Thiodicarb is a carbamate insecticide that acts by inhibiting acetylcholinesterase activity. It was evaluated by JMPR in 1985 and 1986 (Annex 1, references 44 and 47). An ADI of 0–0.03 mg/kg bw was established in 1986. It was considered by the 2000 JMPR within the periodic review programme of the Codex Committee on Pesticide Residues.

The thiodicarb used for biochemical and toxicity testing was usually supplied as an off-white powder with a purity of 91–99.9%. The sponsor commented that there have been no significant changes in the composition of technical-grade thiodicarb over time. The compound is also known as ‘Larvin technical’ and ‘UC51762’.

A number of studies on the toxicokinetics of thiodicarb that were reviewed by the 1985 JMPR (Annex 1, reference 46) were not resubmitted. The findings of the studies were consistent with those of the more recent work evaluated at the present Meeting. For completeness, and to provide a comparison with the newer studies, sections of text from the 1985 JMPR monograph are reproduced here.

Thiodicarb, which consists essentially of two methomyl moieties joined through their amino nitrogen by sulfur, is rapidly degraded to S-methyl-N-[(methylcarbamoyl)oxy]thioacetamide (methomyl) in the rat stomach. Over 30% of an oral dose of 16 mg/kg bw was converted to methomyl in the gut within 15 min. Only 40% of the dose was found in the gut at 15 min, indicating that the compound had been rapidly absorbed. Approximately 26% of the radiolabel in the administered dose was found in other body fractions, the remainder of the dose (34%) apparently having been lost as volatile compounds.

In rats given radiolabelled thiodicarb as a single oral dose of 40 mg/kg bw, 80% of the dose was eliminated within 48 h. After 4 days, 48% of the dose had been eliminated in the respiratory gases, 32% in the urine, and 4.5% in the faeces; 11% of the dose remained in the carcass because of incorporation of radiolabelled carbon dioxide and acetic acid metabolites into natural products.

Thiodicarb is degraded in the stomach not only to methomyl but also to some other unstable intermediates, including methomyl methylol, methomyl oxime, methomyl sulfoxide, and methomyl sulfoxide oxime, which are subsequently converted to acetonitrile and carbon dioxide and eliminated primarily by respiration and in the urine. Acetonitrile is the only metabolite retained to some extent in body tissues and fluids; a small fraction of the acetonitrile is further degraded to carbon dioxide, acetic acid, and acetamide, which is suspected to be carcinogenic in mice and rats. The ultimate metabolic fate of methomyl in animals depends on its isomeric configuration. In rats, the stable and predominant form is the syn isomer, which is metabolized primarily to carbon dioxide, while partial conversion from the syn isomer to the anti isomer leads primarily to acetonitrile, most of which is respired unchanged.

On the basis of the finding that 5.7% of administered syn-methomyl is converted to acetonitrile, a maximum of 5.7% of the administered [¹⁴C]thiodicarb might therefore be available for conversion to acetamide, since thiodicarb is metabolized predominantly to syn-methomyl. On a weight–percent basis, the maximum amount of acetamide that could be produced after ingestion of thiodicarb residues is 1.9% of the administered dose (53:l thiodicarb:acetamide, weight:weight).

An extensive study of the toxicokinetics of thiodicarb in rats was performed. The study was divided into three phases: determination of the T_max and C_max in blood, plasma, and erythrocytes; a mass balance investigation of excretory pathways; and an investigation of tissue concentrations and metabolites.

In phase 1, groups of five male and five female Sprague-Dawley rats received a single dose of [acetimide-¹⁴C]thiodicarb (23 mCi/mmol per L) by gavage in Emulphor EL620 (8 ml/kg bw) at 2.4 or 18 mg/kg bw. The purity of the radiolabelled sample varied with analytical procedure from 88% (by high-performance liquid chromatography [HPLC]) to 93% (by thin-layer chromatography [TLC]), the remainder being primarily methomyl. Samples of urine and faeces were taken 6, 12, 24, and 48 h after dosing, and blood samples were taken from the tail vein 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48, 72, and 96 h after dosing. At terminal sacrifice (96 h), samples of 12 tissues and the carcass were taken. Trapping of volatile compounds was not performed. Radiolabel was measured by liquid scintillation counting (LSC) after appropriate processing of samples. Animals receiving 18 mg/kg bw showed a range of clinical signs, including tremors, during the first 3 h. The results (Table 1) showed that thiodicarb at 2.4 mg/kg bw was absorbed very rapidly, but the plasma T_max was slightly longer at 18 mg/kg bw, indicating saturation of absorption. However, as the ratio of plasma to erythrocyte C_max was similar, there appeared to be no significant alteration of key kinetics with increasing dose. The T_max for erythrocytes was later than that for plasma, which, with the high concentration of radiolabel present in erythrocytes at 96 h, indicates that the compounds responsible for the radiolabel in plasma might be metabolized further to the molecules associated with the erythrocytes. Females had a higher erythrocyte C_max than males and delayed urinary excretion, indicating potential sex differences in metabolism. (The high concentration of radiolabel in eythrocytes may be associated with the haematological effects seen in the studies of toxicity, although no work has been performed to investigate the mechanism of this effect.)

