DICHLORVOS First draft prepared by A. Moretto University of Padua, Padua, Italy EXPLANATION Dichlorvos was previously evaluated by the Joint Meeting in 1965, 1966, 1967, 1970 and 1977 (Annex I, references 3, 6, 8, 14, 28). An ADI of 0-0.004 mg/kg bw was allocated in 1966 and was maintained at subsequent Meetings. The compound was re-evaluated by the present Meeting on the basis of the CCPR periodic review programme. Since 1977 many reviews on the toxicological aspects of dichlorvos have been published. The most relevant are the IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans (IARC, 1979, 1991) and the IPCS Environmental Health Criteria 79 on Dichlorvos (WHO, 1989). This monograph summarizes new or not previously reviewed data as well as relevant data from previous monographs and monograph addenda on dichlorvos. BIOCHEMICAL ASPECTS Many studies have been performed on the biochemical aspects of dichlorvos and have been summarized by WHO (1989). Summaries of the most relevant data are given here. Absorption, distribution and elimination Dichlorvos is readily absorbed via all routes of exposure. In rats orally dosed with 32P-dichlorvos, 60-70% of administered radioactivity was recovered in the urine and about 10% in the faeces within 6 days after dosing. Radioactivity in bones slowly increased with time because of the incorporation of 32P into the normal phosphate pool. After the oral administration of methyl-14C-dichlorvos to rats and mice, the excretion of radioactivity was rapid. The major routes of elimination after 4 days were urine (approximately 60%) followed by expired air (approximately 16%). Carcass contained about 5% of the administered radioactivity 4 days after dosing. When vinyl-14C-dichlorvos was used, less radioactivity was recovered in urine (10-30%) while higher levels were recovered in liver, skin and carcass. In carcass, levels of radioactivity were 14-34% after 1-4 days from dosing mice, rats and Syrian hamsters. The higher levels found in carcass indicate that the vinyl moiety enters the 2-carbon metabolic pool. Similar results were obtained using the inhalation and parenteral routes. After a single i.p. dose (10 mg/kg bw), the dichlorvos concentration in brain peaked after 1 min. and than disappeared with a half-life of less than 1 min (Nordgren et al., 1978). Experiments with pregnant rabbits and pigs showed that dichlorvos readily crosses the placenta (WHO, 1989). Biotransformation A scheme of the metabolites of dichlorvos in mammals is given in Figure 1. Two main pathways are responsible for the rapid degradation of dichlorvos. Desmethyl-dichlorvos is produced by a glutathione-dependent enzymatic system while dimethylphosphate and dichloroacetaldehyde are the hydrolysis products of an A-esterase which is present mainly in plasma and liver (Reiner et al., 1975). This appears to be the dominant pathway. The rate of hydrolysis of dichlorvos by rat plasma has been determined to be 12 µmol/hour/ml at 37 °C (Reiner et al., 1980). The metabolism of dichlorvos is rapid and similar in the various species, including humans. Differences between species are related to the rate of metabolite formation rather than to the nature of the metabolites. The metabolism of dichlorvos is so rapid that the half-life in blood could not be determined in experimental animals, and it appears to be shorter than 15 minutes. No evidence of the accumulation of dichlorvos or potentially toxic metabolites has been found (WHO, 1989).Effects on enzymes and other biochemical parameters In vitro studies showed that rodent plasma ChE is more sensitive than erythrocyte and brain ChE to inhibition by dichlorvos. Calculated I50s (concentrations which inhibit 50% of enzyme activity) at 37 °C, pH 7.4-7.6, were 10-8 and 10-7 mol/l, respectively, for an incubation time of 20 minutes (Asperen & Dekhuijzen, 1958; Skrinjaric-Spoljar et al., 1973; Lotti & Johnson, 1978). The rate of phosphorylation of rat brain ChE was calculated to be 1.5 x 105 mol/l/min at 37 °C, pH 7.4-7.6 (Skrinjaric-Spoljar et al., 1973). Half-life of in vitro reactivation (at 37 °C, pH 7.4-7.6) was found to be about 100 minutes; that of aging was found to be about 400 min. This means that inhibited AChE almost completely reactivates within a few hours, leaving a small fraction (20% or less) irreversibly inhibited (i.e. aged) (Skrinjaric-Spoljar et al., 1973). TOXICOLOGICAL STUDIES Acute toxicity studies The results of acute toxicity tests of dichlorvos administered by various routes to different animal species are summarized in Tables 1 and 2. Typical cholinergic signs are observed. WHO has classified dichlorvos as highly hazardous (WHO, 1992). Short-term toxicity studies Mice In a range-finding study, groups of 10 B6C3F1 mice/sex were administered by corn oil gavage 0, 5, 10, 20, 40, 80, or 160 mg dichlorvos/kg bw/day, 5 days per week for 13 weeks. Animals were observed twice daily for clinical signs and body weight was recorded weekly. Necropsy was performed on all animals. Oesophagus and gastrointestinal tract of all animals dying after day 46 were examined histologically. All vehicle controls and all animals in the highest dose group with survivors were examined histologically. All mice at 160 mg/kg bw/day and 5 male mice at 80 mg/kg bw/day died before the end of the study. Final mean body weights in all treated groups were comparable to controls. No gross or microscopic pathologic effects were observed (Chan, 1989). Rats In a range-finding study, groups of 10 male and 10 female F344/N rats were administered by corn oil gavage 0, 2, 4, 8, 16, 32, or 64 mg dichlorvos/kg bw/day, 5 days per week for 13 weeks. Animals were observed twice daily for clinical sign and body weights were recorded weekly. Necropsy was performed on all animals. Oesophagus and gastrointestinal tract of all animals dying after day 46 were examined histologically. All vehicle controls and all animals in the highest dose group with survivors were examined histologically. All rats at 32 and 64 mg/kg bw/day and 1 male and 4 females at 16 mg/kg bw/day died before the end of the study. Final mean body weights of all dose groups were comparable to vehicle controls. No gross or microscopic pathologic effects were observed (Chan, 1989). Table 1. Acute toxicity of dichlorvos (WHO, 1989) Species Route LD50 (mg/kg bw) Mouse oral 68-275 i.p. 28-41 i.v. 8-10 s.c. 13-33 Rat oral 30-110 dermal (24 h) 75-107 i.p. 18 s.c. 72 Guinea-pig s.c. 28 Hamster i.p. 30 Rabbit oral 13-23 dermal 205 Cat oral 28 Dog oral 100-316 Chicken oral 15 Swine oral 157 Table 2. Acute inhalation toxicity of dichlorvos (WHO, 1989) Species Mode of exposure Time of exposure LC50 (hours) (µg/l) Mouse whole body 4 13 head only 4 > 218 Rat whole body 4 15 whole body 1 140 head only 4 340 head only 1 455 head only 4 > 198 Dogs Groups of 3 male and 3 female beagle dogs received 0, 2.3, 6.9, 12 or 23 mg dichlorvos/dog (93% in corn oil by gelatin capsule) daily, for 90 days. The mean doses per group were equivalent to 0.3, 1, 1.5 or 3 mg/kg bw/day. No effects were observed on mortality, growth, haematology, liver and kidney function, organ weights, or at gross and histopathological examination. In the two highest dose groups, the dogs showed excitement, increased activity and aggression. Plasma and erythrocyte ChE activities, measured initially and at intervals of approximately 2 weeks, were normal in the lowest dose group (0.3 mg/kg bw/day) but inhibited in the other dose groups (up to about 60% in the highest dose group). Inhibition was lower from day 70 onwards. Brain ChE activity at termination was decreased (to 33%of control activity) only in the highest dose group. The NOAEL in this study was 1.5 mg/kg bw/day based on reduction in brain ChE activity at termination. However, taking into account the behavioural changes, a more conservative NOAEL can be considered to be 1 mg/kg bw/day (Hine, 1962). Pigs Young swine (35 days old) were fed a PVC-resin formulation of dichlorvos (10%) in dosages equivalent to 1, 4, or 16 mg dichlorvos/kg bw/day (divided over 2 daily doses) for 30 days. No effects were found on body-weight gain, haematology, or clinical chemistry when compared with control animals fed a blank PVC formulation. Plasma and erythrocyte ChE activities were significantly inhibited in the 4 mg/kg bw/day group (37 and 34% of controls, respectively) and in the 16 mg/kg bw/day group (33 and 21% of controls, respectively). The NOAEL in this study was 1 mg/kg bw/day based on inhibition of erythrocyte ChE activity (Stanton et al., 1979). Monkeys Rhesus monkeys (4/sex) were continuously exposed to dichlorvos vapour at an average actual concentration of 0.05 mg/m3 for 3 months. The control group consisted of 4 males and 1 female. No adverse effects were observed in appearance, behaviour, or haematological and clinical chemical determinations. Plasma ChE activity was slightly reduced (up to 28% inhibition), while erythrocyte ChE activity inhibition was 36%. No changes in nerve maximum conduction velocities or muscular evoked action potentials were induced by exposure to dichlorvos (Coulston & Griffin, 1977). Long-term toxicity/carcinogenicity studies Mice Groups of 100 male and 100 female C57BI/6/Bln mice (5-6 weeks old) received by gavage 0.2 mg dichlorvos per mouse (97% in 0.2 ml water), freshly prepared, either twice or 3 times per week for 50 weeks. Control groups received by gavage either 0.2 ml water, 3 times per week (about 50 males and 50 females), or no treatment (35 males and 35 females). Surviving animals were sacrificed after 110 weeks. This strain of mice is known for the spontaneous occurrence 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 decreased in both test groups (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 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 animals with dichlorvos (Horn et al., 1987). Dichlorvos was not co-carcinogenic in C57BI/6/Bln mice when administered by gavage three times/week at 0.2 mg/animal to mice subcutaneously injected with 50 µg N-nitrosodiethylamine per animal weekly for 50 weeks followed by an observation period of up to 110 weeks (Horn et al., 1990). Two groups of 50 male and 50 female B6C3F1 mice were fed 1000 or 2000 ppm dichlorvos (purity > 94%) in corn oil in the diet for 2 weeks. Due to severe signs of intoxication, doses were lowered to 300 and 600 ppm for the following 78 weeks. Samples of the diets analyzed during the study showed that time-weighted average concentrations were 318 and 635 ppm. 