PHOSPHAMIDON EXPLANATION Phosphamidon was evaluated for acceptable daily intake by the Joint Meetings in 1963, 1965, 1966, 1968, 1982, and 1985 (Annex 1, FAO/WHO, 1964, 1965a, 1967a, 1969a, 1983a, and 1986a). Toxicological monographs were published after the 1963 and 1965 Meetings (Annex 1, FAO/WHO, 1964, and 1965b), and monograph addenda were published after the 1966, 1968, and 1982 Joint Meetings (Annex 1, FAO/WHO, 1967b, 1969b, and 1983b). An ADI was established at the 1966 Meeting on the basis of no-effect levels taken exclusively from data reported by Industrial Bio-Test Laboratories (IBT), which could not be validated. For this reason the 1982 Meeting made the ADI temporary and requested replacement studies. The required data were not available to the Meeting in 1985 and, because of the lack of support for maintaining the temporary ADI, the safety factor was doubled. The data have now been provided and are summarized and discussed in this monograph addendum. EVALUATION FOR ACCEPTABLE INTAKE BIOLOGICAL DATA Toxicological studies Special study on reproduction Rats Groups of 15 male and 30 female albino rats (CD-CRL, 7 weeks old) were fed diets containing 0, 5, 30, or 50 (80 for the first 2 weeks) ppm phosphamidon (technical, 92.1% pure) in a 2-generation (2 litters/generation) study. Weanlings from the second litter were selected to become parents of the next generation. In the parental generations unthriftiness, hyperactivity, ocular and/or nasal discharge, and tremours were observed in animals of the 50 and/or 30 ppm dose groups. There were no compound-related effects on mortality or food intake. Body weights were significantly reduced in F0 parental males at 50 ppm and in F1 parental males at 50 and 30 ppm. F1 maternal body weights were slightly reduced at 50 ppm. In some cases F1 parental body weights were also decreased at 5 ppm, but this occurred in a non-dose-related manner. F0 and F1 parental absolute organ weights were significantly affected at 50 ppm. Relative brain and heart weights were significantly increased in F1 males at 50 and 30 ppm. No treatment-related histopathological findings were observed in F0 or F1 parental animals. Reproductive performance, as expressed in indices for mating, fecundity, and male and female fertility, was decreased at 50 ppm. Mean litter size and pup survival were decreased in all generations (significantly for F2b litters and pups) at 50 ppm. Pup weights (at day 21) were decreased in F1a, F1b, F2a, and F2b pups at 50 ppm, and a tendency to lower pup weights was observed in F1a, F1b, and F2b pups at 30 ppm. Significantly-reduced absolute organ weights were observed in F1b and F2b offspring (in females only liver and kidney weights were reduced) at 50 ppm; F1b males receiving 30 ppm phosphamidon also had decreased liver, kidney, and testes weights. Relative brain weights of F1b males and females were increased in the 30 and 50 ppm dose groups and of F2b offspring at 50 ppm. The increase in relative brain weight reflects the decreased body weights of these animals. This was also true for heart and kidney weights in F2b weanlings. After macroscopic and microscopic examinations of F1b and F2b pups, no treatment-related findings were observed. Examination of craniofacial sections from F2a pups was performed because of the finding of hydrocephalus in the first teratogenicity experiment. On day 4 or day 21 postpartum no malformations or variations were noted. The NOEL in this study was 5 ppm (Holson, 1985a). Special studies on teratogenicity Rats Groups of 30 pregnant albino Sprague-Dawley rats were dosed by oral gavage with 0, 1, 2, or 4 mg phosphamidon (92.1% pure) in CMC/kg b.w./day from days 6 through 15 of gestation. All dams were sacrificed on day 20 of gestation and the fetuses were removed, weighed, and examined for sex and for external, visceral, skeletal, and head abnormalities. In the 2 and 4 mg/kg b.w./day groups increased incidences