CHLORFENVINPHOS First draft prepared by M.F.A. Wouters & P.H. van Hoeven-Arentzen National Institute of Public Health and Environmental Protection, Bilthoven, Netherlands 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 Reproductive toxicity Embryotoxicity and teratogenicity Genotoxicity Special studies Skin and eye irritation and skin sensitization Cholinesterase inhibition and related pharmacological and neurotoxic effects Hormone concentrations Porphyrigenic potential Observations in humans Comments Toxicological evaluation References Explanation Chlorfenvinphos was evaluated by the Joint Meeting in 1971 (Annex I, reference 16), which allocated an ADI of 0-0.002 mg/kg bw. The technical material contains not less than 90% chlorfenvinphos with an approximate E:Z isomer ratio of 1:8.6; a typical sample contains 9.5% E and 82.3% Z isomer. The compound was re-evaluated by the present Meeting as a result of the CCPR periodic review programme. This monograph summarizes the data received since the previous evaluation, comprising short-term studies in mice and dogs, long-term studies in mice and rats, a study of reproductive toxicity in rats and studies on embryotoxicity and teratogenicity in rats and rabbits. It also includes relevant studies from the previous monograph (Annex I, reference 17). Evaluation for acceptable daily intake 1. Biochemical aspects (a) Absorption, distribution and excretion Six male and six female rats (strain not specified) given a dose of 2 ppm of [14C-vinyl]-chlorfenvinphos (3.0 µCi/mg) excreted 52-77% of the radiolabel in urine within 24 h. An additional 6.1-26% was excreted during the next 24 h. Faecal elimination comprised 11% of the radiolabel, and a further 1.4% was excreted via the lungs within 96 h, by which time the entire dose had been excreted. In the same study, two male and two female dogs (strain not specified) fed capsules containing 0.3 ppm [14C-vinyl]-chlorfenvinphos (8.0 µCi) excreted 86% (83-91%) of the administered radiolabel in urine and 4.1% in faeces within 24 h (Hutson et al., 1967; Annex I, reference 17). After administration of 25-30 ppm to young rats (strain not specified), unchanged chlorfenvinphos was detected in peripheral blood. A dog that received 88 mg/kg in feed had similar concentrations of unchanged chlorfenvinphos in peripheral blood after a similar interval (Hutson & Hathway, 1967; Annex I, reference 17). After intramuscular injection of a single 233-mg dose of 14C-chlorfenvinphos (647 µCi) into a lactating cow, only 0.2% of the radiolabel appeared in the milk, mainly during the first two milkings. Unchanged chlorfenvinphos represented 75% of the radiolabel (Hunter, 1969a; Annex I, reference 17). Oral administration of a single 12.5-mg dose of 14C-chlorfenvinphos (35.1 µCi) to an adult man resulted in rapid elimination of radiolabel in the urine: 72% of the dose was excreted within 4.5 h and 94% within 24 h (Hutson, 1969; Annex I, reference 17). (b) Biotransformation Oral administration of 2 ppm 14C-chlorfenvinphos to rats (strain not specified) was followed by complete metabolism of the compound. The urinary metabolites were 2-chloro-1-(2,4- dichlorophenyl)vinylethyl hydrogen phosphate (32% of administered radiolabel), [1-(2,4-dichlorophenyl)ethyl-ß-D-glucopyranosid]uronic acid (41%), 2,4-dichloromandelic acid (7.0%), 2,4-dichlorophenylethanediol glucuronide (2.6%) and 2,4-dichlorohippuric acid (4.3%) (Hutson et al., 1967; Annex I, reference 17). 14C-Chlorfenvinphos was also completely metabolized in dogs (strain not specified) that received 0.26 ppm. The proportions of urinary metabolites were as follows: 2-chloro-1-(2,4- dichlorophenyl)vinylethyl hydrogen phosphate, 70%; [1-(2,4- dichlorophenyl)ethyl-ß-D-glucopyranosid]uronic acid, 3.6%; 2,4-dichloromandelic acid, 13%; 2,4-dichlorophenyl-ethanediol glucuronide, 2.7%. 2,4-Dichlorohippuric acid was not detected (Hutson et al., 1967; Annex I, reference 17). The milk of a lactating cow given an intramuscular injection of 0.58 mg/kg bw 14C-chlorfenvinphos contained 0.2% of the radiolabel, of which 75% was unchanged chlorfenvinphos; there were also small amounts of 2,4-dichloro-acetophenone, 1-(2,4-dichlorophenyl)ethanol and 2,4-dichloromandelic acid. The urinary metabolites comprised 1-(2,4-dichlorophenyl)ethanol and 1-(2,4-dichlorophenyl)ethanediol. The glucuronides of these compounds were not detected (Hunter, 1969a; Annex I, reference 17). Five metabolites were identified in the urine of an adult man following administration of a single oral dose of 12.5 mg 