Pesticide residues in food - 2002 - Joint FAO/WHO Meeting on Pesticide Residues
First draft prepared by
Tim Marrs
Chief Toxicologist, Food Standards Agency, London, England
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Biochemical aspects: Effects on enzymes and other biochemical parameters |
Oxydemeton-methyl (S-2-ethylsulfinylethyl O,O-dimethyl phosphorothioate) was evaluated toxicologically by the JMPR in 1973, 1982, 1984 and 1989 (Annex 1, references 21, 39, 43 and 58). In 1989, the Meeting established a group ADI of 0–0.0003 mg/kg bw for demeton-S-methyl and related compounds, which included oxydemeton-methyl, demeton-S-methyl and demeton-S-methylsulfone. Residues of these compounds are measured after oxidation to demeton-methyl.
Rats
Groups of five Sprague-Dawley-derived rats of each sex received diets containing oxydemeton-methyl (purity, 54.8%) at a concentration of 0, 3, 9 or 50 ppm for 14 days, resulting in intakes of 0, 0.22, 0.6 and 3.2 mg/kg bw per day for males and 0.21, 0.56 and 3 mg/kg bw per day for females. Rats were observed for deaths and clinical signs, and body weight and food consumption were measured. Plasma and erythrocyte cholinsterase activity was measured before the start of the study and after 7 and 14 days. The animals were examined grossly at termination, and plasma, erythrocyte and brain cholinesterase activity was determined by a modication of the Ellman et al. (1961) method (Hackathorn et al., 1983). No deaths or clinical signs of toxicity were observed. Reduced body-weight gain was seen for males at 9 and 50 ppm; no such effect was seen in females. No treatment-related effects were seen on gross examination. In males at 9 and 50 ppm, cholinesterase activity in both plasma and erythrocytes was decreased by > 20% at day 7 in comparison with concurrent controls. At 14 days, the plasma activity in males was decreased by > 20% only at the highest dietary concentration, but erythrocyte cholinesterase activity was decreased by > 20% in males at 9 and 50 ppm. In females, plasma cholinesterase activity at day 7 was inhibited by > 20% in comparison with concurrent controls only in those at the highest dietary concentration, while erythrocyte activity was inhibited by > 20% at the two higher concentrations. At 14 days, the activity of the enzyme in plasma and erythrocytes was inhibited by > 20% in females at the two higher dietary concentrations. Brain cholinesterase activity was < 80% that of concurrent controls in animals of each sex at 9 and 50 ppm, and brain cholinesterase activity was 80% that of concurrent controls in males at 3 ppm. The NOAEL was 3 ppm, equal to 0.21 mg/kg bw per day, on the basis of inhibition of erythrocyte and brain cholinesterase activity at the next highest dose and reduced body-weight gain in males (Hayes, 1987a).
Oxydemeton-methyl (technical-grade; purity, 94.6%) was given to Sprague-Dawley-derived rats by gavage at a dose of 0, 0.15, 0.45 or 2.5 mg/kg bw for 14 days. The animals were observed daily for deaths and clinical signs of toxicity, and body-weight gain and food intake were measured. Plasma and erythrocyte cholinesterase activity was measured before the start of the study and after 7, 14 and 21 days, and brain cholinesterase activity was determined at termination. A modification of the Ellman et al. (1961) method (Hackathorn et al., 1983) was used in these assays. The animals were examined grossly at termination. No deaths occurred, and no clinical signs of toxicity were observed. Body weight and weight gain were unaffected by treatment. Depressions in plasma, erythrocyte and brain cholinesterase activity > 20% compared with concurrent controls were found in both sexes at 2.5 mg/kg bw per day. Less consistent inhibition was seen at 0.45 mg/kg bw per day. Plasma cholinesterase activity was not significantly inhibited at the lowest dose, while inhibition by 9–31% was seen at the intermediate dose and greater inhibition at the highest dose. No significant (> 20%) depression of erythrocyte cholinesterase activity was seen in either sex at either time at the lowest dose. In males at 0.45 mg/kg bw, the activity was 80% that of concurrent controls at 7 days, and 71% that of concurrent controls at 14 days. In females at this dose, inhibition of erythrocyte cholinesterase did not reach 20% of concurrent control activity at either time. At the highest dose, erythrocyte cholinesterase activity was consistently inhibited by 51–57% in comparison with concurrent controls. Brain cholinesterase activity was significantly inhibited at 0.45 mg/kg bw per day, the activity in males being 71% that of concurrent controls and that in females being 77%. Even greater inhibition was seen at 2.5 mg/kg bw per day, the activity in males being 32% that of concurrent controls and that in females 34%. Brain cholinesterase activity was not significantly inhibited at the lowest dose. The NOAEL was 0.15 mg/kg bw per day on the basis of inhibition of erythrocyte and brain cholinesterase activity at the next highest dose (Hayes, 1987b).
Technical-grade hexachlorocyclohexane, oxydemeton-methyl 25 EC (purity unspecified) or both were applied in acetone to the skin of groups of 30 female Wistar rats. A control group of 30 rats received acetone alone. The hexachlorocyclohexane used was predominantly the alpha isomer (72%) and also contained 12% beta and 11% gamma isomer. The animals were killed after 7, 15 or 30 days of treatment. At sacrifice, blood was taken for clinical chemistry, including estimation of erythrocyte cholinesterase activity. Selected organs were weighed and processed for histopathological examination, and cholinesterase activity was measured in brain homogenate. Treatment-related changes in clinical chemical parameters were observed with both pesticides. Concurrent dermal administration of hexachlorocyclohexane did not significantly alter the inhibitory effect of oxydemeton-methyl on erythrocyte or brain cholinesterase activity. Both substances caused histopathological changes in the liver, fatty infiltration and hypertrophy in the case of hexachlorocyclohexane and necrosis in the case of oxydemeton-methyl, while the combined treatment produced more severe effects. Both compounds and the combined treatment caused changes to the Purkinje cells of the cerebellum (Raizada et al., 1993). The design of the study precluded any conclusion about an interaction between the two pesticides on the end-points studied, as it was not designed to identify a NOAEL.
