TRIAZOLYL ALANINE EXPLANATION Triazolyl alanine is a plant metabolite of various fungicides of the triazole family. Since the formation of triazolyl alanine is not observed in animals, it became necessary to examine the toxicology of this compound separately in order to assess the safetycof possible residues in edible crops. Triazolyl alanine is considered for the first time by the present meeting. EVALUATION FOR ACCEPTABLE DAILY INTAKE BIOLOGICAL DATA Biochemical aspects Absorption, distribution and excretion [3,5-14C]-Labelled D,L-triazolyl alanine was administered orally by gavage at single doses of 0.5 and 50 mg/kg bw to 2 groups of 4 male and 4 female Tif:RAIf rats. Administration resulted in rapid, nearly complete absorption from the gastro-intestinal tract and very rapid elimination mainly via the kidneys. Within 24 hours (at both dose levels) 95-105% of the radioactivity applied was eliminated from the animal, 85-103% of which was found in the urine. Seven days after dosing 88-108% and 2-12% of the dose was recovered with the urine and faeces, respectively. Less than 0.5% was found in the expired air. Residues in organs and tissues 7 days after administration of 0.5 mg/kg bw were all below the detection limit. After a dose of 50 mg/kg bw, residues were only found in liver, kidneys, blood, stomach, muscle and remaining carcass and were below 0.02 mg/kg in each case (Hamboeck, 1983a). Biotransformation The excreta of rats obtained in the previously described study after a single oral dose of 0.5 or 50 mg/kg bw 14C D,L-triazolyl alanine were used for isolation of metabolites. The major radioactive urinary component (69-86% of dose applied) was identified as unchanged D,L-triazolyl alanine, 8-19% of the dose was eliminated in the urine after metabolic transformation to N-acetyl- D,L-triazolyl alanine. The pattern of metabolites in feces was quite similar with 1-2% found as unchanged parent compound and <1% as the N-acetyl metabolite. The remaining radioactivity consisted of 4 polar metabolite fractions (1-3% of dose each) which were not further investigated (Hamboeck, 1983b). Short-term studies In a two weeks-range finding study groups of 10 male rats were administered 0, 3000 or 10000 ppm triazolyl alanine (purity ca 100%) in the drinking water. At all dose levels no treatment related effects were observed on appearance and behaviour, food and water consumption, body weight, organ weight and macroscopy. A slight but not significant increase was observed in liver and kidney weight at 10000 ppm (Bomhard, 1982). Toxicological studies Acute toxicity TABLE 1. ACUTE TOXICITY OF TRIAZOLYL ALANINE SPECIES SEX ROUTE PURITY LD50 LC50 REFERENCE Mouse M&F oral ? >5000 Mihail, 1982 Rat ? oral ? >2000 Henderson & Parkinson, 1981 Rat M&F oral ? >5000 Mihail, 1982 Rat M&F i.p. ? >5000 Mihail, 1982 Groups of 20 male and 20 female rats (Bor: Wisw SPF/Cpb) were administered by gavage triazolyl alanine for 28 days at dose levels of 0, 25, 100 or 400 mg/kg bw. Groups of 10 male and 10 female rats were kept for a 4 week recovery period. Observations included mortality and signs of toxicity, body weight, food and water consumption, haematology, clinical chemistry and urinalysis, microsomal N-demethylase and O-demethylase and P-450 in the liver, organ weight, macroscopy and histopathology. There were no effects on mortality, appearance, body weight, food and water consumption, urinalysis, liver biochemistry, macroscopy and histopathology during dosing and recovery period. Significantly decreased values for creatinine and urea (males only) were observed at 400 mg/kg bw after the dosing as well as after the post-observation period (urea values only). Liver weight was significantly decreased in females after 4 weeks at 400 mg/kg bw. The NOAEL in this study is 100 mg/kg bw (Mihail & Vogel, 1983). Groups of rats [Bor:WisW (SPF CPB)] (20/sex/group) were orally administered 0, 1250, 5000 or 20000 ppm triazolyl alanine (purity 97.5%) in the diet (equal to 90, 370 or 1510 and 100, 