Table 1. Toxicokinetics of [acetimide-¹⁴C]thiodicarb in groups of five rats of each sex (means)

In phase 2, similar groups of rats received a single dose of the same preparation of [acetimide-¹⁴C]thiodicarb by gavage at 2 or 16 mg/kg bw. Samples of urine and faeces were taken 6, 12, 24, 48, 72, 96, 120, 144, and 168 h after dosing, and blood samples were taken at terminal sacrifice (168 h) with samples of 12 tissues and the carcass. Trapped volatile compounds were sampled at the same times as urine by use of a 2:1 mixture of methyl-cellosolve:ethanolamine to collect carbon dioxide and of butylcellosolve at –10 °C to trap acetonitrile. Clinical signs, including tremors, were seen in animals given 16 mg/kg bw. The total recovery of radiolabel was about 90%, which is considered acceptable for a compound that gives off a large amount of volatile components. The results (Table 2) showed that thiodicarb was absorbed rapidly, 18–50% of the administered dose being excreted within 12 h, mainly as exhaled volatile compounds. The elimination of carbon dioxide was considerably more rapid than that of acetonitrile. The ratio of exhaled carbon dioxide:acetonitrile varied with sex and dose, but the pattern was not consistent and no reliable conclusions can be drawn from this observation. The amount excreted in the urine of males was independent of dose, but in females urinary excretion was more rapid and extensive at 16 mg/kg bw than at 2 mg/kg bw. The radiolabel in tissues at 168 h represented 8% of the administered dose, and the proportion was independent of dose and sex. The highest concentrations of radiolabel were associated with erythrocytes; the major organs had lower concentrations than whole blood (Table 2).

Table 2 (continued)

In phase 3, groups of three rats of each sex received a single dose of [acetimide-¹⁴C]thiodicarb at 2.4 or 18 mg/kg bw. The animals were killed at the times of the plasma T_max/2, T_max (on the basis of the results of phase 1), 24, or 48 h. Samples of urine and faeces were taken immediately before sacrifice or 6, 12, 24, and 48 h after dosing from appropriate groups. Blood samples were taken at terminal sacrifice, with samples of 12 tissues and the carcass. Clinical signs, including tremors, were seen in animals receiving 18 mg/kg bw, and salivation was seen in two animals receiving 2.4 mg/kg bw. The results (Table 3) were generally consistent with the findings in phases 1 and 2, with the highest concentrations of radiolabel in erythrocytes and higher concentrations of residues in females than in males. The value for plasma T_max at 2.4 mg/kg bw was 1 h, as in phase 1, but at 18 mg/kg bw the T_max was 24 h in males and 2 h in females, compared with 4 h in phase 1. The concentrations in major organs were similar to those in whole blood and plasma, the kidney, liver, and spleen having the highest concentrations, which would be expected for a compound that is extensively metabolized, rapidly excreted in urine, and a fraction of which is associated with erythrocytes. Thiodicarb and its metabolites penetrated to only a limited extent into the brain (Hiles, 1987).

The purpose of this study was to verify the metabolic pathway observed in rats and to quantify the metabolites (especially acetamide) in a species more closely resembling humans. The absorption, distribution, metabolism, and excretion of [acetimide-¹⁴C]thiodicarb (radiochemical purity, > 97%; specific activity, 17.7 mCi/mmol per L) and its metabolites were studied in four male cynomolgus monkeys (Macaca fascicularis) aged 1–2.5 years. The animals were placed into individual metabolic chambers immediately after a single oral dose at a nominal rate of 5 mg/kg bw administered by gavage in 1% carboxymethyl cellulose at 4 ml/kg bw in combination with unlabelled thiodicarb (purity, 99.95%). Urine was collected 6 and 24 h after dosing and then every 24 h up to 168 h. Faeces, cage debris, and cage washings were collected daily up to 7 days (168 h). Expired gases were trapped in two methanol traps, two potassium hydroxide traps, and a cold (–70 °C) trap sequentially, and analysed 6, 24, and 48 h after dosing. Seven days after dosing, the monkeys were killed, and lungs, brain, gastrointestinal tract, liver, and samples of fat, muscle, and skin were collected. The radiolabelled compounds were analysed and quantified by a combination of TLC, HPLC, and LSC, with comparison to authentic standards. The presence of conjugates was investigated by incubation with glucuronidase and sulfatase preparations. The efficiency of extraction and capture was determined for each procedure and found to be > 97%.

Approximately 59% of the radiolabel was excreted during the first 24 h. Over the 7-day period, a mean of 31% of the administered dose was excreted in urine (including cage wash). Absorption appeared to be essentially complete, only 4.6% of the applied dose being eliminated in the faeces. During the first 48 h, a mean of 35% of the dose was eliminated as volatile components. At sacrifice 168 h after dosing, < 5% of the dose was found in the tissues (Table 4). Of the radiolabel retained in the tissues, most was in fat (1.3%; 0.5 µg equivalents/g), muscle (1.1%), and skin (1.1%; 0.5 µg equivalents/g). Liver (1.2 µg equivalents/g) had the highest concentration of radiolabel at 7 days, with 0.2 µg equivalents/g in whole blood and 0.15 µg equivalents/g in brain. The total radiolabel recovered in the four animals represented 74–81% of the administered dose. The apparently low recovery might be due in part to the fact that volatile compounds were collected for only 48 h (Hawkins et al., 1993).

Table 4 Excretion of radiolabel by cynomolgus monkeys after a single oral dose of [acetimide-¹⁴C]thiodicarb (5 mg/kg bw)

Table 4 (continued)

Table 4 (continued)

The metabolic fate of [acetimide-¹⁴C]thiodicarb (specific activity, 18 mCi/mmol; radiochemical purity, 98.4%) was studied in two lactating goats after 7 days of dosing at 5–6 mg/kg bw per day by capsule twice daily after milking. A third animal served as a control. Volatile compounds were collected for about 10 h on day 6 of the dosing regime. Faeces, urine, and milk were collected twice daily during treatment and for 7 days after completion of dosing. Blood, gut contents, and tissue samples were taken for analysis within 18 h of the last dose. Volatile compounds, milk, urine, faeces, tissues, blood, and gut contents were analysed for total residues.

