The toxicity of chlorpropham was evaluated by the Joint Meeting in 1963 (Annex 1, reference 2) and 1965 (Annex 1, reference 3); an ADI could not be allocated at either Meeting. A full data package was submitted for consideration by the present Meeting.

In a range-finding study, groups of two male and two female Sprague-Dawley rats received a single oral dose of 5, 100, 300, or 500 mg/kg bw of [ring-¹⁴C]chlorpropham dissolved in corn oil. The radiolabel in urine (including cage rinse) and faeces was determined for 3 days after dosing, and expired [¹⁴C]carbon dioxide was collected for 48 h from the group given 5 mg/kg bw. A gross profile of metabolites was established by high-performance liquid chromatography (HPLC) and/or thin-layer chromatography (TLC) in 24-h urinary and faecal samples from animals of each sex in all groups (see section on biotransformation). Males and females excreted an average of 0.02% and 0.01% of the administered dose as [¹⁴C]carbon dioxide in the expired air, respectively, within the first 24 h after dosing; no [¹⁴C]carbon dioxide was expired during the next 24 h. The group given 5 mg/kg bw excreted > 90% of the administered radiolabel in urine and faeces within 24 h, while mice at the higher doses excreted approximately 80%. Urine was the main route of excretion at all doses (73–92% of the dose after 72 h). Animals at 300 and 500 mg/kg bw excreted more activity in the faeces within 72 h (12–15% for males; 13–21% for females) than those at the lower doses (9–13% for males; 5–8% for females) (Robinson & Liu, 1991).

In a study performed according to GLP and to FIFRA Guideline 40 CFR 158.340 reference 85-1 (essentially the same as OECD Guideline No. 417), four groups of five male and five female Sprague-Dawley rats received a single oral dose of 5 or 200 mg/kg bw of [ring- ¹⁴C]chlorpropham in corn oil, oral doses of 5 mg/kg bw per day of unlabelled chlorpropham in corn oil for 14 days followed by 5 mg/kg bw of [ring- ¹⁴C]chlorpropham in corn oil on day 15 or a single intravenous injection of 0.5 mg/kg bw of [ring- ¹⁴C]chlorpropham in 0.9% NaCl. A control group of two male and two female rats received a single oral dose of the vehicle. Urine (including cage rinse) and faeces were collected for 7 days after dosing with the labelled compound, and then the animals were killed. Radiolabel was determined in excreta, several tissues, blood, plasma, and carcass. Over the 7 days after dosing, 89–97% of the adminstered radiolabel was excreted in urine and 4–7% in faeces, mostly within the first 24 h. The excretion pattern was not affected by route of administration, sex, or the frequency or size of the dose. The percentages recovered are shown in Table 1. No radiolabel was detected in brain, pancreas, testis, bone, fat, uterus, or seminal vesicles in any of the treated groups 7 days after dosing. In the group treated with 0.5 mg/kg bw intravenously, no radiolabel was detected in any of the other tissues examined or in blood or plasma (limit of detection not reported). The amounts of radiolabel in blood, plasma, and other tissues in these groups are shown in Table 1 (Robinson & Liu, 1991).

Bobik et al. (1972) reported the results of two studies that were not performed according to GLP or any guideline. In the first, groups of six male Wistar rats weighing about 250 g each received an oral dose of 3.5 mg of [¹⁴C-ring]- or [¹⁴C-side-chain (isopropyl)]chlorpropham (purity, > 98.5%) as a suspension in methylcellulose mucilage or an intraperitoneal dose of 3.3 mg of [¹⁴C-ring]- or [¹⁴C-isopropyl]chlorpropham in 50% aqueous ethanol. Groups of three animals were given neomycin 24 h before the oral dose of labelled chlorpropham to examine the possibility that chlorpropham might be hydrolysed to isopropanol in the gut by neomycin-susceptible microflora. Excretion of radiolabel in urine, faeces, and expired carbon dioxide was determined (Table 1). In the second study, three male Wistar rats given a single intravenous dose of 1.0 mg of [¹⁴C-ring]- or [¹⁴C-isopropyl]chlorpropham (purity, > 98.5%) excreted 40 and 38%, respectively, of the administered radiolabel in bile within 6 h. The extensive (about 40%) biliary excretion seen in this study combined with the low faecal excretion in the first study indicated almost complete reabsorption of biliary metabolites.

Fang et al. (1974) reported the results of five studies of excretion and distribution that were not performed according to GLP or any guideline. In the first, groups of two to four adult female Wistar rats weighing an average of 270 g received a single oral dose of 0.94, 11, 21, or 51 mg of [¹⁴C-side-chain (isopropyl)]chlorpropham (purity, > 99%) or 0.91 or 21 mg of [¹⁴C-ring]-chlorpropham (purity, > 99%) in corn oil by oral gavage, providing doses ranging from 4 to 200 mg/kg bw. Excretion of radiolabel in urine, faeces, and carbon dioxide was determined for 72 h after dosing (see Table 2). No difference in excretion pattern was seen by dose.