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. Animals were observed twice daily for clinical signs. Alopecia and rough hair coats were noted in many treated animals, particularly in the male groups, beginning at week 20 and persisting throughout the study. The average body weights of the high-dose mice of both sexes were 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 increase 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 one in a high-dose female), 1 papilloma of the oesophagus in a high-dose female and 3 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 ppm in the diet, equivalent to 95 mg/kg bw/day) was not demonstrated to be carcinogenic in this study (NCI, 1977; Weisburger, 1982). Studies in B6C3F1 mice given 0, 400 or 800 mg/litre dichlorvos in drinking-water for two years and in CFE rats exposed to 0, 0.05, 0.48, or 4.7 mg/m3 of dichlorvos 23 hours/day for 2 years were summarized in WHO (1989). There was no evidence of carcinogenic effects in these studies (Blair et al., 1976; Konihishi et al., 1981). Groups of 50 B6C3F1 mice were given dichlorvos (99% purity) by corn oil gavage at doses of 0, 10 or 20 (males) or 0, 20 or 40 (females) mg/kg bw/day daily, 5 days per week, for 103 weeks. The dose volume of the corn oil was 10 ml/kg bw/day. Body weights were recorded once weekly for the first 12 weeks and then monthly. Animals were observed twice per day for clinical signs. Necropsy and histological examinations were performed on all moribund animals or at the end of the study. Body-weight gain and survival did not significantly differ between treated and control groups. No compound-related clinical signs were observed. Blood cholinesterase activity was not determined during the study. However, a separate study showed inhibition of plasma ChE activity by 60-90% in all dose groups while erythrocyte ChE activity was normal (see "Special studies on Cholinesterase activity" for detailed description and comments). The incidences of forestomach squamous cell papillomas were 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. Two forestomach squamous cell carcinomas were seen in high-dose females and none in the other groups. No increase in the incidence of forestomach hyperplasia was seen in the dosed mice compared with vehicle controls (10-20%). 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. Based on the increased incidence of forestomach papillomas, the NOAEL was 10 mg/kg bw/day (Chan, 1989; Chan et al., 1991). Rats Groups of 40 male and 40 female weanling CD rats were fed diets containing nominal concentrations of 0, 0.1, 1, 10, 100 or 500 ppm dichlorvos (93% purity) for 2 years. Diets were prepared weekly. Five males and 5 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 (DCA) content (average concentrations ranging from 0.01 to 28.6 ppm. The average actual concentrations of dichlorvos in each diet were 0.05, 0.5, 4.7, 47 or 230 ppm. No cholinergic signs were observed. No effects were seen on behaviour, mortality rate, weight gain, food consumption, terminal body and organ weights, haematology or urinalysis. Plasma and erythrocyte ChE activities were measured 13 times during the study. In the 100 ppm group, plasma and erythrocyte ChE activities were reduced to 60-90% and to 50-90% of control activities, respectively; in the 500 ppm group, the activities were reduced to 20-70% and 20-60%, respectively. Activities were higher toward the end of the study. Brain ChE activity was decreased in the highest dose group by 45-47%, 24-43%, 24-38% and 5-15% after 6, 12, 18 and 24 months, respectively. Histological examination of major organs (liver, heart, lungs, kidneys, spleen, brain, gonads, pituitary, adrenals, and thyroids) revealed hepatocellular fatty vacuolization in all 500 ppm rats, and in most females and several males at 100 ppm. No effect was seen on serum total proteins or albumin: globulin ratio, or on hexobarbital sleeping time. The tumour incidence was comparable with that of the control group. The NOAEL, based on brain ChE inhibition, was 100 ppm (actual concentration 47 ppm, equivalent to 2.4 mg/kg bw/day) (Witherup et al., 1967). Groups of 50 male and 50 female weanling CFE rats were exposed (whole body) to nominal air concentrations of 0, 0.05, 0.5 or 5 mg dichlorvos (97% purity)/m3 for 23 hours per day for two years. The average actual dichlorvos concentrations were 0.05, 0.48, or 4.7 mg/m3. Body-weight gain was reduced in the two highest dose groups. After two years of exposure, plasma and erythrocyte ChE activities were reduced by 20-30% at 0.5 mg/m3 and by > 60% in the highest dose group; brain ChE activity was reduced by 10% (statistically significant) at 0.5 mg/m3 and by 80% in the highest dose group. No effects attributable to dichlorvos were seen on appearance, food consumption, haematological or blood chemistry values, organ weights, or gross or microscopic examinations of major organs. Ultrastructural examinations of bronchi and alveoli of rats exposed to 0 or 5 mg/m3 showed no differences between the two groups. It was concluded that 2-year exposure to 0.05 mg/m3 dichlorvos did not cause observable adverse effects to CFE rats (Blair et al., 1976). 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 & Blair, 1977). Osborne-Mendel rats (50/sex) were fed 1000 ppm dichlorvos (purity > 94%) in corn oil for 3 weeks. Due to severe cholinergic signs, the dose was reduced to 300 ppm for the remaining 77 weeks. Another group (50 males and 50 females) was fed 150 ppm dichlorvos for 80 weeks. Samples of the diets analyzed during the study showed that time-weighted average concentrations were 330 and 150 ppm, respectively. Matched controls consisted of 10 rats of each sex; the pooled controls from simultaneous studies of other compounds consisted of 60 rats of each sex. Animals were observed twice daily for clinical signs. All surviving rats were killed after 110-111 weeks. 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 and type of tumours attributable to dichlorvos in either sex. Dichlorvos (up to 330 ppm in the diet, equivalent to about 30 mg/kg bw/day) was not demonstrated to be carcinogenic in this study (NCI, 1977; Weisburger, 1982). Groups of 70 and 100 rats per sex received 0.1 mg dichlorvos (97%)/rat in 0.2 ml water by gavage twice or 3 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 3 times per week 0.2 ml water. From the age of 10 months onwards, the incidence of focal hyperplasia of urinary bladder and of renal pelvis increased in males but decreased in females from both test groups compared to controls. No neoplastic lesions were found which could be attributed to treatment (Horn et al., 1988). A study in Fischer 344 rats given drinking-water containing 0, 140 or 280 mg/litre dichlorvos for 108 weeks was summarized by WHO (1989). There was no evidence of carcinogenicity (Enomoto et al., 1981). Groups of 50 male and 50 female F344/N rats were administered dichlorvos by corn oil gavage at 0, 4 or 8 mg/kg bw/day (99% purity), 5 days per week for 103 weeks. Body weights were recorded once weekly for the first 12 weeks and then monthly. Animals were observed twice daily for clinical signs. Necropsy and histologic examinations were performed on all moribund animals or at the end of the study. Mild diarrhoea was observed in treated animals. ChE activities were not determined during the study. However, a separate study showed inhibition of plasma ChE activity by 50-80% in all dosed groups but no erythrocyte ChE inhibition (see "Special studies on cholinesterase activity" for detailed description and comments). No significant differences in mean body weights or survival were observed between any groups of either sex. Increased incidence of cytoplasmic vacuolisation of liver was observed in dosed males and of cortical cytoplasmic vacuolisation of 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, 30/50 and 33/50 in males and 2/50, 3/50 and 6/50 in females, respectively. The increased incidence in treated males was statistically significant. The incidence of mononuclear cell leukaemia (11/50, 20/50, 21/50 in control, low and high-dose groups, respectively) was significantly increased in the treated male rats compared with controls. The incidence of mammary gland adenomas or fibroadenomas in female rats was 11/50 in controls, 19/50 in the 4 mg/kg bw/day group and 17/50 in the 8 mg/kg bw/day group. The incidence in the low-dose group was only slightly higher than the historical control values. Two mammary gland