Dogs
In a study evaluated by the 1984 JMPR, groups of six male and six female beagle dogs were given oxydemeton-methyl (purity, 51.1% [see Hoffman & Rühl-Fehlert, 1987]) by gavage at a dose of 0, 0.025, 0.25 or 2.5 mg/kg bw per day, resulting in intakes of 0, 0.0125, 0.125 and 1.25 mg/kg bw (see Hoffman & Rühl-Fehlert, 1987; Hartmann, 2002). The animals were examined daily, and pupil reactions, corneal reflex, patellar reflex and stretch, righting and bending reflexes were tested in weeks 3, 6, 13, 26, 39 and 52. Ophthalmic examination was carried out before the start of the study and during weeks 13, 26, 39 and 52. Haematological and clinical chemical analyses were undertaken on blood samples drawn in weeks 3, 6, 13, 26, 39 and 52. These measurements included estimation of plasma and erythrocyte cholinesterase activity and, at termination, brain cholinesterase activity, by the method of Ellman et al. (1961). Urine was also analysed. At examination post mortem, selected organs were weighed and processed for histopathological examination, and bone marrow smears were examined by the May-Grünwald-Giemsa staining technique.
No deaths occurred, and no clinical sign of toxicity was seen at any dose, nor was any abnormality found on neurological or ophthalmic examination. Haematological, clinical chemical (except cholinesterase activity) and urine end-points showed no treatment-related-change. At 2.5 mg/kg bw per day, moderate inhibition of plasma cholinesterase was seen from 3 weeks onwards; e.g. at 6 weeks, the activity was 62% that of concurrent controls in males and 59% in females. Erythrocyte cholinesterase activity was inhibited at the highest dose at 3 weeks (activity, 63% of concurrent controls in males and 60% in females) and remained significantly depressed in animals at this dose throughout the study. Although depressions of about 20% were seen on occasion in animals at the intermediate dose (e.g. by 80% in males and 76% in females at 26 weeks), the activity in this group was at most marginally depressed, and significant depression was not seen at earlier times. Brain cholinesterase activity was 54% that of concurrent controls in males and 53% in the females at the highest dose; depressions in activity > 20% were not seen at lower doses. Intergroup differences in adrenal, lung and ovarian weights were seen (see Hoffman & Mager, 1990), but these did not appear to be treatment-related. No histopathological change related to treatment was recorded. The NOAEL was 0.025 mg/kg bw per day, corresponding to 0.0125 mg/kg bw per day expressed as oxydemeton-methyl active ingredient, on the basis of inhibition of erythrocyte cholinesterase activity. The NOAEL for inhibition of erythrocyte and brain cholinesterase activity was 0.25 mg/kg bw, corresponding to 0.125 mg/kg bw per day as active ingredient (Hoffman & Rühl, 1984; Hoffman & Rühl-Fehlert, 1987; Hoffman & Mager, 1990; Eigenberg, 1993).
Mice
Groups of 50 Crl:CD-1 (ICR)BR mice of each sex were given diets containing technical-grade oxydemeton-methyl (purity, 87.2%) at a concentration of 0, 3, 15 or 50 ppm, equal to 0.5, 2.3 and 7.8 mg/kg bw per day for males and 0.6, 2.9 and 8.9 mg/kg bw per day for females. The mice were examined once or twice daily and examined in more detail for clinical signs of toxicity weekly. Body weight and food consumption were determined weekly and body weight again just before sacrifice. Blood for determination of haematological parameters and plasma and erythrocyte cholinesterase activity was obtained at 12, 18 and 21 months from the first 10 surviving animals of each sex per dietary concentration. At the end of the study at 21 months, brain cholinesterase activity was determined in the first 10 surviving mice of each sex per dietary concentration. Cholinesterase was measured by a modification of the method of Ellman et al. (1961), in which the Ellman reagent was replaced by 6,6’-dithionicotinic acid (Hackathorn et al., 1983). Surviving animals were killed at 21 months and examined grossly. Selected organs were weighed, and designated organs were processed for histopathological examination.
Survival was similar in all groups. Oxydemeton-methyl at the highest dietary concentration was associated with clinical signs (decreased activity, urine staining of fur, rough coat and convulsions) in both sexes. Body weight was decreased in males at the highest dietary concentration only. The food consumption of males was unaffected, whereas females at all concentrations showed small declines. Sporadic differences in haematological variables between treated and control animals were seen, but these were considered to be unrelated to treatment. Plasma cholinesterase activity was depressed by > 20% in comparison with concurrent controls in males at 15 and 50 ppm and females at all dietary concentrations at 12 and 18 months. At termination, plasma cholinesterase activity was depressed in animals of each sex at 15 and 50 ppm. Erythrocyte enzyme activity was depressed by > 20% at 12 and 18 months and at termination in animals of each sex at the two highest dietary concentrations. Brain cholinesterase activity was inhibited in animals at 15 and 50 ppm at termination. No treatment-related effects on organ weights were noted. An increased incidence of cytoplasmic vacuolation of the epididymides was seen at 15 and 50 ppm, the incidences being 5/50 in controls, 4/50 at 3 ppm, 44/50 at 15 ppm and 49/50 at 50 ppm. The NOAEL was 3 ppm,, equal to 0.5 mg/kg bw per day on the basis of depressed erythrocyte and brain cholinesterase activity and an increased incidence of cytoplasmic vacuolation of the epididymides at the next highest dietary concentration (Christenson et al., 1992).
Rats
A two-generation study of reproductive toxicity was carried in which albino CD Sprague-Dawley rats received diets containing oxydemeton-methyl 50% concentrate (actually 54.8% active ingredient) at a concentration of 0, 1, 3, 9 or 50 ppm, equivalent to 0, 0.067, 0.2, 0.6 and 3.3 mg/kg bw per day expressed as active ingredient, or technical-grade oxydemeton-methyl (purity, 94.6%) or methyl isobutylketone at a dietary concentration of 50 ppm, equivalent to 3.3 mg/kg bw per day). The dietary concentrations of oxydemeton-methyl were about 5% less than nominal.
