forestomach hyperplasia in the dosed mice compared with controls (11/50, 5/50 and 9/50 in control, low- and high-dose males, respectively and 6/49, 7/49 and 5/50 in control, low- and high-dose females, respectively). In female mice the incidence of adenomas and adenomas or carcinomas (combined) of the pituitary gland (12/45, 6/45 and 6/44 in control, low and high-dose groups, respectively) and the incidence of lymphomas (16/50, 11/50 and 9/50 in control, low and high-dose groups, respectively) showed a significant negative trend with dose. Based on the increased incidence of forestomach papillomas, the NOAEL was 10 mg/kg b.w. per day (Chan, 1989; Chan et al., 1991).

Dietary study:

Two groups of 50 male and 50 female B6C3F1 mice were fed 1000 or 2000 mg/kg dichlorvos (purity > 94%) in corn oil in the diet for 2 weeks. Due to severe signs of intoxication (including tremors and diarrhoea), doses were reduced to 300 and 600 mg/kg for the following 78 weeks. Samples of the diets analysed during the study showed that time-weighted average concentrations were 318 and 635 mg/kg (equivalent to 48 mg/kg b.w. and 95 mg/kg b.w.,



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respectively). Matched controls consisted of 10 mice of each sex; the pooled controls from simultaneous studies with other compounds consisted of 100 male and 80 female mice. All surviving mice were killed at 92-94 weeks. The average body weight of the high-dose mice of both sexes was slightly decreased compared with controls. The low-dose female group showed 74% survival at 90 weeks compared to 84% and 90% in high-dose and control groups, respectively. There was no significant change in the incidence of tumours attributable to dichlorvos in either sex. Two squamous-cell carcinomas of the oesophagus (one in a low-dose male and on in a high-dose female), one papilloma of the oesophagus in a high-dose female and three cases of focal hyperplasia of the oesophageal epithelium in low-dose males were recorded in the treated mice. The significance of the findings in the treated mice was considered uncertain because of insufficient information concerning the spontaneous incidence of these lesions and lack of statistical significance within the experiment. Dichlorvos, up to 635 mg/kg, equivalent to 95 mg/kg b.w. per day, was not demonstrated to be carcinogenic in this study (NCI, 1977; Weisburger, 1982).

Drinking water study:

Groups of 50 male and 50 female B6C3F1 mice were given drinking water containing 0, 400 or 800 mg dichlorvos/l ad libitum (equal to 58 and 95 m/kg b.w. per day in males and 56 and 102 mg/kg b.w. per day in females). The drinking-water solutions were renewed daily. All surviving mice were killed during week 102. Dose-dependent inhibition of body weight increases were observed throughout the study in both sexes at both dichlorvos concentrations. Final mean body weights of all doses groups were significantly reduced relative to controls (reduction of 9% and 14.2% in males and 15% and 14.6% in females, in low and high dose groups, respectively). There was no adverse effect on mortality. The survival rates at week 102 (in controls, low- and high-dose groups, respectively) were 62%, 66% and 84% in males and 66%, 50% and 80% in females. The corresponding tumour incidences were 22.4%, 39.1% and 23.4% in males and 29.3%, 16.2% and 9.1% in females. The more common tumours found were in the lung, liver, spleen, thymus and salivary gland. These tumours occurred in all three groups. There were no statistically significant differences in the incidence of tumours at any site in any group. Squamous-cell papillomas of the glandular stomach were observed in one low dose and one high dose male and in two control group and one low dose female. The incidences of these lesions were not increased to a statistically significant degree; however, 4/47 low dose and 3/47 high dose males and 1/43 high dose females had squamous-cell hyperplasia of the glandular stomach. No forestomach tumours were observed (Konishi et al., 1981 and Konishi et al.,, 1989).

Special study:

Dichlorvos was not co-carcinogenic in C57Bl/6/Bln mice when administered by gavage three times/week at 0.2 mg/animal to mice injected subcutaneously with 50 µg N-nitrosodiethyl-amine per animal weekly for 50 weeks followed by an observation period of up to 110 weeks (Horn et al., 1990).

1.4.2 Rats

Gavage study 1:

Groups of 70 and 99 BD IX/Bln rats per sex received 0.1 mg dichlorvos (97% pure)/rat in 0.2 ml water by gavage (equivalent to 0.25 mg/kg b.w.) twice or three times per week, respectively, for 60 weeks. Thereafter, the animals were observed for another 51 weeks. A control group of 60 animals of each sex received 0.2 ml water three times per week. An increase in focal hyperplasia was seen in the forestomach of both treated sexes. Decreased incidences of adrenals tumours (32/39, 24/49 and 37/70 in control, 0.2 and 0.3 mg/animal/week, respectively) and mammary gland adenocarcinomas (7/34, 0/46 and 2/64 in control, 0.2 and 0.3 mg/animal/week) were observed in treated female groups compared to the control group. No increased incidences of neoplastic lesions were found which could be attributed to treatment (Horn et al., 1988).



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Gavage study 2:

Groups of 50 male and 50 female F344/N rats were administered dichlorvos (99% pure) in corn oil by gavage at 0, 4 or 8 mg/kg b.w. per day (actual doses 0, 4.14 or 7.82 mg/kg b.w. per day), 5 days per week for 103 weeks. Dose selection was based on toxicity in a 13-week pilot study. No significant differences in mean body weight or survival were observed between any groups of either sex. An increased incidence of cytoplasmic vacuolization of the liver was observed in dosed males and of cortical cytoplasmic vacuolization of the adrenal glands in all dosed males and in low-dose females. The incidence of pancreatic adenomas (cross and horizontal tissue sections were analyzed) in control, low and high-dose groups was 25/50, 30/49 and 33/50 in males and 2/50, 3/48 and 6/50 in females, respectively. The increased incidence in treated males was statistically significant (p<0.05). The incidence of mononuclear cell leukaemia (11/50, 20/49, 21/50 in control, low and high-dose groups, respectively) was significantly increased in the treated males compared with controls. Incidences of mononuclear cell leukaemia in females were not significantly different between control and treated groups (17/50, 21/48 and 23/50 in control, low and high-dose groups, respectively). The incidence of mammary gland adenomas or fibroadenomas (combined) in female rats was 9/50, 19/48 and 17/50 in control, low and high-dose groups, respectively. Two mammary gland carcinomas were observed in control and low-dose females. The incidence of mammary gland tumours in both treated groups was within the historical control values. In males, three alveolar/bronchiolar adenomas occurred in the high-dose group but the difference between control and high dose groups was not statistically significant (Chan, 1989; Chan et al., 1991).

Dietary study 1:

Groups of 40 male and 40 female CD rats were fed diets containing nominal concentrations of 0, 0.1, 1, 10, 100 or 500 mg/kg dichlorvos (93% pure) for 2 years. Diets were prepared weekly. Five males and five females from each group were killed after 6, 12 or 18 months. Analysis of diet samples showed a considerable loss of dichlorvos associated with a gradual increase in dichloroacetaldehyde content (0.01 to 28.6 mg/kg). The average actual concentrations of dichlorvos in each diet were 0.05, 0.5, 4.7, 47 or 230 mg/kg (equivalent to 0.0025, 0.025, 0.235, 2.35 or 11.5 mg/kg b.w. per day). The tumour incidence was comparable with that of the control group. The NOAEL, based on brain choline esterase inhibition, was 100 mg/kg (actual concentration 47 mg/kg, equivalent to 2.4 mg/kg b.w. per day (Witherup et al., 1967).

Dietary study 2:

Osborne-Mendel rats (50/sex) were fed diets containing 150 mg/kg and initially 1000 mg/kg dichlorvos (purity > 94%) in corn oil for 80 weeks. Due to severe cholinergic signs of toxicity, the high dose was reduced to 300 mg/kg after 3 weeks for the remaining 77 weeks. Thereafter the animals were maintained on control diet until termination at 110-111 weeks. Samples of the diets analyzed during the study showed that time-weighted average concentrations were 330 and 150 mg/kg (equivalent to 16.3 and 7.5 mg/kg b.w. per day), respectively. Matched controls consisted of ten rats of each sex; the pooled controls from simultaneous studies of other compounds consisted of 60 rats of each sex. The average body weights of the high-dose rats of both sexes were slightly decreased compared with controls. There was no significant increase in the incidence or type of tumours attributable to dichlorvos in either sex. Malignant fibrous histiocytomas were observed in multiple organs in all groups (2/58, 1/10, 4/48 and 8/50 in pooled and matched controls, low and high-dose males, respectively and 4/59, 1/10, 3/48 and 3/49 in pooled and matched controls, low and high-dose females, respectively). Dichlorvos concentrations of up to 330 mg/kg in the diet (equivalent to about 30 mg/kg b.w. per day) were not carcinogenic in this study (NCI, 1977; Weisburger, 1982).

Drinking water study:

Groups of 50 male and 50 female F344 rats were given drinking water (renewed daily) containing 0, 140 or 280 mg dichlorvos/l ad libitum (equal to 8.3 and 18 mg/kg b.w. per day in males and 10 and 22 mg/kg b.w. per day in females). All surviving animals were killed in week 108 (104 weeks of exposure followed by a 4-week recovery). Slight inhibition of body weight



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increase was observed in males in the high-dose group, but there was no influence on mortality. The survival rates in week 108 were 82%, 76% and 73% in males and 82%, 67% and 80% in females, respectively, in control, low and high dose groups. The overall tumour incidences were 100%, 96% and 98% in males and 37%, 31% and 33% in females, respectively, in control, LD and HD groups. High incidences of interstitial cell tumours of the testes in males (49/51, 40/48 and 48/48, respectively) were observed in all three groups. The incidence of mononuclear cell leukaemia and lymphocytic leukaemia (combined) was marginally increased in low dose male rats (p = 0.05) but mononuclear cell leukaemia alone was not statistically increased (6%, 15% and 13% in control, LD and HD groups). There was a positive trend for the incidence of bronchiolar adenomas in dosed females (p = 0.044) but there were no significant differences when the data were analyzed by pair-wise comparison. There was a slight increase in the incidence of dosed animals with ulceration of the glandular portion of the stomach. There was no evidence of treatment-related proliferative changes in either the oesophagus or forestomach (Enomoto et al., 1981; 1989).

