INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY WORLD HEALTH ORGANIZATION Toxicological evaluation of certain veterinary drug residues in food WHO FOOD ADDITIVES SERIES 39 Prepared by: The forty-eighth meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA) World Health Organization, Geneva 1997 THIABENDAZOLE (addendum) First draft prepared by Dr G. Roberts Chemical Products Assessment Section Commonwealth Department of Health and Family Services Canberra, Australia 1. Explanation 2. Biological data 2.1 Toxicological studies 2.1.1 Short-term toxicity 2.1.2 Long-term toxicity and carcinogenicity 2.1.3 Genotoxicity 2.1.4 Reproductive toxicity 2.1.4.1 Multigeneration reproductive toxicity 2.1.4.2 Developmental toxicity 2.1.6 Special study on thyroid function 3. Comments 4. Evaluation 5. References 1. EXPLANATION Thiabendazole is a benzimidazole compound used both as a broad-spectrum anthelmintic in various animal species and for the control of parasitic infestations in humans. It was evaluated at the fortieth meeting of the Committee under the name 'tiabendazole' (Annex 1, reference 104), when an ADI of 0-100 µg/kg bw was established on the basis of reduced body-weight gain in a two-year study in rats and reduced fetal weight in a study of developmental toxicity in rats, by applying a safety factor of 100 to the NOEL of 10 mg/kg bw per day. The Committee requested the opportunity to review further toxicological studies known to be under way, in order to bring the database on thiabendazole up to date. These studies are summarized in this monograph addendum. 2. BIOLOGICAL DATA 2.1 Toxicological studies 2.1.1 Short-term toxicity Dogs Groups of four male and four female beagle dogs were given thiabendazole (purity, 99%) in capsules at doses of 0, 10, 40, or 160 mg/kg bw per day for one year. The dogs were observed daily for mortality and clinical signs and were weighed weekly. Ophthalmological examinations were conducted during weeks 27 and 50. Haematology, serum biochemistry and urinalysis were studied in weeks 4, 12, 26, and 52, and electrocardiograms were recorded during weeks 14, 25, and 50. At necropsy, the major organs were weighed, and an extensive array of organs and tissues were examined histopathologically. The study was conducted according to US EPA good laboratory practice (GLP) guidelines (40 CFR Part 160). An increased incidence of emesis was noted in the animals at 160 mg/kg bw per day, particularly during the first half of the study. One dog at 40 mg/kg bw per day died in week 3, but a relationship with treatment is unlikely. The body-weight gain of animals at 160 mg/kg bw per day was initially compromised, but there were no lasting effects on body weight or food consumption. Ophthalmological examinations revealed no significant treatment-related effects. The dose of 160 mg/kg bw per day decreased erythrocyte counts, haemoglobin levels, and the haematocrit, but increased activated partial thromboplastin time and the numbers of platelets and nucleated erythrocytes. The serum cholesterol level was higher in all treated animals, but there was no dose-response relationship and the values were within the range in historical controls. Urinary parameters and electrocardiograms were not significantly affected. Autopsy revealed mucosal discolouration of the gall-bladders of dogs at 40 and 160 mg/kg bw per day, and increased cytoplasmic vacuolation and inspissated bile in the villi of the gall-bladder in all treated animals. Other changes in animals at the two highest doses were increased liver weight, bile-duct vacuolation, distal tubular vacuolation in the kidney, cytoplasmic inclusions in the urinary bladder epithelium, and increased haemosiderin and erythropoiesis in the spleen. The dogs at 160 mg/kg bw per day also showed increased thyroid weight, follicular-cell hypertrophy, and increased bone-marrow erythropoiesis. The findings in the gall-bladder had been noted previously in dogs treated with thiabendazole and in untreated animals, including concurrent controls, thus casting doubt on their toxicological significance. The NOEL was 10 mg/kg bw per day on the basis of changes indicative of haemolytic anaemia (Lankas et al., 1993). 2.1.2 Long-term toxicity and carcinogenicity Rats Groups of 50 male and 50 female Sprague-Dawley Crl:CD BR rats were fed thiabendazole (purity, 99%) admixed in the diet at concentrations targeted to achieve doses of 0 (two groups), 10, 30, or 90 mg/kg bw per day for two years. Clinical observations were made daily, and body weights, the results of physical examinations, and food consumption were recorded weekly. Ophthalmological examinations were conducted during weeks 52 and 104; haematology, serum biochemistry, and urinalysis were studied in 15 fasted rats of each sex per group during weeks 14, 27, 53, 79, and 105. At necropsy, the major organs from 10 rats of each sex per group were weighed, and an extensive array of organs and tissues from all