PESTICIDE RESIDUES IN FOOD - 1997 Sponsored jointly by FAO and WHO with the support of the International Programme on Chemical Safety (IPCS) TOXICOLOGICAL AND ENVIRONMENTAL EVALUATIONS 1994 Joint meeting of the FAO Panel of Experts on Pesticide Residues in Food and the Environment and the WHO Core Assessment Group Lyon 22 September - 1 October 1997 The summaries and evaluations contained in this book are, in most cases, based on unpublished proprietary data submitted for the purpose of the JMPR assessment. A registration authority should not grant a registration on the basis of an evaluation unless it has first received authorization for such use from the owner who submitted the data for JMPR review or has received the data on which the summaries are based, either from the owner of the data or from a second party that has obtained permission from the owner of the data for this purpose. CHLORMEQUAT (addendum) First draft prepared by J.-J. Larsen Institute of Toxicology, Danish Veterinary and Food Administration, Ministry of Food, Agriculture and Fisheries, Soborg, Denmark Explanation Evaluation for acceptable daily intake Biochemical aspects Absorption, distribution and excretion Biotransformation Effects on enzymes and other biochemical parameters Toxicological studies Acute toxicity Short-term toxicity Long-term toxicity and carcinogenicity Genotoxicity Reproductive toxicity Special studies: Dermal and ocular irritation and dermal sensitization Comments Toxicological evaluation References Explanation Chlormequat (2-chloroethyltrimethylammonium chloride) was evaluated by the Joint Meeting in 1970, 1972, and 1994 (Annex 1, references 14, 18, and 71). In 1972, an ADI of 0-0.05 mg/kg bw was established on the basis of the NOAEL in a study of reproductive toxicity in rats. In 1994, this ADI was withdrawn owing to the inadequacy of the database in comparison with acceptable contemporary standards. The compound was reviewed at the present Meeting in response to a request from the manufacturer. New data on the absorption, distribution, excretion, and biotransformation of chlormequat and on its long-term toxicity in rats and dogs, carcinogenicity in mice and rats, reproductive toxicity in rats, and skin sensitization potential in guinea-pigs were reviewed. Evaluation for acceptable daily intake 1. Biochemical aspects (a) Absorption, distribution, and excretion Experiments are described in articles published in the open literature in which the absorption, distribution, and excretion of chlormequat were investigated. The level of detail in these papers did not permit a complete evaluation by the present Meeting. In the first study, the bulk (61%) of an oral dose of 14C-chlormequat administered to male rats was excreted in the urine within 4 h, and 96% was eliminated within 47 h; faecal excretion accounted for 2.3%, and less than 1% was expired as 14C-carbon dioxide. The remainder was found in the tissues, with the largest amounts in the carcass (0.25%), intestines (0.11%), and liver (0.08%). Analysis of urine samples by four different thin-layer and paper chromatographic systems showed that all of the radiolabel was on chlormequat (Blinn, 1967). In a second study, rats were given a single oral dose of 60 mg 15N-chlormequat or received 2 mg of the labelled compound daily for 100 days. After the single dose, the amount of compound in the brain decreased quickly, but there was considerable accumulation in the kidneys over the 20 days of the investigation. After continuous administration, chlormequat was found particularly in active muscles such as those of the heart and diaphragm (Bier & Ackermann, 1970). In a third study, a lactating cow received a single oral dose of 1000 mg 15N-chlormequat. The compound was found in the milk and urine 3 h after administration; most (490 mg) was found 15-39 h after administration. Only 22 mg were excreted in the milk, and the concentration never exceeded 1 ppm; the peak concentration was found 12-60 h after administration (Lampeter & Bier, 1970). In a more recent study, 136 male and female rats were treated intravenously with a single dose of 0.1 mg/kg bw or orally with a single dose of 0.5 or 30 mg/kg bw 14C-chlormequat (radiochemical purity, 96.8%). Urine, faeces, organs, bile, and expired air were collected from all animals at various intervals up to 168 h after treatment; 82-103% of the radiolabel was recovered. Most was excreted during the first 24 h. More than 85% of the radiolabel was excreted with urine and < 6% with faeces; < 1% was eliminated as volatile compounds. The maximum blood level was reached about 2 h after oral administration of either the low or the high dose. Less than 1% of the radiolabel was eliminated in bile during the first 24 h. The maximum radiolabel appeared 2-5 h after oral administration. Little radiolabel was found in organs or tissues after 168 h, the highest concentrations being found in liver and kidney (Giese & Hoffmann, 1989). (b) Biotransformation Only chlormequat and two other compounds, which may have been other salts of chlorcholine, were reported in the urine of rats that had received 200 mg/kg bw of chlormequat orally. Choline itself was not identified (Bronisz & Romanowski, 1968). A group of 74 male and females rats were given a single intravenous dose of 0.1 mg/kg bw or a single oral dose of 0.5 mg/kg bw (with or without preteatment with unlabelled chlormequat for 14 days) or 30 mg/kg bw 14C-chlormequat (radiochemical purity, 96.8%). Urine, faeces, bile, liver, kidney, gastrointestinal tract, brain, muscle, spleen, bone, lung, heart, fat, testes, and uterus were examined for radiolabel for up to 168 h after