One goat became ill during the study after an injury to the oral cavity considered to be due to the balling gun used to deliver the capsules. The results (Table 5) showed that the primary route of excretion was as volatile compounds (carbon dioxide and acetonitrile). About 50% of the administered dose was recovered from both goats,; the deficit was probably due to incomplete capture of volatile compounds in the indirect respiration chamber used. The concentration of total radiolabel was fairly constant from 3 days after dosing in faeces, urine, and milk. The peak concentrations in milk were 15 and 20 mg/kg. Radiolabel was extensively distributed to tissues, and the highest mean concentrations were found in liver (25 mg/kg), kidney (13 mg/kg), and blood (11 mg/kg) (Hanlon & Norris, 1993).

Table 5. Total recovery of radiolabel from goats given [acetimide-¹⁴C]thiodicarb for 7 days

Groups of two lactating Holstein cows were given diets containing [acetimide-¹⁴C]thiodicarb (radiochemical purity, 98.5%; specific activity, 29 mCi/mg) at a concentration of 0.1, 10, 30, or 100 ppm, equivalent to 0.004, 0.4, 1.4, and 4 mg/kg bw per day, for 21 days. Milk and urine were collected twice daily, and blood and faeces were collected at 3–4-day intervals. Volatile compounds were not collected. One cow at each dose was killed 12 h after the last treatment, and liver, kidney, lung, spleen, heart, brain, ovary, udder, tongue, foreleg muscle, hindleg muscle, neck muscle, and omental fat were collected for analysis. The remaining four cows (one at each dose) were kept for an additional 7 days after termination of treatment, and milk and urine were collected twice, blood and faeces were collected daily, and tissues were collected at termination. The concentration of radiolabel, determined by LSC, increased with increasing dose in all samples of tissue, milk, blood, urine, and faeces. The concentration in milk increased with duration of dosing, reaching a plateau after about 2 weeks at 0.1 and 10 ppm and after 1 week at 30 and 100 ppm. After 21 days of treatment, the concentrations of radiolabel in tissues were highest in liver and kidney (Table 6). Seven days after the last dose, the concentrations were lower in all tissues except liver and muscle. After treatment, the concentrations of radiolabel in milk declined, with an initial half-time of 3–4 days (Feung et al., 1980).

Table 6. Concentrations of radiolabel (10³ dpm/g) in tissues of lactating cows given [acetimide-¹⁴C]thiodicarb for 21 days

A lactating Holstein cow received a single dose of [acetimide-¹⁴C]thiodicarb in a capsule at a dose of 7 mg/kg bw. During 72 h after treatment, 66% of the administered radiolabel was eliminated in respired air, 11% in faeces, 5.0% in urine, and 4.6% in milk; 10% was retained in the tissues, the highest concentration being found in liver (9 µg equivalents/g). Blood samples taken up to 24 h showed higher concentrations of radiolabel in plasma than in erythrocytes, but the trend was reversed subsequently. The peak concentration of radiolabel in milk was 7.3 ppm 18 h after dosing. Expired radiolabel was found mainly in carbon dioxide and acetonitrile in a ratio of 8:1 (Khasawinah & College, 1978).

The metabolic fate of [acetimide-¹⁴C]thiodicarb (21.4 mCi/mmol per L) in groups of three laying hens was studied after 21 days of dosing by capsule with a concentration of 15, 29, or 100 ppm, equivalent to 1, 1.5, and 6 mg/kg bw per day. Faeces and eggs were analysed during treatment and for 7 days after the end of dosing. A range of tissues were taken from one hen in each group 6 h, 3 days, and 7 days after the last dose. Samples of tissues, faeces, eggs, and blood were analysed for total radiolabel by LSC after appropriate extraction and processing. In hens given the highest dose, the concentration of total radiolabel reached a plateau within 1 day in faeces (14 µg equivalents/g), 2 days in egg white (2 µg equivalents/g), and 10 days in egg yolk (14 µg equivalents/g) (Table 7). Significant concentrations were present in eggshell, indicating incorporation as carbonate. The detectable concentrations of radiolabel in eggs declined during the withdrawal period with a half-time of < 1 day. The concentration of total radiolabel in eggs and tissues was proportional to the dose administered. Six hours after the last dose, the highest concentrations of radiolabelled residues in tissues were in erythrocytes, liver, kidney, skin, and fat. The concentrations in the various muscles were similar. The detectable concentrations of radiolabel declined during the withdrawal period with a half-time of 3–4 days (Table 8; Andrawes & Bailey, 1980).

Table 8. Distribution of radiolabel in tissues of hens fed capsules containing [acetimide-¹⁴C]thiodicarb for 21 days, at 6 h and 3 and 7 days after the last dose (µg equivalent/g)

Samples obtained from phase 3 of the study of Hiles (1987; see above) were subsequently processed by solvent extraction, incubation with bovine peptidase or beta-glucuronidase and sulfatase from Helix pomata, and acid hydrolysis. The samples were then analysed by TLC with gas chromatography or HPLC, and the Rf values were compared with standards of a range of postulated thiodicarb metabolites. Most of the urinary radiolabel (86–102%) was extracted into an aqueous solvent, although trace amounts (< 7%) were extracted into dichloromethane, a pattern that was independent of dose and time of sampling. The main components of the extracts are shown in Table 9. Many were not characterized, as they did not co-chromatograph with standards and were volatile. The pattern of metabolites in organic extracts differed by sex. Incubation with the glucuronidase and sulfatase mix did not alter the pattern of metabolites, indicating that no conjugates were present in urine. The residues in liver and kidney were extracted mainly (58–78%) into an aqueous fraction, with traces (< 4%) in chloroform; 18–38% of the radiolabel was unextractable. Erythrocytes showed no residue in the chloroform layer and small amounts in the aqueous layer (6% for females and 14% for males); about 75% of the residue remained in the sample even after acid (6 M HCl) hydrolysis. None of the constituents of the liver, kidney, or erythrocyte residues was identified (Hiles, 1987).