carcinomas were observed in control and low-dose groups. The authors concluded that there was evidence that dichlorvos is carcinogenic to rats (Chan, 1989; Chan et al., 1991). The Meeting observed that the incidence of pancreatic adenomas in male control rats was unusually high and therefore the higher incidence found in treated animals was considered of questionable biological significance. The increased incidence of mononuclear cell leukaemia, which is usually high and variable in this strain of rats, was also of questionable biological significance (Haseman et al., 1985). Dichlorvos at 8 or 16 mg/kg bw/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 bw/day died of leukaemia during the last week of dosing. The severity of the mononuclear cell leukemia 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). Dogs Groups of beagle dogs (3/sex) were fed diets containing nominal concentrations of 0, 0.1, 1, 10, 100 or 500 ppm dichlorvos (93% purity) for 2 years. The average actual concentrations were 0, 0.09, 0.32, 3.2, 32 or 260 ppm dichlorvos; the average dichloroacetaldehyde concentrations at the three highest dosages were 0.6, 6.4 and 20 ppm. Since diet analysis was performed weekly on a mixture of diet samples taken the previous 7 days, it is likely, given the high volatility of dichlorvos, that at the end of the week the actual concentration was much lower than 30% of the nominal concentration. No effects were seen on general appearance, survival, weight gain, food consumption, haematology or urinalysis. Erythrocyte ChE activity was reduced up to 50% of controls in the 10 ppm group with recovery to control values at the end of the feeding period. At the highest dose level, inhibition was more than 90% but complete recovery was observed at 24 months. A similar pattern was observed for plasma ChE activity. Brain ChE activity, measured at the end of the study was similar to that of the controls in all treated groups. Relative liver weights were slightly increased in males at 100 ppm and in both sexes at 500 ppm. Histological examination of major organs revealed slight dose-related cytoplasmic vacuolization and enlargement of hepatocytes in animals at the two highest levels. No differences were seen in serum alkaline phosphatase, transaminase activities, total serum proteins or albumin:globulin ratios (Jolley et al., 1967). Reproduction studies See also under special studies on testes. Mice Male and female Crl:CD3-1 mice were exposed to dichlorvos concentrations of 0, 1.9, 3.0 or 4.6 mg/m3, generated from dichlorvos-impregnated PVC strips in their cages. Exposure began 4 days prior to formation of breeding groups (3 females and 1 male) and continued throughout pregnancy. Signs of intoxication were not observed. Plasma ChE activity was significantly inhibited (by 90%, 93% and 95% in the 1.9, 3.0 and 4.6 mg/m3 groups, respectively) when measured on day 4 after beginning of treatment. Gestation length, number of litters, litter frequency and mean litter size were comparable to controls. No grossly detectable congenital anomalies were detected in any of the offspring (Casebolt et al., 1990). Rats A 3-generation, 2-litter/generation reproduction study in rats summarized by WHO (1989) was negative at doses up to 235 ppm in the diet, equivalent to 12 mg/kg bw/day (Witherup et al., 1965). Domestic animals Several studies have been carried out with pregnant sows but no adverse effects on piglets were observed with doses which inhibited plasma and erythrocyte ChE activity in the sows (WHO, 1989). Special studies on delayed neurotoxicity Several studies have shown that a single dose of dichlorvos does not produce delayed neurotoxicity in pre-medicated hens, whether it is administered orally or subcutaneously (WHO, 1989). However, Caroldi & Lotti (1981) reported mild signs of ataxia in pre-medicated hens 2 weeks after a single massive subcutaneous dose (100 mg/kg bw) and severe (> 80%) inhibition of NTE in peripheral nerve, spinal cord and brain. Johnson (1978) did not observe ataxia in pre-medicated hens given the same dose in the same way. However, in this experiment spinal cord NTE inhibition was below the threshold. When the dose was repeated 1-3 days after the first dose, spinal cord NTE inhibition increased and the hens became ataxic. In a 90-day study, white leghorn hens were given dichlorvos (99.9% purity) either dermally or orally. For oral administration, a 10-20% solution of dichlorvos in corn oil in gelatin capsules was used whereas, dermally, 1-20% emulsifiable concentrates in technical grade xylene (containing 2% Triton X-100) were used. Dichlorvos at doses greater than 1 mg/kg bw/day (dermal) or 6 mg/kg bw/day (oral) led to cholinergic symptoms and death after 2-3 days. None of the animals developed organophosphate-induced delayed neuropathy (Francis et al., 1985). In summary, it is possible to produce clinical neuropathy in hens, but the doses required are far in excess of the LD50. This is consistent with the low in vitro ratio of AChE I50/NTE I50 for dichlorvos (Lotti & Johnson, 1978). Special studies on embryotoxicity and teratogenicity Several embryotoxicity/teratogenicity studies have been performed with dichlorvos in mice, rats and rabbits. These studies are summarized in WHO (1989). Dichlorvos given orally, by inhalation or intraperitoneally, was not teratogenic at doses which were toxic to the pregnant animals. However, in a study in rats where a single i.p. dose of 15 mg/kg bw was given on day 11 of gestation, 3/41 fetuses in the treated group had omphaloceles (0/50 in controls) (Kimbrough & Gaines, 1968). This finding has not been confirmed in other studies. Special studies on cholinesterase activity This section summarizes studies in which the effect of dichlorvos on ChE activity was the only parameter investigated. Studies on the inhibition of ChE activity resulting from a single oral or parenteral dose of dichlorvos in relation to the time elapsed after dosing are summarized in Table 3. In general, the maximum inhibition occurred within one hour followed by rapid recovery. Mice/Rats Groups of 8-week old B6C3F1 mice and F344/N rats (10/sex) were administered dichlorvos (99%) by corn oil gavage at daily doses of 0, 5, 10, 20, or 40 mg/kg bw (mice) and 0, 2, 4, 8, or 16 mg/kg bw (rats), five days per week for one week. Plasma and erythrocyte ChE activities were measured on days 10 or 11, 25 or 26 and 32 or 33; blood was obtained about 3 hours after treatment. Plasma ChE activity was significantly inhibited in all dose groups of mice (50 and 90% inhibition in low and high-dose groups, respectively) and rats (25 and 80% inhibition in low and high-dose groups, respectively). At all time-points, erythrocyte ChE activity in dosed and vehicle-control mice and rats was similar. However, the timing for determination of the enzyme activities might have underestimated the inhibition (Chan, 1989). Rats Reiner & Plestina (1979) compared in vivo reappearance of ChE activity in rats after treatment with either dichlorvos (2.5 mg/kg bw i.v.) or metrifonate (300 mg/kg bw i.v.). Half-lives of recovery of brain and plasma ChE (acetylcholine was the substrate) were found to be 2 and 2.5 hours, respectively, both in dichlorvos-treated and in metrifonate-treated rats. This was consistent with in vitro data (Skrinjaric-Spoljar et al., 1973). In a study on the influence of temperature on ChE activity, rats were injected i.p. with a single dose of 6.3 mg/kg dichlorvos and kept at either 28 °C or 5 °C. The maximum inhibition of whole blood ChE activity (40% and 50% in the two groups, respectively) occurred after 0.5 hour. The animals kept at 5 °C showed slightly less inhibition of whole blood ChE activity than those at room temperature (Chattopadhyay et al., 1982). Table 3. Time-related inhibition of ChE activity in animals after administration of a single dose of dichlorvos Species Route Dose Time Mean % inhibition Reference (mg/kg bw) plasma erythr. brain ChE AChE AChE Mouse, male i.p. 10 15 min 70 Nordgren et al., (1978) (n = 6-8) 60 min 50 2 hours 20 Mouse, male i.p. 15 2 hours 20 Cohen & Ehrich, 1976 Mouse, male i.p. 30 15 min 63 Cohen & Ehrich, 1976 (n = 4) 1 hour 50 5 hours 35 18 hours 10 Rat, male oral 1.6 2 hours 0 Pacheka et al., 1975 (n = 6) 10 2 hours 15 40 1 hour 60* Rat, male oral 40 1 hour 70 Teichert et al., 1976 (n = 1) Rat, male oral 40 5 min 45 Pacheka et al., 1975 (n = 6) 15 min 80 1 hour 85 2 hours 70 8 hours 35 24 hours 25 48 hours 6 Rat, male oral 50 15 min 68 80 97.91** Modak et al., 1975 (n = 6) 3 hours 73 37 68-70** 24 hours 39 24 31-38** Table 3 (contd) Species Route Dose Time Mean % inhibition Reference (mg/kg bw) plasma erythr. brain ChE AChE AChE Rat, male i.v. 2.5 30 min 60 85 Reiner & Plestina, 1979 (n = 5-12) 90 min 40 65 3 hours 7 44 12 hours 0 17 48 hours - 10 Dog, greyhounds oral 11 1 hour 90 93 Snow & Watson, 1973 1 male 2 hours 80 70 1 female 24 hours 15 35 72 hours 8 25 Dog, 2 male oral 22 fatal 90 95 66*** Snow & Watson, 1973 greyhounds, 1 female crossbred Dog, greyhounds oral 22 1 hour 88 83 Snow & Watson, 1973 and crossbred 3 hours 76 66 (n = 7-9) 6 hours 60 50 24 hours 30 30 48 hours 13 30 72 hours 5 35 Dog, beagle oral 50 2 hours 68 37 Ward & Glicksberg, 1971 sex not specified 24 hours 37 26 (n = 15) 5 days 8 22 14 days 5 20 21 days 0 8 Table 3 (contd) * Daily dosing for 14 days caused 60% AChE inhibition when measured 24 hours after the last dose. ** Determinations were performed in striatum, hippocampus, medulla and cortex. No