of oral and ocular discharge, hypoactivity, and tremours were observed. There was no mortality and none of the test animals delivered prematurely or aborted during the study. At 2 and 4 mg/kg b.w./day, mean body weight (actual and corrected for uterus weight) was decreased (significantly at 4 mg/kg b.w./day). The percentages of pregnant dams, number of corpora lutea, number of implantations, and number of pre-implantation losses were comparable among control and treated groups. Prenatal viability and death, mean fetal body weight, and sex ratios were not affected at any dose level. At 4 mg/kg b.w./day an increased incidence of fetuses with a body weight 30% less than the controls was noted, which was probably due to maternal toxicity. The incidence of external malformations was increased at 4 mg/kg b.w./day. Visceral and skeletal malformations were not observed. Hydrocephalus was observed in 2, 2, and 3 fetuses of the 1, 2, and 4 mg/kg b.w./day groups, respectively. Although the incidences were not statistically- significantly increased and no external observations of dome-shaped head were noted in any of the fetuses with suspected hydrocephalus, the finding of hydrocephalus was considered to be equivocal (Holson, 1985b). In order to clarify the equivocal findings of hydrocephalus, a repeat study was initiated in which groups of 30 pregnant female albino Sprague-Dawley rats were dosed by oral gavage with 0, 0.5, 2, 4, or 6 mg phosphamidon (92.1% pure) in CMC/kg b.w./day from days 6 through 15 of gestation. All animals were killed on gestation day 20. Maternal gross lesions were observed and recorded as well as external, visceral, skeletal, and Bouin's head examinations on the removed fetuses. In the 2, 4, and 6 mg/kg b.w./day groups, increased incidences of salivation, ocular discharge (4 and 6 mg/kg b.w./day only), and tremours were observed. Twenty-four animals in the 6 mg/kg b.w./day group and 5 animals in the 4 mg/kg b.w./day group died during the dosing period. Pink pancreas and haemorrhages of the stomach were observed after necropsy and laparohysterectomy of animals in the 2 highest dose groups. Statistical analysis was not carried out on maternal or prenatal data generated from the 6 mg/kg b.w./day group, due to the limited number of dams (less than 50%) surviving until scheduled death. The percentages of pregnant dams, number of corpora lutea, number of implantation sites, and number of pre-implantation losses were comparable among controls and all teated groups. Mean body weights (actual and corrected for uterine weight) of the dams were significantly reduced in the 2 and 4 mg/kg b.w./day dose groups. Mean fetal weight was significantly reduced at 4 mg phosphamidon/kg b.w./day. Significant differences were not observed between control and treated groups in prenatal viability, prenatal death, mean litter size of viable fetuses, or sex ratios. A range of variations was observed during skeletal, external, and Bouin's head examinations. In the 4 mg/kg b.w./day group, a significantly-increased combined incidence of either incompletely ossified sternebra 2, 5, and 6 or absent sternebra 2, 5, and 6 were noted. At the same dose level there was a significant increase in the incidence of fetuses having a body weight 30% less than the mean control. Both effects were probably secondary to the maternal toxicity observed in this dose group. No visceral or skeletal malformations were found, and there was no finding of hydrocephaly after evaluation of the head specimens of all fetuses. Fetotoxicity was not observed at 2 mg/kg b.w./day. Based on these data, the NOEL in this study was 0.5 mg phosphamidon/kg b.w./day for maternal toxicity. There was no evidence of any teratogenic effect in this study (Holson, 1985c). Rabbits Groups of 18 pregnant New Zealand white rabbits were dosed by oral gavage with 0, 1, 3, or 10 mg phosphamidon (91.8% pure) in CMC/kg b.w./day from day 6 to 18 of gestation. The