TOLCLOFOS-METHYL First draft prepared by A. Moretto, Institute of Occupational Medicine, University of Padua, Padua, Italy Explanation Evaluation for acceptable daily intake Biochemical aspects Absorption, distribution and excretion Biotransformation Toxicological studies Acute toxicity Short-term toxicity Long-term toxicity and carcinogenicity Reproductive toxicity Embryotoxicity and teratogenicity Genotoxicity Special studies Skin and eye irritation and ski201 Delayed neuropathy Observations in humans Comments Toxicological evaluation References Explanation Tolclofos-methyl is an organophosphorus fungicide that is effective in controlling soil-borne diseases caused by infection of Basidiomycetes fungi such as Rhizoctonia solani and Corticium rolfsii. It was evaluated for the first time by the present JMPR. Evaluation for acceptable daily intake 1. Biochemical aspects (a) Absorption, distribution and excretion Mice Male and female ICR mice, eight weeks of age, were given an oral dose of 5 mg/kg bw of [14C-4-methyl]-tolclofos-methyl (radiochemical purity, 99%) dissolved in corn oil, and radiocarbon was monitored in urine, faeces and expired air for seven days after administration. Within 24 h, 69-76% of the administered radiolabel was excreted in the urine, 4-6% in the faeces and less than 1% in the expired air. Total radiocarbon residues in the whole body represented less than 1% of the dose seven days after administration (Mihara et al., 1981). Rats Six-week old Sprague-Dawley rats were given an oral dose of 5 mg/kg bw of [14C-4-methyl]-tolclofos-methyl (radiochemical purity, 99%) dissolved in corn oil, and radiocarbon was monitored in urine, faeces and expired air for seven days after administration. Within 24 h, 62-67% of the administered dose was excreted in the urine, 16-21% in the faeces and less than 1% in the expired air. Total radiocarbon residues in the whole body represented less than 1% of the dose seven days after administration. Whole-body autoradiography performed 1 and 6 h after treatment showed the highest accumulation of radiolabel in stomach and intestines, followed by kidney and liver (Mihara et al., 1981). Male and female Sprague-Dawley rats received single oral doses of 5 or 200 mg/kg bw of tolclofos-methyl labelled uniformly with 14C in the benzene ring (radiochemical purity, > 99%). Another group of animals was treated orally for 14 consecutive days with unlabelled tolclofos-methyl at 5 mg/kg bw per day and then with a single oral dose of [14C-phenyl]-tolclofos-methyl at 5 mg/kg bw. The administered radiocarbon was readily excreted, more than 95% of the dose being eliminated in the urine and faeces within 48 h. The amount excreted in urine after seven days was 85-91%; elimination in faeces at that time was: consecutive dose group, 9.3% in males and 12% in females; low-dose group, 20% in males and 19% in females; and high-dose group, 20% in males and 12% in females. Excretion as 14C-carbon dioxide accounted for < 0.1% of the dose in all groups. The concentration of 14C reached a peak within 2 h in almost all tissues. After administration of the low dose, the highest concentrations were found in the kidney; expressed in tolclofos-methyl equivalents, the levels were 4700 ng/g tissue in males and 3450 ng/g tissue in females; the levels in plasma were 1140 ng/ml in males and 1270 ng/ml in females. Those in the liver were 1240 ng/g tissue in males and 1220 ng/g tissue in females, and those in blood were 736 ng/ml in males and 835 ng/ml in females. The concentrations of 14C in various organs 72 h after administration were < 5% of the respective peak concentrations. After seven days, radioactive residues accounted for less than 1% of the administered dose (Krautter et al., 1987, 1988; Esumi & Yokoshima, 1989). In male and female rats with bile-duct cannulas, cumulative excretion of 14C over 48 h was 5.8-12% of the dose in bile, 47-59% in urine and 42-24% in faeces (Esumi & Yokoshima, 1989). (b) Biotransformation Male ICR mice were given 5 mg/kg bw [14C-4-methyl]-tolclofos- methyl orally; metabolites were isolated from the faeces and urine by chromatography and identified by co-chromatography with authentic standards and/or spectroanalysis. The following major metabolites were detected in the excreta: 2,6-dichloro-4-methylphenol (see Figure 1; 9% of administered label), O,O-dimethyl- O-(2,6- dichloro-4-carboxyphenyl)phosphate (11%), O-methyl- O-hydrogen- O-(2,6-dichloro-4-carboxyphenyl)phosphate (12%), 3,5-dichloro-4- hydroxybenzoic acid (12%) and 3,5- O-dichloro-4-hydroxybenzyl glycine (13%). The major biotransformation reactions are oxidative desulfuration to oxon and related derivatives, oxidation of the 4-methyl group to alcohols and acids, cleavage of P-O-aryl and P-O-methyl linkages and conjugation of the resultant acid with glycine. The metabolites found in mice are similar to those seen in rats, except for 3,5- O-dichloro-4-hydroxybenzylglycine (Mihara et al., 1981). Male and female Sprague-Dawley rats were given an oral dose of 5 or 200 mg/kg bw tolclofos-methyl labelled with 14C either in the 4-methyl group or uniformly in the phenyl ring, with or without pretreatment with unlabelled tolclofos-methyl at 5 mg/kg bw per day for 14 consecutive days. Metabolites were isolated from the faeces, urine, bile and major tissues by chromatography and identified by co-chromatography with authentic standards and/or spectroanalysis. More than 10 metabolites were detected in the excreta. No marked differences were seen in relation to sex or dose. The major metabolites detected in the excreta were O-methyl O-hydrogen- O-(2,6-dichloro-4-methylphenyl)phosphate ((see Figure 1; 10-26% of urinary 14C), O-methyl- O-hydrogen- O-(2,6-dichloro-4- hydroxymethylphenyl) phosphorothioate (12-25%), O-methyl O-hydrogen- O-(2,6-dichloro-4-carboxyphenyl)-phosphorothioate (11-35%) and O-methyl- O-hydrogen- O-(2,6-dichloro-4- methylphenyl)-phosphorothioate (12-44%). In rats with bile cannulas, most of the radiolabel excreted into the bile within 24 h after administration was associated with polar metabolites; the major metabolites in the bile were O-methyl- O-hydrogen- O-(2,6- dichloro-4-hydroxymethylphenyl)-phosphorothioate and 2,6-dichloro-4- methylphenol glucuronides. Radiocarbon excreted into the faeces within 24 h after administration was associated only with the parent compound (Esumi & Yokoshima, 1989). Two hours after oral administration, the major metabolites in blood, liver and kidney were O,O-dimethyl- O-(2,6-dichloro-4- carboxyphenyl) phosphorothioate ((see Figure 1), 3,5-dichloro-4- hydroxybenzaldehyde, O-methyl- O-hydrogen O-(2,6-dichloro-4- methylphenyl)-phosphorothioate and O-methyl- O-hydrogen- O-(2,6- dichloro-4-hydroxymethylphenyl)-phosphorothioate. Only a small amount of the parent compound was detected in the liver. The major biotransformation reactions were oxidative desulfuration to oxon and related derivatives, oxidation of the 4-methyl group to alcohols and acids, cleavage of the P-O-aryl and P-O-methyl linkages and conjugation of the resultant acids and phenols with glucoronic acids (Mihara et al., 1981; Krautter et al., 1987, 1988; Esumi & Yokoshima, 1989). The general pattern of metabolism of tolclofos-methyl is shown in Figure 1.2. Toxicological studies (a) Acute toxicity Data on the acute toxicity of tolclofos-methyl (technical-grade and formulations) in laboratory animals are summarized in Table 1. Animals showed decreased spontaneous motor activity, dyspnoea, piloerection, urinary incontinence and ataxia. Recovery was complete by day 10. Brain cholinesterase activity was lower 16 days after treatment in dogs given 1000 mg/kg bw than in animals given lower doses; however, as no control data were reported, conclusions cannot be drawn. No treatment-related gross changes were seen at necropsy in any species. Table 1. Acute toxicity of technical-grade and formulated tolclofos-methyl in laboratory animals Species Strain Sex Route Formulation LD50 Reference (purity) (mg/kg bw) Mouse dd M Oral Technical (97%) 3500 Segawa, 1978 F 3600 Mouse ICR M&F Oral 50% wettable > 5000 Segawa, 1981a powder (98%) Mouse ICR M&F Oral 10% dust (98.8%) > 5000 Segawa, 1981b Mouse dd M&F Dermal Technical (97%) > 5000 Segawa, 1978 Mouse ICR M&F Dermal 50% wettable > 5000 Segawa, 1981c powder (98%) Mouse ICR M&F Dermal 10% dust (98.8%) > 5000 Segawa, 1981d Mouse dd M Intraperitoneal Technical (97%) 1070 Segawa, 1978 F 1260 Mouse dd M&F Subcutaneous Technical (97%) > 5000 Segawa, 1978 Rat SD M&F Oral Technical (97%) approx. 