In samples from the study of Hawkins et al. (1993), extensive metabolic degradation of thiodicarb was observed. During the 48-h period over which volatile compounds were collected, a mean of 28% of the dose was eliminated as carbon dioxide and 8.6% as acetonitrile. More than 18 metabolites were found in the urine, none of which accounted for > 5% of the dose. The metabolites identified corresponded to acetonitrile (1.2–2.9% of dose), acetic acid (0.4–0.9% of dose), and acetamide (0.8–1.0% of dose). No thiodicarb or its major degradation product methomyl was identified. Chromatography with reference standards also confirmed the absence of syn and anti forms of methomyl oxime, methomyl sulfoxide, and methomyl oxime sulfoxide in the urine. After treatment with Escherichia coli beta-glucuronidase, the propotion of radiolabel associated with the major polar component (M1, consisting of six individual compounds) decreased from 12% to 4.2% of the dose. The concentrations associated with eight components increased, including those corresponding to acetic acid (0.5–2.1% of the dose) and acetonitrile (3.2–3.8%), indicating some conjugation. Similar changes were not seen after incubation with glucuronidase and sulfatase from H. pomata. Faecal metabolites were not characterized because they contained too little radiolabel.

The major polar metabolites (M1 and M2) were detected in extracts of whole blood at 0.09–0.16 µg equivalents/g and in liver at 0.5–1.1 µg equivalents/g. In liver, a component corresponding to acetic acid (0.12–0.1 µg equivalents/g) was confirmed by both reverse-phase and ion-exchange HPLC; it was not found in blood. None of the radiolabelled components in whole blood or liver corresponded to thiodicarb, the syn or anti forms of methomyl, methomyl oxime, methomyl oxime sulfoxide, acetonitrile, or acetamide (Hawkins et al., 1993).

Samples from the study of Hanlon & Norris (1993) were investigated by a range of techniques to determine the metabolic profile of [acetimide-¹⁴C]thiodicarb in two goats. Urine samples were analysed directly by HPLC and TLC; milk samples were extracted with acetonitrile and hexane; the stomach contents and faeces were extracted with methanol; and selected tissues were extracted with water and then methanol. The resulting solids were treated with protease to liberate additional residues, and fractions were treated subsequently with various enzymes and/or base, as appropriate. Various extracts were analysed by a combination of HPLC and TLC. The gut contents of the two animals contained acetonitrile (15 and 24%), acetamide (5.8 and 7.1%), thiodicarb (6.3 and 5.5%), and methomyl (3.2 and 7.5%). Faeces were found to contain thiodicarb and methomyl as the major radiolabelled residues. In urine, only acetonitrile and acetamide were identified; no thiodicarb or methomyl was found. Between 70 and 91% of the radiolabelled residues was extractable from liver, kidney, and muscle with water. No thiodicarb, methomyl, or methomyl metabolites were found in any of these tissues. Acetonitrile and acetamide were detected in liver, kidney, and muscle; one of the radioactive components was a water-soluble polar compound which was transformed into acetic acid after alkaline hydrolysis (Table 10).

Table 10. Residues in edible tissues of goats as % total ¹⁴C-residues (TRR) and ppm thiodicarb equivalents

Most of the recoverable radiolabel was extracted from fat and milk samples with acetonitrile and hexane. The main constituents in fat, although there was considerable inter-animal variation, were saponifiable fatty acids and saponifiable lipids, indicating that radiolabel had been incorporated into normal metabolism. Milk (Table 11) contained low concentrations of radiolabel (< 5 ppm of any individual component) incorporated into natural products (lactose, lipids, and fatty acids) and a significant proportion of acetonitrile (Hanlon & Norris, 1993).

Table 11. Residues in milk of goats as % total ¹⁴C-residues (TRR) and ppm thiodicarb equivalents

Samples from the study of Feung et al. (1980; see above) were analysed for metabolites by TLC, infra-red, nuclear magnetic resonance, and gas chromatography after a range of extraction procedures. No parent compound or related carbamates or oximes was found in tissues, milk, or urine. Much of the residue in tissues (40–80%) was not readily extractable, and a significant proportion of the extractable residues could not be identified, although they had properties similar to those of lipids and proteins. The main compounds identified in tissues were acetamide and acetonitrile; the ratio varied with tissue, but was typically 1:1. The main compound identified in milk was acetonitrile, the concentration reaching a peak within 7 days in all groups and approaching 1 ppm at the highest dose. The concentration of acetonitrile remained nearly constant throughout the rest of the 21-day treatment period, then declined during the 7-day post-treatment phase. Acetamide was found in trace amounts (0.0l ppm) in the milk of cows at the highest feeding concentration. About 24% of the aqueous extractable urinary radiolabel was identified as acetamide, with a similar amount of acetonitrile (Feung et al., 1980). These results are generally consistent with those of Khasawinah & College (1978). Although the earlier workers reported significant concentrations of acetamide in the milk from a cow given a single dose equivalent to 327 ppm, the findings were not quantified.