Seven adult female Wistar rats weighing an average of 270 g received a single oral dose of 0.91 mg of [¹⁴C-ring]chlorpropham (purity, > 99%) in corn oil and were killed after 0.5, 1, 2, 4, 6, 12, and 24 h. Determination of radiolabel in dry tissues showed peaks 1–2 h after dosing, except in the intestine, where the concentration declined rapidly. The greatest activity was detected in kidney, intestine, liver, blood, and lungs. Significant amounts of radiolabel crossed the blood–brain barrier and accumulated in the brain. The half-lives were 11 h in brain, 8.9 h in fat, 8.1 h in liver, 6.2 h in kidney, 4.6 h in muscle, 4.3 h in intestine, 4.1 h in lung, 3.7 h in heart, 3.6 h in blood, 3.2 h in spleen, and 2.5 h in stomach.

Five pregnant rats (stage of gestation not reported; average weight, 340 g) received a single oral dose of 0.25 mg of [¹⁴C-ring]chlorpropham (purity, > 99%) and were killed 2, 4, 6, 12, and 24 h after dosing. Radiolabel was determined in tissues and fetuses. The distribution in tissues was similar to that in non-pregnant rats. The radiolabel was readily transferred to fetuses, where the amounts declined more slowly than in the kidneys, liver, lung, and blood of the dams.

Three nursing rats received, just after parturition, a single oral dose of 0.25 mg of [¹⁴C-ring]chlorpropham (purity, > 99%), and radiolabel was determined in the tissues of the pups up to 7 days after dosing. Activity was found in milk in the stomach on days 1–3 and in blood, liver, kidney, intestine, brain, and carcass on days 1–4. By day 7, no radiolabel was detected in any tissue. The radiolabel was evenly distributed throughout all tissues, in contrast to the situation in adult rats. Considerable differences were seen in tissues of pups from different litters.

Seven of the adult female Wistar rats that had received a single oral dose of 0.91 mg of [¹⁴C-ring]chlorpropham in corn oil and were killed after 0.5, 1, 2, 4, 6, 12, and 24 h, respectively, were used to study the intracellular distribution of ¹⁴C in liver and kidneys. In the liver, about 30 and 50% of the ¹⁴C present was in the nuclear and soluble fractions, respectively, 1 h after dosing. After 24 h, the amount of ¹⁴C in the soluble fraction had decreased to 7% and that in the nuclear fraction had increased to 63% of the ¹⁴C present. The proportions in the mitochondrial and microsomal fractions remained at approximately 10% of the total throughout the 24 h after dosing. In the kidneys, more ¹⁴C was found in the soluble fraction and less in the other fractions. The effect of elapsed time on the distribution of radiolabel between subcellular fractions was comparable to that in the liver.

In a range-finding study, groups of two male and two female rats received a single oral dose of 5, 100, 300, or 500 mg/kg bw of [¹⁴C-ring]chlorpropham in corn oil. The gross metabolic profile was determined by HPLC and/or TLC in 24-h urinary and faecal samples fom all animals. The distribution of metabolites in urine from animals given 100 mg/kg bw or more was different from that of rats given 5 mg/kg bw. At the lowest dose, chlorpropham represented a major peak in faeces from females but not in those from males, while no major peak was seen in either males or females at higher doses. Twelve metabolites were identified in urine and faeces from animals at 500 mg/kg bw, and two were identified partially (Robinson & Liu, 1991).

Four groups of five male and five female rats received a single oral dose of 5 or 200 mg/kg bw of [¹⁴C-ring]chlorpropham in corn oil or daily oral doses of unlabelled chlorpropham at 5 mg/kg bw in corn oil for 14 days followed by the same dose of [¹⁴C-ring]chlorpropham on day 15 or a single intravenous injection of 0.5 mg/kg bw of [¹⁴C-ring]chlorpropham in 0.9% NaCl. Urine and faeces collected over the first 24 h were analysed for metabolites. The study was performed according to GLP and to FIFRA Guideline 40 CFR 158.340 reference no. 85-1 (essentially the same as OECD Guideline No. 417). A total of 21 metabolites were detected, 13 of which accounted for 88–95% of the administered dose (see Table 3), mainly in the urine. No appreciable difference in the total profile of metabolites was seen according to dose or sex. In the urine, aryl-O-sulfate conjugates accounted for 58–70% of the administered dose. The main metabolites were M2-O-sulfate (mean percentage of administered dose, 39%), M9-O-sulfate (16%), and M2 (14%). Most of the radiolabel in faeces was in the form of free metabolites. Unchanged chlorpropham was detected in some faecal samples but not in urine. Three major metabolic pathways were proposed (see Figure 1), involving aromatic hydroxylation, oxidation of the isopropyl side-chain, and carbamate hydrolysis followed by rearrangement to 3-chloroaniline.