treatment. Expired air from two males was examined within 0-24 h. A total of 81% of the dose of radiolabel was excreted during the test period. In all cases, > 85% of the radiolabel was found in urine and < 5% in faeces. Chlormequat was excreted mainly unmetabolized. A very polar but unidentified metabolite was found in faeces (Giese & Kohl, 1989). (c) Effects on enzymes and other biochemical parameters General pharmacological tests were carried out to determine the physiological effects of chlormequat injected intravenously. Oligopnoea, salivation, and a tendency to inhibition of intestinal propulsion were observed in mice immediately after they were given 7.4 mg/kg bw. In cats, there was mild inhibition of the vasopressor effect of noradrenaline 30 min after administration of 1 mg/kg bw chlormequat. In rabbits, neuromuscular junctions were blocked by doses of > 1 mg/kg bw; this effect was counteracted by administration of 10 mg/kg bw D-tubocurarine and potentiated by administration of 1 mg/kg bw neostigmine. Coagulation of rat blood was unaffected by concentrations up to 3 mg/ml. In dogs, doses of > 3 mg/kg bw caused a drop in blood pressure; at higher doses, increased respiratory and heart rates were observed. These effects were mitigated by prior intravenous administration of 1 mg/kg bw atropine (Mutoh et al., 1987). The action of chlormequat was tested in vitro with the patch clamp technique for electrophysiological measurements described by Hamill et al. (1981). Muscles were excised from the feet of adult NMRI mice and dissociated enzymatically to obtain individual muscle cells. Chlormequat (purity, 95.6%) activated the nicotinic acetylcholine receptor channel at all concentrations between 10 and 100 mmol/L (Franke & Mellert, 1991). The affinity of chlormequat for subtypes of muscarinic acetylcholine receptors was investigated in vitro on membranes from bovine cerebral cortex, rat heart, and rat submaxillary gland. The results were compared with those obtained for subtype-specific reference substances, and atropine was included as a high-affinity reference compound with no subtype selectivity. Chlormequat had low affinity for the muscarinic receptors in comparison with the reference substances (Weifenbach, 1991). 2. Toxicological studies (a) Acute toxicity Clinical signs of toxicity seen after treatment with chlormequat (Table 1) generally consisted of salivation, writhing, chromodacryorrhoea, decreased activity, tremors, diuresis, and piloerection. Death generally occurred within 24 h of treatment; animals that survived recovered within 48 h. The findings at autopsy were not consistent or related to treatment. Table 1. Acute toxicity of chlormequat Species Route LD50 or LC50 Purity Reference (mg/kg bw or (%) mg/L air) Mouse Oral 215-1020 NR Oettel ( 1965 ) NR Levinskas & Shaffer (1966) NR Ignatiev (1967) 98.0 Hattori (1981) Mouse Intraperitoneal 60-68 NR Shaffer (1970) 98.0 Hattori (1981) Mouse Subcutaneous 88-92 98.0 Hattori (1981) Rat Oral 330-750 NR Oettel (1965) NR Levinskas & Shaffer (1966) NR Ignatiev (1967) NR Stefaniek (1969) 98.1 Hattori (1981) Rat Oral 522 66.1a Fischer & Lowe (1990) Rat Intraperitoneal 53-75 98.1 Hattori (1981) Rat Subcutaneous 113-118 98.1 Hattori (1981) Rat Dermal > 4000 NR Gelbke & Freisberg (1978) Rat Dermal > 5000 98.1 Hattori (1981) Rat Inhalation > 5.2 99 Zeller & Klimisch (1979) Rat Inhalation > 4.6 66.1 Hershman (1990) Hamster Oral 1070 NR Levinskas & Shaffer (1966) Guinea-pig Oral 215-620 NR Oettel (1965) NR Levinskas & Shaffer (1966) Rabbit Oral 60-81 NR Oettel (1965) NR Levinskas & Shaffer (1966) Rabbit Dermal 1250 66.1a Fischer et al. (1990a) Cat Oral 7-50 NR Oettel (1965) NR Levinskas & Shaffer (1966) Dog Oral < 50 NR Levinskas & Shaffer (1966) Sheep Oral 150-200 NR Schulz et al. (1970) Monkey Oral > 800 NR Costa et al. (1967) NR, not reported a Technical material consisting of 66.1% aqueous solution Rabbits, cats and dogs may be more sensitive to the toxic effects of chlormequat than rats and mice. The acute toxicity in monkeys was similar to that seen in rats and mice. (b) Short-term toxicity Rats Two short-term studies of toxicity are described in the 1972 JMPR monograph addendum (Annex 1, reference 19), but detailed reports were not available for evaluation at the present Meeting. The summary of the first study states that groups of 10 male rats were fed chlormequat at dietary levels of 0, 500, 1000, or 2000 ppm (equivalent to 0, 25, 50, or 100 mg/kg bw per day) for 29 days. There were no deaths and no clinical signs of reaction to treatment; body-weight gain and food intake remained undisturbed by treatment, and no gross pathological changes were observed at termination of the study (Levinskas & Shaffer, 1962). In the second study, groups of 20 male and 20 female rats were fed chlormequat at dietary levels of 0, 200, 600, or 1800 ppm (equivalent to 0, 10, 30, or 90 mg/kg bw per day) for 90 days. There were no deaths, no clinical signs of reaction to treatment, and no treatment-related changes in blood chemistry. The body-weight gain of males fed 1800 ppm was slightly depressed in comparison with that of controls. Slightly increased kidney weights were recorded in treated female rats and slightly increased liver weights in treated males, particularly at 1800 ppm; however, histopathological examination of major organs revealed no treatment-related changes (Levinskas, 1965). In a more recent experiment of acceptable scientific quality, groups of five male and five female Wistar rats were fed chlormequat at dietary levels of 0, 500, 1500, 3000, or 4500 ppm (equal to 0, 46, 140, 270, or 410 mg/kg bw per day) for four weeks. The test