The F0 and F1 parents comprised 35 animals of each sex per dietary concentration, except that the group of F1 parents given 50 ppm of oxydemeton-methyl 50% concentrate consisted of 28 females and 26 males, and that given technical-grade oxydemeton-methyl consisted of 32 females and 30 males. The F0 parents received the test diets from 8 weeks of age and the F1 parents from weaning. Then, F0 parents were mated to produce the F1a and F1b litters, and randomly selected F1b pups became the F1 parents. These were mated to produce the F2a and F2b litters. During the study, adults were evaluated for deaths, and clinical signs were evaluated daily. Body weight was measured weekly during the 10 weeks before mating, and food consumption was measured weekly. The body weights of dams were measured on days 0, 6, 13 and 20–24 of gestation and on days 0, 4, 7, 14 and 21 of lactation, while food consumption was measured twice a week during gestation and lactation. Plasma, erythrocyte and brain cholinesterase activity was measured in 10 F0 and F1 adults of each sex per dietary concentration by a modification of the Ellman et al. (1961) method (Hackathorn et al., 1983). The numbers of live pups and stillbirths were recorded for each litter. Pups were culled on day 4 after birth to yield eight pups per litter. Pups were weighed at birth and on days 4, 7, 14 and 21. Offspring were evaluated for sex ratio, viability and clinical signs. Cholinesterase activity was determined in plasma, erythrocytes and brain from one male and one female pup from, if possible, 10 litters per dietary concentration at the time of culling, i.e. 4 days post partum, and at weaning (21 days post partum). Decedents, pups culled on day 4 and weanlings not selected for mating were examined grossly post mortem. Gross lesions and testes, epididymides, seminal vesicles and prostates from male weanlings were preserved in Bouin solution. After their pups had been weaned, the F0 and F1 parents were killed and necropsied. The body weights and the weights of the testes and ovaries were determined. The ovaries, vagina, cervix, uterus and pituitary were collected for histological examination from females and the testes, epididymides, seminal vesicles and prostate and pituitary glands from males.
Treatment-related clinical signs were found in the adults, consisting of an increased number of moribund F2a and F2b pups given technical-grade oxydemeton-methyl. Body weights were increased during the premating period for F0 females at 50 ppm of 50% concentrate and technical-grade oxydemeton-methyl and decreased at some times in male F0 parents. Decreased body weights were observed in male and female F1 animals given 50 ppm of 50% concentrate and technical-grade oxydemeton-methyl during the premating period. Terminal body weights were affected in males given 50 ppm 50% concentrate and technical-grade oxydemeton-methyl, and F0 females in these groups had increased food consumption. There was no significant effect on the estrus cycle. Both groups given 50 ppm of oxydemeton-methyl had reduced body-weight gain during gestation. No reproductive or developmental effects were seen in animals at 1, 3 or 9 ppm oxydemeton-methyl 50% concentrate or the group given methyl isobutylketone. Litter size was reduced in the F1a and F1b breedings given technical-grade oxydemeton-methyl and in the F1b animals given the 50% concentrate. No difference was seen from controls in the F1a animals given 50 ppm 50% concentrate or the F2a and F2b breedings given 50 ppm 50% concentrate or technical-grade oxydemeton-methyl. There were no treatment-related intergroup differences in the occurrence of stillbirth. Reduced viability during lactation were seen for F1a and F1b pups given technical-grade oxydemeton-methyl and for F2a and F2b pups given 50 ppm of either formulation. Birth weights were unaffected by treatment, but impaired weight gain was seen with 50 ppm of 50% concentrate and technical-grade oxydemeton-methyl in the F1a, F1b, F2a and F2b generations.
Effects on cholinesterase activity were seen in both parents and pups. In the parents, plasma cholinesterase activity was inhibited at 9 ppm in all generations and both sexes, except F2a and F2b males. In female F2a adults, this enzyme was inhibited at 3 ppm during the premating period. Erythrocyte cholinesterase activity was inhibited by > 20% in males at 9 ppm. In females, inhibition by > 20% was observed at dietary concentrations >3 ppm in F0 and F1 adults at termination and in F2a and F2b adults during the premating period. In females, the inhibition was more consistent at higher dietary concentrations. Brain cholinesterase activity was inhibited by > 20% in F0 and F1 adults at termination, at concentrations > 9 ppm in males and > 3 ppm in females. At 3 ppm, 20% inhibition was found in F0 females and 21% inhibition in F1 females. In the pups, depressions in cholinesterase activity were seen more consistently at 21 days than at 4 days; nevertheless, decreased activity was seen at 4 days in both groups given 50 ppm oxydemeton-methyl. At 21 days, depressions of plasma cholinesterase > 20% were seen in F1a and F1b males and females at 9 ppm oxydemeton-methyl 50% concentrate and in F2a females; plasma cholinesterase activity was more consistently inhibited at higher concentrations. Erythrocyte cholinesterase activity was consistently inhibited in male and female F1a, F1b, F2a and F2b pups at 50 ppm of 50% concentrate and technical-grade oxydemeton-methyl. Brain cholinesterase activity was inhibited in male and female F1a, F1b, F2a and F2b pups at 50 ppm 50% concentrate and technical-grade oxydemeton-methyl, but less consistently than the erythrocyte enzyme. Compound-related depressions in erythrocyte or brain cholinesterase were not seen at the lower dietary concentrations. Absolute testis weights were reduced in F0 and F1 males at 50 ppm of both oxydemeton-methyl preparations. No treatment-related effects were seen on organ weights in females. Vacuolation of the epidydimis was seen in males and decreased or absent corpora lutea in the ovaries and implantation sites were observed in females at 50 ppm 50% concentrate and technical-grade oxydemeton-methyl.
The NOAEL for parental toxicity was 1 ppm, equivalent to 0.07 mg/kg bw per day, on the basis of inhibition of brain and erythrocyte cholinesterase activity. The NOAEL for offspring was 9 ppm, equivalent to 0.6 mg/kg bw per day, on the basis of decreased pup weight gain and viability and inhibition of erythrocyte and brain cholinesterase activity. The NOAEL for reproductive toxicity was 9 ppm, equivalent to 0.6 mg/kg bw per day, on the basis of vacuolation of the epididymides, decreased or absent corpora lutea in the ovaries and implantation sites and reduced litter size at 50 ppm (Eigenberg, 1990).