Bobik et al. (1972) reported that M2 was the main metabolite excreted in the urine of adult male rats given [¹⁴C-isopropyl]chlorpropham (purity, > 98.5%) orally or intraperitoneally, accounting for two-thirds of the radiolabel in urine. They also reported that 4’-hydroxychlorpropham accounted for 28% of the radiolabel excreted in 6-h bile after a single intravenous dose of 1.0 mg of [¹⁴C-ring]- or [¹⁴C-isopropyl]chlorpropham. Neither study was performed according to GLP.

Fang et al. (1974) determined metabolites in urine from adult female Wistar rats given [¹⁴C-ring]- or [¹⁴C-isopropyl]chlorpropham orally or intraperitoneally (see above). Hydroxylation of the ring was found to be the main reaction in metabolic conversion, and the resulting M2 was conjugated with sulfate. Hydroxylation of the isopropyl moiety yielded both mono- and dihydroxy derivatives of the alkyl chain, which probably underwent further oxidation. Hydrolytic cleavage yielded the corresponding amine, carbon dioxide, and 2-propanol. The relative amounts of the metabolites formed were not affected by dose. The study was not performed according to GLP.

In a study carried out according to GLP and FIFRA Guideline 40 CFR 158.240, reference 171-4, two lactating goats weighing 39–48 kg were given a daily oral dose of 75 mg of [¹⁴C-ring]chlorpropham plus lactose by capsule for 7 days, providing 1.6–1.9 mg/kg bw per day. A control goat received lactose only by capsule. Urine and faeces were collected daily and milk twice daily during treatment and before necropsy, and blood samples were taken on days 1, 3, and 5 of treatnment and before necropsy. The animals were killed 24 h after the last dose, and liver, kidney, heart, loin muscle, leg muscle, and omental and peripheral fat were collected for analysis. The mean amounts of radiolabel excreted in urine, faeces, and milk over 7 days, up to 24 h after the last dose, represented 99, 6.0, and 1.2% of the cumulative dose, respectively; about 1% was transferred to milk and liver and one or two orders of magnituse less to fat and muscle. The amount of radiolabel in milk, expressed as milligrams of chlorpropham equivalents per kilogram, was fairly constant throughout treatment, with maximum concentrations of 0.32–0.44 mg/kg of milk in the two goats. The concentration of radiolabel in tissues was highest in liver (0.18–0.32 mg/kg), followed by kidney (0.051 mg/kg); that in heart, muscle, and fat was below the limit of detection (0.030 mg/kg). The mean concentration in blood was below the limit of detection (0.030 mg/kg) on day 1, < 0.030–0.046 mg/kg on day 3, 0.059 mg/kg on day 5, and 0.092 mg/kg on day 7. In milk, the metabolites were M2 O-sulfate (81% of total residue), M4 aryl sulfate (5.0%), M9 O-sulfate (4.5%), M2 O-glucuronide (3.7%), and M2 (0.89%). In liver, the metabolites were M2 (3.2%), 3-chloroacetanilide (M7; 2.0%), and M4 (1.0%). In kidney, the metabolites were M2 O-sulfate (16%), M4 O-sulfate (4.1%), M9 (3.8%), M2 O-glucuronide (3.5%), and an unknown metabolite, which was also detected in rat urine (3.7%). In fat, mainly chlorpropham (88%) was detected. Metabolites in excreta and blood were not reported.

These results indicate that chlorpropham is readily metabolized, initially by hydrolysis and hydroxylation (oxidation), and is then converted to conjugated metabolites. A small amount of both the parent compound and 3-chloroaniline was tightly and/or irreversibly bound to endogenous macromolecules (4% and 8% of radiolabel in liver and kidney, repectively). The metabolic scheme is shown in Figure 2 (Wu, 1991a).