significant difference was found between these brain areas. *** Measured 20-155 min. after treatment. When pregnant rats were given oral doses of 1.1 or 5.6 mg dichlorvos/kg bw/day during days 14-21 of gestation, plasma ChE activity of the high-dose mothers was inhibited (30-50%) but not that of the young. Brain ChE activity did not show any significant inhibition (Zalewska et al., 1977). Rabbits Dichlorvos, when infused into the ear vein of adult male rabbits, produced dose- and time-related inhibition of whole blood ChE activity during infusion. Spontaneous but incomplete recovery to 60-80% of the normal activity occurred within 60-90 minutes after infusion. Almost complete recovery was obtained by injecting oximes up to 2.5 hours later (Shellenberger et al., 1965; Gough & Shellenberger, 1977-1978; Shellenberger, 1980). The progeny of rabbits, treated orally with 6 mg/kg bw/day for the last 10 days of gestation, showed inhibition of brain ChE activity at one day (30%) and 8 days of life (15%). Plasma ChE activity was higher throughout days 1-16 of life (Maslinska & Zalewska, 1978). Guinea-pigs Groups of 5 male and 5 female guinea-pigs were given daily applications of 0, 25, 50 or 100 mg/kg bw/day dichlorvos (94% purity) on the shorn skin for 8 days. All animals survived. A dose-dependent inhibition of both plasma and erythrocyte ChE activities occurred in all test groups. Recovery of plasma ChE activities was complete within one week of the last exposure, and that of erythrocyte ChE was complete within one week in the females and 2 weeks in the males (Brown & Roberts, 1966). Hens Chickens dosed once orally with 1,3, or 6 mg/kg bw showed a rapid inhibition of plasma ChE activity followed by recovery with an half-life being estimated at three days (Rauws & van Logten, 1973). White Leghorn pullets and hens were fed a diet containing 30 ppm dichlorvos for 35 days followed by a 21-day recovery period. Plasma ChE activity was inhibited by 70% after four weeks of treatment but returned to normal during the recovery period (Pym et al., 1984). Monkeys Thirty-two Rhesus monkeys were given a pelleted PVC-resin formulation containing 20% dichlorvos at dosages ranging from 1 to 16 mg dichlorvos/kg bw once daily or 1.6 and 4 mg dichlorvos/kg bw twice daily for 10 to 21 consecutive days. None of the monkeys showed overt signs of intoxication, although they ate less food and had soft faeces. Plasma and erythrocyte ChE activities were reduced by approximately 80% in all animals (irrespective of the dose) and remained inhibited throughout the study. Plasma ChE activities returned to normal values within approximately 3 weeks and the erythrocyte ChE activities within 50 to 60 days following cessation of exposure (Hass et al., 1972). Special studies on genotoxicity Methylating reactivity In vitro studies In a quantitative colour test in which methylation of 4-(p-nitro-benzyl) pyridine was measured to predict DNA alkylating potential, dichlorvos gave a positive response, the reaction being about 1/3 that of the known alkylating compound methylmethanesulfonate (MMS) (Bedford & Robinson, 1972). Alkylation by dichlorvos of calf thymus DNA, resulting in the formation of N-7-methylguanine, was reported by Lofroth (1970). Methylation by dichlorvos of isolated salmon sperm DNA and of DNA from intact E.coli and from human HeLa cells broadly resembled that by MMS (Lawley et al., 1974). In this study a very high dichlorvos concentration (14 mmol/l) was used with isolated DNA (12.5 mmol/l DNA-P). Dichlorvos concentrations with E. coli and HeLa cells were 1-3 mmol/l. The rate of methylation of guanine-N-7 from salmon sperm DNA or from intact HeLa cell DNA by dichlorvos was 2-3 x 10-4 mol/l/min (calculated assuming guanine as 23-25% of DNA with an average molecular weight of DNA base pair of 649, Lawley et al., 1974). This is about 15 times lower than the rate of methylation by MMS. This rate is to be compared with the rate of reaction with AChE (phosphorylation) (1.5 x 105 mol/l/min at 37 °C) (Skrinjaric-Spoljar et al., 1973). Therefore, the relative rate of phosphorylation is about 9 orders of magnitude higher than that of alkylation. Labelled 7-methylguanine was present in both DNA and RNA isolated from E. coli exposed to [Me-3H]-dichlorvos (1.1 mmol/l for 4 hours). The methylating capability of dichlorvos was less, by a factor of 10-100, than that of strongly genotoxic methylating compounds (Wennerberg & Lofroth, 1974). Incubation of bacteriophage R17 with 0-100 mmol dichlorvos/litre for 90 hours did not result in methylation of the phosphate groups of the RNA to any significant extent (Shooter, 1975). In vivo studies Mice were given i.p. injections of methyl-14C-dichlorvos (1.9 µmol/kg bw, approximately 420 µg/kg bw). The degree of alkylation of guanine-N-7 in DNA isolated from soft tissues amounted to 8 x 10-13 mol methyl per gram of DNA (about 5 x 10-10 mol/mol guanine) (Segerback, 1981; Segerback & Ehrenberg, 1981). From acute toxicity data in mouse (see Table 1) it can be extrapolated that this dose would cause 1 mol/mol phosphorylation of erythrocyte ChE. DNA and RNA from the total soft tissues of male rats exposed to atmospheres containing 0.064 mg/m3 (about 0.1% of the LC50) of methyl-14C-dichlorvos for 12 hours did not show methylation of the N-7 atom of guanine moieties. The exposure period constituted a significant fraction of the half-life of the 7-methylguanine moieties in DNA (Wooder et al., 1977; Wooder & Wright, 1981). Excretion of labelled 7-methylguanine in the urine by NMRI mice and rats injected i.p. with [Me-14C]-dichlorvos, or exposed by inhalation for 2 hours (mice only) was reported by Wennerberg & Lofroth (1974) and Lofroth & Wennerberg (1974). In rat urine, labelled 3-methyladenine and 1-methyl-nicotinamide were also present (Lofroth & Wennerberg, 1974). According to the authors, these results demonstrate that dichlorvos spontaneously methylates guanine and adenine moieties in nucleic acids. However, administration of radiolabelled adenine and guanine to otherwise untreated rats gave rise to the excretion of radiolabelled methylated purines in the urine. Therefore, the detection of radiolabelled purines, per se, in the urine of animals exposed to methyl-labelled methylating agents does not constitute evidence for the spontaneous methylation of the purine moieties of nucleosides or nucleic acids by methylating agents (Wooder et al., 1978; Wooder & Wright, 1981). Moreover, a natural biosynthetic pathway has been demonstrated whereby the methyl carbon atoms of dichlorvos can be incorporated into the heterocyclic rings and the methyl groups of urinary 7-methylguanine after entering the 1-C pools, in vivo (Wright et al., 1979; Wooder & Wright, 1981). Genotoxicity In vitro studies Several studies using bacteria and fungi as test organisms have been carried out (Table 4). In most of the studies, only one, often high, dichlorvos concentration was tested, sometimes resulting in low survival of the test organism. The alkylating properties of dichlorvos (see above) are most probably the cause of the mutagenic action. This is suggested, for instance by data in E. coli strains deficient at four repair loci (Bridges et al., 1973). In vitro studies using mammalian cells are summarized in Table 5. Dichlorvos caused cell transformation, mutations, DNA strand breaks, sister chromatid exchanges and chromosomal aberrations in cultured animal cells. In cultured human cells, dichlorvos induced unscheduled DNA synthesis but did not induce sister chromatid exchanges or chromosomal aberrations. Negative results from chromosomal aberration tests in cultured human lymphocytes were also cited by Fahrig (1974) and Wild (1975). Dichlorvos (0.3 µg/ml) caused a synergised an increase in sister chromatid exchanges in Chinese hamster ovary cells when tested in combination with synthetic pyrethroids or propoxur which, as such, were negative; however, in the combination phenothrin + dichlorvos (0.3 µg/ml) the induction of SCE by phenothrin was negative (Wang et al., 1988). In vivo studies In vivo studies are summarized in Table 6. In Drosophila melanogaster, chromosomal aberrations but not sex-linked recessive lethal mutations were induced. Negative results were obtained in host-mediated, dominant lethal, sister chromatid exchange and micronucleus assays (except on skin after local application at cytotoxic doses). Dichlorvos did not induce in vivo chromosomal aberrations in bone-marrow cells, spermatocytes or spermatogonia, DNA strand breaks or unscheduled DNA synthesis. Special studies on liver microsomal enzymes A decrease in liver microsomal cytochrome P-450 occurred in rats after three daily i.p. injections with 6 mg dichlorvos/kg bw (Purshottam & Kaveeshwar, 1982) but no effect on liver microsomal UDP-Glucuronyl transferase was found in mice 4 hours after a single i.p. injection of 25 mg/kg bw (Yoshida et al., 1976). Pre-treatment of rats with three daily i.p. injections of sodium phenobarbital did not significantly change mortality and plasma ChE inhibition caused by an i.p. injection of dichlorvos (20 mg/kg bw) (Purshottam & Kaveeshwar, 1979). Short- and long-term studies with mice, rats or dogs dosed orally or intraperitoneally with dichlorvos did not show any effect on microsomal drug metabolizing enzymes (Witherup et al., 1967; Uchiyama et al., 1975; Farber et al., 1975). Table 4. Mutagenicity tests on microorganisms in vitro Test system Test object Concentration Purity Results 1 Reference