Rats
In a study reviewed by the 1989 JMPR, groups of 45 mated female Charles River COBS CD rats were given oxydemeton-methyl (purity, 90.6%) at a dose of 0, 0.5, 1.5 or 4.5 mg/kg bw per day on days 6–15 of gestation. Five rats were killed on day 16 of gestation and 28 on day 20 of gestation, and their fetuses were removed surgically. Twelve rats killed on day 21 post partum were examined during delivery, and their pups were observed and subjected to behavioural tests on day 21 post partum. Blood and brain were collected for determination of cholinesterase activity from rats killed at days 16 and 20 of gestation. Fetal brain cholinesterase activity was determined in 20 fetuses delivered on day 20 of gestation. The dams killed on day 20 of gestation were observed for physical appearance and behaviour, and body weight was determined. The fetuses were weighed, sexed, inspected for external abnormalities and examined for visceral and bone malformations.
Tremors were seen in nearly all the animals at the highest dose during treatment, and body-weight gain was significantly reduced in this group. Plasma cholinesterase activity was reduced in all groups killed on day 16 of gestation. Significant reductions in the activity of erythrocyte and brain cholinesterase were seen at the two higher doses and marginal inhibition of brain cholinesterase activity at the lowest dose (activity, 78.5% of concurrent controls). In dams killed on day 20 of gestation, no significant reduction in plasma cholinesterase activity was seen, but erythrocyte and brain acetycholinesterase activity was inhibited at the two higher doses. Fetal brain cholinesterase activity did not show intergroup differences of any significance. The numbers of live fetuses, resorptions, malformations, implants and fetal weights did not differ significantly between the groups. No treatment-related visceral or skeletal abnormalities were observed. In the pups observed after delivery, no intergroup differences in survival, body-weight gain, developmental indices or reflexes were observed. A NOAEL for maternal toxicity could not be identified, as brain cholinesterase activity was inhibited at all doses. The NOAEL for fetotoxicity was 4.5 mg/kg bw per day, the highest dose tested (Clemens et al., 1985).
Rabbits
Oxydemeton-methyl (53.5% in methyl isobutyl ketone) was administered to groups of 17 female inseminated American Dutch rabbits at a dose of 0, 0.1, 0.4 or 1.6 mg/kg bw per day on days 7–19 of gestation. The does were weighed on days 0, 7, 10, 14, 17, 21 and 28 of gestation and killed on day 28. Pregnancy rates were evaluated, and the numbers of corpora lutea, implantations, resorptions, litter size, fetal weights, viability of fetuses, sex ratio and pre- and post-implantation losses were estimated. Fetuses were examined for gross external, visceral and skeletal abnormalities. Three deaths from respiratory infections occurred during the study, one each in controls and animals at the two lower doses. Loose stools were observed in animals at the two higher doses. Maternal weights and all reproductive parameters were similar in all groups. A non-statistically significant increase in resorptions and post-implantation losses was seen at the highest dose. The NOAEL for maternal toxicity was 0.1 mg/kg bw per day, on the basis of loose stools at the intermediate dose. The NOAEL for fetotoxicity was 1.6 mg/kg bw per day, the highest dose tested. Teratogenicity was not seen (Clemens & Hartnagel, 1984; Hartmann, 2002).
A supplemental study was performed with groups of eight inseminated American Dutch does given the same doses of oxydemeton-methyl (50% concentrate; 53.5% active ingredient) as above, two groups of eight does receiving the test material at each dose. Litter parameters were not evaluated. The does were observed daily and weighed on days 7, 10, 14, 17 and 20 of gestation. One group of eight does at each dose was killed at 20 days of gestation, and the remainder were weighed at 28 days and then killed. In each case, blood was taken for estimation of plasma and erythrocyte cholinesterase activity from the first five does found to be pregnant. Brain cholinesterase activity was estimated in half brains after storage at –20 ºC. The method of Ellman et al. (1961) was used to determine cholinesterase activity. No significant clinical effects were observed, and the test material had no effect on body weights. Statistically non-significant inhibition of plasma cholinesterase activity by > 20% was seen at 28 days in does at 0.1 and 0.4 mg/kg bw per day. Biologically and statistically significant inhibition of erythrocyte cholinesterase activity was seen at the highest dose at 20 days (activity, 56.7% of concurrent control), but non-significant inhibition of erythrocyte cholinesterase activity was seen at this dose at 28 days (activity, 70.2% of concurrent control). Biologically and statistically significant inhibition of brain cholinesterase activity was seen at the highest dose at 20 days (activity, 79.3% of concurrent control), but nonsignficant inhibition was seen at this dose at 28 days (activity, 77.9% of concurrent control). The NOAEL for inhibition of erythrocyte and brain cholinesterase activity was 0.4 mg/kg bw per day (Bare et al., 1985).
A further supplemental study was reported in response to a peer review, in which it was concluded that the previous study was unacceptable as insufficient numbers of litters had been examined for skeletal effects. Additional fetal skeletons were evaluated and partially disarticulated, and a table was supplied giving the skeletal findings for 20 litters. Neither the fetal nor the litter incidence of skeletal abnormalities was correlated with treatment (Clemens & Hartnagel, 1993).
Chickens
A study of developmental toxicity was carried out with white Leghorn chick embryos in which oxydemeton-methyl (purity, 89%) was applied to the vitelline membrane directly above the stage 12 embryo at a dose of 0.01, 0.05, 0.1, 1 or 2 mg per embryo. The embros were monitored to stage 41 and then autopsied. At doses > 0.5 mg, survival was reduced. A dose-related increase in musculoskeletal and cardiovascular teratogenic effects was seen in all groups. No NOAEL could be identified (Lenselink et al., 1993).
