ISOFENPHOS EXPLANATION Isophenphos was evaluated for acceptable daily intake by the Joint Meeting in 1981 and reviewed in 1982 (Annex 1, FAO/WHO, 1982a and 1983a). A toxicological monograph was published after the 1981 Meeting (Annex 1, FAO/WHO, 1982b) and a monograph addendum was published after the 1982 Meeting (Annex 1, FAO/WHO, 1983b). In 1981, a temporary ADI of 0 - 0.0005 mg/kg b.w. was established, which was extended in 1982 because of the absence of appropriate delayed neurotoxicity studies and because of questions raised by the multi-generation studies submitted at that time. The requested studies have been submitted and are summarized in this monograph addendum. EVALUATION FOR ACCEPTABLE INTAKE BIOLOGICAL DATA Toxicological studies Special studies on cholinesterase inhibition In vitro studies on purified isofenphos indicate no inhibition of horse serum acetylcholinesterase or human serum butyrylthiocholine- sterase up to concentrations of 100 mM. It was concluded that direct inhibitors are not present as impurities in these isofenphos preparations (Blass, 1982; Wehling, 1983). Special studies on delayed neurotoxicity Experiments using a single dose for delayed neurotoxicity studies were performed using treatment with antidotes (atropine & 2-PAM) for cholinergic symptoms. Isofenphos (74 mg/kg b.w. orally) given to hens after pre-treatment with atropine did not produce clinical or histopathological signs of delayed polyneuropathy. The unprotected oral LD50 in this study was 21 mg/kg b.w. (Kimmerle, 1972; Cherry et al., 1972). In a similar study, isofenphos was given orally (36 mg/kg b.w.) to hens after pre-treatment with atropine. Nevertheless, 17 hens out of 30 died of cholinergic symptoms. The surviving hens did not show clinical or histopathological signs of delayed neurotoxicity (Hixson, 1982). Another study measured the response of NTE in humans to oral dosing with isofenphos (36 mg/kg b.w.) and pre-treatment with atropine. Substantial inhibition of NTE was measured within 1 - 2 days after dosing. No concurrent controls for clinical/histological evaluations were used in this study, and the reference oral unprotected LD50 was 16 mg/kg b.w. isofenphos (Thymen, 1978; Thymen & Eben, 1983). Another study provided evidence for the potential of isofenphos to cause delayed neurotoxicity in hens. The oral unprotected LD50 in tested hens was 3 - 5 mg/kg b.w. isofenphos. After appropriate antidotal treatment (before and several times after dosing) it was raised to 100 mg/kg b.w. One hundred mg/kg b.w. isofenphos given orally caused more than 70% inhibition of brain NTE when measured 3 days after dosing and clinical-morphological signs of delayed neurotoxicity approximately 2 weeks later (Wilson et al., 1984). In a subchronic delayed neurotoxicity study, groups of 10 adult white leghorn laying hens (15 to 20 months old) were treated daily by gavage with technical isofenphos (as an emulsion in water with Cremophor EL) at 0.25, 1, or 2 mg/kg b.w. for 90 consecutive days. Ten hens were used as untreated controls and 10 "dose controls" were given water and Cremophor EL by gavage. After the seventh administration, clinical signs and 1 mortality occurred in the highest-dose group (2.5 mg/kg b.w./day). At 1.25 mg/kg b.w./day 1 bird showed clinical signs after the seventh administration. A significant decrease in body weight and an inhibition of cholinesterase activity in plasma, erythrocytes, and whole blood were observed at 2 mg/kg b.w./day throughout the 90-day treatment period. Slightly-reduced body weights and very slight inhibition of the cholinesterases also occurred at 1 mg/kg b.w./day. Histopathological examination of the brain, spinal cord, and sciatic nerve of all 10 hens in the 2 mg/kg b.w./day dose group revealed minimal changes that were similar in incidence and severity to those noted in concurrent controls. Positive controls (10 hens), treated orally with tri-o-cresyl phosphate (TOCP) at 5 mg/kg b.w./day (as an emulsion in water and Cremophor EL), displayed clinical signs (ataxia from the fifth week onwards) and histopathological lesions, such as severe degeneration of the pathways in the spinal cord and axonal degeneration in the medulla oblongata and cerebellum, which are typical of delayed neurotoxicity. The dose of 0.5 mg/kg b.w./day isofenphos was tolerated without effects (Thyssen, 1978; Flucke & Kaliner, 1985). Special study on embryotoxicity/teratogenicity Rats A study to investigate embryotoxic and teratogenic effects in Long Evans rats of the FB30 strain following dermal application was conducted. Males were between 3 and 6 months of age and weighed 350 - 500 g at the start of the study; females were between 2.5 - 3.5 months of age and weighed 199 - 260 g. Five groups of 25 animals were treated with 0, 0.3, 1.0, 3.0, or 10.0 mg isofenphos/kg b.w. The compound was applied to shaved dorsal skin for a 5-hour contact time, the skin being washed at the end of each exposure. The applied volume was 0.5 ml/kg b.w. in each test