Mitotic A. nidulans strain p ? ? + Morpurgo et al., 1979 non-disjunction and crossing over Recombinant assay B. subtilis H17 Rc+ 2 mg/plate ? - Shirasu et al., 1976 M45 Rec- " + " " Forward mutation A. nidulans strain 35 14 mg/disc ? + Bignami et al., 1977 E. coli B 5-25 mmol/l 95% +2 Wild, 1973 Gal RS ? ? + Fahrig, 1974 K12(5-MT) 0.3-3.2 mmol/l ? + Mohn, 1973 S. coelicolor A 3(2) his A1 5.6 mg/disc 99.9% + Carere et al., 1978a, 1978b Reverse mutation E. coli B/r WP2 5 mg/plate > 97% +3 Moriya et al., 1978 S. typhimurium TA 1535 5 mg/plate > 97% +4 " " E. coli WP2 hcr up to 5 mg/plate ? +3 Moriya et al., 1983 S. typhimurium TA 98 up to 5 mg/plate ? - " " TA 100 " " ? + " " TA 1535 " " ? ? " " TA 1537 " " ? - " " TA 1538 " " ? - " " Table 4 (contd) Test system Test object Concentration Purity Results 1 Reference Reverse mutation (cont'd) E. coli B/r Wp2( ); SR714 0.04-2.3 mmol/l 99% +3 Houk & DeMarini, 1987 CM 561 0.2% " +5 Bridges et al., 1973 CM 571; CM 611 " " -6 " " WP2 " " +5 " " WP2 uvr A " " +5 " " WP2 micro drop of ? -7 Dean, 1972a analytical grade, technical grade or 10% acqueous solution/plate K12HfrH 0.1% 97.5% + Voogd et al., 1972 C. freundii 425 0.05% or 0.1% " +8 " " E. aerogenes 0.1% " + " " K. pneumoniae 0.05% oe 0.1% " + " " S. typhimurium 64-320 " " + " " S. marcescens Hy/alpha 13 1.25-5 mg/disc ? + Dean, 1972a Hy/alpha 21 E. coli WP2 WP2 5 µg/ml ? +9 Green et al., 1976 hcr+/hcr- 20-25 µl/disk 50% commercial +10 Nagy et al, 1975 WP2 5 mg/plate formulation " " hcr+/hcr- ? Table 4 (contd) Test system Test object Concentration Purity Results 1 Reference Reverse mutation S. typhimurium TA 1535 5 mg/plate ? + Shirasu et al., 1976 (cont'd) TA 1536 " ? + " " TA 1537 " ? - " " TA 1538 " ? - " " E. coli WP2 5 µl/plate11 ? + Hanna & Dyer, 1975 WP2 uvr A " ? + " " WP 67 " ? + " " CM 561 " ? - " " CM 571 " ? - " " CM 611 " ? - " " S. typhimurium TA 1530 TA 1535 5 µl/plate ? + Hanna & Dyer, 1975 his C117 " ? + " " his G46 " ? - " " " ? - " " P. aeroginusa PAO 38 0.08 mol/l ? + Dyer & Hanna, 1973 S. typhimurium his C117 0.03 mol/l ? + " " S. typhimurium TA 98 ? ? -3 Braun et al., 1982 TA 100 ? ? +3 " " TA 1535 ? ? -3 " " TA 1536 ? ? -3 " " TA 1537 ? ? -3 " " TA 1538 ? ? -3 " " TA 98 0.1-6.6 mg/plate12 99% -3 Chan, 1989: Zeiger et al., 1988 Table 4 (contd) Test system Test object Concentration Purity Results 1 Reference TA 100 " " " +3 Carere et al., 1978a,b TA 1535 2.8 mg/plate 99.9% -3 " " TA 1536 " " " -3 TA 1537 " " " S. typhimurium TA 1538 2.8 mg/plate 99.9% -3 Carere et al., 1978a,b TA 1535 1.5 mg/ml 99.9% + Schizosaccharmoyces pombe 1.5-14 mmol/plate13 > 99% +3 Gilot-Delhalle et al., ade 6 1983 Gene conversion S. cerevisiae D4 2-8 mg/ml > 97% +14,15 Dean et al., 1972 D4 6-40 mmol/l ? +2 Fahrig, 1973, 1974 632/4 ? ? - Guerzoni et al., 1976 +16 Griffin & Hill, 1978 DNA strand breaks E. coli K-12CR34Co1E1 1 mg/ml ? Growth inhibition E. coli W3110 pol A+/pol A- 6.4 mmol/l ? + Rosenkranz, 1973 P. mirabilis PG 273; PG 713 ? ? +15 Braun et al., 1982 1 Without metabolic activation except where noted. 2 Positive controls (methyl methanesulfonate 1-4 mmol/l) yielded expected positive results. 3 Both with and without metabolic activation. 4 Only without metabolic activation. 5 Methyl methane sulfonate (0.04%) yielded positive responses. 6 Methyl methane sulfoante (0.04%) yielded negative responses. 7 Methyl methane sulfonate, N-methyl-N'-nitro-N-nitroso guanidine yielded positive response. 8 At 0.1% 9 Positive controls (methyl methanesulfonate 0.5-2.0 µg/ml) yielded expected positive results. Table 4 (contd) 10 Positive controls (N-methyl-N'-nitro-nitroso guanidine, N-nitroso-N-methylurethane and acridium chloride) yielded positive responses. 11 Toxic dose. 12 Toxic effect at 3.3 mg/plate. 13 The LD50 was 5.5 mmol/l. 14 Positive controls (ethyl methane sulfonate) yielded positive responses. 15 From 4 mg/ml. 16 Methyl methane sulfonate (2-10 mmol/l), N-methyl-N'-nitro-N-nitroso guanidine (3.4-6.8 mmol/l) yielded positive responses. Table 5. Mutagenicity tests in mammalian cells in vitro Test system Test object Concentration Purity Results Reference Viral transformation Syrian hamster embryo 0.05-0.45 mmol/l ? +1 Hatch et al., 1986 cells/adenovirus SA7 cytotoxic Gene mutation Chinese hamster V79 cells up to 1 mmol/l ? - Wild, 1975 (azaguanine resistance) 1.25-5 mmol/l ? +2 Aquilina et al., 1984 Mouse lymphoma L5178Y cells 6.25-50 nl/m3 ? +4,5 Chan, 1989 (trifluorothymidine resistance) 12.5-200 nl/m6 +5,7 Chan, 1989 DNA strand breaks Chinese hamster V79 cells 0.2% ? +8 Green et al., 1974 Sister chromatid exchange Primary rat tracheal epithelial cells 5-160 µg/ml9 93.9% +10,11 Lin et al., 1988 Chinese hamster ovary cells 0.03 and 0.1 mmol/l 98 + Nishio & Uyeki, 1981 0.1-0.5 mmol/l > 98% + Tezuka et al., 1980 0.3-1000 µg/ml ? +12 Wang et al., 1988 1-50 µg/ml ? +13 Chan, 1989 Human lymphocytes 2.5-10 µg/ml 99% - Nicholas et al., 1978 Human fetal lung fibroblasts 99% - Nicholas et al., 1978 Chromosomal aberrations Rat tracheal epithelial cells 5-160 µg/ml9 93.9% +11,14 Lin et al., 1988 Chinese hamster lung fibroblasts ? ? + Ishidate et al., 1981 Chinese hamster V79 cells 0.1-0.5 mmol/l > 98% +15 Tezuka et al., 1980 Chinese hamster ovary cells 16-160 µg/ml ? +16 Chan, 1989 Human lymphocytes 1-40 µg/ml > 99% -17 Dean, 1972b Table 5 (contd) Test system Test object Concentration Purity Results Reference Unscheduled DNA synthesis Human lymphocytes 5-500 µg/ml 99.8% + Perocco & Fini, 1980 EUE human cells 6.5-650 mmol/l ? +18 Aquilina et al., 1984 DNA (sedimentation coefficient) Calf thymus DNA 0.1% ? + Rosenkranz & Rozenkranz, 1972 DNA (thermolabile regions) Calf thymus DNA 45 mmol/l 99% - Olinski et al., 1980 DNA (resistance to micrococcal) Chinese hamster ovary cells 10 mmol/l ? + Nishio & Uyeki, 1980 1 Positive controls (benzo(a)pyrene 0.001-0.002 mmol/l) yielded expected positive responses. 2 Positive controls (ethyl methan sulfonate 20 mmol/l) yielded expected positive responses. 3 Growth inhibition from 12.5 nl/ml. 4 From 12.5 nl/ml. 5 Positive controls (methyl methanesulfonate 5 nl/ml) yielded expected positive responses. 6 Growth inhibition from 100 nl/ml. 7 From 100 nl/ml. 8 Negative at lower concentrations. 9 50% mortality was observed at 80 µg/ml. 10 From 10 µg/ml. 11 Positive controls (N-methyl-N'-nitro-N-nitroso guanidine 0.25-1 µg/ml, 50% mortality at 0.5 µg/ml) yielded expected positive responses. 12 From 40 µg/ml. 13 From 10 µg/ml. When incubated with S9, positive response at 50 µg/ml. 14 From 80 µg/ml. 15 At 0.5 mmol/l. 16 At 160 µg/ml. 17 Cytotoxicity was observed at 5-40 µg/ml. 18 Positive controls (N-methyl-N'-nitro-N-nitroso guanidine) yielded expected positive responses. 19 Possible index of DNA alkylation. 20 Possibly indicating structural rearrangement of chromatin. Table 6. Mutagenicity tests in vivo Test system Test object Concentration Purity Results* Reference Crossing over/ Drosophila melanogaster 0.035%1 ? - Jayasuriya & Ratnayaka, 1973 recombination ++++/dp b cn bw Sex-linked Drosophila melanogaster 0.035%1 ? - Jayasuriya & Ratnayaka, 1973 recessive lethal Sobels & Todd, 1979 mutation Oregan K 0.01-0.1 ppm in food ? -2 Kramers & Knapp, 1978 commercial formulation Chromosomal Drosophila melanogaster 1 ppm in food ? + Gupta & Singh, 1974 aberration Autosomal recessive Drosophila melanogaster 0.1-0.75 ppm in food for ? + Hanna & Dyer, 1975 levels 18 months Host-mediated assay Salmonella typhimurium 25 mg/kg bw s.c. ? - Buselmayer et al., 1972 G46 His- in mice (NMRI) Salmonella typhimurium 8-10 mg/kg bw p.o. 97.5% -3 Voogd et al., 1972 (64-320) in mice (Swiss) Serratia marcescens 25 mg/kg bw s.c. ? - Buselmayer et al., 1972 (a 21 Leu-) in mice (NMRI) Sacchoromyces cerevisiae 50 or 100 mg/kg bw p.o. > 97% -3 Dean et al., 1972 (D4) in mice (CF1) Table 6 (contd) Test system Test object Concentration Purity Results* Reference Dominant lethal Female mice (CF1) 0, 25 or 50 mg/kg bw p.o. > 97% -4 Dean & Blair, 1976 0, 2 or 8 mg/m3 inhalation, > 97% - Dean & Blair, 1976 from weaning to 11 weeks of age Male mice (ICR/Ha Swiss) 0, 5 or 10 mg/kg bw/day ? - Epstein et al., 1972 p.o. for 5 days (8 weeks of mating) Single i.p. injection of ? - Epstein et al., 1972 13 or 16.5 mg/kg bw (8 weeks of mating) Male mice (Q) 2 ppm in drinking water, 99% - Degraeve et al., 1984a 5 days/week for 7 week 10 mg/kg bw i.p. 99% -5 Moutschen-Dahmen et al., 1981 Male mice (CF1) 30 or 55 mg/m3 inhalation > 97% - Dean & Thorpe, 1972b for 16 h 2.1 or 5.8 mg/m3 inhalation > 97% -6 Dean & Thorpe, 1972b for 23 hours/day for 4 weeks Sister chromatid Male mice (B6C3F1) 5-35 mg/kg bw i.p. 99% -7 Kligerman et al., 1985 exchange peripheral lymphocytes Male mice (B6C3F1) 6-40 mg/kg bw i.p. ? -8 Chan, 1989 bone marrow cells Table 6 (contd) Test system Test object Concentration Purity Results* Reference Micronucleus test Mice (Swiss Webster) 0.0075-0.015 mg/kg bw/day ? -9 Paik & Lee, 1977 i.p. for 2 or 4 days Micronucleus test Mice (HRA/Skh, hairless) skin painting with tech.grade +10 Tungul et al., 1991 (in vitro/in vivo) skin keratinocytes 0-228 µg (in 100 µl acetone) Chromosomal Chinese hamster, both 10-15 mg/kg bw p.o. > 97% -11 Dean & Thorpe 1972a aberrations sexes bone-marrow cells Male mice (Q) 2 ppm in drinking-water, 99% - Moutschen-Dahmen et al., 1981; bone-marrow cells 5 days/week for 7 weeks Degraeve et al., 1984b 10 mg/kg bw i.p. 99% - Degraeve et al., 1984b Degraeve et al., 1984b Male mice (B6C3F1) bone 6-40 mg/kg bw i.p. ? -12 Chan, 1989 marrow cells Female Syrian golden 0, 3, 6, 15 