Samples from the study of Andrawes & Bailey (1980; see above) were analysed for metabolites by TLC, gel permeation chromatography, and gas–liquid chromatography after a range of extraction procedures. The results for eggs produced during the withdrawal period were not presented. There was no evidence that thiodicarb or related carbamate or oxime compounds were present. Much of the radioactivity was unextractable or unidentifiable, as it was associated with lipids (Table 12). The main residue identified was acetonitrile, with lower concentrations of acetamide (Andrawes & Bailey, 1980).

Table 12. Radiolabelled compounds in liver, breast muscle, and eggs of hens given [acetimide-¹⁴C]-thiodicarb at an equivalent of 102 ppm for 21 days (g equivalent/g)

The results of the very extensive work on goats are consistent with the findings in rats, cynomolgus monkeys, cows, and hens. Orally administered thiodicarb is initially hydrolysed to methomyl, which is then hydrolysed to acetonitrile, acetamide, and acetic acid. Further reactions result in incorporation of the acetyl carbon of thiodicarb into normal metabolism (Figure 1).

Thiodicarb is a rapidly reversible inhibitor of acetylcholinesterase. In a study to determine the times to peak effect and recovery, groups of nine male and nine female Sprague-Dawley rats received a single dose of thiodicarb (purity, 95.1%) at 0, 5, 20, or 40 mg/kg bw in 0.5% methyl cellulose by gavage. Three animals of each sex per group were tested in a limited battery of observational tests for function (FOB) at 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, and 24 h. Blood was drawn from the tail vein for determination of cholinesterase activity from three animals of each sex before dosing and 0.5, 1, 2, 4, 6, and 8 h after dosing. No deaths or clinical signs of toxicity were seen other than in the FOB. Reduced body weight was seen between days 1 and 2 in males and females receiving 20 or 40 mg/kg bw. There was considerable variation in the findings of the FOB between animals and over time. Reductions in locomotor activity, degree of arousal, ataxic gait, tremors, lachrymation, and salivation were seen at 0.5 h, but recovery was essentially complete by 3 h. The time to maximal effect varied with the end-point but was generally within 0.5–1.5 h. Plasma and to a lesser extent erythrocyte cholinesterase activity was inhibited at 5, 20, and 40 mg/kg bw. The peak inhibition occurred before 2 h, followed by recovery and a second phase of reduced activity; this was also seen to a certain extent in controls (Table 13). The study was not designed to determine a NOAEL. It shows that blood samples for determination of cholinesterase activity in studies with thiodicarb should be taken within 2 h of the last dose or feed (Beyrouty, 2000a).

The acute toxicity of thiodicarb has been investigated in a large number of studies in which compounds of a range of purity and various vehicles were used. After oral administration, females were generally more sensitive than males, and the toxicity of thiodicarb given by this route was greater when an aqueous vehicle was used rather than corn oil. Thiodicarb was moderately toxic after oral administration and was classified as moderately hazardous by WHO. It was also moderately toxic after inhalation but showed little toxicity when applied dermally. The results are summarized in Table 14.

The deaths that occurred were usually within the first few hours after exposure. In a number of studies, the pattern of deaths was such that an LD₅₀ could not be determined for one or both sexes by standard procedures. The clinical signs of toxicity were consistent with inhibition of acetylcholinesterase activity. A common finding at gross post-mortem examination was haemorrhaged or red lungs.

Thiodicarb has been tested for dermal irritancy in rabbits in three studies on both intact and abraded sites. Thiodicarb was not significantly irritating, the maximum response being slight erythema after 24 h of exposure (Wentz & Wolfe, 1979; Mallory et al., 1982i; Glaza, 1996b).

The ocular irritancy of thiodicarb was examined in three studies in rabbits. Thiodicarb produced transient, moderate irritation in rinsed and unrinsed eyes (Cameron & Wolfe, 1979; Mallory et al., 1982j; Glaza, 1996c). A study in cynomolgus monkeys (Macaca fascicularis) resulted in slight conjunctival redness, which had regressed by day 4 (Weatherholtz et al., 1982).

The dermal sensitizing potential of thiodicarb was investigated in three studies in guinea-pigs given topical or intra-dermal applications. Thiodicarb had some skin sensitizing potential (Table 15).

A repeated-insult patch test for contact sensitivity was performed in 43 female and eight male volunteers aged 16 to > 60 years. Informed consent was obtained. Technical-grade thiodicarb (purity not specified) was diluted in distilled water to make an approximate 50% slurry. Induction involved nine exposures to 8 mg/24 h over 3 weeks. Reactions to the induction patches were scored 48 or 72 h after each application. After 2 weeks’ recovery, a challenge was applied on a naive site with 8 mg, and the scores were recorded 48 and 96 h later. No erythema or oedema was observed, indicating that technical-grade thiodicarb is not a sensitizing agent in humans when applied topically (Carabello et al., 1980).

Groups of 10 male and 10 female ICR-JCL (SPF) mice, 5 weeks of age, were fed diets containing thiodicarb (purity, 92.5%) at a concentration of 0, 30, 100, or 300 ppm, equal to 0, 4.3, 14, and 44 mg/kg bw per day for males and 0, 4.7, 16, and 46 mg/kg bw per day for females, for 4 weeks. Neither haematology nor clinical chemistry was performed. No deaths occurred during the study. Body weight, food consumption, clinical signs, and organ weights (brain, heart, liver, spleen, kidney, testes, and ovaries) were unaffected by treatment. The cholinesterase activity in plasma, erythrocytes, and brain was similar in all groups. Within the limits of the study protocol, the NOAEL was 300 ppm, equal to 44 mg/kg bw per day, the highest dose tested (Yoshida et al., 1983a).