group. Males were not treated; females were treated from days 6 - 15 of gestation. On the 20th day of gestation the animals were killed by CO2 asphyxiation and the fetuses examined by the Wilson technique for visceral malformations and by clearing and skeletal staining for skeletal abnormalities. There were no treatment-related abnormalities in behaviour or physical appearance. One dam in the 3 mg/kg b.w. group was killed during treatment because of evidence of illness, of which it had shown signs before treatment began. All dose groups gained less weight than controls during treatment and throughout gestation. There were no significant differences between control and treated groups with regard to fertilization and pregnancy rates, which corresponded to those known for the rats used in this study. The malformations seen in the various groups are shown in Table 1. Evidence of maternal toxicity was observed, with a significant failure of treated animals to gain weight. The apparent no-effect level for maternal toxicity was 0.3 mg/kg b.w., although the mothers in this group showed poor weight gain during gestation (Schluter, 1981). Special study on reproduction A 3-generation rat reproduction study with isofenphos that was performed at the Huntingdon Research Center in 1976 and 1977, and was summarized in the 1981 toxicological monograph (Annex 1, FAO/WHO, 1982b), was re-evaluated. Twenty animals per group were fed doses of 0, 1, 10, or 100 ppm isofenphos in the diet. Body weights, food consumption, and cholinesterase activity were measured. The F0 generation was mated 3 times and cross-mated (control with 100 ppm group). F1a and F2a rats were mated once. There were no significant effects on any variables examined, including litter size, birth weight, viability, or lactation, in animals fed up to 10 ppm. In the F2a generation successful mating did not occur in the 100 ppm group because of what was described as "reduced gestation and lactation" rates. Comparable gestation and insemination rates between control and test animals were found in the 1 and 10 ppm groups in all 3 generations. Following the second mating of the F0 generation, 90% of the control females were pregnant and 70%, 60%, and 40% of the females in the 1, 10, and 100 ppm groups, respectively, were pregnant. Taking into account the fact that the insemination rates for this mating were reduced in all of the study groups, all inseminated females in the 1 ppm group became pregnant, 12/14 in the 10 ppm group became pregnant, and all inseminated females in the 100 ppm group became pregnant. Reduced pregnancy rates in multi-generation studies are common as a result of reduced willingness to mate, and it occurred in this study in control animals as well as in the test animals. One animal in the control group and 1 in the 1 ppm group repeatedly failed to become pregnant. The low insemination rate exhibited by controls was considered to be comparable with the apparently low gestation rate after the second mating of the F0 animals. The apparent no-effect level in this study was 10 ppm (Palmer et al., 1977; Eiben, 1983). Table 1. Embryotoxic and terotogenic malformations in rats following dermal application of isofenphos. Number of Nature of Group malformed fetuses malformations Control 1 1x BB fissure, rib fusion, microthalmia resp. anophthalmia 1 1x kinky tail 2 2x nodulations on ribs 1 1x oedematous thorax 0.3 mg/kg b.w. 1 1x nodulations on ribs 1 mg/kg b.w. 1 1x nodulations on ribs 3 mg/kg b.w. 1 1x multiple malformation; WS, Ri, Be, extra tail, eyes & umbilical hernia, and general oedema 1 1x nodulations on ribs 1 1x cleft sternlum, abdominal fissure, dysplasia of extremities, cleft palate 10 mg/kg b.w. 1 1x microphthalmia 3 1x hypoplasia of telencephalon, eye malformations, dysplasia of extremities 2x hypoplasia of telencephalon, eye malformations BB = thorax and abdomen WS = vertebral column Ri = ribs Be = pelvic bone COMMENTS In vitro studies indicate that, in the preparations of isofenphos used, direct cholinesterase inhibitors are not present as impurities. The present Meeting reviewed a 2-generation study and a re-evaluation of a 3-generation study performed in 1976 and 1977. The apparent no-effect level in the multi-generation study was 10 ppm. In a dermal study there was some evidence of anomalies associated with embryotoxicity at 3 mg/kg b.w. In the evaluation of the potential of isofenphos to cause delayed polyneuropathy, the ratios between the unprotected LD50 and the dose tested for delayed polyneuropathy were considered and are shown in Table 2. Table 2. Delayed neuropathy in rats dosed with isofenphos. Approximate ratio Delayed Unprotected LD50/ neuropathy Reference Neurotoxic dose < 0.3 no Kimmerle, 1972; Cherry et al., 1972 < 1 (?) no Hixson, 1982 0.04 yes Wilson et al., 1984 Two studies on the biochemical test for delayed neuropathy (NTE inhibition) can also be compared, as shown in Table 3. Table 3. NTE inhibition in rats dosed with isofenphos. Isophenphos Approximate ratio Brain NTE (oral) Unprotected LD50/ inhibition mg/kg b.w. Neurotox dose (range) Reference 36 approx. 