or 30 mg/kg 50% +13 Dzwonkowska & Hübner, 1986 hamster commercial formulation bone-marrow cells bw i.p. Mice (CF1), both sexes 64-72 mg/m3 inhalation > 97% -11 Dean & Thorpe, 1972a bone-marrow cells for 16h 5 mg/m3 inhalation > 97% > 97% -11 Dean & Thorpe, 1972a 23 hours/day for 21 days Male Chinese hamsters 28-36 mg/m3 inhalation > 97% -11 Dean & Thorpe, 1972a bone-marrow cells for 16h. Table 6 (contd) Test system Test object Concentration Purity Results* Reference Chromosomal Mice (Q) spermatocytes 2 ppm in drinking-water 99% - Moutschen-Dahmen et al., 1981; aberrations 5 days/week for 7 weeks Degraeve et al., 1984a (cont'd) Chinese hamsters 15 mg/kg bw p.o. > 97% -11 Dean & Thorpe, 1972a spermatocytes Mice (Q) spermatocytes 10 mg/kg bw i.p. 99% - Moutschen-Dahmen et al., 1981; Degraeve et al., 1984a Mice (CF1) spermatocytes 64-72 mg/m3 inhalation > 97% -11 Dean & Thorpe, 1972a for 16h 5 mg/m3 inhalation > 97% -11 Dean & Thorpe, 1972a 23h/day for 21 days Chinese hamster 28-36 mg/m3 inhalation > 97% -11 Dean & Thorpe, 1972a spermatocytes for 16 h Mice (CF1) spermatogonia 2 ppm in drinking-water, 99% - Moutschen-Dahmen et al., 1981; 5 days/week for 7 weeks Degraeve et al., 1984b 10 mg/kg bw i.p. 99% - Moutschen-Dahmen et al., 1981; Degraeve et al., 1984b DNA strand breaks Rats (Wistar), both 10 mg/kg bw i.p. 99.8% -14 Wooder & Creedy, 1979 sexes rat liver cell DNA Table 6 (contd) Test system Test object Concentration Purity Results* Reference Unscheduled DNA Male rats (F344) 0, 2, 10 or 35 mg/kg ? - Mirsalis et al., 1989 synthesis hepatocytes bw p.o. Mice (B6C3F1) (both sexes) 0, 10, 20, 40 or > 98% -15 Bedford, 1991 forestomachrats 100 mg/kg bw p.o. 1 Approximate LD50 2 Positive controls (2.5 mmol/l ethyl methanesulfonate) yielded expected positive responses. 3 Positive controls (400 mg/kg bw p.o. ethyl methanesulfonate) yielded expected positive responses. 4 Positive controls (100 mg/kg bw p.o. methyl methanesulfonate) yielded expected positive responses. 5 Positive controls in 2nd and 5th week of mating. 6 Positive controls (2000 mg/kg bw p.o. methyl methanesulfonate) yielded expected positive responses. 7 Positive controls (2-acetylaminofluorene, ethyl methane sulfonate and N-nitroso morpholine) yielded positive results. All animals treated with 35 mg/kg bw i.p. died. 8 Positive controls (ethyl methane sulfonate 100 mg/kg bw p.o.) yielded expected positive responses. 9 Positive controls (cyclophosamide 30-240 mg/kg bw i.p.) yielded expected positive responses. 10 Recovery of plated cells in vitro after a single topical application of dichlorvos was about 50% of controls. 11 Positive controls (endoxan 100-200 mg/kg bw i.p.) yielded expected positive responses. 12 Positive controls (ethyl methane sulfonate 300-375 mg/kg bw p.o.) yielded expected positive responses. 13 LD50 = 30 mg/kg bw i.p. No dose related response. Positive controls (cyclophosphamide 40 s. mg/kg bw i.p.) yielded expected positive responses. 14 Positive controls (methyl methane sulfonate 30-60 mg/kg bw i.p.) yielded expected positive responses. 15 Positive controls (N-methyl-N'-nitro-N-nitroso guanidine 200 mg/kg bw p.o.) yielded weakly positive results. Dichlorvos treatment induced a hyperplastic response similar to that induced by the non-genotoxic carcinogen butylated hydroxyamisole (300 mg/kg bw p.o.). Special studies on metabolites Acute and short-term toxicity studies The intraperitoneal toxicity of metabolites of dichlorvos in female mice is considerably less than that of dichlorvos. The LD50 (mg/kg bw) was 250 for dichloroacetic acid, 440 for dichloroacetaldehyde and 890 for dichloroethanol. For other metabolites it was > 1000 mg/kg bw. A short-term inhalation study with dichloroacetaldehyde in rats did not show adverse effects at concentrations up to 2 mg/m3 for 30 days (WHO, 1989). Methylating reactivity In a quantitative colour test in which methylation of 4-(p-nitrobenzyl) pyridine was measured to predict DNA alkylating potential, desmethyl-dichlorvos, dimethyl phosphate, dichloroethanol, dichloroacetaldehyde and dichloroacetic acid gave no reaction (Bedford & Robinson, 1972). Mutagenicity In a rec-type repair test with Proteus mirabilis strains PG713 and PG273, desmethyldichlorvos (10 or 40 µmol/plate) did not induce base-pair substitutions or other DNA damage (Braun et al., 1982). Dichloroacetaldehyde (DCA) appeared to be mutagenic in the Salmonella test using S. typhimurium TA 100 and TA 1535. The mutagenicity decreased in the presence of a microsomal activation system (Lofroth, 1978; Bignami et al., 1980). It was also positive when tested in concentrations of 10-40 µg/plate in a forward and a back mutation system in S. coelicolor and two forward mutation systems in A. nidulans (Bignami et al., 1980). DCA (0.01%) was not mutagenic to K. pneumoniae in a fluctuation test (Voogd et al., 1972). DCA did not induce unscheduled DNA synthesis in the human epithelial-like cell EUE as well as ouabain-resistant mutations in cultured V-79 Chinese hamster cells (Aquilina et al., 1984). In a dominant lethal assay with DCA administered as a single i.p. injection of 176 mg/kg bw to two strains of male mice (AB Jena-Halle and DBA), positive effects were reported (Fischer et al., 1977). It should be noted, however, that the dose used would never be reached after dichlorvos treatment (i.p. LD50 = 28-41 mg/kg bw). Ramel (1981) reported negative results in a dominant lethal study with another strain of mice. No evidence for mutagenicity of dichloroethanol was obtained in S. typhimurium TA 100 and TA 1535 (Lofroth, 1978; Bignami et al., 1980). In S. coelicolor (test doses 60-80 µg/plate) and A. nidulans (test doses 10-50 µg/plate), dichloroethanol was a weak mutagen (Bignami et al., 1980). Dichloroethanol (1 M and 0.1%) was mutagenic to K. pneumoniae in a fluctuation test (Voogd et al., 1972). Special studies on skin sensitization In the guinea-pig maximization test by Magnusson & Kligman, using intradermal and topical induction concentrations of 5% and 25% in water respectively, a 0.5% challenge concentration caused sensitization in 30-40% of the animals but a 0.05% challenge concentration caused no sensitization (Matsushita et al., 1985). Special studies on testes Mice Groups of 14 male NMRI/Han mice received either a single oral dose of 40 mg/kg bw dichlorvos in olive oil or 18 daily oral doses of 0 or 10 mg/kg bw dichlorvos in olive oil. On days 9,18, 27, 36, 54 and 63, two animals from each group were killed and their testes examined histologically. A significant increase in the number of damaged seminiferous tubules (desquamation, decreases in cell population, "holes") was observed in both dichlorvos groups. The supporting Sertoli cells were also damaged, which may have resulted in the above effects. In addition, there was an increase in the number and hypertrophy of Leydig cells. No explanation was given for these effects (Krause & Homola, 1972, 1974). An increased incidence, just above background, of sperm abnormalities, was observed in a screening study on hybrid mice given five daily i.p. injections of 10 mg/kg bw dichlorvos (approximately half the LD50). At lower doses, 1 mg/kg bw, the number of sperm abnormalities was either similar to or lower than those in the controls (Wyrobek & Bruce, 1975). Rats Groups of 16 male juvenile Wistar rats received either 20 mg/kg bw dichlorvos in olive oil on days 4 and 5, 10 mg/kg bw dichlorvos in olive oil daily from days 4 to 23, or 0.1 ml olive oil daily from days 4 to 23. On days 6, 12, 18, 26, 34, and 50 of life, two rats from each group were sacrificed. Histological examination of the testes showed slight reduction in the number of spermatogenic cells and Leydig cells. All changes were reversed by the 50th day of life. It was assumed that a reduction in testosterone synthesis resulted in damage to the spermatogenic cells (Krause et al., 1976). In a subsequent experiment measurement of testosterone concentrations in the testes, and luteinizing hormone (LH) and follicle stimulating hormone (FSH) concentrations in serum showed no differences between treated animals and olive-oil treated controls (Krause, 1977). However, in this study the use of a different dosing regimen (10 mg/kg bw by gavage every other day for 2 weeks) prevented a strict comparison with the earlier study by Krause et al. (1976). Fifty-five male Wistar rats (aged 5 months) were orally administered dichlorvos at levels of 5 or 10 mg/kg bw every other day for 8 weeks. Eleven rats were killed every 4 weeks. No change was seen in body-weight gain or testes weight. The score values of the seminal cellular system decreased after 4-8 weeks of treatment, but were restored 8 weeks after the end of treatment (Fujita et al., 1977). Miscellaneous studies Effects of inhaled ChE inhibitors, including dichlorvos (dissolved in acetone), on bronchial tonus were studied in young adult rats exposed head-only. Bronchoconstriction did not occur at toxicologically significant doses. An increase in response to acetylcholine provocation test was observed (Pauluhn et al., 1987). The effect of diet on the toxicity of dichlorvos was investigated using young male rats kept for 30 days on the following diets: high protein (HPD), low protein (LPD), high fat (HFD), and standard (SD). Growth rates were normal except for a slightly decreased body-weight gain in the HFD group. A single i.p. injection of 50 mg/kg bw dichlorvos led to slightly higher mortality in LPD rats and slightly lower mortality in HPD rats, compared