5000 Segawa, 1978 Rat SD M&F Oral 50% wettable > 5000 Segawa, 1981e powder (98%) Rat SD M&F Oral 10% dust (98.8%) > 5000 Segawa, 1981f Rat SD M&F Oral 50% flowable > 5000 Hiromori et al., 1989a Rat SD M&F Dermal Technical (97%) > 5000 Segawa, 1978 Rat SD M&F Dermal 50% wettable > 5000 Segawa, 1981g powder (98%) Rat SD M&F Dermal 10% dust (98.8%) > 5000 Segawa, 1981h Rat NR M&F Dermal 50% flowable > 2000 Hiromori et al., 1989b Table 1 (contd) Species Strain Sex Route Formulation LD50 Reference (purity) (mg/kg bw) Rat SD M Intraperitoneal Technical (97%) 5000 Segawa, 1978 F 4900 Rat SD M&F Subcutaneous Technical (97%) > 5000 Segawa, 1978 Rat Wistar M&F Inhalation Technical (97.7%) > 3.32 mg/la Hardy et al., 1986 Rat SD M&F Inhalation 50% wettable > 1.9 mg/l Eschbach & powder (98%) Hogan, 1981a Rat SD M&F Inhalation 10% dust (98.8%) > 1.8 mg/l Eschbach & Hogan, 1981b Rat SD M&F Inhalation 50% flowable > 1.07 mg/la Jackson et al., 1990 Dog NR M&F Oral Technical (98.7%) > 1000 Pence et al., 1978 SD, Sprague-Dawley; M, male; F, female; NR, not reported a 4-h whole-body exposure (b) Short-term toxicity Mice Groups of 15 male and 15 female ddY mice were fed diets containing tolclofos-methyl (purity, 97%) at 0, 10, 30, 100 or 3000 ppm, equal to 1.2, 3.8, 12 or 510 mg/kg bw per day for males and 1.4, 4.1, 14 or 560 mg/kg bw per day for females, for nine months. Three satellite groups of five males and five females each were similarly treated and killed at 2, 4 and 13 weeks for determination of cholinesterase activity. Animals were observed daily for mortality and signs of toxicity; at the end of exposure, an ophthalmological examination and clinical chemical analyses (including plasma and erythrocyte cholinesterase) were undertaken, and brain cholinesterase activity was measured. Most organs were weighed and examined microscopically. There were no overt signs of toxicity and no deaths. Body weights were reduced by about 20% and body-weight gain by about 35% in animals of each sex at 3000 ppm, but at this dose food consumption was increased in males and females and water intake was increased in males. No treatment-related ophthalmological changes were observed, and haematological parameters were unaffected by treatment. The cholesterol level was increased by 35% in females at 3000 ppm. A dose-related decrease in plasma cholinesterase activity was found in animals of each sex, with activities that were 63-94% of the control values in mice at 30 ppm, 30-56% at 100 ppm and 4-35% (61% in one case) at 3000 ppm. At the high dose, the activity reached a plateau after four weeks. Erythrocyte cholinesterase activity was decreased to 39-46% of the control level in males at 3000 ppm at weeks 13 and 40 and to 57-75% of the control level in females at this dose at all time points. Brain cholinesterase activity was unaffected by treatment in females but was reduced by about 24% in males at 3000 ppm at weeks 13 and 40. Organ weights in the 3000-ppm group were reduced, but neither gross nor histopathological changes attributable to treatment were observed. The NOAEL was 100 ppm, equal to 12 mg/kg per day, on the basis of inhibition of brain cholinesterase activity and an effect on body weight at the highest dose (Suzuki et al., 1978). Rats Groups of 10 male and 10 female Sprague-Dawley rats were fed diets containing tolclofos-methyl (purity, 97.9%) at 0, 200, 1000, 5000 or 20 000 ppm for 32-34 days. Animals were observed twice daily for mortality and clinical signs, and food consumption and body weight were recorded weekly. Ophthalmoscopic examination, standard haematological tests and clinical chemical analyses were performed at week 4, and certain tests were repeated at termination. Plasma and erythrocyte cholinesterase activities were determined in the control and high-dose groups at week 4 and in all animals at termination, when brain cholinesterase activity was also measured. At terminal sacrifice, relevant organs were weighed and most organs were examined microscopically. There were no treatment-related deaths. Reductions in food consumption (-16% in males and -6% in females) and body-weight gain (-45% in males and -37% in females) were observed at 20 000 ppm. Body-weight gain was slightly reduced in animals of each sex at 5000 ppm during week 1. Haematological parameters were unaffected by treatment. Cholesterol levels were increased (by 116-138%) in animals of each sex at 20 000 ppm, and total protein and albumin levels were slightly higher in males in this group. Other differences between treated and untreated animals were detected, but the values were within the normal range. Plasma cholinesterase activity was reduced by 14% in males and by 50% in females at 20 000 ppm. Erythrocyte cholinesterase activity was reduced by 9-19% in animals of each sex at 5000 and 20 000 ppm; however, no clear dose-response relationship was seen. Brain cholinesterase activity was lower (by 12-31%) than that in controls in males in all treated groups and in females (by 20%) at 5000 and 20 000 ppm. Although the reduction was statistically significant in all male groups, no clear dose-response relationship was found, and the activity in controls was slightly higher than usual. There were no ophthalmoscopic alterations or gross changes at necropsy. The relative weights of livers were increased (by 12%) in females at 5000 ppm and in males and females at 20 000 ppm (by 27% in males and 39% in females). Hypertrophy of hepatocytes was observed in animals of each sex at 20 000 ppm. The relative weights of the kidneys were slightly increased in animals of each sex at 5000 and 20 000 ppm, but no treatment-related histopathological changes were seen. The NOAEL was 1000 ppm, equal to 79 mg/kg bw per day, on the basis of increased relative kidney weights and brain cholinesterase activity at higher doses (Colley et al., 1982). Groups of 12 male and 12 female Sprague-Dawley rats were fed diets containing tolclofos-methyl (96.6% pure) at 0, 100, 1000 or 10 000 ppm, equal to 6.5, 66 or 653 mg/kg bw per day for males and 7.1, 71 or 696 mg/kg bw per day for females, for 13 weeks. Animals were observed daily for mortality and clinical signs, and body weight and food and water consumption were determined weekly. Ophthalmological analysis, urinalysis, standard haematological tests, clinical chemical analyses and measurement of plasma, erythrocyte and brain cholinesterase activities were undertaken at termination, when relevant organs were weighed and most organs were examined microscopically. No deaths and no significant abnormal clinical or ophthalmological signs were noted during the study. Depression of body-weight gain of males (-20%) and females (-15%) at 10 000 ppm was seen throughout the treatment period and was associated with similar decreases in food and water consumption. A slight decrease in food consumption was noted in females at 1000 ppm during week 1. Decreased activities of plasma (16-53%), brain (8-9%) and erythrocyte (19-20%) cholinesterase were noted in animals of each sex at 10 000 ppm; a minimal decrease in erythrocyte cholinesterase activity (10%) was also seen in females at 1000 ppm. Liver weight was increased in animals of each sex at 10 000 ppm, and this was found at histopathological examination to be associated with hypertrophy of hepatocytes. Changes in some clinical chemical parameters were observed at 10 000 ppm, which included increased levels of cholesterol (48% in males, 102% in females) and phospholipids (37% in males, 64% in females) and slightly increased alpha2- and ß-globulin levels in animals of each sex. Other changes, although statistically