Inhalation study:

Groups of 50 male and 50 female CFE rats were exposed (whole body) to nominal air concentrations of 0, 0.05, 0.5 or 5 mg dichlorvos (97% pure)/m3 for 23 hours per day for 2 years. The average actual dichlorvos concentrations were 0.05, 0.48 or 4.7 mg/m3 (equivalent to 0.05, 0.5 or 4.9 mg/kg b.w. per day3). Body-weight gain was reduced in the two highest dose groups. The NOAEL, based on erythrocyte acetyl choline esterase inhibition and reduced body weight gain, was 0.05 mg/m3 (Blair et al., 1976). Electron microscopy of the respiratory tract provided enhanced scrutiny of the site of contact and demonstrated an absence of effects. It should be noted that in this study the rats were not only exposed by inhalation but also via their food, drinking water and by grooming. This resulted in additional oral ingestion of dichlorvos (Stevenson and Blair, 1977).

Special study:

Dichlorvos at 8 and 16 mg/kg b.w. per day was administered by gavage to groups of 8-12 male F344 rats, either with or without leukaemia transplant, for 5 days a week. Other groups of rats were either not treated or given the leukaemia transplant only. At 70-days post-transplant, the animals were killed. The rats dosed with dichlorvos developed the disease earlier and the rate of tumour progression was increased. Three out of 16 transplant recipients dosed with 16 mg/kg b.w. per day died of leukaemia during the last week of dosing. The severity of the mononuclear cell leukaemia in the transplant recipients, as measured by histopathological examination of spleen and liver, was correlated with the changes in tumour growth rates. However, no dose-response was found for spleen weight and WBC count (Dieter et al., 1989).

Comments:

Dichlorvos has been evaluated for carcinogenicity in five long-term studies in mice and in six long-term studies in rats. The studies have been reported with sufficient details for an evaluation, although with some or major deviations from recent guidelines. The substance was administered via the diet, or drinking water, by oral gavage or by inhalation (one study in rats). Most of the studies provide no evidence of carcinogenicity and only in two gavage studies, one in F344/N rats and the other in B6C3F1 mice, both carried out by the Southern Research Institute and reported by the Chan (1989), was there some evidence of carcinogenic effects. In these experiments, increases in the incidence of mononuclear cell leukaemia in male rats, mammary fibroadenomas combined with adenomas in female rats, pancreatic acinar adenomas in male rats and forestomach tumours in male and female mice were observed. It is noted that there was no carcinogenic response in the drinking water study by Enomoto et al., 1981, where the same strain of rat (but from a different source) and a slightly higher dose was used.

3 value (mg/kg b.w. per day) = value (mg/l) x 45 l/kg/hour (rat respiration rate) x 23 hours (daily exposure in this rat study) x 1 (default value for respiratory absorption = 100%)



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Mononuclear cell leukaemia (MCL) is a spontaneously occurring neoplasm of splenic origin and is commonly diagnosed, albeit at highly variable rates, in aging laboratory rats (Moloney et al., 1969; Davey and Moloney, 1970). Its background control rate has been increasing over time. In the leukaemic phase, it metastasizes to the liver, lymph nodes and eventually to the bone marrow. The incidence of MCL in the male corn oil controls of the NTP study (Chan, 1989) with F344/N rat, 22% might be considered low for untreated rats of this strain, the mean historical control incidence having been reported to be 50.5% (range 32%-74%) (Haseman et al., 1998). However, the use of corn oil as vehicle reduces the incidence of MCL. The incidence of MCL in NTP studies using male F344 corn oil controls and performed concurrently with dichlorvos averaged 17.1% (range 2%-44%) and 8.8% (2%-18%) at Southern Research Institute, the laboratory where the experiment with dichlorvos was performed. Haseman also reported a mean incidence of MCL of 26% (range 10-46%) for corn oil gavage studies (Haseman et al., 1998). Therefore, it cannot be said that the control group incidence was particularly low in this experiment. Nevertheless, the incidence of this neoplasm is variable from experiment to experiment and because of this variability, any data relating to MCL must be interpreted cautiously. In this particular experiment, dichlorvos had no effect upon either the severity or progression of MCL in male rats, the increase was not dose-related and there was no increase in incidence in female rats (Manley et al., 1997). Furthermore, there was no significant increase in incidence in either male or female rats of the Enomoto (1981) study with the same strain of rat and in which slightly higher dose levels were used. Therefore, the increased incidence of MCL was of questionable biological significance. Moreover, there are no critical pathognomonic features in common between MCL in rats and leukaemia in humans and MCL in rats.

The reported incidence of mammary gland fibroadenomas and adenomas in female control rats of the NTP study (Chan, 1989) was low (18%) compared with that of historical controls in general at the time of the study (25.6%) and with the incidence for Southern Research Institute (28.3%). When mammary gland neoplasms were evaluated together, only the incidence of the low dose group remained statistically greater than that in the vehicle controls. This lack of a dose-response relationship in the absence of any survival difference weakens the biologic importance of this finding. Moreover, the increase of mammary gland fibroadenomas and adenomas in females was statistically significant only at p < 0.05. For common neoplasms, a better reflection of carcinogenic potential is given by p < 0.01 since the usual p < 0.05 level of significance does not take sufficient account of the likelihood of chance differences, due to the multiplicity of comparisons (Haseman, 1983; Haseman et al., 1986).

Male F344 rats are more sensitive to exocrine pancreas adenoma development than female F344 rats and corn oil gavage alone is reported to increase pancreatic acinar-cell tumours in F344 rats (M: 37.4%, F: 4.6% in corn oil controls compared to M: 14.5% and F: 1.0% in untreated controls) (Boorman et al., 1987; Haseman and Rao, 1992). Thus, the significant increase observed in the incidence of this tumour (p < 0.05) in male F344/N rats could be due to this effect of corn oil (although this effect should also have been expected in the corn oil vehicle control group). It is also noted that there was no significant increase in pancreatic tumours in female rats in this study or in male or female rats of any other rat studies with dichlorvos.

The only other tumours that could be considered as evidence for a carcinogenic effect of dichlorvos are the forestomach squamous cell papillomas in B6C3F1 mice after gavage administration of dichlorvos in corn oil. Two forestomach squamous cell carcinomas were also observed in high dose females. In rodents, the stomach is divided into two parts by the mucoepidermoid junction separating squamous from glandular epithelium. The proximal part, or forestomach, is non-glandular, continuous with the oesophagus, and lined by keratinized, stratified squamous epithelium. There is no forestomach in human beings. The forestomach is a storage organ colonized by microflora and subjected to changes in pH. These factors may have some role in the pathogenesis of tumours induced by specific agents. Chemicals given orally come into direct contact with the forestomach epithelium, sometimes at high concentrations and for prolonged periods. This retention is increased if the vehicle used is corn oil.



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In B6C3F1 mice squamous papillomas of the forestomach are relatively frequent: 13% in males, 1.5% in females, while squamous cell carcinomas are rare: 0.1% in males and 0% in females (Haseman et al., 1998). At Southern Research Institute, the historical control incidences for forestomach tumours in B6C3F1 mice dosed with corn oil by gavage were: 0.8% papillomas (range 3/49-0/50) and 0.3% carcinomas (range 1/49-0/50) in males and 1.0% papillomas (range 2/50-0/50) in females. It is noted that the female control group frequency of squamous cell papillomas in the critical NTP study (Chan, 1989) was 10.2%, almost 7-fold higher than the historical mean for the Program and 10-fold higher for the laboratory.

There was no support for this tumour response from the other studies performed either by gavage using water as vehicle or via the diet or drinking water. Administration by gavage in a corn oil vehicle delivers a very high local concentration of dichlorvos that would remain in the stomach for a longer time, in contrast to the lower local concentrations and shorter transit times resulting from dietary or drinking water vehicles.

There was no evidence of a neoplastic response in the forestomach of rats in the NTP study (Chan, 1989). Administration was also by gavage in corn oil, however, the doses had to be lower than in the mouse study because of systemic toxicity of dichlorvos.

In none of the mouse or rats studies, were hyperplastic lesions of the forestomach observed or increased in treated animals compared to controls.



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Table 1 Studies on Carcinogenicity of Dichlorvos

Strain Experimental Conditions

Observations Statistical significance

Reference

Mice C57Bl/6Bln Oral (gavage)

0.2 mg/mouse (equivalent to 10 mg/kg b.w.), either two or three times/week for 50 weeks surviving animals killed at 110 weeks

Not carcinogenic Horn et al., 1987

B6C3F1 Oral (gavage) M: 0, 10 or 20 mg/kg b.w./day F: 0, 20 or 40 mg/kg b.w./day 5 days/week, 103 weeks

Forestomach papillomas: M: 1/50, 1/50 and 5/50 F: 5/49, 6/49 and 18/50* (hist. control in corn oil vehicle NTP studies: M: 1% ± 2%, F: 0.9% ± 2%) Forestomach carcinomas: F: 0/49, 0/49 and 2/50 (hist. control: NTP: 0%)

trend * p<0.01 trend

Chan, 1989; Chan et al., 1991

B6C3F1 Oral (diet) 1000 or 2000 mg/kg for 2 weeks 300 or 600 mg/kg (actual: 318 or 635 mg/kg, equivalent to 48 or 95 mg/kg b.w./day) for 78 weeks surviving animals killed at 92-94 weeks

Not carcinogenic NCI, 1977; Weisburger, 1982

B6C3F1 Oral (drinking water) 0, 400 or 800 mg/l water (equal to 0, 58 and 95 mg/kg b.w./day in males and 0, 56 and 102 mg/kg b.w./day in females) surviving animals killed at week 102

Not carcinogenic Konishi et al., 1981; 1989

Rats BD IX/Bln Oral (gavage)

0.1 mg/rat (equivalent to 0.25 mg/kg b.w./day), two or three times/week for 60 weeks surviving animals killed at 111 weeks

Not carcinogenic Horn et al., 1988



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F344/N Oral (gavage) 0, 4 or 8 mg/kg b.w./day (actual doses: 0, 4.14 or 7.82 mg/kg b.w./d) 5 days/week, 103 weeks

Pancreatic adenomas: M: 25/50, 30/49* and 33/50* F: 2/50, 3/48 and 6/50 (hist. controls: laboratory: 9%, NTP: 6% (37%a)) Mononuclear cell leukaemia: M: 11/50, 20/50* and 21/50* F: 17/50, 21/50 and 23/50 (hist. controls: laboratory: 9% ± 7%, NTP: 15% ± 9%) Mammary gland fibroadenoma/adenomas: F: 9/50, 19/48* and 17/50* Mammary gland carcinoma: F: 2/50, 2/48 and 0/50 Total mammary neoplasms: F: 11/50, 20/48* and 17/50 (hist. controls: laboratory: 31%)