with SD rats (Purshottam & Kaveeshwar, 1979). In a further study, growing male rats were kept on an HFD or HPD for 30 days. At the end of this period, a single i.p. dose of dichlorvos (20 or 30 mg/kg bw) was administered. No difference in plasma and erythrocyte ChE inhibition was found. In the case of the HPD, the spontaneous recovery of plasma and erythrocyte ChE activity was slightly reduced (Purshottam & Srivastava, 1984). Observations in humans In vitro studies The sensitivity to inhibition by dichlorvos of erythrocyte and brain ChE was higher than that of plasma ChE, the I50s being about 10-8 and 10-7 mol/l, respectively (Skrinjaric-Spoljar et al., 1973; Carter & Maddux, 1974; Lotti & Johnson, 1978; Boyer, 1978; Casale et al., 1989). The rate of phosphorylation of human erythrocyte ChE was similar to that of rat brain ChE (1.2 x 105 and 1.5 x 105 mol/l/min at 37 °C, pH 7.4-7,6, respectively). The rate of dichlorvos hydrolysis by human plasma at 37 °C was found to be 7-11 µmol/hour/ml (Reiner et al., 1980; Traverso et al., 1989). Studies on volunteers Six healthy male volunteers were given single oral doses of 7.5 mg/kg bw of metrifonate. Concentrations of metrifonate (trichlorfon) and dichlorvos, which is a metrifonate rearrangement product, were determined in whole blood at different times for up to 24 hours. The half-life of metrifonate was about 2 hours. The concentrations of dichlorvos closely followed those of metrifonate with a constant ratio of 0.01-0.02. However, a half-life for dichlorvos of 2 hours cannot be accepted because dichlorvos is continuously formed from metrifonate (Abdi & Villen, 1991). Groups of five young men received total daily doses of 1, 1.5, 2 or 2.5 mg dichlorvos in corn oil per person, divided in two gelatin capsules administered at 08.00 h and at 15.00 h for 28 days. A further group of ten men received 1.5 mg/day for 60 days. Control groups of two men per dose level received gelatin capsules with corn oil. Plasma and erythrocyte ChE activities were determined twice weekly before, during and after dosing. Once each week a medical interview, complete blood count, and urinalysis were done on each subject. Before dosing and after the last dose SGOT, ALP, PT, thymol turbidity, and total bilirubin were determined. The 2.5 mg/day dose produced a decrease in plasma ChE activity from the second week of treatment; dosing was discontinued after 20 days when plasma values showed a 30% decrease. The plasma ChE activity returned to control values 15 days after dosing was ended. The 2 mg/day dose produced also a reduction in plasma ChE activity from the second week of treatment which reached a maximum inhibition of 29% the second day after the last dose. The group receiving 1.5 mg/day for 28 days showed no change in plasma ChE activity while in the group dosed for 60 days this activity was inhibited by 27% compared to the control values. None of the groups showed an effect on erythrocyte ChE activity, haematology, clinical chemistry or urinalysis. The NOAEL in this study was the highest dose tested of 2.5 mg/day/man (approximately 0.03 mg/kg bw/day) based on the absence of inhibition of erythrocyte ChE activity (Rider, 1967; Rider et al., 1967, 1968). Two groups of six men (21-45 years of age, 64-106 kg bw) received 0.9 mg dichlorvos three times a day for 21 days, either in a pre-meal capsule filled with cottonseed oil or in a gelatin salad consumed during the course of the meal. Control subjects were dosed with either cotton seed oil capsules or plain gelatin. No cholinergic signs or symptoms were observed. Plasma ChE activity was inhibited by 30-40%, the inhibition being higher when dichlorvos in cotton seed oil was ingested before the meal. The half-life for the regeneration of plasma ChE activity was 13.7 days. Erythrocyte ChE activity was not reduced. The NOAEL in this study was 0.04 mg/kg bw/day, based on absence of erythrocyte cholinesterase inhibition (Boyer et al., 1977). A number of studies have been done with a slow-release PVC formulation of dichlorvos intended for use as an anthelminticum. Single oral doses of above 4 mg dichlorvos/kg bw resulted in inhibition of erythrocyte ChE activity 24 hours after application, the maximum being 46% at 32 mg/kg bw. Plasma ChE activity was affected at single doses of 1 mg/kg bw (50% inhibition) and above. However, no dichlorvos-related symptoms were observed. Repeated oral dosing for 7 days produced clinical symptoms with doses of 8 mg/kg bw/day and above. Plasma ChE activity was inhibited by about 80% at all dose levels (1-16 mg/kg bw/day). Erythrocyte ChE activity showed a dose-related decrease from 5-30% at 1 mg/kg bw/day up to 50-80% inhibition at the highest dose. Blood count, urine, liver function, PT and BUN were normal in all studies (Hine & Slomka, 1968, 1970; Pena-Chavarria et al., 1969; Slomka & Hine, 1981). In children (aged 7-18 years) orally treated with metrifonate (7.5, 10.0 or 12.5 mg/kg bw), the half-lives of recovery of inhibited erythrocyte AChE and plasma ChE were 15 and 6.7 days, respectively (Reiner & Plestina, 1979). It is known that the anticholinesterase activity of metrifonate is due to its decomposition to dichlorvos (Reiner et al., 1975). Similar half-lives for plasma ChE have been described by Boyer et al., (1977) after repeated oral dosing with dichlorvos and by Bisby & Simpson (1975) after cutaneous exposure of a sprayman to dichlorvos. This longer half-life contrasts with faster in vitro half-lives, which are similar to those of the rodent (Skrinjaric-Spoljar et al., 1973). Several inhalation studies have been carried out and are summarized by WHO (1989). These studies confirmed that plasma ChE is more sensitive to inhibition by dichlorvos than RBC ChE. The latter was found inhibited when the dichlorvos dose (concentration x time) was higher than about 1500 mg/m3/min (WHO, 1989). Thirteen men (31-53 years of age) were exposed to dichlorvos strips (20% dichlorvos; 1 strip per 30 m3) for 3 months. No biologically or statistically significant changes were observed in their electromyography results nor in their whole blood ChE activity (Ottevanger, 1975). Poisoning incidents Fournier et al. (1978) reported that dichlorvos was detected in blood in three cases of human intoxication within 24 hours after poisoning (exact timing not reported). A woman who intentionally ingested an estimated 100 mg dichlorvos/kg bw survived following intensive care for 14 days (Watanabe et al., 1974). A suicide with a dichlorvos dose of about 400 mg/kg bw succeeded in spite of treatment (Shinoda et al., 1972). A female patient, aged 35 years, who had accidentally ingested 60 g fluid Divipan (dichlorvos concentration not reported), was comatose for a week and recovered slowly. Clinical and electrophysiological examinations showed a pure motor form of neuropathy, according to the authors (Vasilescu & Florescu, 1980). Three cases of poisoning with dichlorvos taken orally in unspecified but high quantities have been reported from India. The patients first showed severe cholinergic signs for a few days. After recovery, delayed neurotoxicity developed. Nerve conduction studies showed a severe axonal degeneration (Wadia et al., 1985, 1987). Thirteen cases of dichlorvos ingestion, either accidental or deliberate, were reported from a hospital in Beijing, China. The patients included 10 women and three men, aged 11-38 years. Twelve of the 13 patients ingested 25-50 ml of dichlorvos (80% purity). Ten cases were associated with acute pulmonary oedema. Two patients died because OP poisoning was not diagnosed in time. The others recovered completely 2-5 days after treatment (Li et al., 1989). Occupational Exposure A number of fatal and non-fatal dichlorvos poisoning cases have been described and summarized by WHO (1989). Two workers who failed to promptly wash off the concentrated formulation of dichlorvos, which splashed on to their skin, died subsequently. However, in those cases where the spilled solution was washed off immediately, the victims showed symptoms of intoxication but recovered after treatment. Occupational exposure of spraymen entails both dermal and respiratory absorption of dichlorvos. When appropriate protective equipment was not used, inhibition of plasma ChE and less frequently of RBC ChE was found. Sometimes mild, short lasting, cholinergic symptoms and signs have been reported (WHO, 1989). Each of 13 pest-control operators carried out urban pest-control work for one day in 4 houses using 230-330 g dichlorvos as aerosol and 40-50 g dichlorvos as emulsion spray. At the end of the day's work, an operator had an average dichlorvos residue of 0.8 mg/m2 on the back, 0.4 mg/m2 on the chest, and 11 mg/m2 on the respirator filter. Urinary dimethylphosphate excretion in 3 applicators ranged from 0.32 to 1.39 micrograms on the day of treatment, but approached the level of detection by the following morning. Blood and urine analyses revealed no changes in various clinical parameters, including serum cholinesterase levels (Das et al., 1983). COMMENTS Dichlorvos is rapidly absorbed by all routes of exposure and rapidly degraded. The metabolic pathways of dichlorvos are similar in mammalian species, including humans. Metabolites are rapidly excreted or incorporated into natural enzymatic pathways. Dichlorvos has a marked acute oral toxicity with typical cholinergic signs and has been classified by WHO as highly hazardous. Rat erythrocyte and brain cholinesterase inhibited by dichlorvos