400 or 1680 mg/kg bw/day for males and females, respectively) for 97 days. All animals were inspected twice daily for mortality and appearance and behaviour, while body weight, food intake and water consumption were recorded weekly. Ophthalmoscopical observations were performed before the start and at the end of the study while haematology, clinical chemistry and urinalysis were carried out on 10 male and 10 female rats from each group after 1 month and at the end of the study. At the end of the study all animals were killed, selected organs were weighed, and complete gross and histopathological examinations were performed. No toxic symptoms or treatment related deaths occurred. No differences between control and treated animals were noted in food and water consumption, ophthalmoscopy, haematology and urinalysis. Body weight was slightly decreased in males at 20000 ppm from week 2 to the end of the study. Clinical biochemistry revealed statistically significant decreases in triglyceride, bilbirubin and urea values in males at 20000 ppm and in triglyceride values in females at 5000 and 20000 ppm. ASAT values were significantly increased in males at 20000 ppm. Relative heart weight was significantly decreased and relative kidney weight was significantly increased in high dosed males. Based on the effect in females the NOAEL in this study is 1250 ppm in the diet, equal to 100 mg/kg bw (Maruhn & Bomhard, 1984). Dogs Groups of 4 male and 4 female Beagle dogs received triazolyl alanine (purity 97.5%) in the diet at 0, 3200, 8000 or 20000 ppm for 13 weeks. No treatment related effects were observed on mortality, clinical signs, body temperature, pulse rate, ophthalmoscopy and neurological findings, water consumption, hematological and clinical chemical examinations, urinalysis, gross pathology and histopathological liver findings. Female body weight and food consumption were decreased at 20000 ppm. Spleen weight was increased in males at the same dose. The NOAEL in this study is 8000 ppm in the diet, equivalent to 200 mg/kg bw (von Keutz & Groning, 1984). Reproduction study Rats In a preliminary reproduction study groups of 12 female and 6 male rats were fed 0, 150, 625, 2500 or 10000 ppm triazolyl alanine in the diet prior to mating. Male rats were killed after mating, while in females treatment was continued throughout pregnancy, lactation and weaning of offspring, whereupon all females and offspring were killed. No treatment related effects were observed except for an increase in mean pre-coital period and a significantly decreased mean litter weight at day 1 at 10000 ppm (Birtley, 1983). Groups of 15 male and 30 female ALpk:AP rats received triazolyl alanine (purity 97.6-97.8%) in the diet at 0, 500, 2000 or 10000 ppm. After 12 weeks of treatment the rats were paired to start a 2-generation (2 litters/generation) study. Diets were maintained during mating, gestation and lactation for two successive litters (F1a and F1b) and repeated for 2 generations. F1 parents selected from F1b offspring were mated after 11 weeks. No adverse effects were observed on clinical signs and food consumption, parental bodyweight, male and female fertility, pre-coital interval, gestation period, number of viable litters, live and dead fetuses, macroscopy in parental rats and offspring, microscopy in offspring. Initial pup weight was slightly decreased in F1b, F2a and F2b pups at 10000 ppm and the total weight of the F2b litter was significantly decreased at the same dose. No compound related pup abnormalities were observed (Milburn et al. 1986). Special studies on mutagenicity See Table 2 TABLE 2. RESULTS OF MUTAGENICITY ASSAYS ON TRIAZOLYL ALANINE CONCENTRATION TEST SYSTEM TEST OBJECT OF TRIAZOLYL PURITY RESULTS REFERENCE Ames test (both S. tyhimurium 20, 100, 500, ? Negative Herbold, 1983a with and without TA1535, TA100, TA1537 2500 and 12500 (1) activation) TA1538, TA98 ug/pl dissoved in DMSO Ames test (both S. tyhimurium 20, 78, 313, 1250 97.4 Negative Deparade, 1986 with and