AZINPHOS-METHYL. First draft prepared by U. Mueller 1 and A. Moretto 2. Food Standards Australia New Zealand, Canberra, ACT, Australia; and 2

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1 AZIPHS-METHYL First draft prepared by U. Mueller 1 and A. Moretto 2 1 Food Standards Australia ew Zealand, Canberra, ACT, Australia; and 2 Department of ccupational Medicine and Public Health, University of Milan, ICPS spedale Sacco, Milan, Italy Explanation Evaluation for acceptable daily intake Biochemical aspects Absorption, distribution and excretion Biotransformation Effect on enzymes and other biochemical parameters Toxicological studies Acute oral toxicity Short-term studies of toxicity Long-term studies of toxicity and carcinogenicity Reproductive toxicity (a) Multigeneration studies (b) Developmental toxicity Special studies: neurotoxicity bservations in humans Comments Toxicological evaluation References Explanation Azinphos-methyl is the International rganization for Standardization (IS) approved common name for S-3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-ylmethyl,-dimethyl phosphorodithioate (International Union of Pure and Applied Chemistry, IUPAC) or,-dimethyl S-[(4-oxo- 1,2,3-benzotriazin-3(4H)-yl)methyl] phosphorodithioate (Chemical Abstracts Service, CAS; CAS o ). It is a broad-spectrum organophosphorus pesticide. Its toxicity was first evaluated by the 1965 JMPR, when an acceptable daily intake (ADI) of mg/kg bw was established based on a no-observed-adverse-effect level (AEL) of 0.25 mg/kg bw per day for inhibition of cholinesterase activity in serum and erythrocytes in a repeat-dose study in rats. The 1968 JMPR considered a number of additional studies that were not available to the Meeting in The ADI established in 1965 was confirmed. The 1973 JMPR considered new studies that involved human volunteers but, owing to the absence of sufficient information on the conduct of these studies in humans, the existing ADI was re-affirmed. In 1991, the ADI was increased to mg/kg bw on the basis of reduced body-weight gain and fertility observed in the multigeneration study in rats. Azinphosmethyl was reviewed by the present Meeting within the periodic review programme of the Codex Committee on Pesticide Residues (CCPR). All studies previously submitted to JMPR were available

2 140 for c onsideration by the present Meeting. Several new studies, including two double-blind clinical studies in human volunteers, were also considered by the present Meeting. Most studies, excluding some described in previous JMPR monographs, were certified as having been performed in compliance with good laboratory practice (GLP) and in accordance with the relevant rganization for Economic Co-operation and Development (ECD) test guidelines. The studies in humans were conducted in accordance with the principles of good clinical practice and the Declaration of Helsinki, or equivalent statements prepared for use by national and/or multinational authorities. As these guidelines specify the tissues normally examined and the clinical pathology tests normally performed, only significant exceptions to these guidelines are reported here, to avoid repetitive listing of study parameters. Evaluation for acceptable daily intake 1. Biochemical aspects 1.1 Absorption, distribution and excretion The pharmacokinetic behaviour of carbonyl- 14 C-labelled azinphos-methyl was investigated in male Sprague-Dawley rats. The material was almost completely absorbed from the digestive tract, and irrespective of dose and route of administration, 60 70% was eliminated in the urine and 25 35% in the faeces within 48 h. Less than 0.1% of the administered radiolabel was eliminated with the respiratory air within 24 h after dosing, and in rats with biliary fistulas about 30% of the intravenously administered radiolabel was eliminated in the bile within 24 h after dosing. Two days after dosing, the total amount of radioactivity in the animal (excluding digestive tract) was less than 5% of the administered dose; by 4 days this had declined to 2% and by 16 days to 1%. Six hours after dosing, the highest concentrations of radioactivity were found in the organs of elimination (liver and kidney) with relatively high concentrations found in blood. The activity concentrations decayed rapidly in all organs up to 2 days after dosing, but thereafter the activity was more slowly eliminated. At 16 days after dosing, the highest concentration was found in the erythrocytes. In studies in vitro, in which whole blood was incubated with labelled parent compound, there was no evidence for accumulation of radioactivity in the blood constituents (Patzschke et al., 1976; A nnex 1, reference 64). The pharmacokinetic behaviour of benzazimide was investigated in rats using the 14 C-ringlabelled compound. After oral administration, the 14 C was almost completely absorbed from the gastrointestinal tract. Elimination of the radiolabel took place quickly, 24 h after administration only 1.3% of the amount administered was present in the animal not including the gastrointestinal tract. More than 99% of the amount administered was eliminated within 48 h (54 66% in the urine and 33 45% via the faeces) (Weber et al., 1980; Annex 1, reference 64). 1.2 Biotransformation The metabolism of azinphos-methyl was investigated by administration of ring-ul- 14 C-labelled azinphos-methyl to male and female Sprague-Dawley rats. The proposed metabolic pathway of azinphos-methyl in rats is given in Figure 1. Upon absorption, azinphos-methyl is rapidly metabolized by mixed function oxidases and glutathione transferases in the liver and other tissues, which results in the formation of azinphos-methyl oxygen analogue, mercaptomethylbenzazimide, glutathionyl methylbenzazimide and desmethyl isoazinphos-methyl. Further hydrolysis,