Groups of 10 male and 10 female CD-1 mice received thiodicarb (purity, 96%) in the diet at a concentration of 0, 30, 1750, 3500, or 7000 ppm for 4 weeks. The animals were observed for deaths, clinical signs, body weight, and food consumption. Blood samples for analysis of plasma and erythrocyte cholinesterase activity were taken at week 4. Brain samples were removed at necropsy, rapidly frozen, and assayed for acetylcholinesterase activity. No haematological, clinical chemistry, or histological investigations were performed. The homogeneity and achieved concentrations were acceptable; the actual intakes are given in Table 16. Animals at the highest dose were often found to be thin and pale during the first half of the study, and the body-weight gain and food consumption of these animals were reduced. Increases in the absolute weights of the liver and spleen were seen at concentrations ³ 1750 ppm. Cholinesterase activities were similar in all groups, but the wide variation in control values for brain (2.5-fold) and erythrocytes (8-fold) hinders interpretation. Within the limits of the study protocol, the NOAEL was 30 ppm, equal to 6.2 mg/kg bw per day, on the basis of increased spleen weight at 1750 ppm, equal to 350 mg/kg bw per day (Atkinson et al., 1991).

Groups of 28 male and 28 female ICR-JCL (SPF) mice were fed diets containing thiodicarb (purity, 92.5%) at a concentration of 0, 30, 150, or 600 ppm, equal to 0, 3.8, 19, and 77 mg/kg bw for males and 0, 4.3, 22,and 85 mg/kg bw for females, for 13 weeks. Deaths, food consumption, body weight, water consumption, and urinary, haematological, and clinical chemical end-points were determined. Cholinesterase activity was determined in plasma, erythrocytes, and brain at the end of the study, as were organ weights and gross and histopathological changes. Brain acetylcholinesterase activity was depressed in females at 150 and 600 ppm and in males at 600 ppm. Females at the highest dose also had significantly increased relative liver weights. No other compound-related effects were reported. Data on individual animals and details of gross and histopathological examinations were not submitted. Within the limits of the study design and reporting, the NOAEL was 30 ppm, equal to 4.3 mg/kg bw per day, on the basis of inhibition of brain acetylcholinesterase activity at 150 ppm (Yoshida et al., 1983b).

Groups of five male and five female Harlan-Wistar rats were fed diets containing thiodicarb (purity unknown) at a dose of 0, 5, 25, or 100 mg/kg bw per day for 7 days. Slight decreases in body weight and food consumption were reported in males at the highest dose The relative kidney weights were increased in all treated males (Woodside et al., 1975).

Groups of five male and five female Wistar rats were given diets containing thiodicarb (purity, > 99%) at unspecified concentrations, providing doses of 0, 0, 18, 49, and 130 mg/kg bw per day for males and 0, 21, 50, and 130 mg/kg bw per day for females, for 7 days. Observations were made for deaths, food consumption, body weight, and gross changes in the liver and kidney. Body-weight gain was reduced in all treated groups in a dose-related fashion, and body-weight loss was seen during some or all of the dosing period in all groups except males at the low dose. Food consumption was reduced by about 10% in groups at the low dose and by about 50% in animals at the highest dose. Cholinesterase activity and histopatho-logical changes were not examined. No NOAEL could be identified owing to body-weight loss in females at 18 mg/kg bw per day, the lowest dose tested (Woodside et al., 1978).

In a range-finding study, groups of five male and five female Sprague-Dawley rats received thiodicarb (purity, 96%) in the diet at a concentration of 0, 100, 300, 900, or 1800 ppm for 2 weeks. The investigations were limited to deaths, clinical signs, body weight, food consumption, organ weights, gross findings at necropsy, and determinations of cholinesterase activity. Analyses of the diet showed it to be acceptable, and the intakes were 0, 15, 46, 120, and 210 mg/kg bw per day for males and 0, 16, 50, 140, and 210 mg/kg bw per day for females. Body-weight gain was reduced by 15–55% in a dose-related manner in animals at 900 and 1800 ppm. Food consumption paralleled body weight. Plasma cholinesterase activity was reduced by > 30% in males receiving concentrations ³ 300 ppm and in females at the highest concentration. Erythrocyte cholinesterase activity was similar in all groups. Brain acetylcholinesterase activity was reduced in females receiving 900 or 1800 ppm (Table 17); although this reduction was not statistically significant, a comparison of individual values indicates a clear effect in some animals. The weight of the spleen relative to body weight was increased markedly at the two higher concentrations and in males receiving 900 ppm. Within the limits of the investigations, the NOAEL was 300 ppm, equal to 46 mg/kg bw per day, on the basis of reduced body weight and inhibition of brain acetylcholinesterase activity at 900 ppm, equal to 120 mg/kg bw per day (Atkinson & Perry, 1991).

Groups of 10 male and 10 female Fischer 344 rats received thiodicarb (purity unknown) at various (unspecified) concentrations in the diet for 4 weeks to provide an intake of 0, 1, 3, 10, or 30 mg/kg bw per day for 29 days. Blood samples were taken for determination of erythrocyte and plasma cholinesterase activity on days 8, 15, and 29; brain cholinesterase activity was determined in samples taken at necropsy. The animals were not fasted before sampling, and processing for cholinesterase activity was rapid. There were no deaths or clinical signs. Body-weight gain was reduced significantly from the start of dosing in females receiving 10 mg/kg bw per day and in animals of each sex at 30 mg/kg bw per day, by 15% in males and 50% in females. Food consumption was reduced by about 10% in females receiving 10 or 30 mg/kg bw per day. Brain acetylcholinesterase activity was not inhibited. Plasma and erythrocyte cholinesterase activities were decreased only at the two higher doses, the greatest degree of inhibition being seen at day 8, when the findings were statistically significant (p < 0.05). The erythrocyte cholinesterase activity was 70% of the control value on day 8, 80% on day 15, and 95% on day 29 in males, and 75% of control values on day 8, 80% on day 15, and 90% on day 29 in females. The NOAEL was 3 mg/kg bw per day on the basis of reduced body-weight gain in females at 10 mg/kg bw per day (DePass et al., 1982).