14C-chlorfenvinphos. These were 2-chloro-1-(2,4-dichlorophenyl)vinylethyl hydrogen phosphate, 24%; 2,4-dichloromandelic acid, 24%; [1-(2,4-dichlorophenyl)-ethyl-ß-D- glucopyranosid]uronic acid, 2,4-dichlorophenylethanediol glucuronide and 2,4-dichlorobenzoyl glycine (Hutson, 1969; Annex I, reference 17). Pretreatment of CFE rats for 12 days with 200 ppm dieldrin, an inducer of the monooxygenase system, afforded a 10-fold protection against the acute toxic effects of chlorfenvinphos. At a dose of 2.5 mg/kg bw chlorfenvinphos, dieldrin pretreatment brought about minimal changes in the metabolic profile; however, in rats treated with 13 mg/kg bw chlorfenvinphos, animals that had not received dieldrin pretreatment showed clinical signs of intoxication, while the pretreated rats had a four-fold increase in the yield of de-ethyl-chlorfenvinphos and more rapid urinary excretion (66% compared with 16% without pretreatment during the first 12 h) (Hutson & Wright, 1980). The acute oral toxicity of chlorfenvinphos in Fischer 344 rats was also reduced by pretreatment with chlorfenvinphos. Protection was seen after 8 h and became maximal 24 h after oral pretreatment at a dose of 15 mg/kg bw. A maximal three-fold increase in the LD50 was seen, but there were no changes in the type of toxic signs or time of death. The protection was related to a decrease in the plasma concentration of chlorfenvinphos and to reduced inhibition of brain cholinesterase. The same single oral pretreatment increased chlorfenvinphos metabolism to 178% in the hepatic microsomal fraction and increased various indicators of cytochrome P450-related biotransormation activity. Pharmacokinetic analysis of protection against the acute toxicity of succeeding doses suggested that the decrease in plasma concentration was correlated to the induction of the hepatic biotransformation and possibly to a greater uptake of chlorfenvinphos by the liver and reduced uptake by the intestines (Ikeda et al., 1990, 1991, 1992). (c) Effects on enzymes and other biochemical parameters The de-ethylation of radiolabelled chlorfenvinphos in vitro was investigated in liver slices from mice, rats, rabbits and dogs. In all of the preparations, chlorfenvinphos was converted to 2-chloro-1-(2,4-dichlorophenyl)vinylethyl hydrogen phosphate. The catalysing enzyme was located in the microsomal fraction of liver homogenates and was found to be dependent on NADPH and oxygen. The mechanism of dealkylation involved hydroxylation of the alpha-carbon atom of an alkyl group, which was removed as the corresponding aldehyde. An inverse correlation was observed between the oral LD50 and the rate of de-ethylation, the conversion rates being 1 in rats, 8 in mice, 24 in rabbits and 88 in dogs (Donninger et al., 1972). The rates of oxidative metabolism (O-de-ethylation) by liver preparations from rats, rabbits and human beings in vitro have also been compared. As in the study described above, rats had a much lower conversion rate than rabbits. When the results were expressed in terms of cytochrome P450, detoxification of chlorfenvinphos by human liver enzymes was almost as effective as that by rabbit enzymes (Hutson & Logan, 1986). A proposed metabolic pathway is shown in Figure 1. 2. Toxicological studies (a) Acute toxicity A striking intra-species difference in acute toxicity is seen, the oral LD50 values being 9.6-39 mg/kg bw for rats, 117-200 mg/kg bw for mice, 125-250 mg/kg bw for guinea-pigs, 300-1000 mg/kg bw for rabbits and > 12 000 mg/kg bw for dogs. The dermal LD50 was 30-108 mg/kg bw for rats and 412-4700 mg/kg bw for rabbits. The symptoms recorded were typical of anticholinesterase activity (Annex I, reference 17). In male and female Charles River CD rats, the oral LD50 of 93.1% pure chlorfenvinphos was 31 mg/kg bw and the dermal LD50 was 420 mg/kg bw. The effects observed included tremor, fasciculation, salivation, splayed gait, unkempt appearance, chromodacryorrhoea, piloerection, hunched posture, salivation, hypothermia, tachypnoea and twitching (Gardner, 1992). Pretreatment of rats (strain not specified) with 200 ppm dieldrin (equivalent to 10 mg/kg bw per day) for 12 days afforded a 10-fold protection against the acute oral toxic effect of chlorfenvinphos (Hutson & Wright, 1980). (b) Short-term toxicity Mice Groups of 10 male and 10 female NMRI mice were treated with chlorfenvinphos (purity, 92.8%) at doses of 0, 1, 10, 100 or 1000 ppm in the diet (equal to 0.18, 1.9, 18 or 188 mg/kg bw per day for males and 0.21, 2.1, 21 or 226 mg/kg bw per day for females) for 