3 141 methylation and oxidation of mercaptomethyl-benzazimide forms benzazimide, methylthiomethylbenzazimide and its corresponding oxidized metabolites. Hydrolysis of glutathionyl methylbenzazimide may result in the formation of cysteinylmethyl-benzazimide. Subsequent oxidation of cysteinyl-methylbenzazimide forms its corresponding sulfoxide and sulfone (Kao, 1988; Annex 1, r eference 64). The rate of disappearance of azinphos-methyl via a hepatic oxidative desulfurating system and a demethylating system was investigated in liver homogenates from four different species (rat, guinea-pig, chicken and monkey). Azinphos-methyl was metabolized by both systems and homogenates from all species were uniformly active (Rao & McKinley, 1969; Annex 1, reference 64). 1.3 Effect on enzymes and other biochemical parameters The acute oral toxicity of azinphos-methyl, dissolved in propylene glycol, was investigated in groups of female mice and the effect of the oxime antidote, toxogonin (80 mg/kg bw intraperitoneally, 15 min before oral dosing), was determined. Antidote treatment reduced the toxicity of azinphos-methyl by increasing the median lethal dose (LD 50 ) by a factor of 2 (Sterri et al., 1979; Annex 1, reference 64). 2. Toxicological studies 2.1 Acute oral toxicity The results of studies to establish the acute toxicity of azinphos-methyl are summarized in Table 1. The acute oral toxicity of azinphos-methyl technical in mammals was high. Consistent with the cholinergic effects observed with other organophosphorus compounds, signs of acute intoxication with azinphos-methyl included diarrhoea, salivation, lacrimation and vomiting (muscarinic effects), muscular tremors and paralysis (nicotinic effects), and restlessness, ataxia and convulsions (central nervous system effects). 2.2 Short-term studies of toxicity Rats In a dose-range finding study with emphasis on the most sensitive end-point, namely inhibition of cholinesterase activity, groups of five male and five female SPF BR:WISW (SPF/Cpb) rats were fed diets containing azinphos-methyl (purity, 93.3%) at a concentration of 0, 5, 20, or 50 ppm for 28 days. The average doses ingested at 5, 20, or 50 ppm were 0.35, 1.30, and 3.37 mg/kg bw per day for males and 0.46, 1.54, and 3.96 mg/kg bw per day for females, respectively. There were no effects on appearance, behaviour, mortality, body-weight gain, food consumption, and gross pathology. A statistically significant reduction in plasma cholinesterase activity of 44 66%, relative to the control group, was seen in females at 50 ppm. Appreciably less inhibition of plasma cholinesterase activity was observed in males and this was consistent across the entire sampling period. Erythrocyte acetylcholinesterase activity was unaffected in males at 20 ppm, but was significantly reduced (17 22%) in females. Brain cholinesterase activity was significantly in females at 50 ppm (Table 2). The AEL was 20 ppm, equal to 1.30 mg/kg bw per day in males and 1.54 mg/kg bw per day in females, on the basis of inhibition of cholinesterase activity in the brain at 50 ppm (Eiben et al., 1983).

4 142 Figure 1. Proposed metabolic pathway for azinphos-methyl in rats CH 3 C P S CH 2 CH 3 Azinphos-methyl oxygen analog CH 3 CH 3 HS CH 2 C MF Mercaptomethylbenzazimide HS C P S CH 2 CH 3 Desmethyl isoazinphos-methyl MF GSH transferase S P S CH 2 C Azinphos-methyl GSH transferase H C Hydrolysis CH 3 S CH 2 Methylation C H 2 H C CH CH 2 CH 2 C H H C CH 2 H C CH CH 2 S CH 2 C Benzazimide Methylthiomethylbenzazimide Glutathionyl methylbenzazimide MF D-Glutamyl transpeptidase Cysteinyl glycinase CH 3 S CH 2 C Methylsulfinylbenzazimide H H C CH CH 2 S CH 2 Cysteinylmethylbenzazimide C MF MF CH 3 S CH 2 C H 2 H C CH CH 2 S CH 2 C Methylsulfonylbenzazimide Cysteinylmethylbenzazimide sulfoxide MF H 2 H C CH CH 2 S CH 2 C Cysteinylmethylbenzazimide sulfone GSH, glutathione; MF, mixed function oxidases.

5 143 Table 1. Acute oral toxicity of azinphos-methyl and its metabolites Compound Species Strain Sex (n) Vehicle Purity (%) Azinphos-methyl Mouse ICR/SIM Males (20) Rat SD Males and females (4) Rat SD Females (25) Rat S Males (10) Rat Sherman Males and females (S) Rat S Males (10) Rat Wistar Males (10 or 20) LD 50 (mg/kg bw) Reference Corn oil S 15 Simmon (1978) DMS (fasted males) 6.4 (fasted females) Ethanol and propylene glycol Water and c remophor EL Crawford & A nderson (1974); Annex 1, r eference DuBois et al. (1955); DuBois et al. (1957) S 25.4 Flucke (1979); A nnex 1, r eference 64 Peanut oil S 13 (males) 11 (f emales) Water and c remophor EL Water and c remophor EL Rat S Males (5) Water and c remophor EL Rat SD Females (4) Rat Wistar Males and females (15) Rat SD Females (4) Rat CD Males and females (2) Rat S Males (10) Rat SD Males and females (5) CMC (fasted) 10 (nonfasted) Guineapig S Males (S) Dog Beagle Males (1 or 2) S 9.1 (fasted) (nonfasted) (fasted) 12.8 (nonfasted) Gaines (1969); A nnex 1, r eference 64 Heimann (1981); Annex 1, r eference 64 Heimann (1982); Annex 1, r eference 64 S 7.1 (fasted) Heimann (1987); Annex 1, r eference 64 Water and c remophor EL Panasol A-2 Methylene chloride + 10% arabic gum in Tween 80 Water and c remophor EL Polyethylene glycol Ethanol and propylene glycol Water and C remophor EL (fasted males) 4.4 (fasted females) S (two samples) Lamb and A nderson (1974); Annex 1, r eference 64 Mihail (1978); A nnex 1, r eference elson (1968) > (males) 24 ( females) Pasquet et al. (1976); Annex 1, reference Thyssen (1976a); Annex 1, r eference 64 S 9.0 (males) 6.7 (f emales) Crown & yska (1987) DuBois et al. (1957); DuBois et al. (1955) 91.6 > 10 (0/2 deaths), fasted Mihail (1978); Annex 1, r eference 64

6 144 Benzazimide (azinphos-methyl metabolite in rats) Rat SD Males and females (4) DMS (fasted males) 269 (fasted females) Crawford & Anderson (1974); Annex 1, r eference 64 a Rat Males and females (S) CMC S 576 (fasted males) 368 (fasted f emales) 576 (non-fasted males) 487 (nonfasted females) Lamb & A nderson, (1974); Annex 1, reference 64 b Methyl b enzazimide (azinphos-methyl metabolite in rats) Rat SD Males and females (4) DMS S 330 (fasted males and females) Crawford & Anderson (1974); Annex 1, r eference 64 Rat Males and females (S) CMC S 412 (fasted males) 390 (fasted f emales) 524 (non-fasted males) 460 (nonfasted females) Lamb & A nderson (1974); A nnex 1, r eference 64 b CMC, carboxymethyl cellulose; DMS, dimethyl sulfoxide; S, not stated; ZW, ew Zealand White; SD, Sprague- Dawley. a Modified with reference to original data. b riginal data not provided. Groups of 10 male and 10 female CD rats were given azinphos-methyl (purity, 93%; in corn oil) at a dose of 0, 0.22, 0.86, 3.44 mg/kg bw per day by gavage for 13 weeks. All rats were examined at least three times per day for mortality and clinical signs. Food consumption and body weights were recorded weekly, and water consumption was recorded daily. Food conversion ratios were calculated as the amount of food consumed per unit body weight gained. phthalmoscopic examinations were performed on all rats before the commencement of treatment and on rats in the control and the highest dose groups after 12 weeks of treatment. Haematology and clinical chemistry parameters (including cholinesterase activity) were determined at termination, after which all animals were given a macroscopic examination, and selected organs were removed and weighed. Histopathological examination was performed on selected tissues harvested from rats in the control group and in the group at the highest dose, while kidneys, liver and lungs were examined in all rats. There were no treatment-related deaths. Salivation was observed in males at and above 0.86 mg/kg bw per day from approximately week 3, with the majority of rats being affected from week 8 at 0.86 (up to 8 out of 10) and 3.44 mg/kg bw per day (up to 10 out of 10). Salivation was observed shortly after dosing in the majority of males, but occasionally it was seen before dosing or up to an hour after dosing in some animals. Salivation was observed infrequently in females with the exception of 2 out of 20 rats at the intermediate dose only during week 7, and 4 out of 10 rats at the highest dose only during week 9. There were no treatment-related effects on ophthalmoscopy, bodyweight gain, or food and water consumption. However, there was a clear treatment-related effect on the inhibition of plasma butyryl, plasma acetyl, erythrocyte and brain cholinesterase activities in males and females (Table 3).