significant, were minor and of questionable biological significance. At 10 000 ppm, increased relative kidney weights were seen in animals of each sex, increased blood urea nitrogen levels were seen in males only and decreased urinary pH was seen in males and females. At 1000 ppm, relative kidney weights were slightly increased in females and relative liver weights in males. The Meeting considered that the increased weight ratios observed at 1000 ppm were not biologically relevant and that the NOAEL was 1000 ppm, equal to 66 mg/kg bw per day (Kimura et al., 1990). Groups of 15 male and 15 female Sprague-Dawley rats were fed diets containing tolclofos-methyl (purity, 97%) at 0, 300, 1000, 3000 or 10 000 ppm, equal to 16, 51, 164 or 540 mg/kg bw per day in males and 18, 65, 184 or 623 mg/kg bw per day in females, for 28 weeks. Animals were observed daily for clinical signs, and body weight and food consumption were measured weekly. Ophthalmologic examinations were carried out after three months and at termination; standard haematological tests and clinical chemical analyses were performed at termination. Urinalysis was carried out in 12 animals per group after three months and at termination. Plasma and erythrocyte cholinesterase activities were determined after 2, 4 and 13 weeks, and at termination; brain cholinesterase was determined only at termination, when relevant organs were weighed and several were examined histologically. There were no overt signs of toxicity and no treatment-related deaths. Although the body weights of animals of each sex at 10 000 ppm were lower than those of controls throughout the study, their initial body weights were also statistically significantly lower. Body-weight gain was significantly reduced (by 18%) in females at 10 000 ppm. Food consumption was unaffected by treatment. Treatment did not affect ophthalmologic, urinary, haematological or clinical chemical parameters. Plasma cholinesterase activity was lower (by 23-41%) in females at 10 000 ppm than in controls at weeks 2, 4, 13 and 28. Erythrocyte cholinesterase activity was highly variable and was reduced by 15-19% in comparison with controls in males at 10 000 ppm at weeks 2, 4 and 28. Brain cholinesterase activity was unaffected by treatment. Kidney and liver weights were slightly increased in animals of each sex at 10 000 ppm and in females at 1000 and 3000 ppm, but the biological significance of these findings was questionable. No gross abnormalities were seen at necropsy. Bile-duct proliferation was found in the livers of 3/15 females at 10 000 ppm and oval-cell proliferation was seen in 2/15 females at 3000 ppm and 3/15 at 10 000 ppm. No histopathological changes were seen in the kidneys. The NOAEL was 1000 ppm, equal to 65 mg/kg bw per day, on the basis of histopathological changes in the livers of females at higher doses (Hiromori et al., 1978). Rabbits Groups of five male and five female New Zealand white rabbits received tolclofos-methyl (purity, 97.7%) in acetone on the skin at doses of 0, 30, 300 or 1000 mg/kg bw per day for 6 h per day, on five days per week for 21 days. There were no deaths and no treatment-related clinical signs; neither body weight nor food consumption was affected by treatment. Dermal irritation (slight erythema) was seen in some animals in all treated groups from day 6. Eosinophil counts were increased in males at 1000 mg/kg bw per day, and the level of inorganic phosphorus was decreased (by 14-20%) in animals of each sex at this dose. Brain cholinesterase activity was about 15% lower than that in controls in males and about 15% higher in females at 300 and 1000 mg/kg bw per day. These changes were considered not to be of biological significance. Erythrocyte cholinesterase activity was lower than in controls in males at the same two doses, but there was no dose-effect relationship. Plasma cholinesterase activity was lower (by 22-29%) than in controls in animals of each sex at 300 and 1000 mg/kg bw per day. The relative weights of the kidneys were increased (by 20%) in females at 1000 mg/kg bw per day. Macroscopic findings consisted of accumulation of the compound on treated skin; microscopic examination revealed hyperkeratosis, acanthosis and subepidermal pleocellular infiltration of the skin (Gargus, 1986). Dogs Groups of six male and six female beagle dogs were fed diets containing tolclofos-methyl (purity, 98.7%) at 0, 200, 600 or 2000 ppm, equal to 7.4, 23 or 69 mg/kg bw per day in males and 4.1, 21 or 65 mg/kg bw per day in females, for 26 weeks. Animals were observed daily for mortality and weekly for clinical signs, body weight and food consumption. Ophthalmoscopic examinations were performed before treatment and at termination of the study. Standard haematological tests and clinical chemical analyses were performed at regular intervals until termination. Erythrocyte and plasma cholinesterase activities were determined at weeks 2, 4, 8, 13 and 26. Brain cholinesterase was determined at termination, when relevant organs were weighed and most were examined histologically. There were no deaths and no overt signs of toxicity. Body-weight gain was reduced by 54% in males and by 46% in females at 2000 ppm, although food consumption was unaffected by treatment. There were no treatment-related ocular changes. Although the haematocrit was within the normal range, decreased values in comparison with controls (-11% in males and -10% in females) were observed at 2000 ppm. The mean corpuscular volume and mean corpuscular haemoglobin concentration were not affected by treatment. Alkaline phosphatase activity was increased by 139-295% in animals of each sex at 2000 ppm throughout the study. Total bilirubin was not increased in treated animals. At 2000 ppm, plasma cholinesterase activity was decreased by 19-26% in females throughout the study, but no significant decreases were seen in erythrocyte cholinesterase activity in animals of each sex or in plasma cholinesterase activity in males. Brain cholinesterase activity was unaffected by treatment. Urinalysis gave unremarkable results, and there were no gross changes at necropsy. Liver weights were increased (by 59% in males and 43% in females) at 2000 ppm, but there were no concomitant histological changes, and there were no treatment-related histopathological changes. The NOAEL was 600 ppm, equal to 21 mg/kg bw per day, on the basis of increased liver weights, reduced body-weight gain and increased alkaline phosphatase activity at 2000 ppm (Pence et al., 1979a). Groups of six male and six female beagle dogs were fed diets containing tolclofos-methyl (purity, 96.7%) at 0, 80, 400 or 2000 ppm, for 52 weeks. Animals were observed daily for mortality and weekly for clinical signs. Ophthalmoscopic examinations were performed before treatment and at weeks 26 and 52. Standard haematological tests, clinical chemical analyses and determinations of plasma and erythrocyte cholinesterase activities were performed one week before treatment and at weeks 13, 26 and 52. Brain cholinesterase was determined at termination, when relevant organs were weighed and most were examined histologically. No clinical signs of toxicity were observed. Decreased food consumption (-9% in males and -16% in females) and body-weight gain (-32% in males and -54% in females), slightly lower erythrocyte counts (-8% in males and -20% in females), haematocrit value (-7% in males and -17% in females) and haemoglobin concentration (-8% in males and -7% in females), slightly decreased albumin and total protein levels (significantly at some time points) and elevated alkaline phosphatase levels (> 200%) were found at 2000 ppm. Plasma cholinesterase activity was slightly, but not significantly lower than that in controls in females at the highest dose; erythrocyte and brain cholinesterase activities were not affected by the treatment. No treatment-related ophthalmoscopic alterations were observed. Increases in mean liver weights, both absolute (+32% in males and +61% in females) and relative (+52% in males and +74% in females), were seen in dogs at 2000 