28 days. Treatment did not affect survival, body weight or ophthalmoscopic end-points, and macroscopic and microscopic examinations showed no effect. Females at the highest dose had a slight but significant increase in food consumption during the last two weeks of the study, but relative food consumption was not significantly increased. The concentration of serum albumin was slightly increased in males fed 100 or 1000 ppm, but the increase was not related to dose; the total protein concentration was increased in males fed 1000 ppm. Plasma cholinesterase activity was inhibited in both males and females, by 18-19% in those fed 10 ppm, by 79-82% in those treated with 100 ppm and by 94-95% in those at 1000 ppm. Erythrocyte cholinesterase activity was inhibited by 30% in males fed 100 ppm and by 65-66% in males and females fed 1000 ppm. Brain cholinesterase activity was inhibited by 50-54% in animals of each sex fed 1000 ppm; in females, activity was inhibited by about 30% in those fed 100 ppm, by 20% in those fed 10 ppm and by 26% in those fed 1 ppm (not related to dose); in males, 8% inhibition was seen in those fed 100 ppm and 22% in those fed 10 ppm. At 1000 ppm, males had increased relative weights of liver and kidney, and females showed increased relative weights of liver and heart. The NOAEL was 1 ppm, equal to 0.18 mg/kg bw per day (Tennekes et al., 1991). Dogs In a range-finding study, groups of two male and two female pure-bred beagle dogs were fed diets containing 0, 3, 100 or 3000 ppm chlorfenvinphos (equal to 0.12, 3.9 or 105 mg/kg bw per day) for four weeks. No effect was seen on mortality, clinical signs, body weight or ophthalmoscopic or haematological end-points; urinalysis and macroscopic and microscopic examinations also showed no effect. The food intake of two dogs fed 3000 ppm was reduced during the first week of treatment and remained reduced in one dog throughoutthestudy. In dogs fed 100 or 3000 ppm, a dose-related reduction in plasma cholinesterase activity was observed; those fed 3000 ppm also had a reduction in erythrocyte cholinesterase activity and a slight increase in liver weight (Allen et al., 1992). Groups of four male and four female pure-bred beagle dogs were fed diets containing 0, 3, 100 or 3000 ppm chlorfenvinphos (equal to 0.1, 2.8 or 92 mg/kg bw per day for males and 0.1, 3.1 or 93 mg/kg bw per day for females) for 52 weeks. No effects were found on mortality, clinical signs, ophthalmoscopic or haematological parameters or brain cholinesterase activity; urinalysis and macroscopic and microscopic examinations also showed no effect. Food consumption of animals fed 3000 ppm was reduced during the first four to five weeks, and body-weight loss was recorded for most of these animals during the first five to six weeks of treatment; however, both food consumption and body weight subsequently returned to normal. In males fed this dose, the plasma magnesium concentration was decreased at 52 weeks; in females, the plasma chloride concentration was increased at 26 and 52 weeks, and albumin was decreased at 4, 13 and 52 weeks. Plasma cholinesterase activity was inhibited by 71-95% in dogs fed 100 or 3000 ppm, and the effect was dose-related. In animals fed 3 ppm, a significant 31% reduction in plasma cholinesterase activity was observed in males at 52 weeks and a 7-10% reduction in females at weeks 8-13. Erythrocyte cholinesterase activity was markedly reduced (75-83%) in animals of each sex fed 3000 ppm from week 4 onwards. At this dose, the relative adrenal weight was increased in males and the relative thyroid weight was increased in females. The NOAEL in this study was 100 ppm, equal to 2.8 mg/kg bw per day (Allen et al., 1993). Figure 1. Metabolism of chlorfenvinphos in mammals(c) Long-term toxicity and carcinogenicity Mice In a study designed to determine carcinogenic potential, chlorfenvinphos (purity, 92.8%) was administered for 24 months to groups of 74 male and 74 female NMRI mice at 1, 25 or 625 ppm in the diet, equal to 0.15, 3.7 or 93 mg/kg bw per day for males and to 0.2, 5.0 or 119 mg/kg bw per day for females. Twelve mice of each sex from each group were sacrificed after 52 weeks of treatment; all females were killed after 90 weeks of treatment and all males after 104 weeks. Clinical signs, food consumption, body weight, differential leukocyte count, cholinesterase activity and organ weights were observed, and macroscopic and microscopic examinations were undertaken. Plasma cholinesterase activity was inhibited in animals of each sex fed 