material was a technical-grade formulation of 66.7% purity, but the dietary levels were expressed as pure chlormequat. Clinical signs of general deterioration in health were seen in males and females receiving 4500 ppm and, temporarily, in one male and one female receiving 3000 ppm. Reduced body-weight gain and food intake were seen in animals fed 4500 ppm, and slightly reduced weight gain was seen among those fed 3000 ppm. Serum creatinine levels in males and females receiving 4500 ppm and in females receiving 3000 ppm were lower than those of controls. Decreased serum concentrations of total protein (in males) and of urea (in females) were also seen at the high dietary level. Tests of locomotor activity and swimming, gross pathological examinations, organ weighing, and histopathological examination revealed no reaction to treatment. The NOAEL was 1500 ppm, equal to 140 mg/kg bw per day (Schilling et al., 1990). Rabbits In a recent experiment of acceptable scientific quality, groups of 10 male and 10 female New Zealand white rabbits received chlormequat (purity, 99%) by repeated occluded dermal applications on shaven skin, five days per week for three weeks at doses of 0, 20, 50, or 150 mg/kg bw per day. Possible reactions to treatment at the application site were limited to erythema during the first two weeks of the study; however, these reactions were no more severe than reactions frequently seen after repeated occluded dermal applications of control compounds. There were no other clinical signs of reaction to treatment, and body-weight gain and food intake remained undisturbed by treatment. Investigations of haematological parameters and blood chemistry, gross pathology, organ weights, and histopathology revealed no reaction to treatment (Buch & Finn, 1981). Dogs In an experiment for which no detailed report was available to the Meeting (Annex 1, reference 19), groups of two male and two female dogs were fed chlormequat at dietary levels of 0, 20, 60, or 180 ppm (equivalent to 0, 0.5, 1.5, or 4.5 mg/kg bw per day) for 106-108 days. There were no deaths and no clinical signs of reaction to treatment, and body-weight gain and food intake were unaffected. Organ weight analysis and histopathological examination at termination of the experiment revealed no treatment-related changes (Levinskas, 1965). In a study reported in 1967, which was not conducted to currently acceptable scientific standards, groups of three male and three female beagle dogs were fed chlormequat (technical-grade purified twice by recrystallization) at dietary levels of 100, 300, or 1000 ppm (equivalent to 0, 2.5, 7.5, or 25 mg/kg bw per day) for two years; groups of 10 males and 10 females served as controls. Excessive salivation and hindlimb weakness were seen in some animals receiving 1000 ppm, and one male died after 22 days and one female after 38 days. The deaths were considered by the authors to be secondary to the clinical signs of hindlimb weakness. Analysis of blood chemistry and urinalysis revealed no treatment-related changes other than the presence of chlormequat in the urine of treated animals. At termination, gross pathology, organ weight analysis, and histopathology revealed no changes attributable to treatment. The NOAEL was probably 300 ppm (equivalent to 7.5 mg/kg bw per day), but the absence of further investigation into the signs of hindlimb weakness precluded establishment of a definitive NOAEL (Oettel & Sachsse, 1967). Groups of five male and five female beagle dogs were given technical-grade chlormequat (purity, 67.4%) mixed in the diet at doses of 0, 150, 300, or 1000 ppm, equal to 0, 4.7, 9.2, or 31 mg/kg bw per day in males and 0, 5.2, 10, or 32 mg/kg bw per day in females, for 12 months. Food consumption was determined daily and body weight once a week. In addition to clinical, hematological, and ophthalmological examinations, urinalysis and neurofunctional examinations were carried out. At the end of study, all animals were subjected to gross pathological and histopathological examination. Animals at 1000 ppm had diarrhoea, vomiting, salivation, apathy, and other severe clinical symptoms as well as many changes in clinicochemical and haematological parameters. Two dogs at this dose died. Diarrhoea, vomiting, and salivation were also seen at 300 ppm. The NOAEL was 150 ppm, equal to 4.7 mg/kg bw per day, on the basis of diarrhoea, vomiting, and salivation (Mellert et al., 1993). (c) Long-term toxicity and carcinogenicity Mice In a study reported in 1971, the design of which was clearly not in accordance with currently acceptable scientific standards, groups of 52 male and 52 female CFLP mice were fed chlormequat (purity, about 98.5%) at dietary levels of 0 or 1000 ppm (equivalent to 0 or 150 mg/kg bw per day) for 78 weeks. Survival was unaffected, and there were no clinical signs of reaction to treatment, except that treated animals gained less weight than controls. Histopathological examination was initially restricted to 10 males and 10 females from each group but was extended to all tissues from animals in which a treatment-related effect was seen. The incidence of benign lung tumours was higher (20/52) in treated males than in controls (10/51) but was considered to be within the normal range in untreated mice. The incidences of lung tumours in females and of tumours in all other organs examined in animals of each sex were not significantly higher in the treated group than in controls (Weldon et al., 1971). Groups of 50 male and 50 female B6C3F1 mice were fed diets containing chlormequat (purity, 97-98%) at 0, 500, or 2000 ppm (equal to 0, 70, or 290 mg/kg bw per day) for 102 weeks. The