The finding of vacuolation of the corpus of the epididymis in the two-generation study in rats (Eigenberg, 1990) stimulated a study of the toxicity of oxydemeton-methyl to the male reproductive tract. Groups of 40 albino CD Sprague-Dawley rats were fed diets containing a 50% concentrate of oxydemeton-methyl (content, 54.8%) at a concentration of 3, 9 or 50 ppm of active ingredient, equivalent to 0.15, 0.45 and 2.5 mg/kg bw per day for 2 months. A control group of 50 rats was established, of which 10 were sacrificed on day 1. A further group of 40 rats received the diet containing oxydemeton-methyl at a concentration of 50 ppm and were used to study recovery, being killed after various periods on the diets: 10 rats at 0, 3 and 50 ppm were killed at 2 months (no recovery period), three rats from each group were killed at 3.5 months (18 days’ recovery), nine rats were killed at 4 months (2 months’ recovery), and nine were killed at 6 months 3–4 months’ recovery). Nine rats at 0, 9 and 50 ppm were killed at 8 months (5 months’ recovery). Cholinesterase activity was measured by a modification of the Ellman et al. (1961) method. Indicators of reproductive toxicity were determined. An additional group of rats received oxydemeton-methyl at a dietary concentration of 50 ppm, and 9 or 10 animals per group were given control diet for 18 days to 5 months after 3.5, 4, 6 or 8 months, as recovery groups; some of the results of this study were reported separately (Eigenberg & Hastings, 1987). Clinical signs, body weight and food consumption were monitored. Terminal body and testis weights were measured in all animals, and testes, epididymides and any gross abnormalities were examined microscopically. Sperm was collected from the cauda of the epididymis post mortem and examined for sperm count, morphological changes and motility.
No treatment-related deaths, clinical signs or changes in body weight or food consumption were seen. Depressed plasma cholinesterase activity was seen in the groups at 50 ppm at 2 months and in the groups at the two higher dietary concentrations at 4, 6 and 8 months; no inhibition of plasma cholinesterase activity was seen in the animals allowed to recover. At the two higher dietary concentrations, depressed erythrocyte cholinesterase activity was seen at 2, 4, 6 and 8 months; marginal inhibition was observed at the lowest dietary concentration at 3 months (21% depression by comparison with concurrent controls). The activity of this enzyme remained inhibited in the group that received treatment for 3.5 months and was then allowed an 18-day recovery period. Brain cholinesterase activity was depressed at the two higher dietary concentrations at 2, 4, 6 and 8 months. Inhibition by 20% was seen at 3 ppm at 6 months. Inhibition was still present in those rats fed the test diet for 3.5 or 4 months and allowed to recover for 18 days and 2 months, respectively. No significant inhibition was found in the animals allowed a 3- or 4-month recovery period after being fed oxydemeton-methyl at the highest dietary concentration for 6 months. (Interpretation of these data is difficult as there were no concurrent control data.) No significant intergroup differences were seem in testis weights or in sperm counts, sperm morphology or sperm motility. Severe vacuolation of the epithelium of the corpus epididymis was seen in all rats at the highest dietary concentration, from 2 months onwards. This effect was reversible, and only two of nine animals fed oxydemeton-methyl at the highest dietary concentration for 8 months and allowed a 5-month recovery showed minimal vacuolation. Minimal vacuolation was seen from 4 months onwards in one of nine animals given the intermediate dietary concentration. The authors considered the NOAEL to be 3 ppm (equivalent to 0.15 mg/kg bw per day), although there was marginal inhibition of erythrocyte and brain cholinesterase activity at the lowest dietary concentration. No clear NOAEL could be identified (Eigenberg, 1987a).
A study to ascertain whether oxydemeton-methyl or methyl isobutyl ketone, used as a diluent for the 50% concentrate, was responsible for the epididymal vacuolation in the above study was reported as an annex. Diets containing 0 or 50 ppm oxydemeton-methyl or methyl isobutyl ketone were given to groups of 30 male CD Sprague-Dawley rats for up to 2 months. Depression of cholinesterase activity and epididymal vacuolation were seen in the rats fed diets containing oxydemeton-methyl but not in those fed diets containing methyl isobutyl ketone or the controls. It was concluded that oxydemeton-methyl, and not methyl isobutyl ketone, had caused the epididymal vacuolation seen in studies in which animals were given diets containing a mixture of oxydemeton-methyl and methyl isobutyl ketone (Eigenberg, 1987b).
A further study of the reproductive effects of oxydemeton-methyl was undertaken. After preliminary studies, oxydemeton-methyl (purity, 94%) was administered to groups of 125 Crl:CD(SD)BR male rats by gavage at a dose of 0, 0.15, 0.9 or 5 mg/kg bw. Twenty rats in each group, designated ‘breeder’ males, were placed with female rats at seven mating intervals, after which 15 rats in each group were killed for sperm evaluation and histopathological examination of the testes. The 140 female CD rats assigned as mates for each treated and control group were not treated with oxydemeton-methyl. Mating was optimized on the basis of the females’ estrus cycle. The animals were mated by cohabitation for 5 days, the first mating trial beginning on day 1, 24 h after the last day of treatment, and subsequent trials beginning on days 7, 14, 21, 35, 56 and 112, with the same breeder males and different females in each trial. Throughout the study, all males were observed daily for signs of toxicity. They were weighed on days 1, 3 and 5 of treatment and then weekly and at termination. Food consumption was evaluated during the 5-day treatment period in those male animals intended for histopathological evaluation. Pregnant rats were weighed on days 1, 6, 15 and 20 of gestation, and food consumption was recorded. After mating, females were examined for abortion or premature delivery; if these occurred, the dams were killed, and uterine implantations or resorptions and dead fetuses were recorded. Corpora lutea were also counted. The surviving females were killed on day 20 of gestation. At necropsy, the corpora lutea in the ovaries were counted, and the uterus was weighed. The fetuses were removed surgically, and the numbers of implantations, live and dead fetuses and resorptions were recorded. Fetuses were sexed, weighed and examined externally. Males were necropsied, and both testes were weighed, as were the epididymides and the cauda epididymis, the seminal vesicles, with and without contents and the prostate and pituitary glands. Sperm was evaluated by counting spermatids and cauda epididymal sperm. Cauda epididymal sperm morphology and motility were also recorded, and the epididymal fluid was examined for debris and unusual cell types. The right testis and the whole of the epididymis were processed for histopathological examination.