25 ppm (38-50% inhibition) or 625 ppm (87-96%). In males and females fed 625 ppm, erythrocyte cholinesterase activity was decreased by 18-66% and brain cholinesterase activity by 25-46%. Microscopic examination at termination of the experiment showed changes in the adrenal cortex of males fed 625 ppm, which included increased incidence and intensity of ceroid pigment and focal hypertrophy and increased severity of nodular hyperplasia. Tumour incidence was not enhanced. No effects were observed at 25 ppm, equal to 3.7 mg/kg bw per day (Schmid et al., 1993). Rats In a study of both chronic toxicity and carcinogenicity, groups of 50 male and 50 female Wistar rats were fed chlorfenvinphos (purity, 90.5%) at 0.3, 1, 3 or 30 ppm in the diet (equivalent to 0.015, 0.05, 0.15 or 1.5 mg/kg bw per day) for 24 months. The control group consisted of 100 rats of each sex. Clinical signs, body weight and food consumption were observed. At termination, organ weights, haematological parameters and the results of clinical chemistry, urinalysis and macroscopic and microscopic examinations were recorded. Additional groups of 12 rats of each sex were examined after 6, 12 and 18 months for haematological and clinical chemical parameters including cholinesterase activity; after sacrifice, the organs were weighed and animals fed 0, 0.3 or 30 ppm chlorfenvinphos were examined macroscopically and histologically. Plasma cholinesterase activity was inhibited after six months in all treated animals but thereafter only in rats fed 3 or 30 ppm. In males and females fed 30 ppm, erythrocyte cholinesterase activity was inhibited by about 60% and brain cholinesterase activity by about 45%; erythrocyte cholinesterase activity was inhibited by about 17% in females fed 1 or 3 ppm after 12 months' exposure. In all groups and at all intervals during the study, minor but significant changes were seen in the concentrations of alpha-, ß- and gamma-globulins. No dose-related or significant macroscopic or histological changes were observed, and tumour incidences were not enhanced. The NOAEL in this study was 3 ppm, equivalent to 0.15 mg/kg bw per day (Pickering, 1980). (d) Reproductive toxicity Rats In a range-finding study, 10 male and 10 female Wistar rats were exposed to 0, 5, 25 or 125 ppm chlorfenvinphos (purity, 92.8%) mixed with microgranulated feed (equivalent to 0.25, 1.3 or 6.3 mg/kg bw per day) during a three-week period before mating and throughout mating, gestation and lactation. After they had been weaned, F1 pups were reared for a further week on their test diets. The body-weight gain of males and females fed 125 ppm was slightly reduced. Inhibition of erythrocyte and brain cholinesterase activity was observed in females fed 5, 25 or 125 ppm and in males fed 25 or 125 ppm. Plasma cholinesterase activity was inhibited only in females. Reproductive parameters, including mating, gestation, parturition, pregnancy rate, lactation and litter characteristics, were not affected (Dotti et al., 1993a). In a two-generation study of reproductive toxicity, groups of 25 male and 25 female Wistar rats were treated with 0, 1, 10 or 100 ppm chlorfenvinphos (purity, 92.8%) mixed with microgranulated diet, equivalent to 0, 0.05, 0.5 or 5 mg/kg bw per day. The diets were fed to the parental generation during growth, mating, gestation and lactation and to one litter per generation. Clinical signs, body weight, food consumption, cholinesterase activity (only in the parent generation), mating performance and reproductive parameters were observed. The parents were examined histologically; the pups were sexed and examined for gross malformations, and the numbers of stillborn and live pups were recorded. One male fed 100 ppm died on day 10, and reduced body-weight gain and food consumption were noted in males and females of the parental and F1 generations at this dose. A slight decrease in food consumption was observed in F1 males and parental and F1 females fed 10 ppm. Plasma cholinesterase activity was decreased by 14-78% in males and females fed 10 or 100 ppm, and erythrocyte cholinesterase activation was inhibited by 35-63% in males and females fed 100 ppm. Brain cholinesterase activity was decreased by 32% in males and 38% in females fed 100 ppm and by 17% in females fed 10 ppm. In the parental generation, post-implantation loss was higher in animals fed 10 or 100 ppm than in controls, and postnatal loss was increased in rats fed 100 ppm; breeding loss up to the end of lactation was higher in animals fed 10 or 100 ppm. In the F1 