7 145 Table 2. Cholinesterase activity in rats fed diets containing azinphos-methyl for 28 days Time-point Plasma Cholinesterase activity (% inhibition relative to control values) Dietary concentration (ppm) Males Females Males Females Males Females Day / * * 53** * 0 26* 61** Erythrocytes Day * 22** 34** ** 14** 35** Brain Day ** From Eiben et al. (1983) * p < 0.05; ** p < There were no treatment-related effects on absolute organ weights or histopathological abnormalities. The AEL was 0.86 mg/kg bw per day on the basis of salivation in male rats and significant inhibition of brain cholinesterase activity at 3.44 mg/kg bw per day (Broadmeadow et al., 1987). Dogs In a 52-week study of toxicity, groups of four male and four female beagle dogs were given diets containing azinphos-methyl (purity, 91.9%) at a concentration of 0 (control), 5, 25 or 125 ppm (equal to 0.16, 0.74, or 4.09 mg/kg bw per day). Clinical signs of reaction to treatment were confined to a higher incidence of diarrhoea in dogs at 125 ppm. Two males at 125 ppm failed to gain weight during the course of the study, but food intake remained unaffected by treatment. Haematology and urine analysis revealed no indication of any reaction to treatment. Clinical biochemistry tests revealed a depression of cholinesterase activity in plasma and erythrocytes in dogs at 25 and 125 ppm and in brain at termination of dogs at 125 ppm (Table 4). There was also a very slight increase, compared with controls, in liver cytochrome P450 and -demethylase activity at the highest dose and a reduction in albumin levels. Pathological investigations (macroscopic examination, organ weight analysis and histopathology) revealed no evidence of any reaction to treatment with azinphos-methyl. The AEL was 25 ppm (0.74 mg/kg bw per day) on the basis of reduced body-weight gain and inhibition of cholinesterase activity in brain (modified with reference to the original data, Allen, et al., 1990; Annex 1, reference 64). 2.3 Long-term studies of toxicity and carcinogenicity Mice A bioassay of azinphos-methyl (purity, 90%; from manufacturing specification) for possible carcinogenicity was conducted by the ational Cancer Institute (CI). sborne-mendel rats and

8 146 Table 3. Cholinesterase activity in rats given azinphos-methyl by gavage for 13 weeks Cholinesterase Cholinesterase activity (% inhibition relative to control values) Dose (mg/kg bw per day) Males Females Males Females Males Females Plasma butyryl cholinesterase 18*** 3 16** 10 30*** 47*** Plasma acetyl cholinesterase 18** 3 14* 7 42*** 49*** Erythrocyte acetylcholinesterase 8* 9*** 17*** 29*** 77*** 78*** Brain cholinesterase 3 4 9* 3 67*** 64*** From Broadmeadow et al. (1987) * p < 0.05; ** p < 0.01; *** p < Table 4. Cholinesterase activity in dogs a fed diets containing azinphos-methyl for 52 weeks Cholinesterase Plasma cholinesterase Cholinesterase activity (% inhibition relative to control values) Dietary concentration (ppm) Males Females Males Females Males Females Males Females Week * Week ** 58** Week ** 57** Week * 53** Erythrocyte acetylcholinesterase Week ** 86** Week ** 43** 87** 92** Week ** 88** 91** Week * 86** 86** Brain cholinesterase ** 20* From Allen et al. (1990), Annex 1, reference 64, modified with reference to the original data. a n = 4 * p < 0.05; ** p < B6C3F 1 mice were fed diets containing azinphos-methyl. Groups of 50 male and 50 female sborne- Mendel rats were given diets containing azinphos-methyl for 80 weeks, and were then observed for 34 or 35 weeks. Males received azinphos-methyl at a time-weighted average dietary concentration of 78 or 156 ppm (equivalent to 6.25 and 12.5 mg/kg bw per day respectively), while females received 62.5 or 125 ppm (equivalent to 3.12 and 6.25 mg/kg bw per day respectively). Matched controls consisted of 10 male and 10 female untreated rats; pooled controls consisted of matched controls combined with 95 male and 95 female untreated rats from similar bioassays of 10 other chemicals. The study in mice was of similar design; groups of 50 mice were treated for 80 weeks, then observed for 12 or 13 weeks, males received azinphos-methyl at a dietary concentration of 31.3 or 62.5 ppm (equivalent to 4.7 and 9.4 mg/kg bw per day respectively) and females received 62.5 or 125 ppm (equivalent to 9.4 and mg/kg bw per day respectively), matched controls consisted of 10 males and 10 females, pooled controls comprised 130 males and 120 females. Typical signs of organophosphate intoxication (hyperactivity, tremors and dyspnoea) were observed in a few animals of both species