dietary levels were set on the basis of the results of an eight-week study with doses of 1200-20 000 ppm designed to provide a statistical estimate of the dose that would depress body-weight gain by 10%. The control group consisted of 20 males and 20 females. Body-weight gain remained largely unaffected by treatment, and there were no treatment-related clinical signs. Survival was unaffected by treatment and was adequate for assessment of carcinogenicity, as at least 80% of animals in each group survived until termination of the experiment. The incidence of haemangiomas and haemangiosarcomas was slightly increased in treated females (1/20 in controls, 4/50 at 500 ppm, and 5/50 at 2000 ppm), but the authors concluded that there was no clear evidence for the carcinogenicity of chlormequat in these mice (National Cancer Institute, 1979). Groups of 50 male and 50 female B6C3F1 mice were fed diets containing technical-grade chlormequat (purity, 67.4%) at 0, 150, 600, or 2400 ppm, equal to 0, 21, 84, or 340 mg/kg bw per day in males and 0, 23, 91, or 390 mg/kg bw per day in females, for 110 weeks. Satellite groups of 10 male and 10 female mice at each dose were killed after 52 weeks. Food consumption and body weight were determined weekly during the first 14 weeks and every four weeks thereafter. The health of the animals was checked daily, and clinical signs were followed up thoroughly once a week. All animals that died or were killed in a moribund condition during the study or were killed terminally were subjected to gross pathological and histopathological examinations. The body weight of animals in the satellite goup at 2400 ppm was reduced. The incidence and severity of tubular down-growth in the ovaries and the incidence of endometrial hyperplasia were increased in mice at 600 and 2400 ppm. There was no significant increase in the incidence of benign or malignant tumours. The NOAEL was 150 ppm, equal to 21 mg/kg bw per day, on the basis of tubular down-growth in the ovaries and endometrial hyperplasia (Mellert et al., 1994). Rats In 1994, the Meeting reviewed a short summary of a two-year study reported in 1967, the design of which was not in accordance with contemporary scientific standards. Groups of 50 male and 50 female Sprague-Dawley rats were fed chlormequat (technical grade, purified twice by recrystallization) at dietary levels of 0, 500, or 1000 ppm (equivalant to 0, 25, or 50 mg/kg bw per day) for two years. The authors reported that survival was unaffected, there were no clinical signs of reaction to treatment, and food intake and body-weight gain were unchanged. Haematological examinations, analysis of blood chemistry, and urinalysis carried out after three and 12 months and before termination revealed no reaction to treatment. Gross pathological examination, analyses of liver and kidney weights, and histopathological examination revealed no abnormalities attributable to treatment. Normal, age-related pathological changes were seen; in particular, the tumour profiles in treated and control animals were indistinguishable. Detailed evaluation of this report was not possible (Oettel & Froberg, 1967). A summary of another long-term study was available in which groups of 50 male and 50 female Sprague-Dawley rats were fed diets containing chlormequat (technical grade purified twice by recrystallization) and choline chloride in a ratio of 10:7 for two years. The dietary levels of chlormequat were 0, 500, 1000, or 5000 ppm (equivalent to 0, 25, 50, and 250 mg/kg bw per day). The control group consisted of 100 male and 100 female rats. Survival was unaffected by treatment; the survival rates after two years were 72-82% in male rats and 48-64% in females. There were no clinical signs of reaction to treatment, and body-weight gain and food intake remained unaffected. Haematological examination, limited analyses of blood chemistry, and urinalysis carried out after three and 12 months and before termination revealed no indication of reaction to treatment. Gross pathological examination, analysis of liver and kidney weights, and histopathological examination of a range of organs and tissues revealed no abnormalities attributable to treatment. Normal, age-related pathological changes were seen; in particular, the tumour profiles in treated and control animals were indistinguishable (Oettel & Sachsse, 1974). In a study designed to assess the tumorigenicity of chlormequat in rats, groups of 50 male and 50 female Fischer 344 rats were fed diets containing chlormequat (purity, 97-98%) at 0, 1500, or 3000 ppm (equivalent to 0, 75, or 150 mg/kg bw per day) for 108 weeks. The dietary levels were set on the basis of the results of an eight-week study with doses of 3150-14 700 ppm designed to provide a statistical estimate of the dose that would depress body-weight gain by 10%. The control group consisted of 20 males and 20 females. The body-weight gain of treated rats was slightly lower than that of controls, but there were no treatment-related clinical signs. Survival was unaffected by treatment and was adequate for assessment of carcinogenicity, as at least 64% of animals in each group survived until termination of the experiment. The incidence of leukaemia or malignant lymphoma was slightly increased in treated females (3/20 in controls, 11/50 at 1500 ppm, and 14/50 at 3000 ppm), and there was an apparently dose-related increase in the incidence of islet-cell adenomas of the pancreas in treated males (0/18 in controls, 1/47 at 1500 ppm, and 7/45 at 3000 ppm); however, the authors concluded that there was no clear evidence for the carcinogenicity of chlormequat in these rats (National