generation, breeding loss was higher than that in controls in animals fed 100 ppm. At this dose, retardation of body-weight gain was noted in both F1 and F2 pups during the lactation period. The NOAEL in this study was 1 ppm, equivalent to 0.05 mg/kg bw per day (Dotti et al., 1993b). (e) Embryotoxicity and teratogenicity Rats Groups of 25 pregnant specific-pathogen-free CrL:COBS CD(SD)BR rats were treated by gavage with chlorfenvinphos (purity, > 90%) at 0, 0.3, 1 or 3 mg/kg bw per day in corn oil on days 6-15 of pregnancy. The doses were determined in a preliminary study in which doses of 3, 6 and 10 mg/kg bw per day caused dose-related loss of body weight. The rats were sacrificed on day 20 of gestation. No effects were found on food consumption, pregnancy rate or litter parameters, including numbers of fetal deaths, implants, corpora lutea and pre- and post-implantation losses, litter weight, fetal weight and gravid uteri, or on the incidences of malformations, visceral and skeletal anomalies and variations. The only clinical effect seen was the occurrence of small faeces in animals fed 3 mg/kg bw per day from days 3-4 until the end of treatment; body-weight gain was slightly decreased in animals fed 3 mg/kg bw per day on days 6-20. There was no indication of irreversible structural effects. The NOAEL for maternal toxicity in this study was 1 mg/kg bw per day, and that for developmental toxicity was 3 mg/kg bw per day (Mayfield & John, 1986). Rabbits Pregnant banded Dutch rabbits (21-22 per group) received three capsules containing chlorfenvinphos (purity not specified) at 25, 50 or 100 mg/kg bw per day in corn oil (total volume, 200 µl) on days 6-18 of gestation. A control group of 32 females was used. The rabbits were sacrificed on day 28 of gestation. No effects were found on mortality, clinical signs or mean numbers of corpora lutea, implantations, resorptions or fetal deaths. Body-weight gain and food consumption were reduced in dams fed 100 mg/kg bw per day on days 6-12 of gestation. Plasma and erythrocyte cholinesterase activities were decreased in dams at all doses, erythrocyte activity being decreased by 55-75%. Brain cholinesterase activity was not measured. Pre-implantation losses occurred in dams fed 25 or 100 mg/kg bw per day. No differences in the incidences of visceral or skeletal malformations or variations between the groups were observed that could be attributed to treatment. There was no indication of irreversible structural effects. No NOAEL could be established for maternal toxicity; the NOAEL for embryo- and fetotoxicity was 100 mg/kg bw per day (Dix et al., 1979). In a limited experiment with rats, rabbits and hamsters, doses of 50 mg/kg bw per day and higher increased the incidence of open eyes and oedema in hamster dams and decreased the weight and length of hamster fetuses (Dzierzawski & Minta, 1979). (f) Genotoxicity The results of tests for the genotoxicity of chlorfenvinphos are summarized in Table 1. (g) Special studies (i) Skin and eye irritation and skin sensitization Chlorfenvinphos (purity, 93.1%), administered to three New Zealand white rabbits as a 4-h semi-occluded topical application of 0.5 ml undiluted compound, caused very slight erythema within 1 h after removal of the dressing. The erythema persisted for at least 48 h after treatment, but all overt effects disappeared within seven days. The compound was considered slightly irritating to rabbit skin (Gardner, 1992). The skin sensitizing potential of chlorfenvinphos was examined in two studies in guinea-pigs. In the first study, 10 male and 10 female Tunstall breeding unit 'P' guinea-pigs received an intradermal injection of 0.5% (mol/volume) chlorfinvenphos (purity, 91.3%) during the induction period, a topical application of the undiluted test compound one week later and a challenge application of undiluted compound two weeks after that. A control group of five male and five female animals was used. No effect was seen in the test animals 24 or 48 h after removal of the challenge patches (Rose, 1982). In the second study, 10 male and 10 female Dunkin-Hartley guinea-pigs were given an intradermal induction dose of 2% (mol/volume) chlorfenvinphos (purity, 93.1%) and undiluted compound for the topical and challenge applications. A control group of five male and five female animals was used. Ten of 20 test animals showed a positive response after 24 h and nine of the same animals after 48 h. None of the control animals responded (Gardner, 1992). Instillation of 0.1 ml undiluted chlorfenvinphos (purity, 93.1%) into the eyes of three New Zealand white rabbits caused marked constriction of the pupil, slight conjunctival redness, slight chemosis and ocular discharge. All signs were reversed within 24 h. The compound is not considered to irritate the eye (Gardner, 1992). Table 1. Results of tests for the genotoxicity of chlorfenvinphos End-point Test system Concentration Purity Results Reference of chlorfenvinphos (%) In vitro Reverse mutation S. typhimurium TA98, 25-2025 µg/platea NR Negativeb,c Arni & Müller, 1979 100, 1535, 1537 Reverse mutation E. coli WP2, WP2 hcr, 250 µg/plate NR Negatived Shirasu et al., 1976 S. typhimurium TA1535, 1536, 1537, 1538 Reverse mutation S. typhimurium TA98, Up to 5000 µg/plate NR Negativec,d Moriya et al., 1983 100, 1535, 1537, 1538 Reverse mutation S. typimurium TA100 Up to 5000 µg/plate NR Positivec,e Moriya et al., 1983 Reverse mutation S. typhimurium TA98, Up to 5000 µg/plate NR Negativec,e Moriya et al., 1983 1535, 1537, 1538 Reverse mutation S. typhimurium TA98, 0.2-2000 µg/plate 90.5 Negativeb,c Brooks, 1978 100, 1538 Reverse mutation E. coli B/r WP2 NR > 99 Negativec,d Dean, 1971 Reverse mutation E. coli WP2, WP2 uvrA 0.2-2000 g/plate 90.5 Negativec Brooks, 1978 Reverse mutation E. coli WP2 hcr Up to 5000 µg/plate NR Negativeb,c Moriya et al., 1983 Mitotic gene Saccharomyces 0.001-5.0 mg/ml; toxic 90.5 Negativec,d Brooks, 1978 conversion cerevisiae JD1 at 0.5, 1.0, 5.0 mg/ml Chromosomal Human lymphocytes 18.8-300 µg/platea,d 93.1 Negativeb,c Strasser & Arni, aberrations 15.6-250 µg/platea,e 1988 Table 1 (contd) End-point Test system Concentration Purity Results Reference of chlorfenvinphos (%) In vivo Chromosomal Chinese hamster 25 or 50 mg/kg bw 90.5 Negativec Dean, 1978 aberrations bone marrow per day for 2 days Dominant lethal CD1 mice (male) 10, 20 or 40 mg/kg bw 90.5 Negativec Dean & Hend, 1978 mutation in DMSO NR, nor reported; DMSO, dimethyl sulfoxide a It is not clear whether chlorfenvinphos was tested: the substance was coded C8949. b In the presence and absence of metabolic activation c Positive controls were used. d In the absence of metabolic activation e In the presence of metabolic activation (ii) Cholinesterase inhibition and related pharmacological and neurotoxic effects After oral administration of chlorfenvinphos at 15 mg/kg bw to Fischer 344 rats, brain cholinesterase activity was maximally inhibited (to about 20% of the control value) at 4 h; the inhibition lasted more than 24 h after administration (Ikeda et al., 1990). Groups of 10 male and 10 female Wistar rats were fed dietary concentrations of 0, 0.3, 1, 3 or 30 ppm chlorfenvinphos for four weeks, or received the same treatments and then received control diet for four weeks. Administration of 30 ppm resulted in decreased activity of plasma, erythrocyte and brain cholinesterase. Feeding of the control diet for four weeks reversed the inhibition of plasma, erythrocyte and brain cholinesterase activities, resulting in values of 100, 88 and 91%, respectively (Pickering, 1978). In a comparison of the lethality of chlorfenvinphos after intravenous and oral administration, Sprague-Dawley rats given lethal doses by either route showed typical signs of anticholinesterase poisoning and marked reductions in brain and erythrocyte cholinesterase activity. Anaesthetized and conscious rats administered chlorfenvinphos intravenously had hypertension and apnoea. After oral administration, breathing stopped before cessation of heart beats (Takahashi et al., 1991). The effects of chlorfenvinphos on spontaneous electroencephalographic, electromyo-graphic and cholinesterase activity in brain and red blood cells was examined in male Wistar rats. Single oral doses of 1 mg/kg bw had no effect. Doses over 2 mg/kg bw induced dose-related inhibition of the cholinesterase activity in brain and erythrocytes. Maximal inhibition of brain cholinesterase activity was seen 3 h after treatment and lasted for more than 72 h. Electroencephalography showed a prominent arousal pattern, and the appearance of slow-wave sleep and para-sleep was markedly depressed. The duration of the arousal pattern was proportional to the dose; however, the awake-sleep cycle returned to the control rate on the second day, and an increase in para-sleep occurred on the third day (Osumi et al., 1975). In 30 Sprague-Dawley rats fed a diet containing 150 ppm chlorfenvinphos (equivalent to 7.5 mg/kg bw per day) for three months, no effect was seen on the amplitude of the muscular response to nerve