In a study carried out according to GLP and FIFRA Guideline 40 CFR 158.240, reference 171-4, 10 white Leghorn laying hens weighing 1.4–1.8 kg were given a daily oral dose of 6 mg of [¹⁴C-ring]chlorpropham plus lactose by capsule for 7 days, representing 3.3–4.2 mg/kg bw. Five control hens received lactose only by capsule. During treatment, excreta were collected once daily and eggs twice daily. All hens were killed 8 h after the last dose, and blood, breast and thigh muscle, fat, heart, gizzard, kidney, liver, skin, eggs (white and yolk), and blood were collected for analysis. During treatment and 8 h after the last dose, a total of 83% of the cumulative dose was recovered from excreta and 0.03% from total egg production, with 0.01% in egg white and 0.02% in yolk. No radiolabel was detected in egg yolk during the first 3 days, but the concentration increased steadily from 0.10 mg/kg on day 4 to 0.23 mg/kg on day 7, when a steady state had not yet been reached. In egg white, the concentration ranged from 0.007 to 0.074 mg/kg, and a plateau was reached on day 6. The concentrations in liver and kidney, were 0.47 and 0.46 mg/kg, and those in skin and fat were 0.15 and 0.19 mg/kg, respectively. Low concentrations were found in gizzard, heart, and blood (0.089, 0.039, and 0.094 mg/kg, respectively), while those in thigh and breast muscle were 0.015 and 0.006 mg/kg, respectively.

The main metabolites (expressed as per cent of total residue) were M6 O-sulfate (22%) and M2 O-sulfate (7.7%) in egg white; M6 O-sulfate (32%) and chlorpropham (20%) in egg yolk; M2 O-sulfate (4.3%), M2 (3.7%), chlorpropham (0.51%), and enzyme- or acid-releasable 3-chloro-4-hydroxyaniline-related metabolites (64%) in liver; M2 O-glucuronide (9.2%), M9 O-glucuronide (8.1%), chlorpropham (7.4%), M4 (5.0%), M9 O-sulfate (3.7%), M6 (3.4%), M6 O-sulfate (3.4%), M3 (3.0%), M9 (0.40%), and enzyme- or acid-releasable 3-chloro-4-hydroxyaniline-related metabolites (25%) in kidney; chlorpropham (92%) in fat; and chlorpropham (68%) and M2 O-sulfate (19%) in skin.

These results indicate that chlorpropham is readily metabolized, initially by hydrolysis and hydroxylation (oxidation), and then converted to conjugated metabolites (Wu, 1991b).

Bobik et al. (1972) reported that studies of the hydrolysis of chlorpropham by supernatants (10 000 x g) of liver and kidney homogenates from untreated and phenobarbital-pretreated rats showed that the liver was a possible site of this reaction. Phenobarbital pretreatment had no effect.

Additional data on metabolism in rat hepatocytes are cited from a review, as the original publication was not submitted. After incubation of rat hepatocytes with chlorpropham, 33% of the dose remained unchanged. 4'-Hydroxychlorpropham sulfate conjugate and 4’-hydroxychlorpro-pham glucuronide conjugate accounted for 35 and 20% of the dose, respectively; 3% of the dose was present as free 4’-hydroxychlorpropham. About 10% of the dose was split hydrolytically, yielding 2% 3-chloroaniline, which was further metabolized to 3-chloroacetanilide, representing 5% of the dose (Ministry of Agriculture, Fisheries and Food, 1993).

These studies are summarized in Table 4.

After oral administration to rats, the mortality rate was 10%, 60%, 60%, and 90% at 3000, 4000, 5000, and 6000 mg/kg bw, respectively, and death occurred 6–72 h after dosing. The signs of toxicity were hypoactivity, lethargy, ataxia, and salivation (Krohmer, 1990a). In a limit test in rats, no deaths occurred. Lethargy and uncoordinated movements were seen in all animals and hunched posture and piloerection in one female on day 1, but the signs had disappeared by day 2. No effect on body-weight gain was seen, and no macroscopic changes were observed (Pels Rijcken, 1996a).

No deaths or signs of systemic or local respiratory toxicity were observed after exposure of rats by inhalation. The mean body-weight gain was unchanged during the first week of observation but increased during the second week. No macroscopic changes were observed (Debets, 1985).

In a study of dermal application, no deaths occurred. Red staining of the neck was seen in one female on day 9 (Pels Rijcken, 1996b). Very slight, transient erythema was observed in three of five males and three of five females 30 min after the end of a 24-h dermal application of chlorpropham to rats (Krohmer, 1990b). In a study of dermal irritation (Krohmer, 1990c), one female rabbit showed very slight erythema (score 1 on the Draize scale) 24 h after removal of the patch, but the reaction had disappeared after 48 h. No oedema was seen. In another study (Pels Rijcken, 1996c), very slight skin erythema was seen in all three rabbits after 1 h, accompanied by very slight oedema in one animal. After 24, 48, and 72 h, very slight erythema was seen in three, two, and none of the animals, respectively; no oedema was observed.