Cancer Institute, 1979). Groups of 20 male and 20 female Wistar rats were fed diets containing technical-grade chlormequat (purity, 67.4%) at 0, 280, 940, or 2800 ppm, equal to 0, 13, 43, or 140 mg/kg bw per day in males and 0, 17, 56, or 170 mg/kg bw per day in females, for 78 weeks. Food consumption and body weight were determined once a week during the first 14 weeks and then at four-week intervals. The animals were observed daily and inspected thoroughly once a week for clinical signs. At the start and end of the study, controls and those at the highest dose were examined for ophthalmological signs. All animals were subjected to haematological examination, blood chemical analyses, and urinalysis at 3, 6, 12, and 18 months. All rats still alive at the end of study and rats that died intercurrently were subjected to gross pathological and histopathological examination. Statistically significant reductions in body weight (18% in males and 10% in females) and a minor reduction in food intake were observed in animals at 2800 ppm. Pathological examination revealed no substance-induced changes in any organ. Higher incidences of tubular mineralization and tubular atrophy in the kidneys of treated males were considered to be toxicologically insignificant. Tumour incidences were not enhanced. The NOAEL was 940 ppm, equal to 43 mg/kg bw per day, on the basis of reduced body weight in males (Schilling et al., 1992). Groups of 50 male and 50 female Wistar rats were fed diets containing technical-grade chlormequat (purity, 67.4%) at 0, 280, 940, or 2800 ppm (equal to 0, 13, 42, or130 mg/kg bw per day in males and 0, 16, 55, and 170 mg/kg bw per day in females) for two years. Food consumption and body weight were determined once a week during the first 14 weeks and then at four-week intervals. The animals were observed daily and inspected thoroughly once a week for clinical signs. At the end of the study, all surviving rats (40 controls, 34 at 280 ppm, 36 at 940 ppm, and 32 at 2800 ppm) were subjected to haematological examination, blood chemical analysis, and urinalysis and then to gross pathological and histopathological examination. Reduced body weights (14% in males and 22% in females) and food consumption (10% in males and 6% in females) were observed in animals at 2800 ppm at the end of the study. No signs of carcinogenicity were observed. The NOAEL was 940 ppm, equal to 42 mg/kg bw per day, on the basis of reduced body weight (Mellert et al., 1992). (d) Genotoxicity The results of tests for the genotoxicity of chlormequat are summarized in Table 2. All of the assays for gene mutation carried out in bacteria and mammalian cells gave negative results. No cytogenetic anomalies were found in human lymphocytes in vitro, and unscheduled DNA synthesis was not seen in rat hepatocytes. Neither dominant lethal mutation nor micronuclei were seen in mice in vivo, and no chromosomal aberrations occurred in rats. (e) Reproductive toxicity Mice The results of three experiments in mice were summarized in the 1972 monograph addendum (Annex 1, reference 19), but detailed reports were not available for evaluation by the present Meeting. In the first experiment, groups of pregnant mice received intraperitoneal injections of chlormequat at 30 mg/kg bw on days 14 and 15 or days 11-15 of gestation. Another group of mice received 200 mg/kg bw per day by gavage on days 11-15 of gestation. All mice were killed on day 19. The number of fetuses per dam, fetal size, frequency of resorptions, and incidence of malformations were similar in the test and control groups. In the second experiment, pregnant mice were fed chlormequat at dietary levels of 0, 1000, or 10 000 ppm (equivalent to 0, 150, or 1500 mg/kg bw per day) on days 1-15 of gestation or 25 000 ppm (equivalant to 3750 mg/kg bw per day) on days 11-15. All animals were killed on day 19. The number of fetuses per dam, fetal size, and frequency of resorptions were similar in the test and control groups. Dams fed 10 000 or 25 000 ppm had slightly more malformed fetuses than controls. In the third experiment, groups of mature male and female mice were fed chlormequat at dietary levels of 0, 1000, or 5000 ppm (equivalent to 0, 150, or 750 mg/kg bw per day) and mated after 1, 3, 4, and 10 weeks. All mice were killed on day 19 of gestation. Feeding of chlormequat was found to have no effect on fertility, and no evidence of teratogenicity was seen in the offspring (Shaffer, 1970). Rats In a study conducted between 1965 and 1967 which was not in accordance with currently acceptable scientific standards, groups of 20 male and 20 female rats received diets containing chlormequat at 0, 100, 300, or 900 ppm (equivalent to 0, 5, 15, or 45 mg/kg bw per day) throughout three generations. No abnormalities were seen in the appearance, behaviour, food intake, body-weight gain, fertility, gestation, lactation, or viability of the offspring, and no fetal Table 2. Results of tests for the genotoxicity of chlormequat End-point Test system Concentration Purity Result Reference of chlormequat (%) In vitro Reverse S. typhimurium TA98, < 5000 µg/plate 66.1a Negativeb Traul & Mulligan mutation 100, 1535, 1537, 1538 (1990) E. coli WP2uvrA Reverse S. typhimurium TA98, < 2500 µg/plate 92.4 Negativeb Zeller & Engelhardt mutation 100, 1535, 1537, 1538 (1979) Reverse Chinese hamster < 5000 µg/ml 66.1a Negativeb Traul & Johnson mutation ovary cells, hprt locus (1990) Reverse Chinese hamster V79 < 5000 µg/ml 94.5-98.9 Negativeb Debets et al. (1986) mutation cells, hprt locus Chromosomal Human lymphocytes < 5000 µg/ml 94.5-98.9 Negativeb Enninga et al. (1987) aberration Unscheduled Rat