An increased mortality rate was seen among breeder males at the highest dose. The group mean body weight was decreased during treatment but recovered subsequently. Diarrhoea was seen in all treated groups, especially at the two higher doses, and tremors were seen at the highest dose. A significant change in mortality rate was not seen among the males destined for histopathological examination. Animals at the highest dose showed decreased body weight during treatment up to day 14. These animals also had decreased food consumption. On day 35, a decreased percentage of motile sperm was found in all treated groups, but no inter-group differences were seen at other times. Sperm morphology and spermatid and cauda epididymal sperm counts showed no treatment-related effects, and no differences were seen in testicular, epididymal, cauda epididymal or prostatic weights or those of the pituitary glands. Histopathological examination showed vacuolation of the epithelium of the corpus epididymis at day 112 in males given 5 mg/kg bw per day, but the statistical significance of this finding was not tested. No significant increase in vacuolation was seen at earlier times or in epididymides from animals at lower doses, nor was it found in the caput or cauda epididymis. No histopathological changes were seen in the testes. No differences in weight or in food consumption were found between females mated with treated males and those mated with control males. On days 7, 56 and 112, marginal decreases in fertility were found in females mated with males at 5 mg/kg bw per day. There were no differences in gravid uterine weight, dam body weight or weight change between females mated with treated and those mated with control males. There was some indication of increased pre-implanation loss and decreased post-implantation loss in females mated with treated males. The incidence of dead fetuses did not appear to be related to treatment of the paternal animals. Although some malformed fetuses were observed, the incidence did not appear to be treatment-related. The NOAEL was 0.9 mg/kg bw per day on the basis of clinical signs of toxicity and decreased body weight in males during and after treatment. Furthermore, decreased food consumption and vacuolation of the epithelium of the corpus epididymis at day 112 were observed in males at the highest dose intended for testis evaluation (Table 1). The marginal decrease in fertility in females mated with males at 5 mg/kg bw per day was considered possibly significant. A decrease in sperm motility at day 35 in males at the two higher doses was considered to be incidental, as a similar effect was not seen at other times (Filler, 1989).
Table 1. Vacuolation in the epididymides of male rats killed after 112 days of treatment with oxydemeton-methyl
|
Dose (mg/kg bw per day) |
Vacuolation |
||
|
Caput epididymis |
Corpus epididymis |
Cauda epididymis |
|
|
0 |
2 |
5 |
0 |
|
0.15 |
1 |
8 |
0 |
|
0.9 |
3 |
6 |
0 |
|
5.0 |
3 |
13 |
0 |
From Filler (1989)
No statistical evaluation was undertaken.
A study of male fertility was undertaken in albino CD Sprague-Dawley rats to determine whether the reduction in fertility seen in the two-generation study had been due to an effect on the males. Groups of 40 rats, 7 weeks old, received a diet containing oxydemeton-methyl (purity, 92.5%) at a concentration of 0 or 50 ppm, equivalent to 2.5 mg/kg bw per day, for 10 weeks. At 10 weeks, 10 animals per group were killed and evaluated for plasma, erythrocyte and brain cholinesterase activity and for effects of the diet on the testes and epididymides. The remaining 30 males were bred for up to 3 weeks with females on the control diet, while the males continued to receive test or control diet, as appropriate, during the breeding period. On day 20 of gestation, the females were killed, and the numbers of fetuses, corpora lutea, resorptions and implantation sites and the mating index, fertility index, pre-implantation loss, post-implantation loss and litter size were determined. The breeding males were killed and evaluated for effects on the testes and epididymides. No treatment-related clinical signs were observed. At 10 weeks, plasma cholinesterase activity in the treated group was 41% that of concurrent controls, and erythrocyte cholinesterase activity was 0% and brain cholinesterase 23% that of concurrent controls (see also Eigenberg & Hastings, 1992b). There was no effect on the mating index, fertility index, pre-implantation loss, post-implantation loss or litter size. There were no intergroup differences in body weights or testicular weights in those animals killed before breeding. The terminal body weight of treated breeder males was lower than that of the control group. The absolute but not the relative testis weights were reduced in treated breeder males, and treatment-related vacuolation of the epididymal tubular epithelium, particularly the proximal corpus, was found at sacrifice both before and after mating. No NOAEL could be identified (Eigenberg & Hastings, 1992a).
In a study of sperm motility, groups of male albino CD Sprague-Dawley rats received diets containing technical-grade oxydemeton-methyl (purity, 91.6–93.6%) at a nominal concentration of 0, 3, 9 or 50 ppm, equivalent to 0.15, 0.45 and 2.5 mg/kg bw per day, for 1, 2 or 3 months. The dietary concentrations were up to 10% less than nominal. Ten rats at each dietary concentration were killed at 0, 4, 8 and 13 weeks. The animals were observed for clinical signs once or twice daily. Cholinesterase activity was measured with a modification of the Ellman et al. (1961) method (Hackathorn et al., 1983). Plasma and erythrocyte enzyme were measured within 1 week of termination, and brain cholinesterase activity was measured at termination. At sacrifice, fluid was removed from the cauda epididymis of the right testis, and sperm motility was estimated and expressed as a fraction of the sperm count. Gross necropsies were carried out and terminal body and testicular weights determined. Any gross lesions and the testes and epididymides were examined histologically.
No treatment-related clinical signs were observed. Plasma cholinesterase activity was significantly depressed at all three times in animals at dietary concentrations > 9 ppm, and inhibition by 20% was seen at the lowest dietary concentration at the earliest time (25 days). Erythrocyte cholinesterase activity was depressed at dietary concentrations > 9 ppm, and at 58 days inhibition of 26% was seen at 3 ppm. Brain cholinesterase activity was diminished in rats at 9 and 50 ppm. At 3 ppm, depression was found at 59 and 93 days but not at 28 days. Terminal body weight was decreased only at the highest dietary concentration and only at the 1-month sacrifice. No treatment-related effect was seen on sperm motility or on testis weight, and no treatment-related gross lesions were seen at necropsy. Treatment-related vacuolation in the epididymides was seen at the highest dietary concentration at 4, 8 and 13 weeks, becoming more severe with time. There was some indication of this change at 9 ppm at the latest time of sacrifice, but the prevalence in this group was not statistically significantly different from that in concurrent controls. A NOAEL could not be identified. The LOAEL was 3 ppm, equivalent to 0.15 mg/kg bw per day, on the basis of inhibition of erythrocyte and brain cholinesterase activity at 3 ppm. The NOAEL for vacuolation in the epididymides was 3 ppm, equivalent to 0.15 mg/kg bw per day (Eigenberg, 1991).