9 147 at the highest dose. In both species, body-weight gain for treated males and in females at the highest dose was lower than in animals in the control groups. In rats there was some evidence of decreased survival at the highest dose compared with controls, but this was not seen in mice. In both sexes at all doses, survival to termination was adequate for assessment of effects on late-appearance tumours. The report concluded that the incidence of tumours of the pancreatic islets, and of follicular cells in the thyroid in males suggested, but did not clearly implicate, azinphos-methyl as a carcinogen in rats. There was no similar evidence in female rats, and in male and female mice sex there was no increased incidence of tumours that could be related to the administration of azinphos-methyl ( ational Cancer Institute, 1978; Annex 1, reference 64, amended with reference to original data). In a study of carcinogenicity in mice, groups of 50 male and 50 female CD-1 mice received azinphos-methyl (purity, 88.6%) at a dietary concentration of 0 (control), 5, 20, or 40/80 ppm (equal to 0.79, 3.49, or mg/kg bw per day in males and 0.98, 4.12, or mg/kg bw per day in females) for 2 years. The study was initially used 80 ppm as the highest dietary concentration, but this was reduced to 40 ppm after 1 week, owing to severe reaction (including mortality) to treatment at 80 ppm. After the reduction in the highest dietary concentration, there were no clinical signs and mortality remained unaffected by treatment. Weight gain and food intake remained unaffected by treatment at dietary concentrations up to and including 40 ppm. Haematological investigations revealed no indication of any reaction to treatment. Measurement of cholinesterase activity revealed that at 5 ppm, plasma, erythrocyte and brain cholinesterase activities remained comparable with control values. At 20 and 40 ppm there was a dose-related inhibition of cholinesterase activity in plasma and erythrocytes. A similar effect was noted in brain, except that males were only affected at 40 ppm, while females exhibited a depression of brain cholinesterase activity at 20 and 40 ppm (Table 5). Pathological investigations revealed no evidence of any reaction to treatment, in particular there was no evidence of any carcinogenic effect of azinphos-methyl at any dose. The AEL was 5 ppm (equal to 0.79 and 0.98 mg/kg bw per day in males and females, respectively) on the basis of inhibition of cholinesterase activity in plasma, erythrocytes and brain at 20 ppm (Hayes, 1985; A nnex 1, reference 64, amended with reference to original data). Rats In a combined long-term study of toxicity and carcinogenicity in rats, groups of 60 male and 60 female Wistar rats received diets containing azinphos-methyl (purity, 87.2%) at a concentration of 0 (control), 5, 15 or 45 ppm (equal to 0.25, 0.75, or 2.33 mg/kg bw per day for males and 0.31, 0.96, or 3.11 mg/kg bw per day for females, respectively). From each group, 10 males and 10 females were killed after 1 year, while all survivors were killed after 2 years of continuous treatment. There were no clinical signs of reaction to treatment and survival was unaffected by azinphos-methyl. Bodyweight gain of males at the highest dose was slightly less than that of the controls (7%), but growth in other groups was not affected by treatment. Clinical biochemistry (apart from investigations of acetylcholinesterase activity), haematology and urine analysis revealed no indication of any reaction to treatment. Determinations of cholinesterase activity in erythrocytes, plasma and brain revealed a marked inhibition, relative to controls, in males and females from the group at the highest dose (erythrocytes, plasma and brain) and a less marked effect at 15 ppm (males, erythrocytes; females, erythrocytes and plasma). Cholinesterase activity in brain from rats at 15 ppm and in erythrocytes, plasma and brain from rats at 5 ppm remained unaffected by treatment with azinphos-methyl (Table 6). Pathological examinations (including gross examination, organ-weight analysis and histological examination of tissues) revealed no evidence of any reaction to treatment; in particular, there was no evidence of any carcinogenic effect. The AEL was 15 ppm, equal to 0.75 and 0.96 mg/kg bw per day in males and females, respectively, on the basis of effects on body-weight gain and brain acetylcholinesterase activity (Schmidt, 1987; Annex 1, reference 64, modified with reference to the original data).

10 148 Table 5. Cholinesterase activity in mice fed diets containing azinphos-methyl for 104 weeks Time-point Cholinesterase activity a Dietary concentration (ppm) Males Females Males Females Males Females Males Females Plasma (µmol/ml per min): 6 months year years Erythrocytes (µmol/ml per min): 6 months year year Brain (µmol/g per min): 2 years 14.7/ / From Hayes (1985); Annex 1, reference 64, amended with reference to original data. a Results of statistical analysis not specified in the report. 2.4 Genotoxicity The results of studies of genotoxicity with azinphos-methyl are summarized in Table 7. An effort was made to evaluate the genotoxicity of a variety of pesticides, with the specific objectives of comparing different assays in vivo and in vitro, examining the spectrum of genetic activity displayed by the selected pesticides and examining the test results in relation to other biological and chemical features of the pesticides. In this research programme, azinphos-methyl was used in 14 teats for mutagenicity, examining point or gene mutations, DA damage and chromosomal effects. Positive results for azinphos-methyl were seen in only two tests: an assay for forward mutation in mouse lymphoma L5178Y cells (only in the presence of metabolic activation) and an assay for mitotic recombination in Saccharomyces cerevisiae strain D3. Azinphos-methyl gave negative results in tests for point/gene mutation and DA damage in prokaryotes and showed no positive results in tests for chromosomal effects (Waters et al., 1982; Annex 1, reference 64). 2.4 Reproductive toxicity (a) Multigeneration studies Rats In a two-generation (two litters per generation) study of reproduction, groups of 12 male and 24 female Wistar rats were given diets containing azinphos-methyl (purity, 87.2%) at a concentration of 0 (control), 5, 15 or 45 ppm (equal to 0, , and mg/kg bw per day in males and 0, , and mg/kg bw per day in females). At 45 ppm,