dams were sacrificed on day 30 of gestation and removed fetuses were examined for external, skeletal, and visceral malformations. One dam in each dose group died, 1 animal in each of the mid- and high-dose groups gave premature birth, and 1 animal in the 10 mg/kg b.w./day group aborted. At 10 mg/kg b.w./day, mean body weights of the dams were significantly decreased. The number of pregnant dams, corpora lutea, implantations, and pre-implantation losses, as well as litter sizes, number of resorptions, prenatal viability, and fetal body weights were not affected by phosphamidon treatment. No treatment-related malformations were observed during external, visceral, or skeletal examinations. The NOEL in this study was 3 mg phosphamidon/kg b.w./day, based on maternal toxicity. Phosphamidon did not show teratogenic activity at the doses tested (Holson, 1985d). Special studies on mutagenicity For the results of mutagenicity studies with phosphamidon, see Table 1. Additionally, it was found that phosphamidon was mutagenic in several plant systems (Wuu & Grant, 1966; Reddy & Rao, 1969; Panda & Sharma, 1979; Srivastava & Sarma, 1979; Singh et al., 1979; Sharma et al., 1983). Special study on delayed neurotoxicity Twelve domestic hens, 12 months old and protected by atropine sulphate (10 mg/kg), received orally 30 mg phosphamidon (92.1% pure)/kg b.w. After an interval of 21 days, the same procedure was repeated. The dose of 30 mg/kg b.w. was chosen based upon the LD50 of phosphamidon in laying hens ranging from 25 to 36 mg/kg b.w. A negative control group of 10 hens was treated with distilled water alone. A positive control group of 4 hens received a single oral dose of 1000 mg TOCP/kg b.w. No spontaneous mortality occurred, but 2, 2, and all 4 hens of the vehicle, dose, and positive control groups, respectively, were sacrificed before the end of the study, due to poor general condition. The animals of the TOCP group showed signs of severe ataxia. In the phosphamidon-treated animals, ataxia, hyper-irritability, trismus, ventral body position, and diarrhoea were observed, but after both applications (days 1 and 22), they recovered within 6 days. There were no indications of delayed neurotoxicity. In all hens of the positive control group, weight loss was observed, as well as progressive ataxia in the second week after administration. After neuro-pathological examination, no treatment-related changes were observed in the test group, whereas the positive control group developed neuropathy (Schoch & Krinke, 1985). Short-term study Dogs Groups of beagle dogs (8/sex/group) were orally administered in gelatine capsules 0, 0.05, 0.1, or 2.5 mg phosphamidon (91.8% pure)/kg b.w./day for 1 year. Interim sacrifice of 2 dogs/sex/group was carried out after 3 months of treatment. Two male and 2 female control dogs, and 1 male and 2 female dogs of the high-dose group, were sacrificed after a 4 week recovery period. Table 1. Special studies on the mutagenicity of phosphamidon Test system Test object Concentration Purity Results Reference used non-activated activated In vitro Ames test S. typhimurium 25 - 2025 µg/plate 91.8% negative negative Arni & Müller, strain: TA98 1982 TA100 TA1535 TA1537 Mitotic gene conversion S. cerevisiae 80 - 10,000 91.8% negative questionable Arni & Müller, test strain D7 g/plate 1983 Back mutation test S. cerevisiae 80 - 10,000 91.8% negative questionable Arni & Müller, strain D7 g/plate 1983 Spot test reverse E. coli not given not negative negative Nagy et al., 1975 mutation WP2 given Mouse lymphoma forward mouse L5178Y 20.6 -33.0 nl/ml 91.8% negative Beilstein & mutation assay Tk+/- cells 65.0 - 1040 nl/ml 91.8% negative Müller, 1983 DNA repair test human fibroblasts 2, 10, 50, & 250 91.8% negative Puri & Müller, nl/ml 1983a rat hepatocytes 1, 5, 25 & 125 nl/ml 91.8% negative Puri & Müller, 1983b Table 1. (cont'd). Test system Test object Concentration Purity Results Reference