In a preliminary study, groups of five male and five female Fischer 344 rats were given diets containing thiodicarb (purity, 99.8%) at unspecified concentrations to provide an intake of 0, 5, 15, or 45 mg/kg bw per day for 13 weeks. An additional group received 90 mg/kg bw per day for 80 days. The animals were observed for deaths, food and water consumption, organ weights, and erythrocyte, plasma, and brain cholinesterase activity. No haematology or histology was performed. Blood samples were taken for determination of cholinesterase activity by cardiac puncture 24 h after the last feed. At necropsy 24 h after the last feed, the brain was removed, weighed, and assayed for acetylcholinesterase activity. The relative weights of the liver, adrenal gland, and spleen were increased in all treated males, and that of the spleen was increased in females at the two higher doses. Body weights were reduced in a dose-related manner throughout the study in all treated males, but those of females were unaffected by treatment. The only effect on cholinesterase activity was increased activity in erythrocytes in males and females at the high dose, although it is likely that significant reactivation occurred between the last feed and sampling. The reporting and investigations in this study were limited, and no NOAEL could be identified, as effects on liver and spleen weights and body-weight gain were seen in males at 5 mg/kg bw per day, the lowest dose tested (Woodside et al., 1979a).

Groups of 10 male and 10 female Fischer 344 rats were given diets containing thiodicarb (purity, 99%) at unspecified concentrations to provide an intake of 0, 0, 1, 3, 10, or 30 mg/kg bw per day for 13 weeks. The animals were observed for deaths, clinical signs, food and water consumption, body weight, organ weights, haematological, clinical chemical, and urinary end-points, and plasma, erythrocyte, and brain cholinesterase activity, and underwent gross and microscopic examination. Samples for determination of cholinesterase activity were taken after 24 h on control diet, which may have resulted in a significant degree of reactivation. Animals at 1 or 3 mg/kg bw per day were not examined for haematological or clinical chemical end-points. Histological examination was limited to controls and animals at the highest dose. At both 10 and 30 mg/kg bw per day, body weight and water consumption were significantly decreased among females and slightly increased in males. At 30 mg/kg bw per day, the relative weight of the spleen was significantly increased in males, and the relative weight of the kidney was increased in females. Urine volume was increased in males and decreased in females, and the specific gravity of urine was decreased in females at this dose. Other clinical parameters were unaffected by treatment. Slightly (7%) decreased mean haemoglobin concentrations were seen in males at the high dose. Cholinesterase activities were comparable in all groups, although the control values showed wide variation. No remarkable gross or microscopic findings were made. Within the limits of the protocol, the NOAEL was 3 mg/kg bw per day (Homan et al., 1978a).

Groups of 10 male and 10 female 4-week-old Sprague-Dawley rats received diets containing thiodicarb (purity, 96%) at a concentration of 60, 250, or 1200 ppm for 13 weeks. The animals were observed routinely for clinical signs, body weight, and food consumption. During week 13, samples were taken for haematology, clinical chemistry, determination of cholinesterase activity, and urine analysis. All animals underwent gross necropsy; 12 organs were weighed, and a wide range of tissues from controls and animals at the highest dose, the liver, kidney, and lungs from animals at the intermediate and low doses, and the spleens of females at the intermediate and low doses were examined histologically. The homogeneity and achieved dietary intakes were acceptable; the actual intakes are listed in Table 18. There were no deaths or clinical signs of toxicity. Body-weight gain, food consumption, urine volume and pH, and cholinesterase activities were all reduced in animals at the highest dose. Erythrocyte parameters were affected variously in females at the highest dose (Table 18). The serum potassium concentration was reduced in a dose-related manner in females, and this finding is considered to be of potential biological significance, as electrolyte concentrations are normally tightly controlled. The relative weight of the spleen was statistically significantly increased in rats at the highest dose and in males at the low dose. The frequency of pigment deposition in the spleen was increased in animals at the highest dose. The NOAEL was 60 ppm, equal to 6 mg/kg bw per day, on the basis of the dose-related, statistically significant decrease in serum potassium concentration at the next highest dose in females. The increased spleen weight in males at 60 ppm was considered not to be biologically adverse as the effect was not related to dose and was seen in only one sex (Atkinson & Robb, 1992).

Groups of four male and four female 9-month-old beagles were fed diets containing thiodicarb (purity, 99.8%) at a concentration of 0, 400–500, 1300–1800, or 2500–3500 ppm for 13 weeks to provide targeted doses of 0, 15, 45, and 90 mg/kg bw per day. Owing to a high mortality rate at the highest dose, it was reduced to 76 mg/kg bw per day for females and 86 mg/kg bw per day for males. The animals were examined routinely for physical and behavioural changes, food and water consumption, changes in body weight, and haematological, (limited) clinical chemical, and urinary parameters. Organ weights and gross and histological changes (excluding nerves) were determined at necropsy. Plasma and erythrocyte cholinesterase activity was determined twice before treatment and on days 28, 63, 79, and 93 of the study. Brain acetylcholinesterase activity was determined at sacrifice. The time between last exposure and sampling for cholinesterase activity was not reported.