11 149 Table 6. Cholinesterase activity in rats fed diets containing azinphos-methyl for 2 years Tissue Plasma Cholinesterase activity (% of values for controls) Dietary concentration (ppm) Males Females Males Females Males Females 1 month ** 44** 3 months ** 60** 35** 6 months * 57** 34** 1 year ** 54** 33** 1.5 years * 55** 46** 2 years ** 38** Erythrocytes 1 month ** 87 76** 74* 3 months ** 88** 88** 77** 72** 6 months * 90 86** 80** 77** 1 year * 81** 73** 69** 1.5 years ** 80** 73** 73** 2 years 88** 98 78** 84** 63** 71** Brain 1 year 130** ** ** 2 years ** 68** 45** From Schmidt (1987); Annex 1, reference 64, modified with reference to the original data. * p < 0.05; ** p < there was a decrease in fertility of F 0 and F 1b dams and the total number of delivered pups (Table 8). The viability index was significantly reduced in F 1a, F 1b and F 2a litters at 45 ppm, while the effect was equivocal at 15 ppm (Table 9). At 45 ppm, there was an increased mortality of dams in the F 0 generation and reduced mean pup viability from days 5 to day 28 of lactation in the F 1a and F 1b litters. As a consequence of these effects, at 45 ppm only five females were available for mating in the F 1b generation. During mating of the F 1b generation, fertility was again slightly reduced at 45 ppm but not to as great an extent as it was for the F 0 generation. At all stages of the study, there was no evidence of treatment-induced malformations and food intake remained unaffected. Clinical signs of reaction to treatment, including cholinergic signs, and reduced body-weight gain were observed at the highest dose tested (45 ppm). The AEL was 15 ppm, equal to 1.5 mg/kg bw per day, on the basis of reduced body-weight gain in F 0 and F 1b parental animals and decreased pup body weight and viability at 45 ppm (Eiben & Janda, 1987; Annex 1, reference 64, modified by reference to original data). Another study was conducted to investigate the effects on reproductive performance noted in the study described above. The objectives of this additional, one-generation study were to investigate whether the slight effect on fertility and pup viability observed at 15 ppm could be confirmed, and, if reproducible, to determine whether the effect was attributable to treatment of the male or the female and to determine whether reproductive effects were associated with treatment-induced inhibition of cholinesterase activity. Groups of 18 male and 46 female Wistar rats were given diets containing azinphos-methyl (purity, 92.0%) at a concentration of 0 (control), 5, 15, or 45 ppm (equal to 0, 0.43, 1.30,

12 150 Table 7. Results of studies of genotoxicity with azinphos-methyl End-point Test object Concentration Purity (%) Results Reference In vitro Reverse mutation (Ames) Reverse mutation (Ames) Reverse mutation (Ames) Reverse mutation (Ames) Reverse gene mutation DA damage Unscheduled DA synthesis Clastogenicity Sister chromatid exchange In vivo Micronucleus formation Chromosomal aberration Dominant lethal mutation S. typhimurium TA98, TA100, TA1535, TA1537, TA1538 S. typhimurium TA98, TA100, TA1535, TA1537 S. typhimurium TA98, TA100, TA1535, TA1537 S. typhimurium TA98, TA100, TA1535, TA1537, TA1538 S. cerevisiae S138 and S211α E. coli W3110, E. coli p3478 Rat hepatocytes Chinese hamster ovary cells-(k1) Human lymphocytes Chinese hamster lung cells (V79) Mouse Rat, femoral bone marrow Mouse µg/plate S egative Evanchik (1987) µg/plate 92.3 egative Herbold (1978) µg/plate 92.5 egative Herbold (1988) µg/plate 88.8 egative Lawlor (1987) µg/ml 91.1 egative Hoorn (1983) µg/plate 91.1 egative Herbold (1984) µg/ml, in DMS µg/ml Approx µg/ml (+S9) µg/ml ( S9) in DMS 91.1 egative Myhr (1983) Dose-related chromatid breaks and exchanges 91.9 Clastogenicity observed at cytotoxic concentrations +S9. o effect S9 Alam et al. (1974) Herbold (1986) 5 25 µg/ml 98.7 egative Chen et al. (1982a) µg/ml Chen et al. (1982b) and 2 5 mg/kg bw single oral dose in Cremophor/water 5 mg/kg bw, single IP dose in Cremophor/water 6.28 mg/kg bw, single oral dose in methylcellulose or 0.25 mg/kg bw, single IP dose in corn oil 1 4 mg/kg bw, single oral dose in Cremophor/ water 92.3 egative Herbold (1979a) 92.2 egative Herbold (1995) 92 egative Henderson et al. (1988) S egative Arnold (1971) 92.3 egative Herbold (1979b) DMS, dimethyl sulfoxide; IP, intraperitoneal; S, not specified; S9, 9000 g supernatant from livers of rats. and 3.73 mg/kg bw per day in males and 0.55, 1.54, and 4.87 mg/kg bw per day in females). Treated males and females were paired, and dams allowed to rear litters to weaning. Additional treated males were paired with untreated females.

13 151 Table 8. Fertility of F 0 and F 1b rats fed diets containing azinphos-methyl Parameter Dietary concentration (ppm) Mating 1/2 Mating 1/2 Mating 1/2 Mating 1/2 F 0 Mated females (o.) 24/24 24/24 24/23 23/19 Insemination index (%) 100/ / / /94.7 Fertility index (%) 91.7/ / / /83.3 Gestation index (%) 100/ / / /93.3 Duration of gestation (days) 22.5/ / / /22.8 F 1b Mated females (o.) 24/24 24/24 24/23 5/5 Insemination index (%) 100/ / / /100 Fertility index (%) 91.7/ / / /80.0 Gestation index (%) 100/ / / /75.0 Duration of gestation (days) 22.1/ / / /22.3 From Eiben & Janda (1987); Annex 1, reference 64, modified by reference to original data. At 45 ppm, two females that were sacrificed in extremis exhibited clinical signs consistent with cholinergic effects, such as poor general condition, bloody noses, inertia and a stumbling gait. Males at the same dietary concentration showed no clinical signs. There were no treatment-related effects on fertility parameters (insemination, fertility and gestation indices, and duration of gestation) (Table 10). At 15 ppm, when males and females were treated, the viability index was slightly reduced but it was in the absence of any corresponding reduction in pup body-weight gain. In contrast, dams at 45 ppm delivered pups that had significantly reduced viability and body-weight gain by postnatal day 5. After treatment of male parental animals only, reproductive parameters remained unaffected, even at 45 ppm (Table 11). Investigations of cholinesterase activity in parental animals revealed a depression in activity in plasma and erythrocytes at all dietary concentrations, and a depression in activity in brain at 45 ppm in males and at 15 and 45 ppm in females (Table 12). There were no treatment-related clinical signs observed in F 1 pups at birth or during the 4-week rearing period. Similarly, no treatment-related pathological changes were observed. At 45 ppm, brain cholinesterase activity in pups was also depressed. The AEL was 5 ppm, equal to 0.55 mg/kg bw per day, on the basis of depression of brain cholinesterase activity at 15 ppm (Holzum, 1990; Annex 1, reference 64, modified by reference to original data). (b) Developmental toxicity Mice and rats The effects of azinphos-methyl (purity, 90.6%) on development in rats and mice were investigated in a series of experiments. n the basis of preliminary studies of toxicity, doses of 0, 1.25, 2.5 or 5.0 mg/kg bw per day were selected for two-phase studies of developmental toxicity in both species. During the first phase, pregnant rats and mice were treated for 10 days, starting on day 6 of gestation. During the second phase, pregnant rats were treated from day 6 of gestation to postnatal day 21. In the first phase, maternal toxicity was seen only in rats receiving the highest dose. When dams and fetuses were examined (day 18 of gestation for mice, day 20 for rats) there was no