stimulation. Electrophysiological effects were signalled firstly by a prolonged negative potential after the direct muscle response to a single nerve impulse and secondly by repetitive activity. These abnormalities became more marked with time. Double and repetitive stimulation reduced or abolished the prolonged negative potential and repetitive activity (Maxwell & LeQuesne, 1982). Oral administration to male Wistar rats of chlorfenvinphos at 50% of the LD50 caused maximal inhibition of brain cholinesterase activity (to about 15% of the control value) between 2 and 3 h after treatment. A significant reduction in brain norepinephrine level was seen within 15 min, but no consistent diminution was seen subsequently. During exposure at 5% of the LD50 for 12 weeks, brain cholinesterase activity was inhibited to 60-80% of control values and brain norepinephrine content was significantly decreased, with maximal depletion at week 8 (Brzezinski & Wysocka-Paraszewska, 1980). Cholinesterase activity in plasma, erythrocytes and different parts of the brain, open-field behaviour and response to change in a 'T' maze were investigated in two groups of male Wistar rats after administration of a single intraperitoneal injection of 1 or 3 mg/kg bw chlorfenvinphos. Cholinesterase activity was strongly inhibited, to a similar degree, in the blood and brain, with a mean inhibition of 80% in rats at the high dose and 50% in those at the low dose 3 h after treatment. Reversal of the inhibition proceeded at similar rates in the blood and brain and was complete within 96 h with the low dose and within 14 days with the high dose. No changes in negotiating the 'T' maze that suggested impairment of short-term memory were observed up to 14 days after treatment. In the open-field test, a decrease in short-term memory was manifested by the absence of increased locomotor and exploratory activity in response to a new object, only in rats administered 3 mg/kg bw. This finding suggests that behavioural disturbances last longer than recovery of cholinesterase activity (Socko et al., 1989). (iii) Hormone concentrations In Wistar rats given chlorfenvinphos as a single oral dose of 6.15 mg/kg bw, a significant increase in corticosterone concentration was seen after 1 and 3 h and in aldosterone concentration 1-6 h after treatment. Plasma corticosteroid levels were maximal within 1 h, when brain cholinesterase activity was only slightly inhibited (Osicka-Koprowska et al., 1984). (iv) Porphyrigenic potential Chlorfenvinphos induced porphyria in primary cultures of chicken embryo liver cells. Its porphyrigenic potential was markedly increased by prior treatment of the cells with a compound that induces drug metabolizing enzymes (Koeman et al., 1980). 3. Observations in humans Workers in India were studied while applying chlorfenvinphos to paddy rice over a period of six days; they were not wearing protective clothing. Workers applying an emulsifiable concentrate had decreased plasma cholinesterase activity but no change in erythrocyte cholinesterase activity. Workers applying granules containing 10% chlorfenvinphos had no reduction in cholinesterase activity (Blok et al., 1977). A group of 33 workers employed in a chlorfenvinphos manufacturing plant were monitored medically for eight years (1967-75). Two control groups were used. The exposed group had slightly lower haemoglobin levels and slightly lower cholinesterase activity in plasma and erythrocytes. Some changes in neurophysiological parameters (lower electromyographic voltages, associated with slower conduction velocities) were observed, but these were reversed when the workers were removed from exposure (Ottevanger, 1976). Workers involved in the manufacture of chlorfenvinphos were monitored in 1985-86 in a routine programme in which blood and urine were collected and cholinesterase activity was measured in whole blood, plasma and erythrocytes. A few cases of depressed plasma cholinesterase were recorded, which were not, however, correlated with increases in the urinary concentrations of diethylphosphate and monoethylphosphate metabolites (Eadsforth, 1987). Comments The main step in the biotransformation of chlorfenvinphos is detoxification by de-ethylation into the corresponding phosphodiester; microsomal enzymes play an important role. Interspecies differences in the rate of metabolism of chlorfenvinphos were found in vitro. Oxidative metabolism by human liver enzymes was comparable to that of rabbit liver enzymes. Single oral doses of chlorfenvinphos were very toxic to rats, toxic