ppm. Microscopic examination of the livers revealed increased incidences of hepatocytic hypertrophy and intracytoplasmic homogeneous material and an increased amount of hepatocytic pigment in these animals. The amount of this pigment was also increased at 400 ppm, and it was present in control animals. Its nature has not been elucidated, but it does not contain iron or bilirubin. These dogs also had decreased absolute (-46%) and relative (-40%) weights of the prostate, and the absolute and relative weights of the pancreas were increased by 16-27% and 28-49%, respectively. The NOAEL was 400 ppm, equal to 11 mg/kg bw per day, on the basis of reduced body-weight gain, increased liver weights (with hepatic hypertrophy) and slight anaemia at 2000 ppm (Cox et al., 1988; Moore, 1993). (c) Long-term toxicity and carcinogenicity Mice Groups of 70 male and 70 female B6C3F1 mice were fed diets containing tolclofos-methyl (purity, 94.3%) at 0, 10, 50, 250 or 1000 ppm, equal to 1.3, 6.5, 32 or 134 mg/kg bw per day in males and 1.3, 6.8, 34 or 137 mg/kg bw per day in females, for up to 104 weeks. Animals were observed twice daily for clinical symptoms and mortality. Body weight was measured weekly during the first 13 weeks and then once every four weeks. Food intake was measured weekly. Standard urinalysis was performed in 10 animals from each group at 6, 12 and 18 months and in all survivors at termination of the study. Standard haematological tests were performed in 10 animals from each group at 12 months and in all survivors at termination. Standard clinical chemical analyses and measurements of serum, erythrocyte and brain cholinesterase activities were done in 10 animals from each group at 6 and 12 months and in all animals at termination. Animals in the control and 1000-ppm groups were subjected to ophthalmologic examination at 12 and 24 months. Ten animals from each group were necropsied at 6 and at 12 months, at termination or when found dead or moribund. Relevant organs were weighed and examined microscopically. There were no treatment-related effects on mortality and no overt signs of toxicity, including ophthalmologic changes. Body-weight gain was slightly reduced in females at 1000 ppm up to week 52; food consumption was also slightly decreased in this group after 52 and 104 weeks. Food conversion efficiency, water intake, urinary parameters and the haematological profile were unaffected by treatment. Serum cholinesterase activity was decreased in animals of each sex at 250 ppm (by 25-52%) and at 1000 ppm (by 43-81%) at both times, and erythrocyte cholinesterase activity was decreased by 11-28% at 250 ppm and by 13-47% at 1000 ppm; inhibition of both enzymes was greater at termination. A dose-related decrease was found in brain cholinesterase activity in females (by 7-24% at 250 ppm and 9-33% at 1000 ppm) and males (by 10-13% at 250 ppm and 17-26% at 1000 ppm). Serum and brain, but not erythrocyte, cholinesterase activities were lower (by 12 and 18%, respectively) in females at 50 ppm at week 28 than in controls, which had higher levels than at week 52 and at termination. Glucose levels were increased in males and were slightly increased in females at 1000 ppm at week 104. Absolute and/or relative kidney weights were increased in animals of each sex at 1000 ppm and in males at 250 ppm at week 52, and in females at 250 ppm at week 104. The weights of the thymus were decreased in females at 1000 ppm at weeks 52 and 104, and pituitary weights were increased in females at this dose at week 104. No treatment-related changes were seen at necropsy or on histopathological examination at any time. Tolclofos-methyl was not found to be carcinogenic. The NOAEL was 50 ppm, equal to 6.5 mg/kg bw per day, on the basis of reduced brain cholinesterase activity and increased absolute and relative kidney weights at higher doses (Satoh et al., 1983). Rats Groups of 65 male and 65 female Fischer 344 rats were fed diets containing tolclofos-methyl (purity, 94.9-98.7%) at 0, 100, 300 or 1000 ppm for 122 weeks (males) or 129 weeks (females). At week 52, 10 males and 10 females from each group were killed. Animals were observed twice a day for mortality and signs of morbidity. Body weight and food consumption were recorded weekly (weeks 0-26), every two weeks (weeks 27-52) or every four weeks until termination. Ophthalmoscopic examinations were performed every 26 weeks and at termination. Standard haematological tests, clinical chemical analyses and measurements of erythrocyte and plasma cholinesterase activities were conducted in 10 animals of each sex in each group one week before treatment, at weeks 4, 13, 26, 52, 78 and 104 and at termination. Brain cholinesterase activity was determined in 10 animals of each sex per group at week 52 and at termination. All animals killed or found dead were necropsied, and relevant organs were weighed; most organs were then examined histologically. There were no treatment-related effects on mortality and no overt signs of toxicity. A slight decrease in body-weight gain was observed in males at 1000 ppm. Food consumption and food conversion efficiency were unaffected by treatment, as was the haematological profile. There was a dose-related trend to decreased alkaline phosphatase activity (by 9-46%) in all treated groups: The activity was significantly decreased in males at 1000 ppm throughout the study, in males at 300 ppm from week 13, and occasionally in the other treated groups. Brain cholinesterase activity was decreased in all treated groups at the time of the interim sacrifice, but there was no dose-response relationship. At the time of terminal sacrifice, brain cholinesterase activity was reduced in males at the middle dose (by 52%) and in females at the low (by 57%) and middle doses (by 49%), but not in animals at the high dose. Erythrocyte and plasma cholinesterase activities were also inconsistently lower in treated groups, although no dose-response relationship was seen. Urinalysis revealed no remarkable findings, organ weights were unaffected by treatment, and there were no treatment-related gross changes at necropsy. Toclofos-methyl was not carcinogenic in this study. No NOAEL could be identified, given the variability of the data on cholinesterase activity (Pence et al., 1982). Groups of 30 male and 30 female Fischer 344 rats were fed diets containing tolclofos-methyl (purity, 98.3-97.3%) at 0, 100, 300 or 1000 ppm for 104 weeks, and brain, erythrocyte and plasma cholinesterase activities were assessed. Ten males and 10 females from each group were sacrificed at week 52. Animals were observed daily for mortality, and they were examined for body weight, food consumption and gross signs of toxicity weekly until week 26, every two weeks during weeks 26-52 and every four weeks during weeks 52-104. Blood samples were taken for determination of plasma and erythrocyte cholinesterase activities at initiation of the study, at weeks 5, 14, 27, 53, 79 and at termination. Brain cholinesterase activity was determined at week 52 and at termination. There were no treatment-related effects on survival, and the incidences of clinical signs and palpable tissue masses were comparable among the groups. Mean plasma cholinesterase activities were slightly decreased (by < 25%) in males when compared with controls at 300 and 1000 ppm at weeks 27 (statistically significant), 53, 79 and 105. Mean erythrocyte and brain cholinesterase activities were comparable among the groups throughout the study. Plasma cholinesterase activity was reduced (by < 20%) at certain times in treated animals but no dose-response relationship was found. Gross pathological findings were unrelated to treatment. Doses < 1000 ppm did not depress erythrocyte or brain cholinesterase activity or induce gross pathological effects (Miyamoto, 1985; Pence et al., 1985a). The combined NOAEL in these two studies was 1000 ppm, equal to 41 mg/kg bw per day, on the basis of the absence of any significant findings. (d) Reproductive toxicity Rats A three-generation study of reproductive toxicity was performed in