14 152 Table 9. Pup parameters in rats fed diets containing azinphos-methyl Parameter Dietary concentration (ppm) F 1a Live /dead pups at birth (o.) 252/1 247/0 204/8 197/9 Pups, males/females (%) 52/48 53/47 49/51 52/48 Litter size at day 0/day 5 (o.) 11.5/ / /8.7* 10.1/3.9** Viability index (%) ** 38.7** Pups at day 5 a /week 4 (o.) 175/ / /134 62/17 Lactation index (%) ** Body weight at day 0/week 3 (g) 5.8/ / / /25.8** F 1b Live/dead pups at birth (o.) 235/1 236/11 175/1 133/0 Pups, males/females (%) 52/48 50/50 51/49 55/45 Litter size at day 0/day 5 (o.) 10.6/ / / /2.8** Viability index (%) ** Pups at day 5 a /week 4 (o.) 165/ / /117 39/18 Lactation index (%) * 46.2** Body weight at day 0/week 3 (g) 5.7/ / / **/27.2** F 2a Live/dead pups at birth (o.) 259/3 270/0 230/0 43/0 Pups, males/females (%) 55/45 55/45 54/46 51/49 Litter size at day 0/day 5 (o.) 11.7/ / / */7.0* Viability index (%) ** Pups at day 5 a /week 4 (o.) 176/ / /134 29/21 Lactation index (%) ** 72.4** Body weight at day 0/week 3 (g) 5.7/ / / /22.4** F 2b Live/dead pups at birth (o.) 223/1 244/2 214/3 25/0 Pups, males/females (%) 51/49 55/45 49/51 56/44 Litter size at day 0/day 5 (o.) 10.6/ / / /6.2 Viability index (%) * 88.6* 100 Pups at day 5 a /week 4 (o.) 143/ / /123 22/20 Lactation index (%) * Body weight at day 0/week 3 (g) 5.8/ / / /27.0** From Eiben & Janda (1987); Annex 1, reference 64, modified by reference to original data. a After reduction. * p < 0.05; ** p < 0.01 dose-related increase in anomalies or malformation in rats or mice. In the second phase, dams in the group at the highest dose were more sensitive to azinphos-methyl in the latter stages of gestation and signs of anticholinesterase intoxication, including mortality, were observed. As a result, only 1 litter out of 13 survived to weaning in this group. The authors concluded that azinphos-methyl had little primary effect on development in rats and mice (Short et al., 1978; Short et al., 1980; Annex 1, reference 64, modified by reference to original data).

15 153 Table 10. Fertility parameters in rats fed diets containing azinphos-methyl Parameter Dietary concentration (ppm) Males, females Males, females Males, females Males, females 0, 0 5, 5 / 5, 0 45, 45 / 45, 0 15, 15 / 15, 0 Mated females (o.) 36 36/20 36/20 31/20 Insemination index (%) / / /100 Fertility index (%) / / /100 Gestation index (%) / / /95.0 Duration of gestation (days) / / /22.5 From Holzum (1990); Annex 1, reference 64, modified by reference to original data. Table 11. F 1 pup parameters in rats fed diets containing azinphos-methyl Parameter Dietary concentration (ppm) Males, females Males, females Males, females Males, females 0, 0 5, 5 / 5, 0 15, 15 / 15, 0 45, 45 / 45, 0 Pups at birth (o.) / / / 211 Pups dead at birth (o.) 13 4 / 2 3 / 1 3 / 3 Live birth index (%) * / 99.1* 98.9** / 99.5** 98.6* / 98.6* Male pups (%) 48.8 / / / Litter size (o.) / / / 10.9 Viability index (%) / 98.1* 86.0* / ** / 95.7 Lactation index (%) / 69.8 / 57.7 / Body weight (g): Day / / / 5.7 Day / / * / 8.7 Day / 19.9 / 21.4 / Day / 49.5 / 46.3 / From Holzum (1990); Annex 1, reference 64, modified by reference to original data. * p < 0.05; ** p < Rats Groups of 33 mated Crl:CD BR CD rats were given azinphos-methyl (purity, 87.7%) at a dose of 0 (control), 0.5, 1.0 or 2.0 mg/kg bw per day in 6% Emulphor (EL-719) by gavage on days 6 to 15 of gestation (presence of spermatozoa was counted as day 0 of gestation). From each group, five dams were killed on day 16 of gestation and the remaining dams were killed on day 20. Appearance, behaviour, feed consumption, and body-weight gain of the dams were not adversely affected by treatment at any of the doses tested. n day 16 of gestation, cholinesterase activities in plasma, erythrocytes and brain were depressed, relative to values for controls, in dams at the highest dose only (fetal tissues were not examined). By day 20 of gestation, there was indication of recovery in cholinesterase activity in all previously affected tissues and fetal brain cholinesterase activity was comparable with control values (Table 13). Azinphos-methyl did not affect any maternal reproductive parameters and there was no indication of treatment-related embryotoxicity, fetotoxicity or teratogenicity at any dose. The AEL for

16 154 Table 12. Inhibition of plasma, erythrocyte and brain cholinesterase activity in F 0 and in F 1 pups of rats fed diets containing azinphos-methyl Dietary concentration (ppm) Plasma Cholinesterase activity (% of values for controls) F 0 males F 0 females F 1 pups End of m ating End of pretreatment Post-coital day 11 Postnatal day D D 26* 5 Postnatal day *** 25* 18 46*** 39** 45 43*** 62*** 60*** 66*** 63*** Erythrocytes 5 19** D D 25** 47*** 15 69*** 46*** 52*** 75*** 84*** 45 94*** 71*** 81*** 91*** 89*** Brain Postnatal day * D D 12 D D 15 D 4 21** 38** 48*** 1 14 D D Postnatal day ** 55*** 69*** 66*** 68*** 17* 46*** From Holzum (1990); Annex 1, reference 64, modified by reference to original data. * p < 0.05 ; ** p < 0.01 ; *** p < D, not determined. D D Table 13. Cholinesterase activity in dams (and their fetuses) given azinphos-methyl by gavage on days 6 15 of gestation Tissue Day 16 Cholinesterase activity Dose (mg/kg bw per day) Plasma, ku/l (% of control) 1.73 (100%) 1.56 (90%) 1.64 (95%) 1.08 (63%) Erythrocytes, ku/l (% of control) 0.38 (100%) 0.34 (90%) 0.34 (90%) 0.08* (21%) Brain, U/g (% of control) 2.53 (100%) 2.70 (107%) 2.55 (101%) 1.55* (61%) Day 20 Plasma, ku/l (% of control) 1.44 (100%) 1.47 (102%) 1.40 (97%) 1.32 (92%) Erythrocytes, ku/l (% of control) 0.58 (100%) 0.59 (103%) 0.61 (106%) 0.44 (77%) Brain, U/g (% of control) 2.67 (100%) 2.72 (102%) 2.46 (92%) 1.91* (72%) Fetal brain, U/g (% of control) 1.04 (100%) 0.96 (91%) 1.04 (100%) 1.00 (96%) From Kowalski et al. (1987a; 1987b); Annex 1, reference 64, modified by reference to original data; Astroff & Young (1998). * p < developmental toxicity was 2.0 mg/kg bw per day, on the basis of the absence of any effects at the highest tested dose. The AEL for maternal toxicity was 1.0 mg/kg bw per day, on the basis of the inhibition of brain cholinesterase activity seen on day 16 of gestation at 2.0 mg/kg bw per day (Kowalski et al., 1987a, 1987b; Annex 1, reference 64, modified by reference to original data; Astroff & Young, 1998).