to mice and guinea-pigs and moderately to slightly toxic to rabbits and dogs. The clinical signs observed were consistent with cholinesterase inhibition and included tremors, fasciculation and salivation. Pretreatment with enzyme inducers can increase the acute oral LD50. Interspecies differences in the acute oral LD50 reflect the activity of the hepatic metabolizing enzymes involved in the detoxification of chlorfenvinphos. WHO (1992) has classified chlorfenvinphos as extremely hazardous. In a four-week study in which mice were fed 0, 1, 10, 100 or 1000 ppm chlorfenvinphos in the diet, plasma and erythrocyte cholinesterase activities were inhibited in animals fed 100 or 1000 ppm. Brain cholinesterase activity was inhibited in males fed 10 or 1000 ppm and in females at all doses; thus, no NOAEL could be established for female mice. The NOAEL in males was 1 ppm, equal to 0.18 mg/kg bw per day. In a one-year study in which dogs were fed 0, 3, 100 or 3000 ppm in the diet, inhibition of erythrocyte cholinesterase activity and increased relative adrenal weight were seen in males; in females, increased relative thyroid weight was seen in those fed the highest dose. The NOAEL was 100 ppm, equal to 2.8 mg/kg bw per day. In a study of the carcinogenicity of chlorfenvinphos, in which mice were fed 0, 1, 25 or 625 ppm in the diet, no increase in tumour incidence was observed. Erythrocyte and brain cholinesterase activity was inhibited in mice fed the highest dose. The NOAEL was 25 ppm, equal to 3.7 mg/kg bw per day. In a two-year study of toxicity and carcinogenicity in rats fed diets containing 0, 0.3, 1, 3 or 30 ppm, erythrocyte and brain cholinesterase activity was inhibited in those fed 30 ppm. There was no evidence of carcinogenicity. The NOAEL was 3 ppm, equivalent to 0.15 mg/kg bw per day. In a two-generation study of reproductive toxicity in rats fed diets containing 0, 1, 10 or 100 ppm, reduced brain cholinesterase activity, post-implantation loss and breeding losses were observed in animals fed 10 or 100 ppm. The NOAEL was 1 ppm, equivalent to 0.05 mg/kg bw per day. In two studies of teratogenicity--one in rats and one in rabbits--doses that were maternally toxic were not embryotoxic and there was no indication of teratogenicity. In rats, the NOAEL was 1 mg/kg bw per day for maternal toxicity and 3 mg/kg bw per day, the highest dose tested, for developmental toxicity. In rabbits, no NOAEL could be established for maternal toxicity (< 25 mg/kg bw); the NOAEL for developmental toxicity was > 100 mg/kg bw per day. Chlorfenvinphos was mutagenic in only one study, in Salmonella typhimurium TA100 in the presence of an exogenous metabolic system at doses of at least 1000 µg per plate. It was inactive in other bacterial tests and did not induce mitotic conversion in yeast or chromosomal aberrations in human lymphocytes in vitro. Chlorfenvinphos did not induce chromosomal aberrations in Chinese hamster bone-marrow cells or dominant lethal effects in male mice in vivo. The Meeting concluded that chlorfenvinphos was not genotoxic. Delayed neurotoxicity in chickens has not been evaluated. Observations in humans were not suitable for use in estimating an ADI. An ADI was established on the basis of an NOAEL of 0.05 mg/kg bw per day in a two-generation study of reproductive toxicity in rats and a 100-fold safety factor. Toxicological evaluation Levels that cause no toxic effect Mouse: 25 ppm, equal to 3.7 mg/kg bw per day (two-year study of carcinogenicity) Rat: 3 ppm, equivalent to 0.15 mg/kg bw per day (two-year study of toxicity and carcinogenicity) 1 ppm, equivalent to 0.05 mg/kg bw per day (two-generation study of reproductive toxicity) 1 mg/kg bw per day (maternal toxicity in a study of teratogenicity) Rabbit: < 25 mg/kg bw per day (maternal toxicity in a study of teratogenicity) Dog: 100 ppm, equal to 2.8 mg/kg bw (one-year study of toxicity) Estimate of acceptable daily intake for humans 0-0.0005 mg/kg bw Studies that would provide information useful for continued evaluation of the compound 1. Study of delayed neurotoxicity in chickens, with estimation of neuropathy target esterase 2. Further observations in humans 3. Studies of pharmacokinetics in mammals in vivo References Allen, T.R., Corney, S.J., Frei, T., Luetkemeier, H., Biedermann, K. & Springall, C.J. 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See Also: Toxicological Abbreviations Chlorfenvinphos (ICSC) Chlorfenvinphos (WHO Pesticide Residues Series 1) Chlorfenvinphos (Pesticide residues in food: 1984 evaluations)