The homogeneity, stability, and achieved intakes from the diets were not reported. Two females at the high dose died after 26 and 36 doses, and dose-related signs including anorexia, vomiting, and loose stools were observed in animals at the intermediate and high doses. Cholinesterase activity was not markedly affected at any time during the study; a 10–15% reduction in brain acetylcholinesterase activity in all groups of treated males did not show a dose–response relationship and was within the range of variation in controls. Food consumption was decreased by 20% in females at the intermediate dose and 35% in those at the high dose. Decreased body weight was seen in females at these two doses and in males at the highest dose. Water consumption was increased in animals at the intermediate and high doses, with a doubling in males. Urine volume was increased in males at the intermediate and high doses and in females at the high dose. Decreases in the erythrocytes count, packed cell volume, and haemoglobin were seen in animals at the intermediate and high doses, while the reticulocyte counts and mean corpuscular volume were increased in animals at the high dose. These changes, with bone-marrow hyperplasia and congested spleens seen at the high dose, suggest macrocytic anaemia and compensatory haematopoiesis. The relative weight of the liver was increased by 15–20%, and serum alanine aminotransferase activity was elevated by fourfold in males and 10-fold in females at the high dose. Males at this dose had increased incidences of splenic congestion, hepatocellular vacuolation, and pigmented Kupffer cells. Two animals in each treated group had focal, diffuse inflammation of the liver, with no dose–response relationship, while the incidence in controls was zero. In the absence of other findings and the absence of a dose–response relationship, the hepatic lesions seen at the lowest dose were considered not to be adverse. The NOAEL was 15 mg/kg bw per day on the basis of clinical signs and alterations in erythrocyte parameters at higher doses (Homan et al., 1978b).

Groups of six male and six female beagles, 6–7 months old, were given diets containing thiodicarb (two batches; purity, 99.8% and > 95%) at variable concentrations (not specified) to provide targeted doses of 0, 5, 15, and 45 mg/kg bw per day, for 26 weeks. The animals were observed routinely for survival, clinical appearance, food consumption, ophthalmic end-points, body weight, haematological end-points (mean corpuscular volume not determined), and clinical chemical and urinary parameters. Organ weights and gross and microscopic lesions were determined at terminal sacrifice. Cholinesterase activity was determined twice before treatment and in samples from fasted and non-fasted animals during weeks 8, 17, and 26. Brain cholinesterase activity was determined at sacrifice.

The homogeneity and the achieved dietary levels were not reported. Thiodicarb did not significantly affect body weight, food consumption, urinary parameters, or the gross or microscopic appearance of tissues. Males, but not females, showed a dose-related increase in the combined incidence of soft stools, mucoid stools, and diarrhoea. The absolute and relative weights of the liver were increased in males at the highest dose, but no histopathological correlates were found. The treatment-related changes in haematological end-points included significantly decreased erythrocyte volume fraction, haemoglobin, and erythrocyte count in males at the high dose and in females at the two higher doses (Table 19). Dogs receiving the highest dose had elevated serum alanine aminotransferase and plasma and erythrocyte cholinesterase activity at week 26 and decreased concentrations of total protein and globulin, with a correspondingly increased albumin:globulin ratio. There were no notable differences in cholinesterase activity in samples from fasted and non-fasted animals The NOAEL was 5 mg/kg bw per day on the basis of altered erythrocyte parameters at 15 mg/kg bw per day and above (Wolfe, 1981).

Groups of six male and six female beagle dogs aged 29–33 weeks were given diets containing thiodicarb (purity, 91.6%) at a concentration of 0, 160, 490, or 1500 ppm, equal to 4.4, 13, and 38 mg/kg bw per day for males and 4.5, 14, and 40 mg/kg bw per day for females, for 1 year. The observations included deaths, body weight, appearance and behaviour, and food and compound consumption. Clinical chemical (before treatment and at weeks 13, 26, and 52) and ophthalmic end-points, gross findings at necropsy, organ weights, and detailed histological appearance were determined. Blood samples for measurement of erythrocyte and plasma cholinesterase activity were taken from non-fasted animals on three occasions before treatment and at weeks 5, 13, 26, and 52. Brain acetylcholinesterase activity was assayed in samples obtained at termination.

The homogeneity and achieved incorporation were acceptable. No effects were seen on mortality rate, body-weight gain, urinary parameters, or water consumption. No consistent clinical signs were seen, but tremors occurred in one male and one female receiving the highest dose. Food consumption was reduced by < 10% in males at the beginning of the study and occasionally in females at the highest dose. Slight decreases in erythrocyte count, erythrocyte volume fraction, and haemoglobin were seen in animals at the high dose at all three observation times (Table 20). Plasma, erythrocyte, and brain cholinesterase activity was significantly inhibited (> 25%) in animals at the high dose, and animals at the intermediate dose had significant reductions in erythrocyte cholinesterase activity at week 13. Supplementary investigations showed that the time to peak inhibition of erythrocyte cholinesterase activity was 2 h, indicating that sampling at week 52 (3.5 h after feeding) was less than optimal. No changes were seen that would suggest a compound-related effect on any other measured clinical parameter. The weights of the liver and spleen in treated females were increased in a dose-related manner when compared with controls, and increases were also seen in the organ:body, and organ:brain weight ratios in females, with statistical significance only at the high dose. Such changes were not seen in males. No effects were observed on ophthalmic end-points or gross or histological appearance. As thiodicarb is a carbamate, the Meeting assumed that the inhibition of cholinesterase activity would not worsen with duration of dosing and that the late sampling at week 52 therefore did not compromise the study significantly. The NOAEL was 160 ppm, equal to 4.4 mg/kg bw per day, on the basis of inhibition of erythrocyte cholinesterase activity at 490 ppm (Hamada et al., 1986).