* p<0.05 * p<0.02 trend = 0.08 * p<0.05 trend * p<0.02 trend = 0.07

Chan 1989; Chan et al., 1991

CD Oral (diet) 0, 0.1, 1, 10, 100 or 500 mg/kg (0, 0.05, 0.5, 4.7, 47 or 230 mg/kg equivalent to 0, 0.0025, 0.025, 0.235, 2.35 or 11.5 mg/kg b.w./day) 2 years

Not carcinogenic Witherup et al., 1967

Osborne-Mendel

Oral (diet) 150 mg/kg (equivalent to 7.5 mg/kg b.w./day) : 80 weeks 1000 mg/kg for 3 weeks + 300 mg/kg (actual 326 mg/kg) (equivalent to 16.3 mg/kg b.w./day) for 77 weeks Surviving animals killed at 110-111 weeks

Not carcinogenic NCI, 1977; Weisburger, 1982

F344 Oral (drinking water) 0, 140 or 280 mg/l water (equal to M: 0, 8.3 or 18 mg/kg b.w./day, F: 0, 10 or 22 mg/kg b.w./day) exposure 104 weeks surviving animals killed at 108 weeks

Not carcinogenic Enomoto et al., , 1981; Enomoto et al., , 1989

CFE Inhalation (whole body) 23 hours/day, 2 Not carcinogenic Blair et al.,



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years 0, 0.05, 0.5 or 5 mg/m3 (actual: 0, 0.05, 0.48 or 4.7 mg/m3) (equivalent to 0.05, 0.5 or 4.9 mg/kg b.w./day)

1974; 1976; Stevenson and Blair, 1977

a : retrospective evaluation NTP studies, corn oil gavage (Eustis and Boorman, 1985)



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1.4.3 Non-Genetic Effects of Dichlorvos on Forestomach Tissue 5 In the 1989 NTP-study (Chan, 1989) evidence was obtained that dichlorvos is carcinogenic to mouse forestomach. In separate studies attempts have been made to determine whether, apart from a high local dose, gavage administration produces epigenetic effects that would have contributed to the localization of dichlorvos induced tumours (Benford, 1992; 1993, Benford et al., 1994). Since local tissue irritation upon repeated gavage administration could 10 promote cell repair and cell proliferation and may contribute to the development of local cancer in forestomachs of mice, B6C3F1 mice received single doses of 10, 20, 40, and 100 mg/kg b.w. dichlorvos (in corn oil, volume 10 ml/kg body weight) by gastric gavage and were killed 2 and 4 hours later for evaluation of unscheduled DNA synthesis (UDS), at 6, 8, 10, 12 hours later for replicative DNA synthesis (RDS) and 48 hours later for evaluation of hyperplasia. Dichlorvos 15 did not induce UDS but induced RDS and subsequent hyperplasia in the forestomach tissues (Benford et al., 1994). RDS of forestomach epithelial cells was observed in male and female mice, but not in a dose related fashion. Focal hyperplasia and cell hypertrophy were observed in male and female mice with a more marked response in female mice. There was diffuse hyperplasia in the female forestomach at the highest dose of 100 mg/kg b.w. at which dose 20 three animals died. It was concluded that the absence of UDS indicates non-genotoxicity of dichlorvos in the experiments. A control alkylating compound, 1-methyl-3-nitro-1-nitrosoguanidine (MNNG), but not the non-genotoxic forestomach carcinogen butylhydroxyanisole (BHA) induced UDS.

Comment: 25 These studies were performed with only a single dosing of dichlorvos which treatment was negative in studies of alkylation and frequency of λLacZ mutations in MutaMouse (Pletsa et al., 1999a).The results might have been different had multiple doses been administered, as in other studies on the local genotoxicity of dichlorvos in mice. Nevertheless, these studies do indicate that dichlorvos has only very weak, if any, genotoxicity in vivo and that local high doses 30 can cause significant local irritation leading to increases in cell division. The lack of a dose-response in these studies, however, does give rise to some uncertainty as to the reliability of the findings.

RDS and UDS were measured by the same autoradiographic method based on the incorporation of radioactive thymidine into the cell nuclei. UDS, as an indicator of DNA repair 35 and genotoxicity, however, is overlayed in a replicative tissue by DNA synthesis arising from cell proliferation. Minimal increases in the proportions of cells entering the S-phase would be indistinguishable from a large proportion of cells undergoing repair. The conflicting influence of changes in S-phase synthesis is a particular problem when it is essential to distinguish between genotoxic and non-genotoxic mechanisms, as the latter commonly involves increased cell 40 turnover. Normally PCNA (proliferating cell nuclei antigen) staining is used to indicate proliferating cells entering the S-phase. This was not performed here. Instead the authors have used a different method to detect UDS in cell nuclei of proliferating tissue and performed counting in tissue sections prepared early (4 hours) after gavage administration. Such tissue sections, however, show thymidine incorporation only at the respective section level, whereas in 45 the normal UDS-test with non-proliferating hepatocytes the nucleus itself is scored.

1.5 Chemical Reactivity of Dichlorvos

1.5.1 Protein Phosphorylation

Based on its acute oral and dermal toxicity to rats the IPCS (1992) has ranked dichlorvos to be a “highly hazardous” organophosphate (OP) as the LD50 values are between 20 – 200 mg/kg b.w. 50 for a liquid OP, namely oral LD50 is 80 mg/kg b.w. male rat and 55 mg/kg female rat; dermal LD50 is 107 mg/kg b.w. male rat and 75 mg/kg b.w. female rat. Oral LD50 in male and female mice is 135-148 mg/kg b.w.) (Chan, 1989). All acute neurotoxicity (and lethality) of dichlorvos relates to the phosphorylation of neuronal acetylcholine esterase. The reaction follows phosphorylation of type B-esterases (also typed serine-esterases) on which a phenomenon 55



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occurs called aging, involving the non-enzymatic loss of an alkyl group from the OP leading to the formation of a negatively charged irreversibly phosphorylated and functionally blocked enzyme (Lotti, 2000). Apart from this reaction, dichlorvos is bound by A-esterases (non-serine esterases, arylesterases) which hydrolyse OPs and thereby are involved in dichlorvos detoxification. These enzymes occur 60 preferentially in blood, but also in liver and many other tissues (Wright et al., 1979; Lotti, 2000).

1.5.2 Alkylating Activity of Dichlorvos: General Considerations

Alkylating agents may initiate cancer by alkylation of purine and pyrimidine bases. Alkylation imposes a genetic risk as it may cause base excision, single strand breaks, base mutation, base pair change, frame shift mutation or other disturbances of the genetic matter. 65 Dichlorvos is an alkylating agent as identified by the NBP-test (NBP= 4-(4-nitro-benzyl) pyridine test) (Bedford and Robinson, 1972). In this colourimetric test dichlorvos gave a positive result, the reaction being about 1/3 that of the known alkylating compound methyl methanesulfonate (MMS). The PPR panel identified several reports, some quite old, on the alkylating activity of dichlorvos in vitro and in vivo (Löfroth, 1970; 1978; Dicowsky and Morello, 1971; Page et al., 70 1972; Bridges et al. 1973; Lawley et al., 1974; Wennerberg and Löfroth, 1974; Wooder et al., 1977; Segerbäck, 1981; Segerbäck and Ehrenberg, 1981; Ramel et al., 1980; Pletsa et al., 1999a). Data from the in vivo studies are considered in detail in chapter 1.7.2.

Dichlorvos methylates nucleic bases at the N-7 atom of guanine and only weakly at O-6, the latter mutation playing an important role in carcinogenicity (Barbin and Bartsch, 1989), as this 75 leads to a directly miscoding lesion. Strongly mutagenic and carcinogenic methylating agents, e.g. nitrosomethyl compounds, generate an O-6-meG / N-7-meG ratio of approximately 1 : 11 (Pletsa et al., 1999b). The ratio of O-6- versus N-7 methylguanines produced by dichlorvos is about 1:250 (Lawley et al., 1974). The methylation pattern indicates that dichlorvos alkylates by the SN2 (substitution nucleophil bimolecular) mechanism. This type of reaction means that 80 the reaction rate depends of the concentration of both partners, the methylating agent and the nucleophile (e.g. guanine).

Methyl methanesulfonate (MMS) methylates by the same reaction type but with much greater potency. This compound has been used as a marker compound to define genetic potency of dichlorvos (Fahrig, 1973). With MMS the ratio between O-6- versus N-7 methylguanines is also 85 1 : 250. However, because MMS is a stronger alkylating (and mutagenic) agent, it alkylates proteins to a similar extent as DNA (Segerbäck and Ehrenberg, 1981), whereas dichlorvos alkylates proteins 30 times more than DNA (Wennerberg and Löfroth, 1974). With MMS the relative amount of O-6-methylguanine product of total methylated DNA products was 0.33 % in salmon sperm DNA (Lawley and Shah, 1972). 90 A general reaction scheme showing alkylation and phosphorylation of a nucleophilic macromolecule Yi- with dichlorvos is given by Segerbäck and Ehrenberg (1981) (Fig. 2) :



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Figure 2 95 100 105

Yi- = nucleophilic macromolecule; MeYi = methylated macromolecule

Reaction 1 shows the methylation reaction, yielding a methylated macromolecule and 110 demethyldichlorvos. The reaction was quantitatively determined from incorporation of radiolabelled methyl-groups arising from [Me-14C]-dichlorvos and also from [Me-3H]-dichlorvos into DNA, RNA, and proteins of E. coli after a 4 hours incubation (Wennerberg and Löfroth, 1974). A total of 136 µCi [Me-14C]-dichlorvos (3.4 µCi/ml; 1.1 mmol/L) was applied to the bacteria. At the end 3 mg DNA and 2.6 mg RNA were isolated. After 4 hours the DNA contained 115 44 x 10-6 µCi/mg DNA (almost all as N-7-methylguanine) and a similar amount of radiolabelled RNA (46 x 10-6 µCi/mg RNA) almost equally distributed between N-7-methylguanine (60%) and N-7-methyladenine (40%) (Wennerberg and Löfroth, 1974). Thus about 10-6 of compound-derived radioactivity was found in each nucleic acid fraction indicating that incorporation of the methyl group into bacterial DNA and RNA was very low (see same study given in chapter 120 1.6.2.1, study 1a, for methylation of mammalian DNA).