without TA1535, TA100, TA1537 and 5000 ug/0.1 (1,2) metabolic activation) TA98, TA102 ml in DMSO Gene mutation test Chinese hamster cells 500, 1000, 2000, 97.4 Negative Dollenmeier, 1986 (both with and V79 4000, 6000, 8000 and (1) without metabolic 10000 ug/ml in activation) bidist. water Transformation assay BHK 21 C13 cells 500, 1000, 2000 ? Negative Richold et al. 1981 (without metabolic 4000 and 8000 ug/pl (1) activation) in DMSO Transformation assay BHK 21 C13 cells 1000, 1000, 4000 ? Positive Richold et al. 1981 (with metabolic 8000 and 16000 at toxic activation) ug/pl in DMSO doses toxic >= 16000 (1) Transformation assay Mouse 62.5, 125, 250, 500 ? Negative Beilstein, 1984 (with and without embryofibroblasts and 1000 ug/ml in (1) metabolic activation) BALB/3T) bidistilled water TABLE 2 (contd.) CONCENTRATION TEST SYSTEM TEST OBJECT OF TRIAZOLYL PURITY RESULTS REFERENCE Micronucleus test Male mice (CBC F1) 2500 and 5000 mg/kg ? Negative Watkins, 1982 i.p. in 0.5% Tween (1) 80 (administered twice, 24 hr apart) Micronucleus test Mice (NMRI strain) 8000 mg/kg bw ? Negative Herbold, 1982, 1983b orally in 0.5% (1) cremophor emulsion Micronucleus test Chinese hamster 5000 mg/kg oral 97.4 Negative Strasser, 1986 in 0.5% CMC (1) DNA repair test E. coli pol A1 62.5, 125, 250 ? Negative Herbold, 1983c 500 and 1000 ug/pl (1) in DMSO DNA repair test Rat hepatotes 0.08, 0,4, 2 and 97.4 Negative Puri, 1986 10 mg/ml (1) 1) Positive control yielded positive results. 2) Precipitations occurred at concentrations of 78 µg/0.1 ml and above. Special study on teratogenicity Rats Groups of 24 female rats (Alderley Park AlpK/AP) received 0, 100, 300 or 1000 mg triazolyl alanine (purity 84,8%)/kg bw by gavage from day 7-16 of gestation. Dams were observed for clinical observations, bodyweights and food consumption. On day 22 of pregnancy the animals were sacrificed and their uteri weighed and examined for deaths and live fetuses. The fetuses were sexed, weighed and examined for developmental abnormalities. No clinical signs of maternal toxicity were noted. Maternal body weight, food consumption and reproductive parameters (pregnancy rate, survival and growth of the foetuses in utero) were comparable to those of the controls. At 1000 mg/kg bw ossification was delayed (7th cervicial vertebrae, 13th thoracic centrum and 5th sternebrae). The incidence of not ossified odontoid processes was significantly increased at 300 and 1000 mg/kg bw. The effect at 300 mg.kg bw is minimal and 100 mg/kg b.w is a no observed adverse effect level (Clapp et al. 1983). COMMENTS Triazolyl alanine is rapidly absorbed and excreted, mainly as the unchanged parent compound in the urine. A small proportion is excreted as the N-acetyl metabolite. The substance has a low acute toxicity in the species examined. In a 90-day study in rats at 20 000 ppm, growth inhibition was found. In addition, triglyceride and urea in blood were decreased, and aspartate aminotransferase was increased. A decrease in triglyceride was also observed in females at 5000 ppm. The NOAEL was 1250 ppm, which is equal to 100 mg/kg bw. In a 90-day study in dogs, 20 000 ppm in the diet resulted in decreased body weight and decreased food consumption. The NOAEL was 8000 ppm, equivalent to 200mg/kg bw/day. In a 2-generation 2-litter per generation reproduction study in rats, pup and litter weights were reduced at the highest dose level of 10 000 ppm (equivalent to 500 mg/kg bw day). No teratogenic effects were induced in rats, but a delay in ossification at the highest dose levels was reported. The NOAEL was 100 mg/kg bw/day. After reviewing all available in vitro and in vivo short-term tests, the Meeting concluded that there was no evidence of genotoxicity. The Meeting concluded from the available data that residues of triazoylalanine arising from the use of triazole fungicides do not present a toxicological hazard. REFERENCES All reports are send to WHO on behalf of the triazolyl alanine group (Bayer, Ciba-Geigy, ICI and Rohm & Haas) by