In a study of ocular irritation (Krohmer, 1990d), effects were noted (score 1) on corneal opacity and area at 24 and 48 h in one rabbit, and conjunctival redness and discharge were seen in the same animal after 24 h. No effects were seen at 72 h. In another study (Pels Rijcken, 1996d), grade 1 irritation of the conjunctivae, consisting of redness, chemosis, and/or discharge, was seen 1 h after instillation. The effects had disappeared within 24 h in two animals and within 72 h in one animal.

Chloropham had no effect in a Buehler test (Krohmer, 1990e), an open epicutaneous test (Dickhaus & Heisler, 1989), or a Magnusson Kligman test for skin sensitization (Ministry of Agriculture, Fisheries and Food, 1993). In a split adjuvant test, challenge on day 21 elicited positive reactions in three of 20 guinea-pigs, and a weak reaction, extending beyond the exposed skin area, was seen in another four animals. Challenge on day 28 resulted in positive reactions in six animals (Weterings, 1985).

In a 28-day study, performed according to GLP and OECD Guideline 407, groups of five male and five female Wistar rats (aged about 6 weeks) received diets containing technical-grade chlorpropham (purity, 98.6%) at a concentration of 0, 600, 3000, or 15 000 mg/kg, equal to 0, 63, 350, and 2200 mg/kg bw per day for males and 0, 67, 360, and 2400 mg/kg bw per day for females. The values for intake of the test substance in mg/kg bw per day should be interpreted with care, however, as food scatter resulted in overestimates. After correction for food scatter, the intake during week 2 was 0, ± 60, ± 300, and ± 1400 mg/kg bw per day. The weight of the thyroid was not determined.

Animals at the highest dose showed decreased food consumption and significantly decreased growth. Haematological determinations showed significant decreases in red blood cell count, haemoglobin concentration, and erythrocyte volume fraction at all doses. The anaemia resulted in increased haemosiderosis in tissues at the two higher doses. New (immature) red blood cells were formed, as indicated by increased numbers of reticulocytes at all doses. Significantly increased cholesterol, serum bilirubin, and albumin concentrations were observed at the highest dose. The weights of the liver (at 15 000 mg/kg of diet) and spleen (at 3000 and 15 000 mg/kg of diet) were significantly increased. The leukocytes were small at all doses and were extremely small at the highest dose. Macroscopic examination showed enlarged and/or black, discoloured spleens in all animals at the two higher doses. Two males and three females at the highest dose had red–brown or black–brown discoloured kidneys. Microscopic examination of the spleen revealed increased haematopoiesis and haemosiderosis, slight to moderate congestion, and follicular atrophy in all animals at the two higher doses. The livers of all animals at the highest dose showed multiple foci of erythropoiesis. In the kidneys of all animals at this dose, intracytoplasmic hyaline resorption bodies were seen in the tubules, the effect being more pronounced in males than in females. A NOAEL could not be identified in this study. The lowest dose of 600 mg/kg of diet (equivalent to 60 mg/kg bw per day) was a LOAEL. The author claimed that the NOAEL was 600 mg/kg of diet, as the observed anaemia was only minimal at this dose and no concurrent changes were observed in spleen or liver (see Table 5) (Schoenmakers et al., 1998a).

In a 14-day range-finding study in dogs performed according to OECD Guideline 409, groups of two male and two female beagles aged about 7 months received a gelatin capsule containing technical-grade chlorpropham (purity, 98.6%; ground to powder) at a dose of 0, 25, 125, or 625 mg/kg bw daily 1 h after feeding in the morning, for 14 days. The dogs were offered 0.3 kg of standard maintenance pelleted food once daily and water ad libitum. No deaths occurred. One male and one female at 625 mg/kg bw per day vomited after dosing and showed retching, nausea, excessive salivation, and shaking of the head. At this dose, orange staining of the faeces was seen in all animals from day 9 onwards. No effect on body weight or body-weight gain was seen, and food consumption was normal in all groups. Evaluation of 12 haematological parameters before treatment and after 14 days showed decreased red blood cell count, haemoglobin concentration, erythrocyte volume fraction, and mean corpuscular volume in one male (the only one of the two male dogs analysed) and both female dogs at the highest dose. At this dose, the red cell distribution width was increased in one female and the platelet count in both females. A high leukocyte count in one female at this dose was considered to be incidental. Evaluation of 22 clinical chemical parameters before treatment and after 14 days showed increased bilirubin, cholesterol, phospholipid, and triglyceride concentrations in all animals at the highest dose, whereas increases in cholesterol, phospholipid, and triglyceride concentrations at 125 and 25 mg/kg bw per day were marginal and were considered to be of no biological relevance. At 625 mg/kg bw per day, one male and both females had decreased glucose concentrations. High lactate dehydrogenase activity seen in one male at this dose may have been incidental. The absolute and relative weights of the liver, spleen, and thyroid were increased in males and females at the highest dose, whereas the increases in thyroid weights at 125 and 25 mg/kg bw per day were slight and appeared to be of no biological relevance. Macroscopic examination showed enlarged, black discoloured spleens in one male and both females at the highest dose. Yellowish contents of the small intestines was seen in both females at 625 mg/kg bw per day and one female at 125 mg/kg bw per day. Microscopy of the thyroid gland showed follicular-cell hyperplasia in males and females at the highest dose. The spleens of males and females at this dose showed moderate congestion. The NOAEL was 125 mg/kg bw per day on the basis of changes in haematological parameters, organ weights, and histopathological appearance at the next dose (Schoenmakers & Nesselrooy, 1998). The Meeting noted that reticulocytes in blood were not counted and that OECD Guideline 409 requires groups of four male and four female animals. As only two animals of each sex were used, the effects observed could not be analysed statistically, and only the biological significance of the effects could be evaluated.