17 155 Groups of 22 mated Sprague-Dawley-derived CD rats were given azinphos-methyl (purity, 92.7%) at a dose of 0.4, 1.2, or 3.6 mg/kg bw per day by gavage in maize oil daily from day 6 to day 15 of gestation. All rats were observed daily for clinical signs and body weights were recorded on days 0, 3, 6-15, 17 and 20 of gestation. Food consumption was measured twice weekly. n day 20, the animals were killed and necropsied. o deaths or treatment-related clinical signs were observed during the study. Food consumption during gestation was similar in control and treated females. The group mean body weight of females at 3.6 mg/kg bw per day was slightly lower than controls during the period before treatment, but body weight and body-weight gain were unaffected by treatment. There were no treatment-related changes in gravid uterine weights, the number or sex of live fetuses, or the number of resorptions. Skeletal observations revealed retarded ossification in fetuses at 3.6 mg/kg bw per day, with reduced or absent ossification seen in the supraoccipital, pubic and hyoid bones. An increased incidence of supernumerary ribs (14th, lumbar) was also observed at 3.6 mg/kg bw per day (4.7%; p < 0.01), and the incidence of this finding was outside the range for historical controls in the testing laboratory (0 3.1%). In the absence of any maternal effects, the AEL for maternal toxicity was 3.6 mg/kg bw per day. The AEL for developmental toxicity was 1.2 mg/kg bw per day on the basis of increases in the incidence of supernumerary ribs (14th, lumbar) and delayed ossification (pubic, hyoid, and supraoccipital bones) in fetuses at 3.6 mg/kg bw per day (Rubin & yska, 1988). Rabbits In a teratology study in Himalayan rabbits, groups of 11 or 12 pregnant animals received azinphos-methyl (purity, 92.4%) as a daily oral dose at 0 (control), 0.3, 1.0 or 3.0 mg/kg bw per day from day 6 to day 18 of gestation (day of insemination was counted as day 0). Caesarean section was carried out on day 29 of gestation. Azinphos-methyl induced no evidence of maternal toxicity at any dose and there were no detectable effects on embryonic or fetal development (Machemer, 1975; A nnex 1, reference 64, modified with reference to the original data). In a further study of teratology in American Dutch rabbits, groups of 20 inseminated does received azinphos-methyl (purity, 87.7%) as a daily oral dose at 0 (control), 1, 2.5 or 6 mg/kg bw per day from day 6 to day 18 of gestation (day of insemination was counted as day 0). Plasma and erythrocyte cholinesterase activities were determined on days 19 and 28 of gestation, and in brain on day 28 of gestation. Ataxia in four does at the highest dose and tremors in two of these represented clinical signs of reaction to treatment. At the intermediate and highest dose, plasma and erythrocyte cholinesterase activity on day 19 of gestation was depressed compared with controls. By day 28 of gestation there was clear evidence of recovery in plasma and erythrocyte cholinesterase activity, although activity in brain was depressed, compared with controls, at the highest dose. Azinphos-methyl did not affect any maternal reproductive parameters and there was no evidence of any treatmentrelated effect on embryotoxicity, fetotoxicity or teratogenicity at any dose. The AEL for maternal toxicity was 2.5 mg/kg bw per day on the basis of the inhibition of brain cholinesterase activity seen on day 28 of gestation at 6 mg/kg bw per day (Clemens et al., 1988; Annex 1, reference 64, modified with reference to the original data). In a study of developmental toxicity, groups of mated ew Zealand White rabbits were given azinphos-methyl (purity, 92.7%) at a dose of 0 (vehicle control), 1.5, 4.75, or 15 mg/kg bw per day by oral gavage in maize oil from day 7 to 19 of gestation. To ensure that at least 12 pregnant rabbits per group were available at terminal sacrifice, the group sizes were adjusted to 16, 18, 15, and 18, at doses of 0, 1.5, 4.75 and 15 mg/kg bw per day, respectively. Rabbits were observed daily for clinical signs and weighed on days 0, 3, 7 19, 22, 25 and 29 of gestation. Food consumption was measured twice per week. Cholinesterase activity in erythrocytes and plasma was measured before pairing and again after 11 days of dosing.