spontaneously reactivates with a half-life of about two hours both in vitro and in vivo. Several carcinogenicity studies in mice and rats using routes other than gavage were negative, even when doses causing signs of toxicity were used. It should be noted that two squamous-cell carcinomas of the oesophagus have been observed in treated mice in one study. In a carcinogenicity study in mice dichlorvos administered by corn oil gavage (0, 10 or 20 mg/kg bw/day to males, and 0, 20 or 40 mg/kg bw/day to females), caused forestomach papillomas (statistically-significant positive trend with increased incidence in the high-dose female group). Elements of the mechanism by which these papillomas might arise have not been established, but the induction of hyperplasia in the forestomach was demonstrated. Additionally, genotoxic effects might occur at high local concentrations of dichlorvos (see below) as can be obtained in gavage dosing but not in dietary exposure. Based on the increased incidence of forestomach papillomas, the NOAEL was 10 mg/kg bw/day. In a two-year feeding study in rats (0, 0.1, 1, 10, 100 or 500 ppm), no neoplastic lesions were attributed to treatment. The NOAEL, based on brain cholinesterase inhibition, was 100 ppm (actual concentration 47 ppm, equivalent to 2.4 mg/kg bw/day). In a carcinogenicity study in Fischer 344 rats, dichlorvos administered by corn oil gavage (0, 4 or 8 mg/kg bw/day) caused an increased incidence of pancreatic adenomas (statistically significant in males only), mononuclear cell leukemias (statistically significant in males only, no dose response) and mammary gland adenomas or fibroadenomas (females only, no dose response, statistically-significant in the low-dose group only). The Meeting observed that the incidence of pancreatic acinar adenomas in male control rats was unusually high and therefore the higher incidence found in treated animals was considered of questionable biological significance. The increased incidence of mononuclear cell leukaemia which is usually high and variable in this strain of rats, was also of questionable biological significance. The doses used significantly inhibited plasma, but not erythrocyte, cholinesterase activity when measured three hours after treatment. However, given the rapid recovery of erythrocyte cholinesterase activity after inhibition by dichlorvos, the timing might have underestimated the inhibition. Dichlorvos has been adequately tested in a series of in vitro and in vivo genotoxicity assays. These data indicate that dichlorvos is genotoxic in bacteria and cultured mammalian cells, but that it is not clastogenic in vivo except under conditions where an unusually high tissue dose can be attained. Dichloro- acetaldehyde, a major metabolite of dichlorvos, is a weak bacterial mutagen. Positive results have been reported in mice given a dose of dichloroacetaldehyde far greater than that which could derive from sublethal doses of dichlorvos. Dichlorvos has been shown to methylate DNA in vitro at a rate that is 8-9 orders of magnitude lower than the rate of phosphorylation. Therefore, DNA alkylation is not likely to occur at doses of dichlorvos which are not inhibitory to erythrocyte/brain cholinesterase. A three-generation reproduction study in rats was negative at doses up to 235 ppm in the diet, equivalent to 12 mg/kg bw/day. A one-litter, one-generation study in mice in which dichlorvos was administered by inhalation at doses which caused > 90% plasma cholinesterase inhibition, but no signs of toxicity, was negative. Dichlorvos caused reversible damage of seminiferous tubules, Leydig and Sertoli cells at oral doses of 10 mg/kg bw daily for 18 days in mice and at 5 mg/kg bw and above every other day for 8 weeks in rats. Dichlorvos appeared not to be teratogenic in mice, rats and rabbits at doses which caused maternal toxicity. Dichlorvos caused delayed polyneuropathy in hens at doses much higher than the unprotected LD50. Cases of delayed polyneuropathy also have been reported in humans after severe intoxications. In humans, the rate of dichlorvos hydrolysis by plasma is similar to that in rats. The rate of recovery of inhibited erythrocyte and plasma cholinesterase activity in humans given dichlorvos is much slower than in rats. Half-lives of recovery are about 15 days in humans and about two hours in rats. A daily dose of 1 mg/kg bw to male human volunteers for seven days caused 5-30% inhibition of erythrocyte cholinesterase. The NOAEL in humans, based on absence of erythrocyte cholinesterase inhibition in 12 volunteer males for 21 days was 0.04 mg/kg bw/day. In 1986, the Joint Meeting discussed the significance of carcinogenicity studies for organophosphorus pesticides and the requirements for further studies (Section 3.1 of Annex 1, reference 47). At that time none of the organophosphorus pesticides had caused a carcinogenic response in experimental animals. That Joint Meeting recommended that, depending upon future evaluation on a case by case basis, further consideration should be given to the need for carcinogenicity studies for organophosphates. In assessing the potential hazard to humans of residues of dichlorvos, the following considerations were taken into account in view of the weakly positive results in the gavage carcinogenicity study in mice. Organophosphorus esters used as insecticides react with biological molecules by means of phosphorylation of serine hydrolases and of alkylation of macromolecules. Phosphorylation of acetylcholinesterase and alkylation of DNA are considered to account for the acute cholinergic toxicity and initiation of the carcinogenic process, respectively. These biochemical reactions occur at different rates. When the rate of phosphorylation is substantially higher than the rate of alkylation, in vivo genotoxic effects are unlikely to occur because effective doses cannot be achieved due to acute toxicity. Dichlorvos meets these criteria, the rate of phosphorylation of acetylcholinesterase being much faster (eight orders of magnitude) than that of alkylation of several macromolecules, including DNA. Hence positive mutagenicity tests were seen only in vitro and, as indicated in the 1986 Joint Meeting report, carcinogenicity studies are unlikely to give more information. The weak carcinogenic response of dichlorvos obtained in mice in a corn oil gavage study should be interpreted as a local effect of dichlorvos. Information on comparative cholinergic toxicity might be of critical relevance for the extrapolation of toxic effects (other than acute effects) of organophosphates in experimental animals to humans. The characteristics of the interactions of a given compound with acetylcholinesterase (rates of phosphorylation, spontaneous reactivation and ageing) from different species can be compared in vitro. Also, the in vivo rate of reappearance of blood acetylcholinesterase activity can be measured. In some cases, metabolic degradation of organophosphates can be assessed comparatively by measuring the level of serum A esterase, which hydrolyses a given compound. All these data enable an improved assessment of cholinergic toxicity of organophosphates in different species. This knowledge may be of special significance in the case of dimethyl phosphates since the rates of in vivo reactivation vary substantially across species. Therefore, chronic dosing is more critical for extrapolation from animal data to humans. In a repeated dose regime, the longer the half-life of reactivation the more rapid and/or more toxic will be the resulting effect (i.e. in a chronic dosing regime, humans will survive much lower doses of dichlorvos causing, when given alone, peak erythrocyte/brain cholinesterase inhibition than those which can be reached in rodents). Therefore, comparison between the in vivo rates of recovery of enzyme activity will enable an assessment of the repeated doses of compounds and the resulting cholinesterase inhibition, which would represent the limiting factors for other toxicities (including mutagenicity and carcinogenicity). In the case of dichlorvos, the Meeting considered the extrapolation of carcinogenicity data derived in rodents and its applicability to human safety, and concluded that the compound would not result in chronic human health hazards at doses below those which result in acetylcholinesterase inhibition. The Meeting maintained the ADI, which is based on studies in humans with a NOAEL of 0.04 mg/kg bw/day, using a 10-fold safety factor. TOXICOLOGICAL EVALUATION Level causing no toxicological effect Mouse: 10 mg/kg bw/day (two-year study) Rat: 47 ppm in the diet, equivalent to 2.4 mg/kg bw/day (two-year study) Human: 0.04 mg/kg bw/day (21-day study) Estimate of acceptable daily intake for humans 0-0.004 mg/kg bw Studies which will provide information valuable in the continued evaluation of the compound Further observations in humans. REFERENCES Abdi, Y.A. & Villen, T. (1991) Pharmacokinetics of metrifonate and its rearrangement product dichlorvos in whole blood. Pharmacol. 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See Also: Toxicological Abbreviations Dichlorvos (EHC 79, 1988) Dichlorvos (HSG 18, 1988) Dichlorvos (ICSC) Dichlorvos (FAO Meeting Report PL/1965/10/1) Dichlorvos (FAO/PL:CP/15) Dichlorvos (FAO/PL:1967/M/11/1) Dichlorvos (FAO/PL:1969/M/17/1) Dichlorvos (AGP:1970/M/12/1) Dichlorvos (WHO Pesticide Residues Series 4) Dichlorvos (Pesticide residues in food: 1977 evaluations) Dichlorvos (IARC Summary & Evaluation, Volume 53, 1991)