In a 28-day range-finding study performed according to GLP and FIFRA Guideline 40 CFR 158.340, reference 83-1, groups of one male and one female beagle dogs (aged about 7 months) received diets containing chlorpropham (purity, 97.1%) at concentrations providing doses of 0, 5, 50, and 500 mg/kg bw per day for 28 days. The test substance was melted at 45–47 °C and used as a liquid, or was allowed to recrystallize, ground to a fine powder, and used as a solid. It was dissolved in corn oil and mixed into the diet. The control diet contained the same amount of corn oil. Fresh diets were prepared weekly and were analysed for chlorpropham. Physical examinations, including ophthalmoscopy, before treatment and before termination showed no abnormalities. No treatment-related deaths or adverse clinical signs of systemic toxicity were seen. At the highest dose, food consumption was negligible during week 1, probably because of unpalatability, was normal during week 2, and decreased progressively during weeks 3 and 4. As a consequence, the intake of the compound at the highest dose was reduced considerably. Marked body-weight loss was seen among animals at the highest dose during week 1 and also after 4 weeks of treatment. Examination of 13 haematological parameters and 19 clinical chemical parameters in all dogs before treatment and during week 4 showed decreased haemoglobin concentration and erythrocyte volume fraction, decreased numbers of platelets, and increased mean corpuscular haemoglobin and haemoglobin concentration in animals at the highest dose and a dose-related increase in serum cholesterol concentration at all doses. Analysis of 13 parameters in urine from animals before treatment and before necropsy revealed no changes. Determination of absolute organ weights and organ:body weight ratios for brain, spleen, liver, kidney, testis with epididymides, and ovaries showed decreased absolute and relative weights of the spleen in animals at 50 and 500 mg/kg bw per day. Macroscopic examination showed no treatment-related changes. The liver, kidneys, spleen, and thyroids and all macroscopically abnormal tissues were examined histologically. The spleen showed slightly increased atrophy in animals at 500 mg/kg bw per day, and the thyroid glands showed moderate and marked activity in animals at 50 and 500 mg/kg bw per day, respectively, and slight activity at 5 mg/kg bw per day and in the control group. The NOAEL was 5 mg/kg bw, on the basis of decreased spleen weight and increased activity in the thyroid gland at 50 mg/kg bw per day (Wedig, 1990a). The Meeting noted that the thyroids were not weighed and the adrenal glands of the control group and animals at the highest dose were not weighed and the testes, adrenals, and heart were not examined histologically.

In a study in mice performed according to GLP and FIFRA Guideline 40 CFR 158.135, reference 82-1 (essentially the same as OECD Guideline No. 408, except for a lack of clinical chemical investigations), five groups of 15 male and 15 female CD-1 mice aged 6 weeks received diets containing chlorpropham (purity, 96.2% during weeks 1–3 and 97.1% during weeks 4–13) at concentrations providing a dose of 0, 100, 210, 420, or 840 mg/kg bw per day for 90 days. The test substance was used as a solid, dissolved in corn oil, and mixed into the diet. The control diet contained the same amount of corn oil. Fresh diets were prepared weekly and were analysed for chlorpropham in weeks 1, 2, 3, 4, 8, and 12.