Ciba-Geigy AG, Basel, Switzerland. Beilstein, P. (1984) Transformation/liver-microsome test (in vitro test for transformation-inducing properties in mammalian fibroblasts). Unpublished Report Test No. 840324 from Ciba-Geigy Ltd. Birtley, R.D.N. (1983) Triazole alanine: Preliminary reproduction study in the rat. Unpublished Report No. CTL/L/470 from Central Toxicology Laboratory, ICI PLC. Bomhard, E. (1982) THS 2212, Preliminary subacute toxicity study on male rats, administration in the drinking water. Unpublished Report No. 11253 from Institut für Toxicology Bayer AG. Clapp, M.J.L. & Killick, M.E. Hollis, K.J. & Godley, M.J. (1983) Triazole alanine. Teratogenicity study in the rat. Unpublished Report No. CTL/P/875 from Central Toxicology Laboratory ICI PLC. Deparade, E. (1986) Salmonella/mammalian-microsome mutagenicity test. Unpublished Report Test No. 860187 from Ciba-Geigy Ltd. Dollenmeier, P. (1986) Point mutation test with chinese hamster cells V79 (OECD conform). Unpublished Report Test No. 860258 from Ciba-Geigy Ltd. Hamboeck, H. 1983a) Distribution, degradation and excretion of D,L-2-amino-3-(1-H-1,2,4-triazol-1-yl)-propanoic acid (D,L-triazolylalanine) in the rat. Unpublished project report 1/83 from Biochemistry Department R & D Plant Protection Agricultural Division Ciba-Geigy Limited. Hamboeck, H. (1983b) The metabolism of D,L-2-amino-3-(1H-1,2,4- triazol-1-yl)- propanoic acid (D,L-triazolylalanine) in the rat. Unpublished project report 11/83 from Biochemistry Department R & D Plant Protection Agricultural Division Ciba-Geigy Ltd. Herbold, B. (1982) THS 2212 Triazolylalanine, Micronucleus test for mutagenic effect on mice. Unpublished Report No. 11054 from Toxicological Institute Bayer AG. Herbold, B. (1983a) THS 2212 Triazolylalanine, Salmonella/ Microsome test for point mutagenic effect. Unpublished Report No. 11388 from Toxicological Institute Bayer AG. Herbold, B. (1983b) THS 2212 Triazolylalanine, Micronucleus test for mutagenic effect on mice. Supplement to unpublished Bayer Report No. 11054: 11054 A from Toxicological Institute, Bayer AG. Herbold, B. (1983b) THS 2212 Triazolylalanine, Pol A1 test on E.Coli during testing for effects harmful to DNA. Unpublished Report No. 11390 from Toxicological Institute Bayer AG. Henderson, C. & Parkinson, G.R. (1981) R152056: Acute oral toxicity in rats. Unpublished Report No. CTL/P/600 from Central Toxicology Laboratory, ICI Ltd. Keutz von, E. & Groening, P. (1984) THS 2212 (tiazolylalanine), subchronic toxicity to dogs on oral administration (13-week feeding study). Unpublished Report No. 12562 from Institute of Toxicology Bayer AG. Maruhn, D. & Bomhard, E. (1984) Triazolylalanine (THS 2212) study for subchronic toxicity to rats (three-month feeding study). Unpublished Report No. 12397 from Institut of Toxicilogy Bayer AG. Mihail, F. Triazolylalanine (1982) (THS 2212) Acute toxicity studies. Unpublished Report No. 11229 from Institute fuer Toxicology Bayer AG. Mihail, F. & Vogel, O. (1983) Triazolylalanine (THS 2212) subacute oral toxicity study on rats. Unpublished Report No. 11491 from Institute of Toxicology Bayer AG. Milburn, G.M. & Birtley, R.D.N. & Pate, I., Hollis, K. & Moreland, S. (1986) Triazole alanine: Two generation reproduction study in the rat. Unpublished Report No. CTL/P/1168 (revised) from Central Toxicology Laboratory, ICI PLC. Puri, E. (1986) Autoradiographic DNA repair test on rat hepatocytes. Unpublished Report Test No. 860184 from Ciba-Geigy Ltd. Richold, M. & Allen, A., Williams, A. & Ransome, S.J. (1981) CTL/C/1085, Cell transformation test for potential carcinogenicity of R152056. Unpublished Report No. 394A/81153 from Huntingdon Research Centre. Strasser, F. (1986) Micronucleus test (chinese hamster). Unpublished Report Test No. 860185 from Ciba-Geigy Ltd. Watkins, P.A. (1982) R152056: 3-(1,2,4-triazol-1-yl) alanine, Micronucleus test in CBC F1 mice-TOM/4. Unpublished Report 156,342 from Central Toxicological Laboratory, ICI PLC.
See Also: Toxicological Abbreviations