Sprague-Dawley rats fed diets containing tolclofos-methyl (purity, 97.9-98.7% pure) at 0, 100, 300 or 1000 ppm, equivalent to 10, 30 or 100 mg/kg bw per day, with two litters per generation. The F0 generation consisted of 30 rats of each sex per group. The F1a, F2a and F3a generations were sacrificed at weaning and underwent gross necropsy. Five rats of each sex in each group were selected at weaning from the F1b, F2b and Fn3b litters for histopathological examination, and 25 rats of each sex from each group were selected from the F1b and F2b litters to breed the following generation. A 15-week growth period was allowed before mating of the 'a' litters, and a minimal 10-day rest period was allowed between weaning of the 'a' litters and mating to produce the 'b' litters. After birth of the 'b' litter, all parental animals were necropsied. There were no treatment-related deaths or overt signs of toxicity. Pregnancy rates were low at times in all groups, but the pattern was not related to dose. The lowest pregnancy rate (52%) was observed in the F1b generation at the middle dose. Pregnancy rates were below 70% in three of the six generations in the controls, in two generations at the low dose, in four generations at the middle dose and in one generation at the high dose. The body weights of F2a and F2b pups at 300 and 1000 ppm were lower than those of controls at the start of the growth period; however, these reductions were not dose-related, and the growth rates and body weights in other groups were comparable throughout the study. There were no treatment-related gross changes at necropsy in any group. The weights of the ovaries were increased in non-pregnant adult F2b females at 1000 ppm, but this was not associated with histomorphological alterations. There was an increased incidence (not statistically significant) of fine cytoplasmic vacuolation of the adrenal cortex in adult F2b females at 1000 ppm that became pregnant at both matings. The etiology and toxicological significance of this finding, given its occurrence in the controls, were unknown. There was no effect on reproductive parameters or on offspring survival and development in any litter, and there were no treatment-related gross or histopathological findings in the litters. The NOAEL for reproductive toxicity was 1000 ppm (equivalent to 100 mg/kg bw per day) on the basis of the absence of significant findings at any dose level (Pence et al., 1985b). (e) Embryotoxicity and teratogenicity Rats Groups of 21-26 pregnant Fischer 344 rats were administered 0, 5, 15 or 50 mg/kg bw per day tolclofos-methyl (purity, 94.9%; dose adjusted to 100%) in 0.5% w/v methylcellulose by gavage on days 6-15 of gestation. There were no overt signs of toxicity, and body-weight gain and food consumption were unaffected by treatment. There were no treatment-related gross lesions in the dams at necropsy. Pregnancy rates were slightly higher in all treated groups (77-87%) than in controls (70%). There were no treatment-related changes in the number of fetal deaths, fetal viability or size or the incidences of visceral or skeletal anomalies or variations. Tolclofos-methyl was not embryotoxic, fetotoxic or teratogenic in rats at doses up to and including 50 mg/kg bw per day. It should be noted, however, that the highest dose was not maternally toxic (Pence et al., 1979b). Groups of 23 pregnant Sprague-Dawley rats were administered 0, 100, 300 or 1000 mg/kg bw per day tolclofos-methyl (purity, 96.7%) in 0.5% methyl cellulose orally on days 6-15 of gestation. On day 20 of gestation, all animals were sacrificed and caesarean section was performed. Rats were observed daily for mortality and clinical signs and weighed. At termination, dams were observed for gross visceral abnormalities, and their uteri were weighed and examined for implantations and resorptions. Live fetuses were weighed, sexed and examined for external, visceral and skeletal abnormalities. All dams survived to the day of scheduled sacrifice with no clinical signs of toxicity. Mean body-weight gain in animals at 100 and 1000 mg/kg bw per day was significantly less than in controls on days 6-11. Both mean body-weight gain (days 6-16) and mean net body-weight gain (days 0-20) in rats at 1000 mg/kg bw per day were lower (by 10 and 14%, respectively) than the corresponding control value. In addition, mean food consumption in animals at 1000 mg/kg bw per day on days 6-16 and 16-20 was slightly below the control value. There were no treatment-related differences in implantation efficiencies, and mean fetal viability, sex ratio and fetal body weight were similar in all groups. The number of fetuses with unossified fifth and/or sixth sternebrae was significantly greater in the 1000-mg/kg bw per day group than in the control group; however, the incidence of total fetal skeletal variations was similar in all groups. Other variations in development were not related to dose. Two to three malformed fetuses were found in each group, but neither the type nor the frequency of malformations indicated a teratogenic or embryotoxic response. Tolclofos-methyl was found to be neither teratogenic nor embryotoxic in this study at doses up to and including 1000 mg/kg bw per day. This dose was slightly toxic to the dams, as indicated by the lower body-weight gain. The NOAEL for maternal toxicity was 300 mg/kg bw per day (Morseth et al., 1987). Rabbits Groups of 13-17 pregnant New Zealand white rabbits were administered 0, 300, 1000 or 3000 mg/kg bw per day tolclofos-methyl (purity, 98.7% pure) in 5% carboxymethylcellulose orally on days 6-18 of gestation. The animals were observed daily for mortality and clinical signs and were weighed. At termination, dams were observed for gross visceral abnormalities, and their uteri were weighed and examined for implantations and resorptions. Live fetuses were weighed, sexed and examined for external, visceral and skeletal abnormalities. One rabbit at 3000 mg/kg bw per day died on day 14 of gestation of an undetermined cause. There were no overt signs of toxicity in any group. Spontaneous abortion occurred in one dam at 1000 mg/kg bw per day and in two at 3000 mg/kg bw per day on or after day 21 of gestation. The mean body weights of animals in the treated groups were significantly lower than the control values throughout the study (including day 0). Body-weight gain was reduced by 76% in animals at 3000 mg/kg bw per day; and at termination, weight gain (days 0-29) was 19% lower than in controls. At 1000 mg/kg bw per day, body-weight gain was reduced by 56% during treatment, but it was similar to that of controls at termination. Food consumption was decreased by up to 38% in animals at 1000 and 3000 mg/kg bw per day during treatment. There were no treatment-related changes in maternal organ weights. One animal at 3000 mg/kg bw per day resorbed her entire litter. There were no other treatment-related changes in implantation efficiency, mean fetal viability, size, sex ratio, fetal body weight or external, visceral or skeletal development. Tolclofos-methyl was not teratogenic in this study at doses up to and including 3000 mg/kg bw per day, which was toxic to dams. The NOAEL for maternal toxicity was 300 mg/kg bw per day (Motoyama et al., 1991). (f) Genotoxicity The results of tests for the genotoxicity of tolclofos-methyl are summarized in Table 2. (g) Special studies (i) Skin and eye irritation and skin sensitization Six male albino Japanese rabbits received 500 mg of tolclofos-methyl (purity, 97%) on clipped intact or abraded dorsal skin for 4 h under an occlusive dressing. No signs of irritation were seen at any of the application sites seven days after treatment (Matsubara et al., 1978). Table 2. Results of tests for the genotoxicity of tolclofos-methyl End-point Test system Concentration Purity Results Reference of tolclofos-methyl (%) In vitro Reverse S. typhimurium TA98, 10, 100, 500, 1000, 2000 97.0 Negativea,b Suzuki & Miyamoto, mutation 100, 1535, 1537, 1538 µg/plate Reverse S. typhimurium TA98, 10, 50, 100, 500, 1000, 98.7 Negativea,c Moriya et al., 1981 mutation 