The second reaction (reaction 2), a dimethylphosphorylation of the nucleophile, tends to decrease the methylation reaction (reaction 1) by competition (Segerbäck and Ehrenberg, 1981). This means that in the presence of proteins there will be less methylation of DNA or RNA. This is reflected by the finding that in the above described experiments on E. coli the 125 dimethylphosphorylation of proteins by dichlorvos, as measured using [3H]methyl-dichlorvos, was 30 times greater than the methylation of DNA at the end of a 4 hours incubation period (Wennerberg and Löfroth, 1974). 36-times more protein labelling than DNA-methylation was also observed in HeLa cells after 1 hour incubation (see Tab. 4, Addendum) (Lawley et al., 1974). 130 1.6. Alkylation of Nucleic Acid Bases by Dichlorvos

1.6.1 In Vitro Alkylation

The methylation of mammalian DNA, RNA and proteins in different systems (HeLa cells, calf thymus DNA) was determined in vitro (Bridges et al., 1973; Fahrig, 1973; Lawley et al., 1974; Wright et al., 1979; Segerbäck, 1981; Ramel et al., 1980). 135 The conclusions from these studies were:

• There is comparable target DNA sensitivity in calf thymus DNA, salmon sperm DNA, HeLa cell and E. coli DNA, as the rate constants of the methylation reaction on N-7 guanine residues are rather similar (Addendum table 3).

• The rate constants for DNA alkylation by dichlorvos are up to 100 times less, if compared 140 with those of the strongly genotoxic methylating agent methyl methanesulfonate (MMS)

CCl2CHOP

OMe

OMe

O

CCl2CHOPOMe

O

O

OCH CCl2-

CHCl2C

H

O

H+

+Yi -

+Yi -

MeYi +

+

1)

2)POMe

O

OMe

Y i



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(Addendum table 3). E.g. the methylation of DNA of intact HeLa cells by dichlorvos proceeds at a rate some 50 times slower than that obtained with MMS.

• According to the reaction equation given by Segerbäck and Ehrenberg (1974) 145

[RYI ] D = dose, ki = rate constant of alkylation

_______ = ki x D [RYi] = concentration of alkylated nucleophile Yi

[Yi -] [Yi -] = concentration of nucleophilic centres R = alkyl group from alkylating compound 150

the alkylation ratio is proportional to dose (a product of rate constant and dose). Thus, the same degree of alkylation as with MMS is achieved with up to 100 times higher dose of dichlorvos. This is only achievable in vitro and with great difficulty in the presence of ADME processes. 155

• The rate of N-7 guanine N-7 methylation for DNA of HeLa cells and salmon sperm by high concentrations of dichlorvos (2.9 and 14 mmol/l, respectively) was 2-3 x 10-4 mol/l/min (JMPR 1993) and thus nine orders of magnitude lower than the rate of reaction with AChE (a phosphorylation reaction) which was 1.5 x 105 mol/l/min at 37°C (Skrinjaric-Spoljar et al., 1973). Assuming that these are also the reaction rates that occur in vivo this means 160 that signs of acute toxicity should occur well before there is any appreciable DNA methylation (but see in Comments and Recommendations of question 1).

• Dichlorvos labels proteins in bacteria at up to 30 fold the rate of any nucleic acid base methylation (up to 20 fold, if DNA and RNA alkylation are considered together) (Lawley et al., 1974) (Addendum table 4). This is a major difference to other alkylating agents, e.g. 165 MMS.

1.6.2 In Vivo Alkylation

The PPR panel identified four (three older and one from 1999) published papers on in vivo alkylation of dichlorvos (Wennerberg and Löfroth, 1974; Wooder et al., 1977; Segerbäck, 1981; Pletsa et al., 1999a). 170 Study 1, Wennerberg and Löfroth (1974): In this study the urinary excretion of radiolabelled 7-methylguanine in mice exposed by a) intraperitoneal administration and b) inhalation of radiolabelled dichlorvos was investigated. Almost 70% of the administered radioactivity was excreted in the urine within 24 hours after i.p. injection.

a) Determination of radiolabelled dichlorvos after intraperitoneal administration: [Me14C]-175 dichlorvos, specific radioactivity 0.7 mCi/mmol, and dissolved in 0.5 to 1.0 ml corn oil was administered by intraperitoneal injection to four 3 to 5 month-old NMRI mice. Total radioactivity dose was 42 to 90 µCi per animal corresponding to 13.26 mg to 28.4 mg dichlorvos per mouse. Peaks of radioactivity amounted to 30 cpm/ml in the effluent of column separated DNA-hydrolysates. About 60% of the total dose (28 to 45 µCi) was excreted in urine in a 180 nonbound form within 24 hours (time period 0 – 24 hours) and only low amounts (2.2% to 4.2%) were excreted over the next 24 hours (time period 24 – 48 hours). Traces of radioactivity, namely 0.58 – 0.94 nCi, were detected in urine as N-7-methylguanine within 24 hours. The N-7-methylguanine fraction amounted to 3 to 4 x 10-5 of the total dose excreted after 48 hours.

b) Determination after inhalation exposure: Total exposure dose by inhalation of [Me14C-185 dichlorvos] was 8.5 µCi (2.6 mg/mouse) to 11 µCi (3.47 mg/mouse) for a time period of 2 hours In this experiment the 7-methylguanine fraction in urine collected over 48 hours was 10 x 10-5 to 14 x 10-5 of the total radioactivity (dose) applied and thus was three to four times higher than after i.p. injection although exposed to lower doses.

Study 2, Segerbäck (1981): In this study the alkylation reaction of dichlorvos was measured in 190 vivo in mice given labelled dichlorvos by intraperitoneal injection. DNA was isolated from soft tissues and incorporation of the [14C]-methyl group into N-7-methylguanine was determined. Only total DNA pooled from various soft tissues was analysed and no differentiation of



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methylation products in individual tissues was obtained. In addition to the in vivo experiments, the rate constant of in vitro methylation of calf-thymus DNA was determined in order to 195 estimate mean retention time in mouse body.

[Methyl-14C]-radiolabelled dichlorvos (99% purity, spec. radioact. 22 mCi/mmol) was administered intraperitoneally to twelve male CBA-mice at a dose of 0.21 mCi/animal (corresponding to 1.9 µmol/kg b.w. corresponding to 0.42 mg/kg b.w.). When measured 5 hours later the amount of guanine N-7 alkylation in pooled DNA isolated from liver, spleen, 200 lung, testes, kidney, brain, and heart, was 8 x 10-13 mol methyl/g DNA which was equal to 7 dpm/70 mg DNA.

Study 3, Wooder et al., (1977): This study was performed to estimate the alkylation of DNA after inhalation of dichlorvos. The limit of detection was one methyl group per 6 x 1011 DNA nucleotide units, equivalent to ca. 0.000001 -% of the administered dose (Wright et al., 1979). 205 The inhalation study was performed with 20 male rats exposed to atmospheres containing 0.064 mg/m3 (0.007 mg/kg, named “practical use concentration”) [Me 14C]-dichlorvos (spec. radioact. 113 mCi/mmol) for 12 hours giving a total inhaled dose of 6 µg/rat (ca. 0.03 mg/kg b.w.). Two replicate experiments were performed in duplicate with five animals in each. At the end of the experiments DNA, RNA, and protein were purified and 7-methylguanine was isolated 210 from DNA and RNA hydrolysates. Urine was collected from animals of the first experiment. Although in both experiments traces of radioactivity in DNA from liver (Exp. 1:0.21 dpm/mg DNA; Exp. 2:0.225 dpm/mg DNA), testis (0.83; 0.79), heart/lung (1.59; 1.61), brain (2.32; 2.2), spleen (9.88; 7.5), and kidney (8.84; 7.8) were detected, no 7-methylguanine was observed in soft tissues (stomach was not analyzed) and also not in the urine collected over 12 hours. The 215 highest specific radioactivity was incorporated into the protein fraction obtained in one experiment from heart/lung (20.8 dpm/mg protein), followed by liver (6.78 dpm/mg) with equal distribution in the protein fractions of spleen, kidney, and testis (3.57, 3.60, 3.32 dpm/mg) and less in brain (1.93 dpm/mg).

In an attempt to validate this study on dichlorvos, experiments were performed with identical 220 conditions and dose on ten rats with radiolabelled [14C]methyl methanesulfonate, 0.065 mg/m3, 12 hours exposure (Wright et al., 1979). The amount of radioactivity measured in the 7-methylguanine fraction from total soft tissue DNA was 678 dpm/441 cpm in 301 mg DNA which contrasted to a non-detectable radioactivity count in the case of dichlorvos.

Study 4, Pletsa et al., (1999a): The study revealed a genotoxic potential of multiple doses of 225 dichlorvos in a transgenic indicator mouse (λlacZ transgenic mouse, MutaTMMouse), but failed to detect somatic methylated DNA adducts following a single dose of the compound. The detection limits for adducts were 5 x 10-6 N-7 meG mol/mol G and 8 x 10-8 mol O-6-meG mol G.

Dichlorvos, dissolved in phosphate buffer, was administered intraperitoneally at a dosage of 4.4 or 11 mg/kg b.w. to 10 weeks old male λlacZ transgenic mice. Either a single dose (4.4 230 mg/kg b.w. and 11 mg/kg b.w.) or multiple doses (11 mg/kg b.w. on day 1 and 2, and three further treatments at intervals of 7 days) were given. As a reference the SN2 methylating agent dimethylsulphate (DMS) was administered i.p. by single dose (30 mg/kg b.w.) or multiple doses (10 x 6 mg/kg b.w.). DNA extraction was performed 4 hours (dichlorvos) or 1.5 hours (DMS) after the final dose. For analysis of mutation frequency (MF), animals were killed 14 235 (dichlorvos) and 15 (DMS) days after the last treatment.

a) Single treatment: No adducts were detected after single dosage in any tissue (liver, bone marrow, white blood cells, spleen, lung, brain, and sperm cells) at either dose of dichlorvos. Also no change in MF was observed in dichlorvos or in DMS treated transgenic mice after a single dose. However, in mice treated with a single dose of DMS, O-6- and N-7 meG adducts 240 were observed at the fmol/µg DNA level in liver and bone marrow but not in lung indicating adduct analysis to be more sensitive than MF analysis.

b) Multiple treatments: Adduct formation was not analysed after multiple treatments with dichlorvos. However, MF of λlacZ-mutant phage DNA increased significantly in liver (145 ± 6.2



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mutations per 106 plaque forming units (P.f.u)) versus controls (49.7 ± 8.7) but MF was not 245 significantly affected in bone marrow (73.3 ± 32.5 after dichlorvos versus 38.0 ± 20.4 in controls). Surprisingly, no change in mutation frequency occurred after multiple doses of DMS, but O-6- and N-7 meG adducts were again observed at the fmol/µg DNA level, albeit at a lower level than after a single dose.