18 156 Deaths occurred in all groups, including controls (one rabbit in the control group, two at 1.5 mg/kg bw per day, one at 4.75 mg/kg bw per day, and three at 15 mg/kg bw per day). Death in the control group and in the group at the lowest and intermediate dose was attributed to intercurrent infections, while at the highest dose it was attributed to accidental lung dosing. o treatment-related clinical signs or changes in body-weight gain were observed. After 11 days of dosing, erythrocyte acetylcholinesterase activity was significantly reduced (p < 0.05) by 27% at 15 mg/kg bw per day. Significant reductions in plasma cholinesterase activity were seen at all doses after 11 days of treatment. However, there was no consistent dose response relationship and the magnitude of the inhibition was 22%, 29%, and 26%, at 1.5, 4.75 and 15 mg/kg bw per day, respectively. Erythrocyte acetylcholinesterase activity was only significantly reduced (27%) at 15 mg/kg bw per day. o treatment-related effects were observed in gravid uterine weight, number of live fetuses, number of resorptions, placental weight or crown rump length. However, the number of fetuses with a bodyweight of less than 30 g was significantly increased at 15 mg/kg bw per day. Skeletal examination revealed a statistically-significant increase in the incidence of reduced ossification of long bone epiphyses in fetuses at 4.75 and 15 mg/kg bw per day. The incidence of reduced ossification (42.4% and 44.3%, respectively) was also outside the range of mean values for historical controls ( %). The AEL for developmental effects was 1.5 mg/kg bw per day on the basis of retarded ossification of the long bones at 4.75 and 15 mg/kg bw per day and reduced fetal body weight at 15 mg/kg bw per day. The AEL for maternal toxicity was 4.75 mg/kg bw per day on the basis of significant i nhibition of erythrocyte acetylcholinesterase activity at 15 mg/kg bw per day (Gal et al., 1988). 2.5 Special studies: neurotoxicity Hens In a published report of experiments designed to investigate the potential relationship between delayed neurotoxicity and copper concentration in the serum of hens, it was reported that azinphosmethyl failed to produce symptoms of neurotoxicity after either single or repeated doses (Kimmerle & Loser, 1974; Annex 1, reference 64). In a test for acute delayed neurotoxicity, a group of 30 white Leghorn hens was given two doses of azinphos-methyl (purity, 85%) at a dose of 330 mg/kg bw, with an interval of 21 days between doses. Hens in the untreated-control, vehicle-control and positive-control (triorthocresyl phosphate, TCP at 600 mg/kg bw) groups, each comprising 10 animals, were also included. To protect against sudden death, atropine (15 mg/kg bw) was administered intramuscularly 15 min before dosing hens with TCP or azinphos-methyl. A total of 11 hens treated with azinphos-methyl survived until sacrifice. These animals appeared normal during the last 12 or 13 days of the study, but exhibited varying degrees of impaired locomotor activity soon after dosing. Histopathological examinations indicated that azinphos-methyl did not increase the incidence or severity of lesions in the nerve tissue compared with untreated and vehicle controls. Investigations of neuropathy target esterase activity were not included in the study (Glaza, 1988; Annex 1, reference 64, modified with reference to the original data). Groups of eight HL hens (age months) were given diets containing azinphos-methyl (purity not stated; prepared as an 80% premix in Silkasil S) at a dose of 0, 75, 150, 300 or 600 ppm (equivalent to approximately 0, 9.4, 18.7, 37.5 and 75.0 mg/kg bw per day) for 30 days. This was followed by a post-treatment recovery period of 4 weeks during which the birds were examined for signs of neurotoxicity (leg weakness, limping and inability to walk). Blood (site or method of blood collection was not specified) cholinesterase activity was determined before the commencement of the study,

19 157 after completion of the feeding trial, and at post-treatment observation periods using an unspecified method. Two birds per group were killed at the conclusion of dosing and the remaining birds were killed 1 day after the completion of the post-treatment observation period. ne bird at 9.4 mg/kg bw day died during the post-treatment observation period as a result of suspected pneumonia, but no details of necropsy were provided. The group mean body weight of birds at 75 mg/kg bw per day was reduced to about 13% of that of the controls by day 14 on study and remained unchanged up until the conclusion of the treatment period. The hens, however, regained weight thereafter and body weights were comparable to those of the controls during the post-treatment observation period. Food consumption was unaffected by treatment. o signs of neurotoxicity or any behavioural changes were noted in any of the treated birds. o intergroup differences in blood cholinesterase activity was seen before treatment or at day 1 of the post-observation period. However, at day 1 after treatment, dose-related depressions in blood cholinesterase activity of about 15% and 27% were observed in birds at 37.5 and 75.0 mg/kg bw per day respectively relative to the controls (Kimmerle, 1964). In a follow up study with essentially the same experimental protocol as described in an earlier study (Kimmerle, 1964), the administered dietary concentrations of azinphos-methyl (purity unspecified) were increased to 900, 1200, 1500, or 1800 ppm (equivalent to approximately 112.5, 150, 187 and 225 mg/kg bw per day respectively). ne bird at 150 mg/kg bw per day died during the last week of the 4-week treatment period. The cause of the death was not specified and necropsy details were not provided. Food consumption in treatment groups was reduced by about 27%, 43%, 40% and 44% (at 112.5, 150.0, and mg/kg bw per day, respectively) relative to the controls during the treatment period. All birds including the controls lost body weight during the treatment period. Hens in the control group and hens at and mg/kg bw per day were about 15%, 23% and 21% respectively lighter relative to the corresponding pre-treatment body weights at the conclusion of the 4-week treatment period. The groups receiving the test substance at and mg/kg bw per day showed a body-weight loss of about 27% at the same observation time. The body-weight data appeared to be consistent with the observations on food consumption. The birds had not achieved their pre-treatment body weights by the completion of the recovery period, though there was some recovery of body weights in all groups. Blood cholinesterase activity was unaffected by treatment. o signs of neurotoxicity or any behavioural changes related to treatment were noted. Similarly, no histopathological findings were made (Grundman, 1965; Kimmerle, 1965). Rats In a study to investigate acute neurotoxicity, groups of 18 male and 18 female fasted Fischer 344 CDF/BR rats were given azinphos-methyl (purity, 92.8%) at a dose of 0 (vehicle), 2, 6 or 12 mg/kg bw for males, and 0, 1, 3 or 6 mg/kg bw for females by oral gavage in 0.5% (w/v) methylcellulose and 0.4% (w/v) Tween 80. f the treated rats, six males and six females in satellite groups were used for determinations of cholinesterase activity and the remainder were involved in testing for changes in n eurobehaviour. Five males (one in the main study and four in the satellite group) and fifteen females (nine in the main study and all in the satellite group) in the group at the highest dose died. Treatment-related clinical signs in males were muscle fasciculations (2 out of 12 and 8 out of 11 at 6 and 12 mg/kg bw respectively), tremors (1 out of 11 at 12 mg/kg bw), uncoordinated gait (1 out of 11 at 12 mg/kg bw), oral stain (3 out of 12 and 10 out of 11 at 6 and 12 mg/kg bw, respectively), and urine stain (1 out of 12, 2 out of 12 and 10 out of 11 at 2, 6 and 12 mg/kg bw, respectively) and red nasal stain (2 out of 12, 2 out of 12 and 7 out of 11 at 2, 6 and 12 mg/kg bw, respectively). ne of the three surviving females at 6 mg/kg bw had oral and urine staining for about 2 days after treatment. All clinical signs that did not involve staining were only observed about 30 min after treatment on the day of dosing. Staining was typically observed from between 1 to 3 days after dosing. There were no treatment r elated d ifferences in group mean body weights of the surviving animals.