0.4 23 - 58% Thyssen, 1978; Thyssen & Eben, 1983 100 approx. 0.04 70 - 80% Wilson et al., 1984 From these studies it is clear that the dose which causes substantial inhibition of NTE is approximately twice the unprotected LD50. This inhibition is, however, below the critical threshold and does not cause delayed polyneuropathy. The dose which causes NTE inhibition above the threshold and thus causes polyneuropathy corresponds to about 25 times the unprotected LD50. A 90-day repeated dosing study in hens was negative for delayed polyneuropathy at doses up to 2 mg/kg b.w./day. On the basis of these results the Meeting concluded that isofenphos has the potential to cause delayed polyneuropathy. However, the dose which produces this toxic effect in hens exceeds the LD50 by more than 2 times. The Joint Meeting believes that isofenphos residues in food are unlikely to cause delayed polyneuropathy in man. The Meeting appreciated the submission of data on NTE inhibition for delayed neuropathy studies, in accordance with the recommendations of the 1984 Joint Meeting. It should be stressed, however, that the mechanism of initiation of delayed neuropathy through inhibition/aging of NTE differs fundamentally from that of acetylcholinesterase inhibition; it is not due to the accumulation of substrate, as with acetylcholine when the scavenger enzyme is inhibited. Therefore, unless particular problems in disposal of the chemical arise, measurement of NTE should be performed 24 - 48 hours after dosing. At that time the inhibition of NTE (> 70 - 80%) correlates with the development of delayed neuropathy. TOXICOLOGICAL EVALUATIONS LEVEL CAUSING TOXICOLOGICAL EFFECT Previous levels of administration free of effect were: Mouse: 1 ppm in the diet, equivalent to 0.15 mg/kg b.w./day Rat: 1 ppm in the diet, equivalent to 0.05 mg/kg b.w./day Dog: 1 ppm in the diet, equivalent to 0.05 mg/kg b.w./day These values were established on the basis of plasma cholinesterase levels. Although some studies suggest that higher doses could be considered no-effect levels, there is not an adequate data base for using an alternative method in this case. ESTIMATE OF ACCEPTABLE DAILY INTAKE FOR MAN 0 - 0.001 mg/kg b.w. STUDIES WHICH WILL PROVIDE INFORMATION VALUABLE IN THE CONTINUED EVALUATION OF THE COMPOUND Observations in man. REFERENCES Blass, W. I50 value of isophenphos. Unpublished report from Bayer AG, 1982 Institut fur Produktinformation und Qualitatsuberwachung. Submitted to WHO by Bayer AG, Bayerwerk, FRG. Cherry, C.P., Newman, A.J., & Urwin, C. Pathology report of SRA 12869 1972 - acute neurotoxicity experiment in hens. Unpublished report from Huntingdon Research Centre, Huntingdon, UK. Submitted to WHO by Bayer AG, Bayerwerk, FRG. Eiben, R. Comments on the three-generation study with SRA 12869. 1983 Unpublished report from Bayer AG, Institute of Toxicology. Submitted to WHO by Bayer AG, Bayerwerk, FRG. Flucke, W. & Kaliner, G. SPA 12869 - Study for subchronic 1985 neurotoxicity. Unpublished report No. 13474 from Bayer AG, Institute of Toxicology, submitted to WHO by Bayer AG, Bayerwerk, FRG. Hixson, E.J. Acute delayed neurotoxicity of technical isofenphos in 1982 hens. Unpublished report from Mobay Chemical Corporation, USA. Submitted to WHO by Bayer AG, Bayerwerk, FRG. Kimmerle, G. SRA 12869 acute neurotoxicity studies on hens. 1972 Unpublished report from Bayer AG, Institute of Toxicology. Submitted to WHO by Bayer AG, Bayerwerk, FRG. Palmer, A.K., Killick, M.E., & Allen, T.R. Effect of SRA 12869 on 1977 reproductive function of multiple generations in the rat. Unpublished report from Huntingdon Research Centre, Huntingdon, UK. Submitted to WHO by Bayer AG, Bayerwerk, FRG. Schluter, G. Evaluation for embryotoxic and teratogenic effects in 1981 rats following dermal application. Unpublished report No. 9801 from Bayer AG, Institute of Toxicology. Submitted to WHO by Bayer AG, Bayerwerk, FRG. Thyssen, J. SRA 12869 acute toxicity studies on hens and quail. 1978 Unpublished report from Bayer AG, Institute of Toxicology. Submitted to WHO by Bayer AG, Bayerwerk, FRG. Thyssen, J. & Eben, A. Isophenphos (SRA 12869) neurotoxic esterase 1983 activity in hen. Unpublished report No. 11402 from Bayer AG, Institute of Toxicology. Submitted to WHO by Bayer AG, Bayerwerk, FRG. Wehling, K. In vitro biochemical test with purified isofenphos for 1983 cholinesterase effect. Unpublished report No. 11418 (P) from Bayer Pharmacology Division. Submitted to WHO by Bayer AG, Bayerwerk, FRG. Wilson, B.W., Hooper, M., Chow, E., Higgins, R.J., & Knaak, J.B. 1984 Antidotes and neuropathic potential of isofenphos. Bulletin of Environmental Contamination and Toxicology, 33, 386 - 394.
See Also: Toxicological Abbreviations Isofenphos (Pesticide residues in food: 1981 evaluations) Isofenphos (Pesticide residues in food: 1982 evaluations) Isofenphos (Pesticide residues in food: 1984 evaluations)