Groups of 12 male and 12 female Sprague-Dawley Crl:CD(SD)BR rats were given oxydemeton-methyl 50% concentrate (containing 51.4% active ingredient) at a single dose of 0, 5, 20 or 100 mg/kg bw by gavage, corresponding to 0, 2.5, 10 and 50 mg/kg bw expressed as active ingredient. Clinical signs were recorded daily, and body weight and food consumption were measured weekly. A battery of functional observational tests (FOB) and a motor activity test were carried out before dosing, on day 1, at the time of presumed peak effects, and on days 7 and 14 after dosing. At the end of the study, six animals of each sex per group were perfused, and the central and peripheral nervous systems were examined histopathologically. Further groups of 30 rats of each sex were dosed as above, and 10 were killed at 0, 7 and 14 days for measurement of cholinesterase activity.
About 50% of those at the highest dose died, and most animals in this group, including the survivors, showed clinical signs of cholinesterase inhibition. At 20 mg/kg bw, less severe signs of cholinesterase inhibition were seen. Changes in the FOB were seen at 20 and at 100 mg/kg bw in both sexes, but no treatment-related changes were seen at 5 mg/kg bw. Reductions in plasma, erythrocyte and brain cholinesterase activity were seen at all doses. At the time of peak effect, brain cholinesterase activity was 55% that of concurrent controls in males and 61% that of concurrent controls in females. At the same time, substantial reductions in erythrocyte cholinesterase activity were seen in animals given the test material at 5 mg/kg bw. A NOAEL could not be identified. The LOAEL was 5 mg/kg bw, corresponding to 2.5 mg/kg bw of active ingredient (Beyrouti, 1995).
Oxydemeton-methyl (purity not stated) was administered to rats of each sex by inhalation as a 1% solution in saline sprayed for 1 h. Exploratory behaviour, barbital-induced hypnosis, electric shock- and pentylenetetrazole-induced seizure activity were compared in treated and control rats. Treated animals showed less exploratory behaviour and had prolonged barbital-induced sleeping time and greater electric shock- and pentylenetetrazole-induced convulsive activity, when compared with the controls (Kansal & Chakrabarti, 2000).
Rats of each sex were exposed to oxydemeton-methyl (purity not stated) by inhalation as a 1% solution in saline sprayed for 0.5 h daily for 7 consecutive days. Exploratory behaviour, barbital-induced hypnosis, haloperidol-induced catalepsy and electric shock- and pentylene-tetrazole-induced seizure activity were compared in the treated groups with data on controls from the study described above (Kansal & Chakrabarti, 2000). Exploratory behaviour was reduced in the treated animals, while barbital-induced hypnosis and haloperidol-induced catalepsy was increased. Electric shock- and pentylenetetrazole-induced seizure activity was greater than in the controls (Kansal & Chakrabarti, 2001).
At least three blood samples for estimation of plasma and erythrocyte cholinesterase activity were taken from 18 men (although the men were described as volunteers, no mention of prior informed consent was made), nine of whom were then given oxydemeton-methyl (purity, 97.5%) orally. The first six subjects received oxydemeton-methyl in corn oil at a dose of 0.0125, 0.025, 0.05, 0.25, 0.5 or 1 mg/kg bw in corn oil, while a seventh received a dose of 1.5 mg/kg bw in a gelatine capsule; two further men received the material in gelatine capsules at a dose of 2 mg/kg bw, but the results for these men were not tabulated or described in detail. Blood samples were then taken for measurement of haematological variables. Blood samples were taken for determination of cholinesterase activity 30 min and 1, 2, 4, 8 and 24 h after dosing. Blood samples were also taken after 24 h from men who showed inhibition of cholinesterase activity. Cholinesterase activity was determined by the manometric method of DuBois & Mangun (1947), with duplicate determinations on each sample. Additionally, enzyme activity in some samples was determined by the automated method of Levine et al. (1965), but the results were not reported, although they were said to be in broad agreement with those obtained with the DuBois method. Urine was also analysed.
No symptoms of anticholinesterase poisoning were observed at any dose, and no treatment-related clinical signs were seen. No differences in haematological or urine parameters were found before and after dosing. Cholinesterase activity was not inhibited at doses up to 0.5 mg/kg bw. At 1 mg/kg bw, both plasma and erythrocyte cholinesterase activity was decreased by > 20% during the first 24 h after dosing when compared with background levels, the nadir in the case of both enzymes being at 24 h (70.5% of control activity for plasma and 69.6% for erythrocyte activity). Inhibition > 20% was still seen in both cases at 48 h, but recovery was evident by 60 h. At 1.5 mg/kg bw, > 20% inhibition of plasma cholinesterase activity was seen at 30 min and 1, 2, 4 and 24 h but not at 8, 48 or 60 h. Erythrocyte cholinesterase activity was inhibited by 20% of the background level only at 60 h. The highest dose (2 mg/kg bw) was reported to have caused approximately 50% inhibition of erythrocyte cholinesterase activity in both men. Blood samples were taken from one of these men for a further 82 days: whereas at 30 days erythrocyte cholinesterase activity was 60% that of controls, it had returned to 98% by 82 days. (These results were not presented in detail.) The NOAEL for inhibition of plasma and erythrocyte cholinesterase activity was 0.5 mg/kg bw (Doull et al., 1973).