used Chromosome aberration human lymphocytes 1.9 - 61 µg/ml not positive Georgian, 1975 test given In vivo Sister chromatid Chinese hamster oral, 1x 2.5, 5, & 91.8% negative Hool & Arni, 1983 exchange test bone marrow cells 10 mg/kg b.w. Nucleus anomaly test Chinese hamster oral, 2x (days 0) 2.5, 91.8% questionable Strasser et al., bone marrow cells 5, & 10 mg/kg b.w. 1983 oral, 2x (days 0, 1) 91.8% questionable Strasser et al., 3.5, 5.0, 7.1, & 1983 10 mg/kg b.w. Chinese hamster bone oral, 2x (days 0, 1) 92.1% negative Strasser et al., marrow cells 1.67, 2.35, & 1985 6.7 mg/kg Chromosome aberration mouse spermatogonia oral, 5x (days 0 - 4) 92.1% negative Strasser & Arni, test 1.12 & 3.35 mg/kg 1985 mouse spermatocytes oral, 5x (days 0, 2, 92.1% negative Strasser et al., 3, 5, & 9) 2.2 & 1986 6.7 mg/kg Table 1. (cont'd). Test system Test object Concentration Purity Results Reference used Micronucleus test mouse bone marrow oral, 2x (days 0, 1) not positive Rani, 1980 5 mg/kg b.w. given Host-mediated assay S. typhimurium i.p. 3x (days 0 - 2) not positive Rani, 1980 G-46, mouse 7.5 mg/kg b.w. given Chromosome aberration rat bone marrow i.p. 0.07 - 9.56 not positive Georgian, 1975 test mg/kg b.w. given mouse bone marrow i.p. 0.07 - 9.56 not positive Georgian, 1975 mg/kg b.w. given One male dog in the 2.5 mg/kg b.w./day group had to be killed during week 24 due to poor general condition. In high-dosed dogs, vomiting was slightly increased. Mean body weight, food consumption, and food conversion were not adversely affected. Ophthalmoscopy and hearing tests showed no abnormal findings. Clinical analysis showed treatment-related effects on haematology and biochemical parameters. In high-dose males erythrocyte counts, haemoglobin concentrations, and haematocrits decreased significantly; alanine aminotransferase activity was increased, but at week 13 only. In males and females of the high-dose group, cholinesterase activity (measured by the Ellman method) was inhibited in plasma (20 - 30%), and erythrocytes (70 - 81%) at weeks 13 and 52; in the same group, brain cholinesterase was depressed (50%) at interim sacrifice and at the end of the administration period. During the 4 week recovery period, plasma and brain cholinesterase activities recovered completely, but inhibition in erythrocytes was still found (50%). Organ-weight ratios were not affected by phosphamidon treatment and no treatment-related effects were observed at macroscopic or microscopic examination. The NOEL was 0.1 mg/kg b.w./day (Kobel et al., 1985). Long-term study Rats Groups of CD Sprague-Dawley (60 - 70/sex/group) rats were fed diets containing 0, 0.2, 0.8, 1, 30, or 80 ppm phosphamidon for 24 months. The groups receiving 0.2 and 0.8 ppm phosphamidon were discontinued after 15 weeks. In the high-dose group, and to a lesser extent in the 30 ppm group, the incidences of crusty muzzle, irritability, foot-pad abnormalities, muscle tremours, yellow/brown-stained fur, and poor coat quality were significantly increased. Survival rates of rats in the treated groups were not significantly different from those of controls. Food consumption at 80 ppm was decreased initially, and increased in the majority of subsequent intervals, up to 1 year for males and up to 2 years for females. In males, growth was decreased at 80 ppm throughout the study, and at 30 ppm during the first 3 months. Only a slight decrease in growth was observed in females at 80 ppm. Haemoglobin, haematocrit, and erythrocyte counts were decreased in males at 80 ppm and in females at 80 ppm after 18 and 24 months. A significant increase in Howell-Jolly bodies in the red blood cells was observed at final sacrifice in the 30 and 80 ppm male groups, and in the 80 ppm female group. Serum cholinesterase and brain cholinesterase activities were markedly inhibited in rats in the 30 and 80 ppm male and female groups. Erythrocyte cholinesterase activity was inhibited to a lesser