One female at 100 and one at 840 mg/kg bw per day died from causes unrelated to treatment. All males at the highest dose had bluish extremities and darkening of the eyes; at the two higher doses, an increased number of animals with darkened blood was seen. Haematological examination showed significantly increased mean corpuscular haemoglobin and haemoglobin concentration and a significantly increased number of reticulocytes. Females at 420 mg/kg bw per day also had an increased number of reticulocytes, but the increase was not significant. Microscopic examination showed increased severity of haematopoiesis, quantitatively increased haemosiderosis in the spleen, and increased severity of bone-marrow cellularity at the highest dose. A quantitiative increase in haematopoiesis was also seen in the liver at this dose. The NOAEL was 420 mg/kg bw per day on the basis of the number of animals with dark blood and the increased number of reticulocytes in females at 420 mg/kg bw per day. The results are shown in Table 6. The darker blood and bluish extremities seen at the highest dose suggested methaemoglobinaemia, which can be caused by metabolites of chlorpropham such as chloroaniline and chloroacetanilide; however, neither methaemoglobin nor Heinz bodies were investigated in blood (Krohmer, 1990f).

Table 6 (continued)

In a study in mice performed according to GLP and to a protocol approved by the sponsor entitled ‘Protocol for a preliminary carcinogenicity study with chlorpropham techn. in mice’ (TNO protocol no. 470842/1926,1927), five groups of 10 male and 10 female CD-1 mice (mean body weight, 28 g for males and 23 g for females) received diets containing technical-grade chlorpropham (purity, 98.6%) at a concentration of 0, 0.1, 0.3, or 1.0% daily for 13 weeks, equal to mean daily intakes of 0, 190, 560, and 2100 mg/kg bw per day for males and 0, 290, 930, and 2800 mg/kg bw per day for females. The test substance was melted at 40–45 °C just before being mixed into the diet. After mixing, the diet was pelleted with skimmed milk powder and tap-water. Analyses for the content, homogeneity, and stability of the substance in the diet showed 10% lower concentrations than intended at all doses owing to the dilution with 10% water before pelleting. The assumption that most of the water would evaporate during or after pelleting turned out to be incorrect. In addition, the figures given for mean intakes of the substance were overestimates, especially for females, because of spillage. All animals were observed daily for clinical signs, general health, and deaths. Body weights and food consumption were determined weekly. After 13 weeks, haematological parameters, organ weights, and organ:body weight ratios were determined for all animals (see Table 7), and all animals were examined macroscopically. No histopathological examination was performed, but samples of adrenals, brainstem, cerebrum, cerebellum, heart, kidneys, liver, lungs (with trachea and bronchi), spleen, testis, thymus, and all gross lesions were preserved for possible future histopathological examination.

Table 7 (continued)

Males at the highest dose showed pale skin, and females at this dose had slightly lower body weights than control females after 13 weeks. A significant, dose-related increase in methaemoglobin concentration (by 1.6, 2.3, 4.5, and 8.4% in males and 2.2, 2.7, 3.6, and 6.4% in females at the four concentrations, respectively) and in the number of Heinz bodies (by 0.0, 7.4, 92, and 320/100 in males and 0.0, 2.6, 6.8, and 150/1000 in females at the four concentrations respectively) was seen at all doses. The weights of the spleen and liver were increased significantly at 1.0% in the diet. A NOAEL could not be established in this study. The LOAEL was 0.1%, equal to 190 mg/kg bw per day (Jonker, 1998).

In a study in rats performed according to GLP and FIFRA Guideline 40 CFR 158.135, reference 82-1 (essentially the same as OECD Guideline No. 408), five groups of 10 male and 10 female Sprague-Dawley rats (body weight, 180–226 g for males and 138–174 g for females) received diets containing chlorpropham (purity, 96.2% in weeks 1–4 and 97.1% in weeks 5–13) to provide a dose of 0, 17, 70, 300, or 1200 mg/kg bw per day for 90 days. The substance was melted and used as a liquid or allowed to recrystallize, ground to a fine powder, and used as a solid. It was dissolved in corn oil and mixed into the diet. The control diet contained the same amount of corn oil. Diets were prepared weekly and were analysed for chlorpropham at weeks 1, 2, 3, 4, 8, and 12. In addition to the guidelines, physical examinations were performed weekly, serum thyroxine and serum cholinesterase were determined, urine was examined before termination, and red blood cell morphology was examined at termination. The stability of chlorpropham in the diet was not determined, but data on stability in the diet were provided in the 24-month study in rats (see below).