100, 1535, 1537, 1538 5000 µg/plate E. coli WP2uvrA Reverse B. subtilis H17 rec+, 1, 10, 100, 1000 µg/disc 97.0 Negatived Suzuki & Miyamoto, mutation M45 rec- 1978 Reverse B. subtilis 20, 50, 100, 200, 500, 98.7 Negativee Moriya et al., 1981 mutation H17 rec+, M45 rec- 1000, 2000, 5000 µg/disc Host-mediated S. typhimurium in 870, 1750 mg/kg 97.0 Negativef Suzuki & Miyamoto, assay male ICR mice 1978 Chromosomal Chinese hamster 10, 20, 40 µg/ml 96.6 Negativea,g Kogiso et al., 1990 aberration K1 ovary cells 37.5, 75, 150 µg/ml Unscheduled Male Sprague-Dawley 0.3, 1, 3, 10, 20, 40 96.6 Negativea,i Hara et al., 1990 DNA synthesis rat hepatocytes µg/mlh Unscheduled Human carcinoma 0.3, 3, 30, 300 µg/ml NR Negativej Monaco & Nunziata, DNA synthesis cells (HeLa) 1981 Gene mutation Chinese hamster 1.5, 15, 150, 1500 µg/ml NR Negativea,k Monaco & Nunziata, V79 lung cells 1981 Table 2 (contd) End-point Test system Concentration Purity Results Reference of tolclofos-methyl (%) In vivo Chromosomal Male ICR mice 500, 1000, 2000, 99.8 Negativei Hara & Suzuki, 1981 aberration 4000 mg/kg i.p.h Dominant Male Sprague- 62.5, 208.3, 625.0 NR Negativej Brusick, 1981 lethal Dawley rats mg/kg orally mutation NR, not reported a With and without metabolic activation. b Positive controls ( N-methyl- N'-nitro- N-nitrosoguanidine and 2-acetylaminofluorene) yielded expected positive results. c Several positive controls yielded expected positive results. d Positive control ( N-methyl- N'-nitro- N-nitrosoguanidine) yielded expected positive results. e Positive (mitomycin C) and negative (Kanamycin) controls yielded expected results. f Positive control ( N-nitrosodimethylamine) yielded expected positive results. g Positive controls (mitomycin C and cyclophosphamide) yielded expected positive results. h At 40 mg/ml, cell viability was less than 20%. i Positive control (2-acetylaminofluorene) yielded expected positive results. j Positive controls (methyl methanesulfonate and urethane) yield expected positive results. k Positive controls (methyl methanesulfonate and N-nitrosodimethylamine) yielded expected positive results. l At 2000 and 4000 mg/kg bw there was > 50% mortality at 48 h. m Positive control (cyclophosphamide) yielded expected positive results. n Positive control (triethylenemelamine) yielded expected positive results. Groups of six male New Zealand white rabbits received 500 mg of either a '50% wettable power' or '10% dust' moistened with saline on 1-inch2 (6.5-cm2) sites on the clipped dorsal intact or abraded skin for 24 h under an occlusive dressing. No irritation, such as erythema and oedema, was observed (Hara et al., 1981a,b). Two male and one female New Zealand white rabbits received 0.5 ml of '50% flowable' tolclofos-methyl on 1 inch2 (6.5 cm2) of clipped dorsal intact or abraded skin for 4 h under an occlusive dressing. No irritation was observed (Nakanishi et al., 1989). Each of eight male albino Japanese rabbits received 50 mg of tolclofos-methyl (purity, 97%) in one eye. Five minutes after the application, the treated eyes of five animals were flushed with 300 ml saline for 2 min. The treated eyes of the remaining animals were similarly flushed 24 h after treatment. There were no corneal, conjunctival or iridal effects up to seven days after treatment (Matsubara et al., 1978). Groups of nine male New Zealand white rabbits received 100 mg of either a '50% wettable power' or '10% dust' in one eye; 30 s after the application, the treated eyes of three animals per group were flushed with 300 ml lukewarm water for 1 min. Slight congestion of the iris was observed 24 h after application. Slight to moderate hyperaemia and slight chemosis and/or discharge in conjunctiva were also observed 1-48 h after application of the '50% wettable power' to unwashed eyes. These changes had disappeared by 72 h after application in all animals. No ocular lesions were found in the washed eyes. The irritation potency of this formulation was judged to be mild. Slight conjunctival hyperaemia and/or chemosis were observed in animals with unwashed eyes and in one with washed eyes 1-24 h after application of the '10% dust'. There were no other signs of irritation at any time. The formulation was classified as minimally irritating to eyes (Hara et al., 1981a,b). One male and two female New Zealand white rabbits received 0.1 ml of '50% flowable' tolclofos-methyl in one eye. Slight redness was observed in conjunctiva after application, which disappeared within 24 h (Nakanishi et al., 1989). The skin-sensitizing potential of tolclofos-methyl (purity, 97%) was assessed in guinea-pigs by the Landsteiner-Draize method. Groups of 10 male Hartley guinea-pigs were given 10 intradermal injections of 1 or 5% tolclofos-methyl (0.05 or 0.1 ml) in corn oil at intervals of two to three days. Two weeks after the final induction, the animals were challenged at a fresh site with 0.05-ml intradermal injections of tolclofos-methyl at the same concentrations used for induction. Negative control animals for both dose groups were given the challenge injection only. Positive control animals were treated with 0.05% 2,4-dinitrochlorobenzene three times before challenge. Slight erythema and swelling were observed in two animals after challenge treatment with 1% tolclofos-methyl, in three after challenge with 5%, and in one animal in each negative control group. Moderate erythema and/or swelling was observed in the animals treated with 2,4-dinitrochlorobenzene. It was concluded that tolclofos-methyl did not sensitize skin in this study (Matsubara et al., 1980). The skin sensitizing potential of a '50% wettable powder' and a '10% dust' of tolchlofos-methyl was tested in guinea-pigs by the Buehler method. Groups of 10 male Hartley guinea-pigs received 500 mg of one formulation, slightly moistened with water, on clipped dorsal skin under an occlusive dressing for 24 h. Induction was performed 10 times at two- to three-day intervals.Positive control animals were similarly treated with 0.5% 2,4-dinitro-chlorobenzene. Negative controls were not subjected to the induction treatment. Two weeks after the final induction, the test animals and positive controls were challenged as in the sensitizing treatment. No skin reactions were observed in the negative control or treated animals. Slight to severe erythema and swelling were observed in the positive controls. The formulations were considered not to sensitize skin (Hara et al., 1981c,d). The skin sensitizing potential of a '50% flowable formulation' was tested in guinea-pigs by the Buehler method. Ten male Hartley guinea-pigs received 0.5 ml of the formulation for 6 h once a week for three weeks on the clipped dorsal skin under an occlusive dressing. Positive control animals were similarly treated with 0.5% 2,4-dinitrochlorobenzene. Negative control animals were not subjected to the induction treatment. Two weeks after the final induction, animals were challenged as in the sensitizing treatment. No skin reactions were observed in the negative control or treated animals. Slight to severe erythema and swelling were observed in the positive controls. The formulation was considered not to sensitize skin (Nakanishi et al., 1990). (ii) Delayed neuropathy Groups of 10 Leghorn hens were administered 0 or 8000 mg/kg bw tolclofos-methyl (purity, 97%) or 500 mg/kg bw tri- ortho-cresyl phosphate orally in corn oil. After a 21-day observation period, a second dose of vehicle or tolclofos-methyl was administered. Animals were sacrificed 21 days after the second dose. There were no deaths in the groups given the vehicle or tolclofos-methyl. Plasma cholinesterase activity in the group treated with tolclofos-methyl was decreased by nearly 50% eight days after the first dose but had recovered to the pre-treatment level 21 days after dosing. Hens treated with tolclofos-methyl had no signs of leg weakness or paralysis and no histopathological changes in the nervous tissues. Birds treated with tri- ortho-cresyl phosphate had the typical clinical and histopathological signs of delayed polyneuropathy (Okuno et al., 1982). 