Comments: 250 The first three studies indicate an extremely low alkylation capacity of dichlorvos in several organs of mice after systemic exposure via single intraperitoneal dose and also after inhalation. Whether this amount of DNA-binding would produce detectable genotoxic activity is uncertain. It is not known whether the incorporation of radioactivity was dose-dependent (single dose studies), but application by inhalation over short time periods at a lower dose increased DNA 255 methylation compared with a higher dose by bolus i.p. administration.

The fourth study has several weaknesses and inconsistencies, namely a) DNA-adducts were analysed only after single but not after multiple dosing (after single dosing MF was not changed with dichlorvos). This is surprising since after multiple dichlorvos dosing the MF changed, which was a less sensitive marker than DNA-methylation. One would expect, if MF is positive also DNA 260 alkylation occurs. b) Background mutation frequency data for bone marrow varied appreciably between animals (from 40 to 105 MF per 106 p.f.u.) and therefore the SD was up to 50% of the mean value. This means that the study had low power to detect changes in this tissue and casts doubt on the reliability of negative data, at least in certain tissues. c) The treatments with the reference compound DMS, which was positive for DNA adduct formation and negative for 265 change in MF, were very different from those with dichlorvos: the single dose was higher, namely 30 mg/kg b.w. DMS versus 11 mg/kg b.w. dichlorvos; for multiple dosing, ten daily doses of DMS at 6 mg/kg b.w. were given without any lag period between doses, whereas with dichlorvos only five doses of 11 mg/kg b.w. were given, with a gap of 7 days between each of the final three doses, which could allow some repair of alkylated DNA. These inconsistencies 270 weaken the value of DMS as a positive control. d) O-6- and N-7 meG adducts did not increase on multiple dosing with DMS versus with a single dose perhaps indicating repair induction over several days. e) Administered doses were 50% of LD50 for dichlorvos and DMS and thus above MTD level.

Although the MutaTMMouse study indicates that dichlorvos has some in vivo mutagenic activity, 275 it cannot be used to indicate that this occurs in the absence of O-6- and N-7 meG adducts, i.e. by a non-alkylating mode of action, because no alkylation measurements were made when the positive MF result was obtained, i.e. after multiple dosing. With respect to DMS, the study showed that adduct formation could occur without any detectable change in mutation frequency (after a single dose). 280 The results of all in vivo alkylation studies are summarised in table 5 (Addendum).

1.6.3 Summary of Direct DNA-Interactions of Dichlorvos

Sufficient evidence is present in the literature to demonstrate that dichlorvos can alkylate nucleic acid bases in DNA and RNA and amino acids in proteins from bacteria and mammalian cells in vitro, although it is of low potency. Methylation of mammalian DNA in vivo when the 285 normal ADME processes would be operative, was reported in mice only after high doses, was very low and was very largely, or perhaps even exclusively, in the N-7-methyl guanine fraction (3-4 x 10-5 of the dose). Thus, the genotoxic potency of dichlorvos in vivo, via systemic exposure is very low. The methylation reaction exhibits the time course and type of products typical of the SN2 mechanism, yielding a ratio of N-7-methyl guanine/ O-6-methyl guanine of 250:1. Under 290 realistic, “practical”, exposure scenarios methylation of DNA could not be detected in mice. Considering the rapid elimination, the type of metabolism, and the high sensitivity to AChE inhibition and the low alkylating potential in vivo the substantial concentrations necessary to cause DNA-modification in vivo in systemically exposed tissues are extremely unlikely to occur because cholinesterase inhibition (NOAEL 0.5 mg/kg b.w. per day in rat) almost entirely limits 295 any such possibility.



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1.7 Studies on Mutagenicity

1.7.1 In Vitro Mutagenicity

The PPR-Panel confirmed and agreed with the statement of the UK Committee on Mutagenicity (COM, 2002) which regarded dichlorvos as a weak methylating agent (compared to methyl 300 methanesulphonate MMS) which “is in-vitro mutagenic, both in the presence and absence of exogenous metabolism to bacteria, yeast cells and in mammalian cell gene mutation assays, chromosome aberrations, the in-vitro micronucleus test and sister chromatid exchange assays”. This statement took account of 107 in vitro mutagenicity studies which are given in the COM-document and are not listed here (COM, 2002). As no further documents were made 305 available to the Panel members the in vitro mutagenicity of dichlorvos in all organisms yet tested (mammalian cell, insects, fungi, and bacteria) was accepted.

1.7.2 In Vivo Mutagenicity

The PPR-Panel identified 28 in vivo studies on the genetic effects of dichlorvos with various 310 endpoints. Six of them reported positive findings. All studies are listed in Table 2.



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Table 2: Results of studies of in vivo genotoxicity conducted with dichlorvos

Endpoint Test object Concentration, route of exposure, harvesting time

Purity (%) Results

Reference

Host-mediated assay Salmonella typhimurium G46 His- in mice (NMRI)

25 mg/kg b.w. s.c. ? - Buselmaier et al., 1972

Salmonella typhimurium (64-320) in mice (Swiss)

8 –10 mg/kg b.w. p.o. 97.5 - Voogd et al., 1972

Serratia marcescens HY (a 21 Leu-) in mice (NMRI)

25 mg/kg b.w. s.c. ? - Buselmaier et al., 1972

Saccharomyces cerevisiae (D4) in mice (CF1)

50 or 100 mg/kg b.w. p.o.

> 97 - Dean et al., 1972

60 or 99 mg/m3, inhal. ? - Dean et al., 1972 Gene mutation assay λ lac Z transgenic

mouse (MutaTMMouse) liver and BM liver BM

11 mg/kg b.w. x 1 i.p. 11 mg/kg b.w. x 5 (over period of 21 days) i.p. 11 mg/kg b.w. x 5 (over period of 21 days) i.p.

?

- + -

Pletsa et al., 1999a

Unscheduled DNA synthesis

Male rats (F344) hepatocytes

0, 2, 10, 35 mg/kg b.w. p.o. sacrifice 2 hours and 12 hours after exposure

? - Mirsalis et al., 1989



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Mice (B6C3F1) forestomach

0, 10, 20, 40 or 100 mg/kg b.w. p.o.

99.8 - Benford, 1993°

Mice (B6C3F1) Forestomach

40, 100 mg/kg b.w., p.o., sacrifice after 2 hours 10, 20, 40, 100 mg/kg b.w., p.o., sacrifice after 4 hours

99.8 - Benford et al., 1994

Sister chromatid exchange

Male mice (B6C3F1) peripheral lymphocytes

5-35 mg/kg b.w. i.p. 99 - Kligerman et al., 1985

Male mice (B6C3F1) BM cells

6.25, 12.5 or 25 mg/kg b.w. i.p.(in PBS) 10, 20 or 40 mg/kg b.w. i.p. (in corn oil)

? - Chan, 1989

Mice (B6C3F1) BM cells

3, 10 or 30 mg/kg b.w. i.p.

98.4 - Ford, et al., 1985a (Task Force)

DNA strand breaks

Rats (Wistar), liver cells

10 mg/kg b.w. i.p. 99.8% - Wooder and Creedy, 1979

Comet assay

Mice No data provided ? + on several tissues Sasaki et al., , 2000

Micronucleus test Mice (Swiss Webster) BM cells

0.0075-0.015 mg/kg b.w./day i.p., for 2 or 4 days

? - (0.03 mg/kg b.w. was lethal)

Paik and Lee, 1977

Male mice (CD1), BM cells

1/8, 1/16 or 1/32 dermal LD50, 50 µl topical (0.051 ml/cm2) in DMSO

? - Schop et al., 1990°

Mice (CD1) 4, 13 or 40 mg/kg b.w., i.p., 2 days

98.4 - Ford, et al., 1985b (Task Force)

ex vivo Mice (HRA/Skh, hairless) skin keratinocytes

Skin painting with 51-1033 nmol (in 100 µl acetone) (11-228 µg)

tech. Grade + Tungul et al., 1991

Nuclear aberrations* Male mice (CD1), hair follicle

1/8, 1/16 or 1/32 dermal LD50, 50 µl

? + at 1/8 LD50 (88.4 µmol/kg b.w.)

Schop et al., 1990



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topical (0.051 ml/cm2) in DMSO

Chromosomal aberrations

Chinese hamster BM cells

10 mg/kg b.w. x 2, p.o. 15 mg/kg b.w. x 1 p.o.

>97 - Dean and Thorpe, 1972a

Male mice (Q), BM cells

2 mg/kg (0.32 mg/kg b.w./day) in drinking water, 5 day/week for 7 weeks

99 - Moutschen-Dahmen et al., , 1981 Degraeve et al., 1984a

10 mg/kg b.w. i.p. 99 - Moutschen-Dahmen et al., 1981 Degraeve et al., 1984b

Male mice (B6C3F1), BM cells

6.25, 12.5 or 25 mg/kg b.w. i.p.(in PBS) 10, 20 or 40 mg/kg b.w. i.p. (in corn oil)

? - Chan, 1989

Female Syrian golden hamster, BM cells

0, 3, 6, 15 or 30 mg/kg b.w. i.p. (LD50: 30 mg/kg b.w.)

50 (commercial formulation)

+ (only if gaps included, not dose-related)

Dzwonkowska and Hübner, 1986°

Mice (CF1), BM cells 64-72 mg/m3, 16 hours inhal.

> 97 - Dean and Thorpe, 1972a

5 mg/m3, 23 hours/day for 21 days


Male Chinese hamster, BM cells

(28-36) 32 mg/m3, 16 hours inhal.


Mice, BM cells 100 mg/kg b.w. p.o. 10 mg/kg b.w. i.p.