hepatocytes < 7.5 µg/ml 66.1a Negative Pant & Law (1990) DNA synthesis Unscheduled Rat hepatocytes < 10 000 nl/ml 72c Negative Cifone & Myhr (1987) DNA synthesis In vivo Dominant Male NMRI mice 1 × 261 mg/kg bw 99.6 Negative Gelbke & Engelhardt lethal mutation (1979) Micronucleus Male and female < 2 × 202.5 94.5-98.9 Negative Geunard et al. (1983) formation NMRI mice mg/kg bw Chromosomal Male and female < 1 × 500 66.1 Negative Sharma & Caterson aberration Sprague-Dawley rats mg/kg bw (1991) a Technical material consisting of 66.1% aqueous solution b With and without metabolic activation c Technical material consisting of 72% aqueous solution malformations occurred that could be attributed to treatment. Histopathological examination of 10 rats of each sex of the F3 generation at each dose after nine weeks of treatment revealed the presence of giant cells in the testicular tubules of four rats treated with 900 ppm and two treated with 300 ppm. The authors suggested that the cells were an expression of delayed maturation during spermatogenesis; however, the Meeting found it difficult to assess the significance of the finding and concluded that the NOAEL was 100 ppm, equivalent to 5 mg/kg bw per day (Leuschner et al., 1967). In a study described in the 1972 monograph addendum (Annex 1, reference 19), groups of pregnant rats were fed chlormequat at dietary levels of 0, 1000, or 5000 ppm (equivalent to 0, 50, or 250 mg/kg bw per day) on days 1-21 of gestation. When the animals were killed the day before parturition, no teratogenic effects were observed (Shaffer, 1970). In a two-generation study of reproductive toxicity, technical-grade chlormequat (purity, 67.4%) was administered to groups of 24 male and 24 female Wistar rats at dietary levels of 0, 300, 900, or 2700 ppm (equal to 0, 29, 86, or 250 mg/kg bw per day in males and 0, 23, 69, or 230 mg/kg bw per day in females). At least 70 days after the beginning of treatment, F0 animals were mated in order to produce an F1a litter and subsequently remated to produce an F1b litter. Groups of 24 males and 24 females from the F1 litter were selected as the F1 parents and kept on the diet for at least 98 days before they were mated to produce an F2 litter. The study was terminated after weaning of the F2 litter. All animals were inspected daily, and the food consumption of the F0 and F1 parents was determined weekly. Pups were observed for the usual parameters and also for physiological development and behavioural effects (gripping reflex, acoustic startle, and pupillary reflex). A statistically significant reduction in body weight was observed in F0 and F1 females at 2700 ppm, and food consumption was moderately reduced in males and females of both generations; transient tremor and hypersensitivity were also observed in F0 and F1 females at this dose, mainly during or after the lactation period. Also at this dose, male fertility was reduced, fewer pups were delivered by each dam, and the growth and development of the F1a, F1b, and F2 pups were retarded. The NOAEL for reproductive toxicity was 900 ppm, equal to 69 mg/kg bw per day, on the basis of reductions in male fertility and number of delivered pups (Hellwig et al., 1993). Hamsters Groups of eight pregnant Syrian golden hamsters were given chlormequat (purity unspecified) by gavage at concentrations of 0, 25, 50, 100, 200, 300, or 400 mg/kg bw once on day 8 of gestation or 100 mg/kg bw daily on days 7, 8, and 9 of gestation. The control group consisted of 15 animals. Clinical signs of toxicity were seen in the groups receiving the higher doses. All animals were killed on day 14 of gestation. Animals fed 100 mg/kg bw or more had fewer fetuses than controls and more fetal resorptions. In animals fed 200 mg/kg bw or more, fetal size and weight were reduced. No abnormalities were observed in animals treated with single doses of 0, 25, 50, or 100 mg/kg bw. Malformations including anophthalmia, microphthalmia, cleft palate, and polydactylism and evidence of developmental retardation were seen in the offspring of dams treated with three doses of 100 mg/kg bw or a single dose of 200, 300, or 400 mg/kg bw. The limited details presented in the publication make it difficult to assess the maternal toxicity; the occurrence of malformations in historical controls was not presented (Juszkiewicz et al., 1970). Rabbits In a study described in the 1972 monograph addendum (Annex 1, reference 19), groups of pregnant rabbits were fed diets containing chlormequat at 0 or 1000 ppm (equivalent to 0 or 30 mg/kg bw per day) on days 1-28 of pregnancy and were killed two days before parturition. No evidence of teratogenicity was seen (Shaffer, 1970). In a study of acceptable design, groups of 15 inseminated female Himalayan rabbits were given chlormequat at 0, 1.5, 3, 6, or 12 mg/kg bw per day by gavage on days 6-18 of gestation. Fetuses were removed after sacrifice of the dams on day 28 of gestation and were examined for abnormalities macroscopically and by X-ray; in addition, the heads of all fetuses were fixed in Bouin's solution, and transverse sections were assessed. Treatment did not affect mortality. Rapid breathing, salivation, and apathy were seen on single occasions in single animals receiving 6 or 12 mg/kg bw per day; the body-weight gain of animals given 12 mg/kg bw per day was reduced temporarily, and food intake was temporarily depressed in all treated groups. Treatment had no effect on the number of fetuses, conception rate, resorption rate, size or weight of the fetuses, or placental weights. No teratogenic effect was seen. Minor variations and developmental retardation were seen to the same extent in