Significantly decreased body-weight gain was observed in animals at the highest dose. Effects on red blood cell morphology were seen at all doses, although the method used to measure changes is sensitive to artefacts. Biochemical analysis revealed treatment-related increases in cholesterol concentrations at the two higher doses. Decreases in cholinesterase activity seen in females at 70, 300, and 1200 mg/kg bw per day, which were significant only at the highest dose, were considered to be not toxicologically relevant as they were of small magnitude (see Table 8). Increased spleen weights, darkened, red or black spleens, and increased liver weights were observed among animals at the two higher doses. Microscopy showed increased haematopoiesis and pigmentation in the liver, increased haematopoiesis, haemosiderosis, and congestion in the spleen, and increased cellularity in the bone marrow at doses from 300 mg/kg bw per day and more. No effects were seen on serum thyroxine or thyroid histology. The changes in red blood cell morphology at 17 mg/kg bw per day were considered to be due to normal variations in the method, and a significant decrease in mean corpuscular haemoglobin concentration only in males at this dose, without changes in other parameters. was considered to be not biologically relevant. The NOAEL was 17 mg/kg bw per day (Wedig, 1990b). Although metabolites of chlorpropham such as chloroaniline and chloroacetanilide may induce methaemoglobinaemia, the presence of neither methaemoglobin nor Heinz bodies in blood was investigated.

Table 8 (continued)

In a study in rats performed according to GLP and OECD guideline 408, four groups of 10 male and 10 female HanIbm: Wistar rats (approximately 6 weeks of age) received diets containing chlorpropham (purity, 98.6%) at a concentration of 0, 120, 600, or 3000 mg/kg of diet daily for 90 days, equal to 0, 10, 47, and 220 mg/kg bw per day for males and 0, 11, 54, and 230 mg/kg bw per day for females. The test substance was ground to a fine powder in an electric grinder and then mixed with powdered feed. Water (approximately 11%) was added to aid pelleting. The pellets were dried for approximately 24 h at 35 ºC before storage. Control animals received similarly prepared pellets but without the test substance. Fresh diets were prepared weekly and were analysed in weeks 1, 6, and 12 for stability, homogeneity, and accuracy. In addition to the guidelines, functional observations (hearing ability, pupillary reflex, static righting reflex, hanging wire test, and activity test) were made during weeks 12–13 of treatment, and the formation of methaemoglobin and Heinz bodies was investigated. Greater food scatter was observed at the highest dose, probably because of unpalatibility; the intake values presented above are corrected for food scatter. Thyroid weights were not determined.

No changes were found in the tests for function. Significant decreases in red blood cell count, haemoglobin concentration, erythrocyte volume fraction, and mean corpuscular haemoglobin concentration and significant increases in mean corpuscular volume and mean corpuscular haemoglobin were seen in animals at the highest dose. Red blood cell counts were also significantly decreased at 600 mg/kg of diet. Significant, dose-related increases in methaemoglobin (by 0.0, 0.2, 0.8 and 3.6% in males and 0.1, 0.4, 1.4, and 5.3% in females at the four concentrations, respectively) and in the leukocyte count were seen in both males and females. At all doses, the reticulocyte counts were significantly increased, but with no dose–response relationship, the reported values at 600 and 120 mg/kg of diet being close to or within control values for this rat strain. Significant decreases in haemoglobin concentration and erythrocyte volume fraction in females at 120 mg/kg of diet, in the absence of a significant decrease at the next higher dose, were considered to be incidental. Increases in platelet counts, seen in males at the two higher doses and in females at all doses, with no dose–response relationship or statistical significance (except in males at 600 mg/kg of diet), were considered to be not biologically relevant. Significant changes in prothrombin time and partial thromboplastin time, with no dose–response relationship, were considered to be incidental. Clinical chemistry showed a significant increase in bilirubin concentration in animals at the highest dose. Dose–response relationships were not seen for other significant changes in clinical chemical parameters (total protein, albumin, sodium), or the changes were within historical control values (urea) and therefore considered not to be biologically relevant. The significant decrease in aspartate aminotransferase activity was also considered to be biologically irrelevant because an increase would be expected in the presence of toxic effects on organs. The absolute weight of the spleen and that in relation to body weight were increased at the two higher doses. The increases were slight at 600 mg/kg of diet but were statistically significant at the highest dose. An enlarged and/or red–brown, discoloured spleen was seen in three males and nine females at the highest dose. As a similar effect was seen in two females at the lowest dose but not at 600 mg/kg of diet, the effect was considered biologically irrelevant. Histopathological examination of the spleen revealed increased haematopoiesis, slight congestion, and follicular atrophy in animals at the highest dose. The NOAEL was 120 mg/kg of diet, equal to 10 mg/kg bw per day, on the basis of the significant decrease in erythrocyte count and the significant, dose-related increase in methaemoglobin at 600 mg/kg of diet. The authors considered the NOAEL to be 600 mg/kg of diet, equal to 47 mg/kg bw per day, as the anaemia observed at this dose was of only minimal severity, and no morphological changes were observed in the spleen (see Table 9) (Schoenmakers et al., 1998b). The Meeting noted that it would have been more appropriate to determine methaemoglobin earlier in the study. Methaemoglobin formation is an acute effect, and adaptation might have occurred when it was determined in this study (at the end of treatment).

Table 9 (continued)