extent at 80 ppm. Organ weights were not affected in a dose-related manner. In the 30 and 80 ppm dose groups, a variety of non-neoplastic alterations was observed. These alterations included ulcerative pododermatitis of the limbs, hyperkeratotic dermatitis of the tail, and inflammatory changes in the lung and liver. There were no differences in the incidence of neoplastic lesions between treated and control groups. The NOEL was 1 ppm phosphamidon in the diet (Mayhew & Wingard, 1986). COMMENTS Phosphamidon induced effects on litter size and pup viability at maternally-toxic dose levels in a rat-multigeneration study. In teratogenicity studies, fetotoxicity and maternal toxicity, but no teratogenic effects, were observed. A study with hens showed that phosphamidon did not induce delayed neurotoxicity. In a 1-year study in dogs, the main effect was inhibition of serum, erythrocyte, and brain cholinesterase activities at 2.5 mg/kg b.w./day. At 0.1 mg/kg b.w./day no effects were found. A long-term toxicity/carcinogenicity study in rats also revealed cholinesterase inhibition as the predominant effect. There was no increase in tumour incidence. In this study no effects were observed at 1 ppm. In mutagenicity studies, phosphamidon was negative in a number of in vitro test systems, except for 1 test on chromosome aberrations. Several in vivo tests on chromosome anomalies have been carried out in rodents. Reports submitted to WHO showed negative or questionable results, whereas literature data showed a positive effect. On the basis of the toxicological data and in view of the negative carcinogenicity study in rats, a full ADI was allocated. TOXICOLOGICAL EVALUATION LEVEL CAUSING NO TOXICOLOGICAL EFFECT Rat: 1 ppm, equivalent to 0.05 mg/kg b.w./day. Dog: 0.1 mg/kg b.w./day. ESTIMATE OF ACCEPTABLE DAILY INTAKE FOR MAN 0 - 0.0005 mg/kg b.w. STUDIES WHICH WILL PROVIDE INFORMATION VALUABLE FOR THE CONTINUED EVALUATION OF THE COMPOUND Observations in man. REFERENCES Arni, P. & Müller, D. C 570 techn.: Salmonella/mammalian-microsome 1982 mutagenicity test. Unpublished report dated 22 November 1982 from Ciba-Geigy Ltd., Basle, Switzerland. Submitted to WHO by Ciba-Geigy Ltd., Basle, Switzerland. Arni, P. & Müller, D. C 570 techn.: Saccharomyces cerevisiae 1983 D7/mammalian-microsome mutagenicity test in vitro. Unpublished report dated 29 September 1983 from Ciba-Geigy Ltd., Basle, Switzerland. Submitted to WHO by Ciba-Geigy Ltd., Basle, Switzerland. Beilstein, P. & Müller, D. C 570 techn.: L5178Y TK+/- mouse lymphoma 1983 mutagenicity test. Unpublished report dated 20 December 1983 from Ciba-Geigy Ltd., Basle, Switzerland. Submitted to WHO by Ciba-Geigy Ltd., Basle, Switzerland. Georgian, L. The comparative cytogenetic effects of aldrin and 1975 phosphamidon. Mutat. Res., 31, 103 - 108. Holson, J.F. C 570 techn.: Two-generation reproduction study in albino 1985a rats. Unpublished Report dated 25 January 1985 from Science Applications, Inc., LaJolla, CA, USA. Submitted to WHO by Ciba-Geigy Ltd., Basle, Switzerland. Holson, J.F. C 570 techn.: Teratology study in rats. Unpublished 1985b Report dated 22 November 1982 from Science Applications, Inc., LaJolla, CA, USA. Submitted to WHO by Ciba-Geigy Ltd., Basle, Switzerland. Holson, J.F. C 570 techn.: Teratology study in rats. Unpublished 1985c Report dated 24 January 1985 from Science Applications, Inc., LaJolla, CA, USA. Submitted to WHO by Ciba-Geigy Ltd., Basle, Switzerland. Holson, J.F. C 570 techn.: Teratology study (SEG II) in albino 1985d rabbits. Unpublished Report dated 29 September 1983 from Science Applications, Inc., LaJolla, CA, USA. Submitted to WHO by Ciba-Geigy Ltd., Basle, Switzerland. Hool, G. & Arni, P. C 570 techn.: Sister Chromatid exchange study, 1983 Chinese hamster. Unpublished report from Ciba-Geigy Ltd., Basle, Switzerland. Submitted to WHO by Ciba-Geigy Ltd., Basle, Switzerland. Kobel, W., Gretener, P. Zak, F., & Malinowski, W. C 570 