? - -

Kurinnyi, 1975

Rat, BM cells 0.97, 1.29, 1.94 mg/kg b.w. p.o., 5 days per week, 6 weeks

? - structural aberrations + numerical aberrations (not dose-rel.)

Nehéz et al., 1994

Mice (Q) spermatocytes

2 mg/kg (0.32 mg/kg b.w./d) in drinking water, 5 day/week for 7 weeks

99 - Moutschen-Dahmen et al., 1981 Degraeve et al., 1984a

Chinese hamster 15 mg/kg b.w. p.o. > 97 - Dean and Thorpe,



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spermatocytes 1972a Mice (Q)

spermatocytes 10 mg/kg b.w. i.p. 99 - Moutschen-Dahmen et

al., 1981 Degraeve et al., 1984b

Mice (CF1) spermatocytes

64-72 mg/m3, 16 hours inhal.


5 mg/m3, 23 hours/days for 21 days


Chinese hamster spermatocytes

(28-36) 32 mg/m3, 16 hours inhal.


Mice (CF1) spermatogonia

2 mg/kg (0.32 mg/kg b.w./day) in drinking water, 5 day/week for 7 weeks

99 - Moutschen-Dahmen et al., 1981 Degraeve et al., 1984a

10 mg/kg b.w. i.p. 99 - Moutschen-Dahmen et al., 1981 Degraeve et al., 1984b

Dominant lethal Female mice (CF1) 0, 25 or 50 mg/kg b.w. p.o.

> 97 - Dean and Blair, 1976

0, 2 or 8 mg/m3 inhalation, 8 weeks

> 97 - Dean and Blair, 1976

Male mice (ICR/Ha Swiss)

0, 5 or 10 mg/kg b.w./day p.o. for 5 days (8 weeks of mating)

? - Epstein et al., 1972

13 or 16.5 mg/kg b.w. i.p. (8 weeks of mating)

? - Epstein et al., 1972

Male mice (Q) 2 mg/kg (0.32 mg/kg b.w.) in drinking water, 5 d/wk for 7 weeks

99 - Degraeve et al., 1984a

10 mg/kg b.w. i.p. 99 - Moutschen-Dahmen et al., 1981

Male mice (CF1) 30 or 55 mg/m3, 16 > 97 - Dean and Thorpe,



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hours inhal. 1972b 2.1 or 5.8 mg/m3, 23

hours/day inhal., 4 weeks

> 97 - Dean and Thorpe, 1972b

Male mice (CD1) 1, 3 or 10 mg/kg b.w. i.p., 5 days

98.4 - Ford, et al., 1985c (Task Force)

Male mice (CD1) 8, 16 or 32 mg/kg b.w., i.p., 5 days

97.5 - Ford and Killeen, 1987 (Task Force)

* Nuclear aberrations: pyknotic and karyorrhectic nuclei, along with chromatin-containing apoptotic bodies and vacuolated, phagocytosed nuclear fragments. ? unknown; no data reported. 315

Data from some original reports (task force), some publications, IPCS (1989), Chan (1989), JMPR (1993), DAR report (2003; 2005).



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1.7.3 Summary on In Vivo Mutagenicity of Dichlorvos

The majority of test systems for in vivo mutagenicity/genotoxicity yielded negative results:

• Dichlorvos was negative in all four studies with host-mediated mutation assays in mice on Salmonella typhimurium, Serratia marcescens and Saccharomyces cerevisiae.

• Dichlorvos was negative in all three studies on unscheduled DNA synthesis. • Dichlorvos was negative in all three studies on sister chromatid exchange and one other

test on DNA-strand breaks. • Dichlorvos was negative in three of four studies using micronuclei tests. • Dichlorvos was negative in all three tests on chromosomal aberrations in spermatocytes

and in five of seven studies of chromosomal aberrations in bone marrow cells.

The reported positive results were:

• Dichlorvos was positive in a gene mutation assay on MutaTMMouse (study 1, see below). • Dichlorvos was positive in a comet assay with unknown experimental design (study 2). • Dichlorvos was positive in a local skin micronucleus assay with ex vivo analysis (study 3). • Dichlorvos was positive in a nuclear aberration test (study 4). • Dichlorvos was weakly positive in two studies on chromosomal aberrations in bone marrow

(study 5 and 6).

Study 1, Pletsa et al. (1999a): This study made use of λlacZ transgenic mice to which dichlorvos was administered by i.p. either once at a dose of 4.4 or 11 mg/kg b.w. (0.5 x LD50) or on five occasions at 11 mg/kg b.w. with an interval of 24 hours between the first two and then 7 days between each for the remaining doses. The mice were killed after 4 hours or 14 days after the end of dosing and were examined for DNA adducts or mutant frequency, respectively (see description of study 4 in 1.4.2.2). Following multiple dosing there was a significant three fold increase in mutant frequency (MF) in liver but not in bone marrow. Single dosing had no effect on MF. The effect on the liver could have been a consequence of the local high exposure of that tissue and hence equivalent to a site of contact effect.

Study 2, Sasaki et al., (2000): This paper reviews the results of 208 different chemicals, lacking description of the particular experiments with dichlorvos. Another drawback of this study is the lack of reporting of any cellular measure of cytotoxicity or apoptosis for the Comet assay study with dichlorvos. Mice were dosed with a 100 mg/kg b.w. dose (ca. 80 % of LD50) and tissues sampled 3 and 24 hours later for assessment of DNA damage in isolated nuclei by single-cell electrophoresis (Comet technique). Positive results were obtained in many tissues. In view of the limitation of the study and report, little weight could be placed on this study.

Study 3, Tungul et al., (1991): In this study dichlorvos was applied once locally on dorsal skin of 5-6 week old male HRA/skh hairless mice at dose levels of 0, 51, 258, 516, and 1033 nmol (0, 0.011, 0.057, 0.11, and 0.23 mg) in 0.1 ml acetone per mouse. 48 hours later skin keratinocytes were isolated and cultured for 4 days, then assessed for micronuclei (MN). In a dose-finding experiment 5165 nmol (1.14 mg/mouse), which adversely affected cell recovery, was topically applied and skin harvested after 1, 6, 12, 48 and 72 h. A significant increase in MN was detectable in cultured cells taken from whole skin as early as 1 hour after application of this high dose and there was also a dose-related increase with all of the lower doses after 48 hours.

Study 4, Schop et al., 1990: The in vivo genotoxicity of dichlorvos was assayed in the bone marrow nucleus test (BM) and the hair follicle nuclear aberration test (NA) in CD1 mice after topical application of dichlorvos at doses of 1/8, 1/16 and 1/32 of LD50-dose in DMSO solution. The treated skin area was 10 x 10 mm. The animals were killed 24 hours after treatment. Only at the highest dose of dichlorvos was an increase in the NA incidence observed, whereas the BM-assay was negative at all doses. At the highest dose the activity of serum cholinesterase was reduced to 70 ± 4.8 %.



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Comment on study 4: The endpoints of the nuclear aberration assay are pyknotic and karyrorrhectic nuclei along with chromatin-condensed apoptotic bodies and vacuolated, phagocytosed nuclear fragments (Schop et al., 1990). As such stages are all seen during apoptosis this assay should be used with caution if used to assess genotoxicity at high doses. In this study the NA test was positive with two known chemical carcinogens, N-methyl-N-nitrosurea and cyclophosphamide, both direct-acting alkylating agents, however, the latter requires metabolic activation. Both compounds increased the incidence of NA in a dose-dependent manner, whereas BM changed only with the highest dose of cyclophosphamide. Although this test is not considered to be a definitive genotoxicity assay, the results might be indicative of a biological effect in the skin following local exposure.

Study 5, Dzwonkowska and Hübner (1986): In this study dichlorvos was administered i.p. to female Syrian hamsters at doses of 0, 3, 6, 15, 30 mg/kg b.w. (½, 1/5, 1/10 and 1 x LD50; LD 50 30 mg/kg b.w.; volume 1ml/100 g b.w. of suspension or water solution) 24 hours before bone marrow was obtained. As a positive control 40 mg/kg b.w. cyclophosphamide was administered. The number of cells with chromosomal changes was increased significantly only at the lowest and two highest doses and only when chromatid breaks and chromatid gaps were combined. Chromatid gaps are not considered indicative of genotoxic potential.

Study 6, Nehéz et al., (1994): The study reports on chromosomal aberrations of bone marrow which was obtained from rats after oral dosing by gavage of 0, 0.97, 1.29, and 1.94 mg/kg b.w. dichlorvos 5 days per week for 6 weeks. No increase in chromosome aberrations occurred at this low level (0.02 x LD50), however, the numbers of cells with numerical aberrations were increased. The latter increase was not dose-related and numerical aberrations are not a reliable indicator of genotoxic potential.

1.7.4 Possible Contribution of Metabolite Dichloroacetaldehyde on In Vivo Mutagenicity Of Dichlorvos

Dichlorvos is metabolized to yield as an intermediate transitory biotransformation product dichloroacetaldehyde, which is further reduced to dichloroethanol or spontaneously decomposes to halogen-free glyoxal at pH 7.4 (Figure 3, Addendum). The acute toxicity of these metabolites (LD50 in mg/kg b.w.) in mice is less than that of dichlorvos (dichlorvos 57 – 108, dichloroacetaldehyde 440, dichlorethanol 890 (IPCS, 1989)). Appreciable amounts of dichloroacetaldehyde may originate from dichlorvos if protein phosphorylation occurs, which indeed is much higher (20 to 30 fold) than DNA-methylation. Because the mutagenic activity of dichlorvos and metrifonate (the drug name of trichlorfon; the latter also yields dichlorvos and dichloroacetaldehyde) was greater than predicted from their respective methylating activity (Segerbäck and Ehrenberg, 1981) the metabolite dichloroacetaldehyde was assumed to contribute to their mutagenicity by a reaction mechanism not involving DNA-alkylation (Segerbäck and Ehrenberg, 1981; Segerbäck, 1981)

The PPR Panel identified one in vivo study (Fischer et al., 1977) and two in vitro studies (Löfroth, 1978; Crebelli et al., 1984) in which the genotoxicity of dichloroacetaldehyde was investigated. In one further study the data were reviewed and commented upon (Wright et al., 1979).