all groups, including controls. The NOAEL for maternal toxicity was 6 mg/kg bw per day, while there was no evidence of fetotoxicity or teratogenicity at the highest dose tested, 12 mg/kg bw per day (BASF, 1979). (f) Special studies: Dermal and ocular irritation and dermal sensitization In two studies conducted between 1978 and 1980, which were not in accordance with current test guidelines, the irritancy of chlormequat chloride to the skin was tested in rabbits. In the first study, about 0.5 ml of the test material (purity unspecified) was applied to intact and abraded sites on each of six Vienna white rabbits and left in place for 24 h under an occlusive dressing. At the intact sites, erythema and oedema were observed at the end of the application period, but these signs were almost fully reversed within two days. More severe signs were observed at the abraded sites; the signs were only partly reversible, and superficial necrosis was seen in three animals after three days (Gelbke, 1978). In the second study, about 500 mg of the same material were tested in the same way in New Zealand white rabbits. Dermal reaction at the treatment sites was limited to very slight or well-defined erythema, which was evident only at the end of the application period. All reaction had resolved completely within 72 h of treatment (Buch & Gardner, 1980). In a study based on current test guidelines, about 0.5 ml of chlormequat (a technical material consisting of a 66.1% aqueous solution) was applied to intact sites on each of six New Zealand white rabbits and left in place for 4 h under an occlusive dressing. Dermal reaction at the treatment sites was limited to barely perceptible or slight erythema, which was evident in three animals 1 h after treatment and in one animal at 24 h. All reactions had resolved completely within 48 h of treatment (Fischer et al., 1990b). The irritancy of chlormequat chloride to the eye was tested in Vienna white rabbits by applying about 0.1 ml of the test material (purity unspecified) to the conjunctival sac of the right eyelid of six rabbits. Conjunctival redness was seen in five rabbits 24 h after treatment. After 48 h, conjunctival redness was seen in two rabbits, one of which had a conjunctival discharge. All reactions had resolved by 72 h after treatment (Gelbke & Grundler, 1981). The irritancy of chlormequat chloride (a technical material consisting of a 66.1% aqueous solution) was also tested in New Zealand white rabbits. About 0.1 ml was applied to the conjunctival sac of the left eyelid of six rabbits and left for 24 h. One hour after treatment, slight reactions were seen in all the rabbits. These signs had resolved by 48 h in two animals and by four days in all rabbits (Lowe & Boczon, 1990). The potential of chlormequat to cause delayed contact hypersensitivity was tested in albino guinea-pigs by the method of Buehler. Induction was performed by applying 0.4 ml of the test material to a shaven flank and leaving it for 6 h under an occlusive dressing. This process was repeated three times per week for a total of nine applications. After a two-week rest period, the animals were challenged by applying the test material to the opposite flank. No erythema or oedema was observed after the challenge, whereas a positive control compound (dinitrochlorobenzene) included in the study gave the expected results. It was concluded that chlormequat is not a skin sensitizer (Ventura & Moore, 1990). The sensitizing effect on the skin of chlormequat was tested in guinea-pigs in the maximization test based on the method of Magnusson and Kligman. After intradermal induction with a volume of 0.1 ml Freund's adjuvant:0.9% saline (1:1), well-defined erythema and slight oedema were observed at the injection sites of both control and test animals. Injection of the test substance in 0.9% saline caused well-defined erythema, and injection of the test substance in Freund's adjuvant:0.9% saline resulted in well-defined erythema and slight oedema. The control animals, treated with 0.9% saline, showed no skin reaction. After percutaneous induction, perfomed only in the test animals, partially open incrustation was observed in additon to erythema and slight oedema. After percutaneous challenge with 50% test substance, no skin reaction was observed in controls or test animals. The authors concluded that chlormequat has no sensitizing effect on the skin of the guinea-pig (Rossbacher & Kirsch, 1992). Comments In experiments with 14C-labelled chlormequat in rats, absorption was rapid, and elimination was essentially complete within 24 h, occurring almost entirely via the urine and mainly as unmetabolized chlormequat. Less than 1% of the administered dose remained in the tissues. Accumulation of 15N-labelled material in the kidneys was reported, but the experimental details were incomplete and detailed evaluation was not possible. Studies of the biotransformation of chlormequat suggested that the only metabolites found in rat urine may have been salts of chlorcholine. An unidentified polar metabolite was found in faeces. Pharmacological tests in mice, rats, rabbits, and cats given chlormequat intravenously revealed a stimulatory effect on the parasympathetic nervous system and a myoneural blocking action. Further work showed that chlormequat is a partial agonist of the nicotinic acetylcholine receptor; the affinity for muscarinic receptors was low and relatively unselective. Chlormequat was of moderate acute oral toxicity in rats, mice, hamsters, guinea-pigs, and monkeys (LD50 = 200-1000 mg/kg bw), but rabbits and dogs appeared to be more sensitive (LD50 = 50-80 mg/kg bw) than the other species. The