3. Observations in humans The medical records of 20 workers in Japan were reviewed. All workers had been engaged continuously in packaging operations since manufacture of technical-grade tolclofos-methyl began in 1988, for an average of 4 h/day. No occupation-related problems were observed or reported. Plasma and erythrocyte cholinesterase activities were not measured (Murayama, 1991). Comments Tolclofos-methyl is excreted rapidly in rats and mice, predominantly in the urine; less than 1% of the dose was retained in tissues after seven days. In both species, metabolism occurred mainly by oxidation of P=S to P=O, oxidation of the 4-methyl group and cleavage of the P-O-aryl and P-O-methyl linkages. There are four main metabolites in mice, one of which is a glycine conjugate, and four in rats, which are excreted as glucuronides. Tolclofos-methyl had low acute toxicity when administered by the oral, dermal, subcutaneous or intraperitoneal route. The overt signs of acute toxicity are not typical of an anticholinesterase, as no chromodaccryorrhoea, lachrymation or fasciculation was seen, although some inhibition of plasma, erythrocyte and brain cholinesterase activities was observed. WHO (1992) has classified tolclofos-methyl as unlikely to present an acute hazard in normal use. In a nine-month study of toxicity in which mice were fed tolclofos-methyl in the diet at 0, 10, 30, 100 or 3000 ppm, the NOAEL was 100 ppm, equal to 12 mg/kg bw per day, on the basis of inhibition of brain cholinesterase activity and effects on body weight at 3000 ppm. In a 32-34-day study of toxicity in which rats were fed diets containing 0, 200, 1000, 5000 or 20 000 ppm, the NOAEL was 1000 ppm, equal to 79 mg/kg bw per day, on the basis of inhibition of brain cholinesterase activity and increased relative kidney weight at 5000 ppm. In a 13-week study of toxicity in which rats were fed diets containing 0, 100, 1000 or 10 000 ppm, the NOAEL was again 1000 ppm, equal to 66 mg/kg bw per day, on the basis of effects on body, liver and kidney weights at 10 000 ppm. In a 28-week study of toxicity in which rats were fed dietary levels of 0, 300, 1000, 3000 or 10 000 ppm, the NOAEL was also 1000 ppm, equal to 65 mg/kg bw per day, on the basis of histopathological changes in the livers of females at 3000 ppm. In a 26-week dietary study in dogs fed levels of 0, 200, 600 or 2000 ppm, the NOAEL was 600 ppm, equal to 21 mg/kg bw per day, on the basis of reduced body-weight gain, an increased serum level of alkaline phosphatase and increased liver weight at 2000 ppm. In a 52-week study of toxicity in dogs given dietary concentrations of 0, 80, 400 or 2000 ppm, the NOAEL was 400 ppm, equal to 11 mg/kg bw per day, on the basis of increased liver weight (with hepatocytic hypertrophy), reduced body-weight gain and slight anaemia at 2000 ppm. In a 104-week study of toxicity and carcinogenicity in which mice were given dietary concentrations of 0, 10, 50, 250 or 1000 ppm, the NOAEL was 50 ppm, equal to 6.5 mg/kg bw per day, on the basis of reduced brain cholinesterase activity and increased absolute and relative kidney weights at higher levels. There was no evidence of carcinogenicity. A 122-129-week study of toxicity and carcinogenicity was performed in which rats were given dietary concentrations of 0, 100, 300 or 1000 ppm. The NOAEL was 1000 ppm, equal to 41 mg/kg bw per day, on the basis of the absence of any significant finding. There was no evidence of carcinogenicity. In a three-generation study (two litters per generation) in rats, tolclofos-methyl was given at dietary levels of 0, 100, 300 or 1000 ppm. The NOAEL was > 1000 ppm, equivalent to 100 mg/kg bw per day, on the basis of the absence of any significant findings. In a study of teratogenicity in which rats were given 0, 5, 15 or 50 mg/kg bw per day of tolclofos-methyl by gavage, the NOAEL was 50 mg/kg bw per day on the basis of the absence of any significant findings. The study was not considered to be fully adequate because the highest dose tested was not maternally toxic. A similar study was conducted at levels of 0, 100, 300 or 1000 mg/kg bw per day. The NOAEL was 300 mg/kg bw per day on the basis of reduced body-weight gain in dams at 1000 mg/kg bw per day. There was no evidence of teratogenicity. A study of teratogenicity was conducted in rabbits given 0, 300, 1000 or 3000 mg/kg bw per day orally. The NOAEL for maternal toxicity was 300 mg/kg bw per day on the basis of reduced body-weight gain in dams at 1000 mg/kg bw per day. There was no evidence of teratogenicity. Tolclofos-methyl was studied in a wide range of tests for genotoxicity in vivo and in vitro. The Meeting concluded that the compound is not genotoxic. Tolclofos-methyl did not cause delayed neuropathy in chickens. The available observations in humans were considered by the Meeting but did not directly contribute to an estimation of an ADI. An ADI was established on the basis of a NOAEL of 50 ppm, equal to 6.5 mg/kg bw per day, in the 104-week study of toxicity and carcinogenicity study in mice, and a safety factor of 100. Toxicological evaluation Levels that cause no toxic effect Mouse: 50 ppm, equal to 6.5 mg/kg bw per day (104-week study of toxicity and carcinogenicity) Rat: 1000 ppm, equal to 41 mg/kg bw per day (122/129-week study of toxicity and carcinogenicity) Rabbit: 300 mg/kg bw per day (maternal toxicity in a study of teratogenicity) Dog: 400 ppm, equal to 11 mg/kg bw per day (52-week study of toxicity) Estimate of acceptable daily intake for humans 0-0.07 mg/kg bw Studies that would provide information useful for continued evaluation of the compound Further observations in humans References Brusick, D.J. (1981) Mutagenicity evaluation of S-3349 T.G. Lot No. 4, rat dominant lethal assay. Litton Bionetics Inc. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Colley, J., Welch, P.J., Heywood, R., Prentice, D.E., Cherry, C.P., Mullins, P.A., Gibson, W.A. & Almond, R.H. (1982) S-3349, toxicity to rats by dietary administration for 4 weeks. Huntington Research Centre Ltd. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Cox, R.H. & Brusick, D.J. (1988) Chronic toxicity study in dogs with S-3349. Hazleton Laboratories America Inc. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Eschbach, J.C. & Hogan, G.K. (1981a) An acute inhalation toxicity study of S-3349 50WP in the rat. Bio/dynamics Inc. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Eschbach, J.C. & Hogan, G.K. (1981b) An acute inhalation toxicity study of S-3349 10d in the rat. Bio/dynamics Inc. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Esumi, Y. & Yokoshima, T. (1989) Study on metabolism of tolclofos-methyl in rats. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Gargus, J.L. (1986) 21-Day dermal toxicity study, in rabbits with Rizolex, technical 97.7%. Hazleton Laboratories America Inc. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Hara, M. & Suzuki, H. (1981) In vivo chromosomal aberration test of S-3349 on bone marrow cells of mice. Sumitomo Chemical Co., Ltd. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Hara, S., Suzuki, T. & Miyamoto, J. (1981a) Primary eye and skin irritation tests of S3349 50% water-dispersible power in rabbits. Sumitomo Chemical Co., Ltd. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Hara, S., Suzuki, T. & Miyamoto, J. (1981b) Primary eye and skin irritation tests of S-3349 10% dust formulation in rabbits. Sumitomo Chemical Co., Ltd. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Hara, S., Suzuki, T. & Miyamoto, J. (1981c) Skin sensitization test of S3349 50% water-dispersible powder in guinea pigs. Sumitomo Chemical Co., Ltd. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Hara, S., Suzuki, T. & Miyamoto, J. (1981d) Skin sensitization test of S3349 10% dust formulation in guinea pigs. Sumitomo Chemical Co., Ltd. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Hara, M., Ota, M., Kogiso, S., Yoshitake, A. & Yamada, H. (1990) In vitro unscheduled DNA synthesis (UDS) assay of Rizolex in rat hepatocytes. Sumitomo Chemical Co., Ltd. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Hardy, C.J., Jackson, G.C., Lewis, D.J. & Gopinath, C. (1986) Technical Rezolex, acute inhalation toxicity in rats, 4-hour exposure. Huntington Research Centre Ltd. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Hiromori, T., Suzuki, T., Okuno, Y., Ito, S., Murakami, M., Kadota, T. & Miyamoto, J. (1978) Six-month oral toxicity study of S-3349 in rats. Sumitomo Chemical Co., Ltd. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Hiromori, T., Misaki, Y., Ito, S., Kohda, A., Kato, T. & Yamada, H. (1989a) Acute oral toxicity study of Rizolex 50 FL in rats. Sumitomo Chemical Co., Ltd. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Hiromori, T., Misaki, Y., Ito, S., Kohda, A., Kato, T. & Yamada, H. (1989b) Acute dermal toxicity study of Rizolex 50 FL in rats. Sumitomo Chemical Co., Ltd. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Jackson, G.C., Hardy, C.J., Morrow, J. & Gopinath, C. (1990) Rizolex 50 FL, acute inhalation toxicity study in rats, 4-hour exposure. Huntington Research Centre Ltd. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Kimura, J., Yosioka, K., Ozaki, K., Adachi, H., Seki, T., Matsuo, M &, Yamada, H. (1990) 90-Day oral toxicity study of S-3349 in rats. Sumitomo Chemical Co., Ltd. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Kogiso, S., Yamamoto, K., Hara, M., Yositake, A. & Yamada, H. (1990) In vitro chromosomal aberration test of Rizolex in Chinese hamster ovary cells (CHO-K1). Sumitomo Chemical Co., Ltd. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Krautter, G.R., Downs, J., Hoglan, N., Marsh, J.D. & Lawrence, L.J. (1987) Metabolism of tolclofos-methyl in the rat. Pharmacology & Toxicology Research Laboratory. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Krautter, G.R., Downs, J., Hoglan, N., Marsh, J.D. & Lawrence, L.J. (1988) Metabolism of tolclofos-methyl in the rat. Final report amendment. Pharmacology & Toxicology Research Laboratory. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Matsubara, T., Hara, S. & Kadota, T. (1978) Eye and skin irritation test of S-3349 in rabbits. Sumitomo Chemical Co., Ltd. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Matsubara, T., Hara, S., Suzuki, T., Kadota, T. & Miyamoto, J. (1980) Skin sensitization test of S-3349 in guinea pigs. Sumitomo Chemical Co., Ltd. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Mihara, K., Ohkawa, H. & Miyamoto, J. (1981) Metabolism of tolclofos-methyl in rats and mice. J. Pestic. Sci., 6, 65-74. Miyamoto, J. (1985) Comments on toxic effects and minimum effect level of S-3349 in chronic oral toxicity studies in rats. Sumitomo Chemical Co., Ltd., Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Monaco, M. & Nunziata, A. (1981) Report of mutagenicity experiment performed on the test substance S-3349, Sumitomo Chemical Co., Ltd. Unpublished report from Centro Ricerca Farmaceutica, Pomezia, Italy. Submitted to WHO by Sumitomo Chemical Co., Ltd. Moore, M.R. (1993) Comments on the toxicological significance of hepatocytic pigment and on the no-adverse-effect-level (NOAEL) in a chronic toxicity study in dogs treated with S-3349 (Hazleton project No. 343-176). Hazleton Washington Inc. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Moriya, M., Ohta, T. & Shirasu, Y. (1981) S-3349, microbial mutagenicity study. Institute of Environmental Toxicology. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Morseth, S.L., Burlew, J.A., Vargas, K.J., Lewis, S.A., Thakur, A.K. & Phipps, N.G. (1987) Teratology study of S-3349 T.G. in rats. Hazleton Laboratories America Inc. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Motoyama, M., Kashima, M. & Takahashi, M. (1991) Teratology study of S-3349 in rabbits. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Murayama, F. (1991) A review on medical examination of factory workers exposed to tolclofos-methyl. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Nakanishi, T., Kohda, A., Kato, T. & Yamada, H. (1989) Primary eye and skin irritation tests with Rizolex 50 FL in rabbits. Sumitomo Chemical Co., Ltd. Unpublished study, submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Nakanishi, T., Kohda, A., Kato, T. & Yamada, H. (1990) Skin sensitization test of Rizolex 50 FL in guinea pigs. Sumitomo Chemical Co., Ltd. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Okuno, Y., Yamada, T., Hosokawa, S. & Miyamoto, J. (1982) Acute delayed neurotoxicity study of S-3349 in hens. Sumitomo Chemical Co., Ltd. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Pence, D.H., Lemen, J.K. & Weatherholtz, W.M. (1978) Acute oral toxicity study in male and female dogs, S-3349. Hazleton Laboratories America Inc. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Pence, D.H., Weatherholtz, W.M., Kundzins, W., Alsaker, R.D., Brown, H.R. & Greenspun, K.S. (1979a) Subacute dietary administration in dogs, S-3349. Hazleton Laboratories America Inc. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Pence, D.H., Pence, L.A., Durloo, R. & Twigg, C.J. (1979b) Teratology study in rats, S-3349. Hazleton Laboratories America Inc. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Pence, D.H., Serota, D., Alsaker, R.D., Koka, M., Banas, D.A., Dawkins, B.G., Kundzins, W. & Hepner, K.E. (1982) Chronic toxicity study in rat, S-3349. Hazleton Laboratories America Inc. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Pence, D.H., Phipps, N., Alsaker, R.D., Hepner, K.E. & Spicer, K.M. (1985a) 104-Week cholinesterase activity study in male and female rats, S-3349. Hazleton Laboratories America Inc. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Pence, D.H., Hepner, K.E., Wolfe, G.W., Voelker, R.W. & Ulland, B.N. (1985b) Three-generation reproduction study in rats, S-3349. Hazleton Laboratories America Inc. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Satoh, R., Kashima, M., Satoh, H., Motoyama, M. & Ishikawa, A. (1983) Twenty-four-month chronic toxicity study of S-3349 in pulverized diet in mice. Nippon Experimental Medical Research Institute. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Segawa, T. (1978) Acute toxicity study of S-3349 in rats and mice. Hiroshima University School of Medicine. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Segawa, T. (1981a) Acute oral toxicity study of S-3349 50% wettable powder in mice. Hiroshima University School of Medicine. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Segawa, T. (1981b) Acute oral toxicity study of S-3349 10% dust in mice. Hiroshima University School of Medicine. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Segawa, T. (1981c) Acute dermal toxicity study of S-3349 50% wettable powder in mice. Hiroshima University School of Medicine. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Segawa, T., (1981d) Acute dermal toxicity study of S-3349 10% dust in mice. Hiroshima University School of Medicine. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Segawa, T. (1981e) Acute oral toxicity study of S-3349 50% wettable powder in rats. Hiroshima University School of Medicine. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Segawa, T. (1981f) Acute oral toxicity study of S-3349 10% dust in rats. Hiroshima University School of Medicine. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Segawa, T. (1981g) Acute dermal toxicity study of S-3349 50% wettable powder in rats. Hiroshima University School of Medicine. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Segawa, T. (1981h) Acute dermal toxicity study of S-3349 50% wettable powder in rats. Hiroshima University School of Medicine. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Suzuki, H. & Miyamoto, J. (1978) Studies on mutagenicity of S-3349 with bacterial systems. Sumitomo Chemical Co., Ltd. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan. Suzuki, T., Okuno, Y., Hiromori, T., Ito, S., Murakami, M., Kadota, T & Miyamoto, Y. (1978) Nine-month feeding study of S-3349 in mice. Sumitomo Chemical Co., Ltd. Unpublished report submitted to WHO by Sumitomo Chemical Co., Ltd, Osaka, Japan.
See Also: Toxicological Abbreviations