Study 1, Fischer et al., (1977): In a dominant lethal test in mice, mutagenic activity of dichloroacetaldehyde (and also of trichlorfon) was reported. After i.p. administration of a single dose of 176 mg/kg b.w. to male mice of two different strains (DBA-mice, inbred AB Jena-Halle) on the day of mating the number of living fetuses/pregnant mouse was decreased on both mouse strains at day 18 of pregnancy (control 8.65 ± 3.43 living fetuses/AB Jena-Halle mouse versus e.g. 6.68 ± 3.43 fetuses/AB Jena-Halle mice mated in the first week after dichlorvos application with treated male mice and control 7.73 ± 1.53 living fetuses/DAB mouse versus e.g. 7.29 ± 1.40, respectively, mated in the first week). Littering was followed during a 5 week observation period in which 65 AB Jena-Halle female mice (non-treated control females were 52) were mated and during a 4 week period in which 86 female DAB mice were mated (control 89). During this period the average number of all living fetuses per mouse of both strains was



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significantly reduced (p < 0.0005 in case of AB Jena-Halle- and p < 0.005 in case of DAB-mice). The embryonic loss after the single dichlorvos treatment was between 14.3% and 38.7% during this 5 week period in AB Halle-Jena mice (control 15.3) and between 7.5% and 19.4% (control 8.0%) during the 4-week observation period on DAB-mice. The mutagenic activity of dichloroacetaldehyde was comparable to that of the organophosphorous compound trichlorfon, which was tested under identical relative dosage. It should be mentioned that dichlorvos is the main metabolite of trichlorfon at pH 7.4 (WHO, 2000). Dichloroacetaldehyde is negative in the NBP-test and is, if at all, only a weak alkylating agent (Fischer et al. 1977). It is assumed to react via its carbonyl group with amino groups of nucleic acid bases as does chloroacetaldehyde (Fischer et al., 1977).

Study 2, Löfroth (1978) : The metabolite dichloroacetaldehyde was four times more mutagenic in the Ames-reversion test with Salmonella typhimurium TA 100 (58 revertants/µmol) than dichlorvos (17 revertants/µmol). The mutagenic activity of dichloroacetaldehyde decreased in the presence of S9 mix. On the other hand, S9 mix had no influence on dichlorvos mutagenicity. This may raise doubts as to the presence of this metabolite in the dichlorvos incubation under these in vitro conditions. The mutagenic activity of chloroacetaldehyde (minus one chlorine atom) was 515 revertants/µmol in this study. Thus in the presence of a dehalogenation process the mutagenic activity of dichloroacetaldehyde is expected to increase.

Study 3 Crebelli et al., (1984): This study was performed to induce somatic segregation of the mould Aspergillus nidulans by 2,2-dichloroacetaldehyde and by some other halogenated aliphatic carbohydrates (1,2-dichloroethane, 1,2-dibromoethane, allyl chloride, 2-chloroethanol, 2,2-dichloroethanol, chloroform and 1,2-dichloropropane), all applied at their maximal possible doses.

Dichloroacetaldehyde was assayed at the highest dosage (10 mmol/l) that did not irreversibly inhibit sporulation. At this dosage segregation of the genetic marker yA2 in the diploid heterozygous A. nidulans strain 35 x 17 was observed, promoting a change of colour from pale green to yellow of the dosed sporulating conidia. The results were interpreted to indicate significant increase of mitotic non-disjunctions producing diploid and haploid segregation. Mitotic crossing-over was not observed.

Study 4, Wright et al., (1979) : This was not an experimental study. Wright et al. (1979) analysed several aspects of the chemical reactivity of dichlorvos and concluded that neither dichloroacetaldehyde nor glyoxal make a measurable contribution to the mutagenicity of dichlorvos in bacterial test systems.

Conclusions on Question 1

Conclusions on Mode of Action of Genotoxicity

The PPR Panel concludes that the available data clearly indicate that dichlorvos is an in vitro mutagen. However, data on its in vivo mutagenicity are far less clear. Studies in vivo, although suffering from some deficiencies, do provide evidence that dichlorvos has only very weak DNA-alkylating activity. There were no studies in which alkylation and mutagenesis could be directly compared in vivo under conditions where a positive mutagenic response had been induced. A number of genotoxicity endpoints in vivo, dependent on systemic exposure, were negative or the interpretation of the results is not possible due to methodological and/or reporting difficulties, e.g. the numerical chromosomal or nuclear aberrations, the comet assay and the chromatid gaps. The Panel concludes from the micronucleus assay on keratinocytes that there is some evidence that dichlorvos is a site of contact in vivo mutagen. Recent data on transgenic mice have shown an increase of the mutation frequency in liver but not of bone marrow following repeated intraperitoneal administration. It is concluded that this effect on the liver could have been a consequence of the local high exposure of that tissue and hence equivalent to a site of contact effect.

The Panel concludes that there is some limited evidence that dichlorvos is a site of contact in vivo mutagen but that the mechanism of this effect is unclear. The Panel also concludes that the contribution of the genotoxic metabolite dichloroacetaldehyde to the in vivo mutagenicity of



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dichlorvos is probably not relevant. One positive study with dichloroacetaldehyde (a dominant lethal assay) contrasts to negative results in all dominant lethal studies with dichlorvos.

Conclusions on Mode of Action of Carcinogenicity

The PPR Panel concludes that with the exception of tumours of the forestomach in the mouse, there was no convincing evidence for a compound-related, relevant tumour response. Tumours observed in other tissues (pancreas, mammary, mononuclear leukaemia) showed no dose-response, were inconsistent between studies and sexes, were reduced in control animals relative to historical control data, or were unique to the experimental conditions of the assay.

The PPR Panel noted that the doses in female mice, in which the most marked response in forestomach was observed, were higher than in male mice or in rats because female mice are less sensitive to the systemic toxicity of dichlorvos. The lack of response in the rat could have been a consequence of the lower dose used in this species, rather than any marked species differences in sensitivity. The tumours in the forestomach are considered to be a site of contact effect, and a consequence of the very high, sustained concentrations of dichlorvos to the forestomach that would be achieved by gavage dosing in corn oil. In particular, this is because the forestomach is a unique structure that retains material appreciably longer than the glandular stomach (Grice, 1988; Clayson et al., 1990; Poet et al., 2003).

On the mode of action for a tumourigenic response the PPR Panel concludes that it is possible that dichlorvos modifies DNA in cells at site of contact, that this results in a mutagenic response and leads to tumours at that site. The only circumstance where the appropriate conditions had been tested experimentally was in rodents following gavage administration. The PPR Panel recognized that the carcinogenic effect of dichlorvos could be due to a mode of action other than direct interaction with DNA, but strong evidence for this was lacking. There is some evidence that dichlorvos can induce local cell proliferation and hyperplasia in the forestomach of mice treated with a single dose by gavage. However, no dose-relationship existed with regard to cell hypertrophy and a dose (100 mg/kg b.w. dichlorvos) greater than the carcinogenic dose was required to generate diffuse hyperplasia. The relevance of these findings to the forestomach tissue carcinogenicity of dichlorvos is unclear, particularly because there was no consistent evidence for any compound-related hyperplasia in the critical mouse carcinogenicity study. Thus, the PPR Panel could not conclude on an alternative or additional mode of action such as contact cytotoxicity and also recognized that tumour promoting activity of dichlorvos (as a possible modifier of N-nitrosodiethylamine carcinogenicity) had already been disproved experimentally (Horn et al., 1990).

If the mode of action was via direct DNA reactivity, in principle it would be possible that tumours could be produced at other sites following systemic exposure. However, the doses necessary are extremely unlikely to occur because cholinesterase inhibition (NOAEL 0.5 mg/kg b.w. per day in rat) almost entirely limits any such possibility. Hence the Panel concluded that there was a threshold for the tumourigenic response observed in mice.

2 Assessment of Question 2 Question 2: Considering these modes of action of the tumourigenic responses is any of them relevant for humans?

As none of the systemic tumours are to be considered to be compound related the only tumour finding on forestomach is discussed below.

2.1 The Relevance of the Finding of Forestomach Tumours to Man

The mouse and rat stomach is anatomically very different from the human stomach. Unlike the human stomach, the rodent stomach has two different regions, the forestomach and the glandular stomach. The significance of rat forestomach tumours to humans was reviewed by Clayson et al., 1990, when reflecting on the tumourigenicity of butylated hydroxyanisole, another selective forestomach carcinogen, in F344 rats after 2 year dietary administration (Ito et al., 1983). Based on the physiological functioning of the forestomach in rodents as a bag



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that retains food and delays food passage from the oesophagus to the stomach, the forestomach of rats, mice, and hamsters receives a much greater overall exposure to any contaminant in the ingested food. This exposure exceeds even that of oesophagus (Grice, 1988), another organ with a squamous epithelium. Due to the fact that the forestomach is a major part of the stomach (in rat the forestomach comprises ca. 60% of the total surface area of the stomach, the other 40% belonging to the glandular stomach), a considerable amount of an orally applied dose of dichlorvos will reside there, and even more will reach an unusual high tissue concentration if applied as a single or repeated bolus dose by gavage in corn oil. A single study was identified in which dichlorvos stomach tissue concentration and its transit kinetics were analysed after single gavage application (Cassida et al., 1962). The results confirmed that the forestomach tissue exposure was the highest of any organ, giving an accumulation of dichlorvos to levels two to five times higher than in the next most highly exposed organs, liver and kidney. Very comparable accumulation ratio and residence in forestomach of rats was reported with [14C]-2-butoxyethanol after single gavage dose, when this tissue retained four to six fold more radioactivity than other tissues, including liver and kidney (Ghanayem et al., 1987). This argues against any exceptional toxicokinetics of dichlorvos. It should be noted that these concentrations were for the entire forestomach, and hence the concentration differential in the target cells for carcinogenicity could have been greater. The high stomach accumulation of dichlorvos lasted ca. 4 hours when levels were comparable with those in other organs. Considering the proposed mode of dichlorvos genotoxicity by direct interaction with DNA, it is conceivable that only in the forestomach are such high concentrations attained sufficient to induce a mutagenic response and thence local tumours. This organ is not present in man and concentrations and time of residence comparable to those in the forestomach of the tested animals cannot occur in any organ or tissue of man.