signs of toxicity may have been due to pharmacological activity, and there were no consistent treatment-related findings at autopsy. WHO has classified chlormequat as slightly hazardous (WHO, 1996). In a four-week study of toxicity in rats at dietary concentrations of 0, 500, 1500, 3000, or 4500 ppm, the NOAEL was 1500 ppm, equal to 140 mg/kg bw per day, on the basis of reduced body-weight gain and depression of serum creatinine concentration. These results are largely in agreement with those of older studies in rats of up to 90 days' duration. In a 12-month study of toxicity in dogs at dietary concentrations of 0, 150, 300, or 1000 ppm, the NOAEL was 150 ppm, equal to 4.7 mg/kg bw per day, on the basis of diarrhoea, vomiting, and salivation. In a 110-week study of toxicity and carcinogenicity in mice at dietary concentrations of 0, 150, 600, or 2400 ppm, the NOAEL was 150 ppm, equal to 21 mg/kg per day, on the basis of tubular down-growth in the ovaries and endometrial hyperplasia. In a 78-week study of toxicity and carcinogenicity in rats at dietary concentrations of 0, 280, 940, or 2800 ppm, the NOAEL was 940 ppm, equal to 43 mg/kg bw per day, on the basis of reduced body weight. Tumour incidences were not enhanced. The potential carcinogenicity of chlormequat was investigated in a 104-week study in rats at dietary concentrations of 0, 280, 940, or 2800 ppm. No carcinogenicity was observed. The NOAEL was 940 ppm, equal to 42 mg/kg bw per day, on the basis of reduced body weight. In a multigeneration study of reproductive toxicity in rats at dietary concentrations of 0, 300, 900, or 2700 ppm, the NOAEL for reproductive toxicity was 900 ppm, equal to 69 mg/kg bw per day, on the basis of reduced numbers of pregnancies and of delivered pups and retarded growth and development of the pups. The developmental toxicity of chlormequat has been investigated in mice after administration by intraperitoneal injection, gavage, or via the diet, in rats by dietary administration, and in hamsters and rabbits by gavage. Many of the study reports were available only in summary form. In a study in mice at dietary concentrations of 0, 1000, or 10 000 ppm on days 1-15 of gestation or 25 000 ppm on days 11-15 of gestation, the number of malformations in animals fed 10 000 ppm or 25 000 ppm was reported to be slightly higher than that in controls; however, the significance of this observation was difficult to assess. In hamsters receiving chlormequat at levels of 0, 25, 50, 100, 200, 300, or 400 mg/kg bw once on day 8 of gestation or 100 mg/kg bw per day on days 7-9 of gestation, malformations and evidence of delayed development were seen with doses > 200 mg/kg bw on day 8 and after the three doses of 100 mg/kg bw per day. Evidence of maternal and fetal toxicity was also seen at these doses. The study in hamsters could not be fully evaluated, owing to lack of detail in the publication. A full report of a well-conducted study in which rabbits were dosed orally with 0, 1.5, 3, 6 or 12 mg/kg bw per day on days 6- 18 of gestation was available. Signs of maternal toxicity were seen at the highest dose, but there was no evidence of developmental toxicity. Chlormequat has been adequately tested for genotoxicity in vitro and in vivo in a range of assays. The Meeting concluded that it was not genotoxic. Chlormequat was not irritating to the skin or eye in rabbits. It did not cause delayed contact hypersensitivity when tested in albino guinea-pigs by the method of Buehler or by the method of Magnusson and Kligman. An ADI of 0-0.05 mg/kg bw was allocated on the basis of the NOAEL of 4.7 mg/kg bw per day for diarrhoea, vomiting, and salivation in the one-year study of toxicity in dogs, and using a safety factor of 100. Toxicological evaluation Levels that cause no toxicological effect Mouse: 150 ppm, equal to 21 mg/kg bw per day (110-week study of toxicity and carcinogenicity) Toxicological criteria for setting guidance values for dietary and non-dietary exposure to chlormequat Human exposure Relevant route, study type, species Results, remarks Short-term Dermal irritation, rabbit Not irritating (1-7 days) Eye irritation, rabbit Not irritating Skin sensitization, guinea-pig Non-sensitizing Inhalation toxicity, rat LC50 > 5 mg/L air Dermal toxicity, rabbit LD50 = 1300 mg/kg bw Oral toxicity, rabbit LD50 = 70 mg/kg bw Oral toxicity, cat LD50 = 7-50 mg/kg bw Medium-term Repeated oral, reproductive toxicity, rabbit NOAEL = 6 mg/kg bw per day: maternal (1-26 weeks) toxicity, no reproductive toxicity Long-term Repeated oral, one year, dog NOAEL = 4.7 mg/kg bw per day: diarrhoea, (> 1 year) vomiting, and salivation Rat: 940 ppm, equal to 42 mg/kg bw per day (104-week study of toxicity and carcinogenicity) 900 ppm, equal to 69 mg/kg bw per day (two-generation study of reproductive toxicity) Rabbit: 6 mg/kg bw per day (maternal toxicity in a study of developmental toxicity) 12 mg/kg bw per day (fetotoxicity and teratogenicity in a study of developmental toxicity) Dog: 150 ppm, equal to 4.7 mg/kg bw per day (one-year study of toxicity) Estimate of acceptable daily intake for humans 0-0.05 mg/kg/bw Studies that would provide information useful for continued evaluation of the compound Study of developmental toxicity in rodents that meets current scientific standards. 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See Also: Toxicological Abbreviations Chlormequat (AGP:1970/M/12/1) Chlormequat (WHO Pesticide Residues Series 2) Chlormequat (Pesticide residues in food: 1976 evaluations) Chlormequat (Pesticide residues in food: 1994 evaluations Part II Toxicology) Chlormequat (JMPR Evaluations 1999 Part II Toxicological)