In one man given oxydemeton-methyl at a dose of 0.4 mg/kg bw per day for 5 days, progressive depression of plasma and erythrocyte cholinesterase activity was seen. By 24 h after the fifth dose, the plasma activity was 50% of the background levels and that of erythrocyte cholinesterase was 35% of background. Similar studies were carried out with two men given a dose of 0.05 or 0.1 mg/kg bw per day. In the man given the higher dose for 120 days, plasma cholinesterase activity fell, and there was a progressive diminution in erythrocyte cholinesterase activity, reaching 50% of background by day 60. Five further men received oxydemeton-methyl at a dose of 0.05 mg/kg bw per day, so that a total of four were treated at this dose for 30 days and two for 60 days. No significant depression of cholinesterase activity was observed at any time. No clinical signs or symptoms were seen at any dose. The NOAEL was 0.05 mg/kg bw per day (Doull et al., 1973).
Pyridinium oxime, pralidoxime and the bis-pyridinium oximes, obidoxime, HLö 7 and HI 6 were reported to be capable of reactivating oxydemeton-methyl-inhibited human cholinesterase in vitro. HI 6 was much less effective than the other oximes, and obidoxime was the most effective (Worek et al., 1996). Reactivation of oxydemeton-methyl-inhibited cholinesterase oximes has been reported subsequently (e.g. Worek et al., 1999). Thiermann et al. (1999) concluded from a case series of five patients that therapy with obidoxime was successful only if instituted soon after poisoning with oxydemeton-methyl.
Oxydemeton-methyl is an organophosphorus compound which inhibits cholinesterase activity. Most of its toxic effects in mammalian species reflect this fact. Oxydemeton-methyl also caused effects on the testes, with equivocal reproductive consequences.
In 14-day studies in rats treated in the diet or by gavage, oxydemeton-methyl inhibited plasma, erythrocyte and brain cholinesterase activity. The inhibition of activity in plasma tended to be greater than that of erythrocyte or brain cholinesterase. On the basis of inhibition of erythrocyte and brain cholinesterase activity, the NOAEL after dietary administration was 0.21 mg/kg bw per day, and that after gavage was 0.15 mg/kg bw per day. In a 12-month study in dogs in which the compound was administered by gavage, the NOAEL was 0.12 mg/kg bw per day at 3 weeks and 0.012 mg/kg bw per day in the overall study; the former NOAEL was based on inhibition of erythrocyte cholinesterase activity and the latter on inhibition of brain and erythrocyte cholinesterase activity.
The Meeting evaluated a long-term study of toxicity in mice that had not been reviewed previously, in which the compound was administered in feed. The effects observed included inhibition of plasma, erythrocyte and brain cholinesterase activity and vacuolation of the cytoplasm of the epididymis. The NOAEL was 0.5 mg/kg bw per day on the basis of inhibition of erythrocyte and brain cholinesterase activity (in animals of each sex) and vacuolation of the cytoplasm of the epididymides.
In a study of developmental toxicity in rats, a NOAEL for maternal toxicity was not identified, as marginal inhibition of brain cholinesterase activity was observed at the lowest dose of 0.5 mg/kg bw per day. Neither fetotoxicity nor teratogenicity was observed, nor did oxydemeton-methyl affect fetal brain cholinesterase activity. Two studies of developmental toxicity in rabbits were available. In the first, the NOAEL for maternal toxicity was 0.1 mg/kg bw per day on the basis of clinical observations (loose stools) at the intermediate dose of 0.4 mg/kg bw per day. Neither fetotoxicity nor teratogenicity was seen. Cholinesterase activity was not measured in this study. In the second, supplementary study, in which plasma, erythrocyte and brain cholinesterase activity was measured in the does, the NOAEL for maternal toxicity was 0.4 mg/kg bw per day. Litter parameters were not measured.
In a multigeneration study of reproductive toxicity in rats, the NOAEL for effects on the parents was 1 ppm, equivalent to 0.07 mg/kg bw per day, on the basis of inhibition of erythrocyte and brain cholinesterase activity. The NOAEL for effects on the offspring was 9 ppm, equivalent to 0.6 mg/kg bw per day, on the basis of decreased weight gain and viability and inhibition of erythrocyte and brain cholinesterase activity. The NOAEL for reproductive toxicity was 9 ppm, equivalent to 0.6 mg/kg bw per day, on the basis of vacuolation of the epididymis, decreased numbers of corpora lutea and reduced litter size at the next highest dose.
Several studies were carried out to investigate the vacuolation of the cytoplasm of the epididymis seen with oxydemeton-methyl and effects on male fertility. These showed that the vacuolation of the cytoplasm of the epididymis was reversible even at the highest dose; the frequency and severity of the effect increased with duration of treatment and dose; the effect was not accompanied by changes in sperm count, morphology or motility; and similar changes were not produced by methylisobutylketone, a common solvent for oxydemeton-methyl in commercial preparations which was used in some of the studies evaluated. There was some indication of decreased fertility of males given high doses (3.3 mg/kg bw per day and above). The Meeting concluded that these effects were not relevant for the establishment of an acute RfD.
In a study of acute neurotoxicity in rats, a NOAEL was not identified. The LOAEL was 2.5 mg/kg bw, the lowest dose tested, at which inhibition of brain and erythrocyte cholinesterase activity was observed.
The NOAEL in a study in volunteers given single doses was 0.5 mg/kg bw on the basis of inhibition of plasma and erythrocyte cholinesterase activity. No other effect was observed. The NOAEL in a study in which oxydemeton-methyl was administered at repeated doses for 30 days or more was 0.05 mg/kg bw per day. In both these studies, a manometric method of analysis for cholinesterase activity was used, which is not very reliable, and in the study with single doses only one subject received each dose.
On the basis of the data reviewed and previous evaluations, the Meeting established an acute RfD of 0.002 mg/kg bw, using the NOAEL of 0.15 mg/kg bw per day in the 14-day study in rats treated by gavage and a safety factor of 100. This acute RfD is supported by the LOAEL of 2.5 mg/kg bw in the study of neurotoxicity in rats given single doses. The Meeting concluded that it would be inappropriate to use the studies in volunteers for establishing an acute RfD because of the limitations described above.
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See Also:
Toxicological Abbreviations
Oxydemeton-methyl (FAO/PL:1967/M/11/1)
Oxydemeton-methyl (FAO/PL:1968/M/9/1)