Forestomach tumours are induced in rats and mice by non-genotoxic compounds such as 2% butylated hydroxyanisole (Grice, 1988) and 2-butoxyethanol (Poet et al., 2003). However, in these cases a marked local irritation (cytotoxicity) and tissue inflammation due to compound contact occurred and regenerative hyperplasia was observed. No such lesions were evident during the generation of forestomach tumours by dichlorvos, although there was some evidence for such effects at high doses after single gavage application (Benford et al., 1994). However, if any repeated systemic exposure by oral uptake or inhalation of humans to dichlorvos would occur, the high acute toxicity (cholinergic effects) would not allow exposure to doses sufficient to induce any local irritation or cytotoxicity in any tissue. Therefore, this mode of action is not relevant for humans.

Conclusions on Question 2

The Scientific Panel on Plant health, Plant protection products and their Residues concludes that only forestomach tumours should be regarded as compound related. The Panel recognized that there was considerable scientific uncertainty as to the mode of action and relevance for humans of the forestomach tumours induced by dichlorvos in the mouse. However, the Panel concluded that whilst it was not possible to exclude DNA interaction as a critical step in the production of mouse forestomach tumours, any such response appeared to be a consequence of the high and sustained local concentrations of dichlorvos that were achieved in this specific exposure situation. As such, the Panel concluded that there was a threshold dose for this response. The Panel also concluded that the weight of evidence suggests that this would not occur at the levels of exposure that would be encountered by the proposed use of the compound, as any such effect would be prevented by the occurrence of severe systemic toxicity.

Documentation provided to the EFSA Panel by the PRAReR unit 1. Letter, dated 7 November 2005 with ref (2005) HK/nb/1336 from H. Koeter (deputy Director of EFSA) requesting the chair of the EFSA Panel a consultation on mutagenic and carcinogenic properties of dichlorvos in relation to the risk assessment.



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2. Report of EPCO experts’ meeting on mammalian toxicology (EPCO 28) on 27 June to 01 July 2005 (extract relating to dichlorvos), pp 1-23.

3. Documents on dichlorvos prepared by Italy 2003 and 2005:

3a. Level 1 – Statement of the subject matter and purpose of the monograph, dichlorvos. Volume 1, Level 1 - 4, including appendixes 1 – 3, August 2003, pp 1 – 91.

3b. Annex B, Active Substance: dichlorvos. B.6 Toxicity and metabolism. August 2003, pp 1-464.

3c. Addendum 1 to the draft assessment report and proposed Decision of Italy prepared in the context of the possibile inclusion of the above mentioned active substance in Annex I of Council Directve 91/414/EEC. Vol. 1, Level 2 and 3, May 2005, page 1- 10.

3d. Addendum 1 to the draft assessment report and proposed Decision of Italy prepared in the context of the possibile inclusion of the above mentioned active substance in Annex I of Council Directve 91/414/EEC. Vol. 3, Annex B, May 2005, pp 1 – 52.

3e. Appendix 3: Listing of endpoints (revised after the experts’ meeting), pp 1 – 18.

3f. Reporting table. Dichlorvos. 16209/EPCO/BVL/04, rev. 1-1, March 2005, pp 1 – 100.

4. COM (UK Committee on Mutagenicity) 2002. Statement from the UK Committee on Mutagenicity, COM/02/S2: 1 – 13.

5. Table on the available studies relating to genotoxicity and long term toxicity evaluated in the Draft Assessment Report (including brief summary on dose levels, effects as well as acceptability).

6. Ford WH, Killeen JC Jr, Baxter RA (1986). L5178 TK +/- mouse lymphoma forward mutation assay with dichlorvos, pp 1 – 22, unpublished report.

7. An in vivo sister chromatid exchange assay in mice with dichlorvos. SDS Biotech Corporation, September 1985, pp 1- 15, unpuplished report.

8. A micronucleus test in the mouse using dichlorvos. SDS Biotech Corporation, September 1985, pages 1 – 6, unpublished report.

9. Benford DJ (1993). Investigation of the genotoxic and/or irritant effects of dichlorvos on mouse forestomach. Stgy No 26/89/TX. Final report No. RI90/0405, Robens Institute of Health and Safety. Unpublished Report.

10. Benford DJ, Price SC, Lawrence JN, Grasso P, Bremmer JN (1994). Investigations of the genotoxicity and cell proliferative activity of dichlorvos in mouse forestomach. Toxicology 92: 203 – 215. Unpublished Report.

11. Ford WH and Killeen JC (1987). A dominant lethal assay in mice with dichlorvos. Microbiological Associates, Unpublished Report N° 1312-86-0043-TX-002 (Task Force).

12. A dominant lethal assay in mice with dichlovos. SDS Biotech Corporation, March 1985, pp 1 – 69, unpublished report.

13. Benford DJ (1992). Investigation of the irritant effects of dichlorvos on mouse forestomach. Study report 14/91/Tx; final report RI91/0405. Robens Institute of Health and Safety. Unpublished Report.

14. Chan PC (1989). NTP Technical Report on the Toxicology and Carcinogenesis Studies of dichlorvos (Cas No. 62-73-7) in F344/N Rats and B6C3F1 Mice (Gavage Studies). NTP Technical Report Series N° 342.

15. Chan, PC, Huff J, Haseman JK, Alison R, Prejean JD (1991). Carcinogenesis studies of dichlorvos in Fischer rats and B6C3F1 mice. Jpn J Cancer Res 82: 157-164.



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89. Witherup S, Stemmer KL, Pfitzer EA, (1967). The effects exerted upon rats during a period of two years by the introduction of Vapona insecticide into their daily diets, Report from Cincinnati (the Kettering Laboratory), prepared for Shell Chemical Company, submitted to WHO 1993 by Bayer AG, Ciba-Geigy Ltd, Temana International Ltd.. Unpublished Report.

90. Wooder MF and Creedy CL (1979). Studies on the effects of dichlorvos on the integrity of rat liver DNA in vivo. Sittingbourne, Shell Research Ltd., TLGR. 79.089, unpublished report.

91. Wooder MF, Wright AS, King LJ (1977). In vivo alkylation studies with dichlorvos at practical use concentrations. Chem Biol Interact 19: 25-46.

92. Wright AS, Hutson DH, Wodder MF (1979). The chemical and biochemical reactivity of dichlorvos. Arch Toxicol 42: 1-18.

Scientific Panel members Damia Barcelo Culleres, Robert Black, Jos Boesten, Alan Boobis, Anthony Hardy, Andy Hart, Herbert Koepp, Robert Luttik, Kyriaki Machera, Marco Maroni, Douglas McGregor *, Otto Meyer, Angelo Moretto, Euphemia Papadopoulou-Mourkidou, Ernst Petzinger, Kai Savolainen, Andreas Schaeffer, John Stenström, Walter Steurbaut, Despina Tsipi-Stefanitsi, Christiane Vleminckx.

*: a conflict of interest was declared



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Appendix Figure 3. Metabolism of Dichlorvos in Mammalian Cells According to Wright et al., (1979)

HC CH=Cl

ClO

Dichlorvos

CH3O

CH3O

P

O

O CH CCl2

CH3O

CH3O

P

O

O

+CHCl2CHO CHCl2CH2OH

CHCl2CH2O glucuronideCOOH

CHO

COOH

CH2NH2

COOH

CHNH2

CH2OH

Protein

CO2

CO(NH2)2

CONHCH2COOH

(1)

(2)

CH3O

O

P

O

O CH CCl2+glutathioneCH3

CH3SCH2CHCOOH

CO2

CH3SCH2CHCOOH

NHCOCH3

NH2

CH3SCH2CHCOOH

O NH2

CH3O

O

P

O

OH

Phosphate

+Methanol

CO2

Reaction (1) shows the methylation reaction on glutathione as a nucleophil; reaction (2) shows the phosphorylation reaction (dimethylphosphorylation) with a protein. In both reactions dichloroacetaldehyde is formed.

Dichlorvos Dichloroacetaldehyde

CH3O

CH3OP

O

O C CCl2



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Comparison of rate constants of in vitro alkylation of mammalian and bacterial DNA by dichlorvos with rate constants by methyl methanesulfonate (MMS)

Table 3 (Segerbäck and Ehrenberg, 1974)

Estimated rate constants K Gua N-7 for the reaction of DDVP and MMS with guanine-N-7 in DNA at 37 OC.

K GuaN-7 (1 • (g DNA)-1• hour-1) 105

DNA origin

DDVP MMS

Reference

Calf thymus DNA 1.2 60

Segerbäck, 1981 Segerbäck et al., 1978

HeLa cells 1.1

52

Lawley et al., 1974 Dito

Salmon sperm DNA 0.9

87

Lawley et al., 1974 Lawley and Shah, 1972

E. coli 1.3 and 1.6 27

Lawley et al., 1974

E. coli 0.6 Wennerberg and Löfroth, 1974



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Comparison of the extent macromolecule alkylation (DNA, RNA, and protein) by dichorvos with methyl methanesulfonate (MMS)

Table 4. (Lawely et al., 1974).

Comparative extent of alkylation of nucleic acids and protein in HeLa cells treated in vitro with methyl- 14C-labelled dichlorvos and [14C]MMS. The values give K1h = (extent of alkylation, µmole alkyl/g cellular constituent) / (concentration of alkylating agent in medium µmole/ml) for 1 hour reaction.

Compound DNA* RNA Protein * Dichlorvos 0.011 0.009 0.40 MMS 0.52 0.39 0.25 * In mammalian DNA from HeLa cells methylation was on position N-7-meGua. Regarding incorporation of radioactivity into proteins neither the nature of the cell proteins nor protein methylation was further analyzed (Lawley et al., 1974). Protein methylation could also mean apart from the transfer of a radioactive methyl group, the dimethylphosphorylation of the protein on a serine-hydroxyl group or another suitable oxyanion residue.



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Table 5. DNA Alkylation In Vivo by Dichlorvos

Animal Result Dose Reference NMRI-mice (unknown sex)

Weak alkylation after 24 hours, indicated by N7-methyl-guanine in urine Four times higher N-7 alkylated guanine in urine

i.p., 1x 13 to 28* inhalation, 2.5 to 3.5*

Wennerberg and Löfroth, 1974

CFE rats, male Negative Inhalation, 0.006* Wooder et al., 1977 CBA mice, male Very weak alkylation

after 5 hours in soft tissues: 8 x 10-13 mol methyl / g DNA

i.p. 1 x 0.42 Segerbäck, 1981

MutaTMMouse, male Negative i.p. 1 x 4 and 1 x 11 Pletsa et al., 1999a Doses: by i.p injection in mg/kg b.w.

* total dose by inhalation in mg per animal