techn.: One 1985 year chronic oral toxicity study in Beagle dogs. Unpublished GU Project No. 820753, report dated 4 February 1985 from Ciba-Geigy Ltd., Basle, Switzerland. Submitted to WHO by Ciba-Geigy Ltd., Basle, Switzerland. Mayhew, D.A. & Wingard, B.L. Twenty four month combined chronic oral 1986 toxicity and oncogenicity study in rats using phosphamidon. Unpublished report from American BioGenics Corp., Decatur, IL, USA. Submitted to WHO by Ciba-Geigy Ltd., Basle, Switzerland. Nagy, Z., Mile, I., & Antoni, F. The mutagenic effect of pesticides on 1975 Escherichia coli WP2 try. Acta Microbiol. Acad. Sci. Hung., 22(3), 309 - 314. Panda, B.B. & Sharma, C. Organophosphate-induced chlorophyll mutations 1979 in Hordeum vulgare. Theor. appl. genet. 55(6), 253-255. Puri, E. & Müller, D. C 570 techn.: Autoradiographic DNA repair test 1983a on human fibroblasts. Unpublished report dated 2 June 1983 from Ciba-Geigy Ltd., Basle, Switzerland. Submitted to WHO by Ciba-Geigy Ltd., Basle, Switzerland. Puri, E. & Müller, D. C 570 techn.: Autoradiographic DNA repair test 1983b on rat hepatocytes. Unpublished report dated 14 June 1983 from Ciba-Geigy Ltd., Basle, Switzerland. Submitted to WHO by Ciba-Geigy Ltd., Basle, Switzerland. Rani, Usha M.V. Mutagenicity studies involving aldrin, endosulfan, 1980 dimethoate, phosphamidon, carbaryl and ceresan. Bull. Environ. Contam. Toxicol. 25(2), 277 - 282. Reddy, M. & Rao, B. The cytological effects of insecticides 1969 (Dimecron-100 and Rogar-40) on Vicia Faba L. Cytologia, 34, 408 - 417. Schoch, M. & Krinke, G. C 570 techn.: Acute delayed neurotoxicity 1985 study in laying hens. GU project No. 831242, unpublished report dated 23 January 1986 from Ciba-Geigy Ltd., Basle, Switzerland. Submitted to WHO by Ciba-Geigy Ltd., Basle, Switzerland. Sharma, C.B. et al. Cytogenetic hazards from agricultural chemicals: 1983 6. Persisting impact of organophosphorus insecticides on the chiasma frequencies in barley. Biol. Zentralbl. 102: 567 - 570. Singh, B. D. et al. Effects of insecticides on germination, early 1979 growth and cytogenetic behaviour of barley (Hordeum vulgare). Environ. Exp. Bot., 19(3): 127 - 132. Srivastava, S. & Sarma, Y.S. Effect of two insecticides - dimecron and 1979 nuvan - on the survival, growth and nuclear cytology of Oedogonium gunnii Witter. J. Cytol. Genet. 14(2), 163-172. Strasser, F. Langauer, M., & Arni, P. C 570 techn.: Nucleus anomaly 1983 test in somatic interphase nuclei, Chinese hamster. Unpublished report dated 21 December 1983 from Ciba-Geigy Ltd., Basle, Switzerland. Submitted to WHO by Ciba-Geigy Ltd., Basle, Switzerland. Strasser, F. & Arni, P. C 570 techn.; Chromosome studies on male 1985 germinal epithelium of mouse spermatogonia. Unpublished report dated 26 November 1985 from Ciba-Geigy Ltd., Basle, Switzerland. Submitted to WHO by Ciba-Geigy Ltd., Basle, Switzerland. Strasser, F., Langauer, M., & Arni, P. C 570 techn.: Nucleus anomaly 1985 test in somatic interphase nuclei of Chinese hamster. Unpublished report dated 23 October 1985 from Ciba-Geigy Ltd., Basle, Switzerland. Submitted to WHO by Ciba-Geigy Ltd., Basle, Switzerland. Strasser, F., Puri, E., & Arni, P. C 570 techn.: Chromosome studies on 1986 male germinal epithelium of mouse spermatocytes. Unpublished report dated 3 February 1986 from Ciba-Geigy Ltd., Basle, Switzerland. Submitted to WHO by Ciba-Geigy Ltd., Basle, Switzerland. Wuu, K.O. & Grant, W.F. Morphological and somatic chromosomal 1966 aberrations induced by pesticides in barley (Hordeum vulgare). Can. J. Genet. Cytol. 8, 481 - 501.
See Also: Toxicological Abbreviations Phosphamidon (ICSC) Phosphamidon (PIM 454) Phosphamidon (FAO Meeting Report PL/1965/10/1) Phosphamidon (FAO/PL:CP/15) Phosphamidon (FAO/PL:1968/M/9/1) Phosphamidon (FAO/PL:1969/M/17/1) Phosphamidon (WHO Pesticide Residues Series 2) Phosphamidon (WHO Pesticide Residues Series 4) Phosphamidon (Pesticide residues in food: 1982 evaluations)