animals were examined histopathologically. The study was conducted in accordance with GLP guidelines as set forth in 40 CFR Part 160 (US EPA) and OECD test guideline 453. There were no effects on survival or clinical signs. Body-weight gain was depressed in males and females at 90 mg/kg bw per day and in males at 30 mg/kg bw per day; absolute food intake was decreased in animals at the highest dose only. Slight decreases in the erythrocyte count, haemoglobin level, and haematocrit were observed in rats at 30 and 90 mg/kg bw per day, but the effects were transient, not dose-related, and rarely significant. The serum cholesterol level was increased in animals at 90 mg/kg bw per day. Urinalysis and ophthalmology showed no abnormalities. At autopsy, the gross pathological findings were similar in all groups. The liver and thyroid weights were increased in rats at 90 mg/kg bw per day, accompanied by microscopic changes. Centrilobular hepatocellular hypertrophy was induced in males given 30 and 90 mg/kg bw per day. The effects on the thyroid included follicular-cell hypertrophy in males and females and follicular-cell hyperplasia in females, both at the 90 mg/kg bw per day dose, with an increased incidence of follicular-cell adenoma in males at 30 mg/kg bw per day and males and females at 90 mg/kg bw per day. Males given 30 mg/kg bw per day and males and females given 90 mg/kg bw per day had increased incidences of renal pelvic epithelial hyperplasia. The NOEL was 10 mg/kg bw per day on the basis of pathological changes in the liver, thyroid, and kidney (Squibb et al., 1993). 2.1.3 Genotoxicity 2.1.4 Reproductive toxicity 2.1.4.1 Multigeneration reproductive toxicity Rats Groups of 33 male and 33 female Sprague-Dawley Crl:CD(SD)BR rats were fed thiabendazole (purity, 99%) in the diet at concentrations adjusted to achieve doses of 0, 10, 30, or 90 mg/kg bw per day. Treatment commenced nine weeks before pairing of animals and continued throughout two mating periods of up to three weeks and lactation, for a total of two generations. Adult rats were observed daily for physical signs, and body weight and food consumption were recorded at least weekly. At appropriate times, females were checked for the presence of sperm in the vagina; parturition was then observed. During lactation, the offspring were observed daily, and body weights were recorded on postnatal days 0, 4, 7, 14, and 21. The litters were culled to four males and four females on postnatal day 4. All adult rats were killed after completion of lactation and subjected to gross examination. The reproductive organs of control animals and those at 90 mg/kg bw per day were also examined for histomorphological changes. The study was conducted in accordance with US EPA GLP guidelines (40 CFR Part 160). Table 1. Results of assays for genotoxicity with thiabendazole End-point Test object Concentration S9 Results Reference In vitro Reverse mutation S. typhimurium 3-6000 µg/plate + Negativea Sina & Lankas TA97, TA98, - Negativea (1992) TA100, TA1535 Reverse mutation E. coli WP2, 3-6000 µg/plate + Negativea Sina & Lankas WP2uvrA, - Negativea (1992) WP2uvrA, pKM101 Inhibition of tubulin Bovine tubulin 100 µmol/litre Positive Parry (1993) polymerization Aneuploidy A. nidulans > 19.87 µmol/litre Positive Parry (1993) S. cerevisiae > 49.7 µmol/litre Positive Hexaploid wheat > 114.2 µmol/litre Positive Aneuploidy Chinese hamster 50 µg/ml Positiveb Natarajan et embryonic al. (1993) fibroblasts Cell-division Chinese hamster > 10 µg/ml Positivec Warr et al. aberration cells; Don, LUC2 (1993) Chromosome Chinese hamster 100 µg/ml Positived Warr et al. enumeration LUC2 cells (1993) In vivo Chromosomal Mouse bone 200-2000 Negativee Galloway & aberration marrow mg/kg bw orally Lankas (1994) Table 1 (continued) S9, 9000 × g fraction of rat liver a Hydrazine sulfate and 2-aminoanthracene used as positive controls b Diethylstilboestrol used as positive control c Colcemid used as positive control d 2-Acetylaminofluorene used as positive control e Mitomycin C used as positive control The adult animals showed no treatment-related physical signs or mortality. Food consumption and body-weight gain were depressed at all stages before parturition in males and females at 90 mg/kg bw per day and in males at 30 mg/kg bw per day. During lactation, however, females at 90 mg/kg bw per day had considerably increased body-weight gains. There were no effects on mating performance, pregnancy rate, length of gestation, implantation rate, litter size or weight, or postnatal survival. The offspring of the rats at 90 mg/kg bw per day had reduced body-weight gain throughout lactation. External examination of pups and histopathological examination of reproductive organs did not reveal treatment-related effects. The findings were consistent in both generations. The NOEL was 10 mg/kg bw per day on the basis of depressed maternal body-weight gain (Lankas & Wise, 1992). 2.1.4.2 Developmental toxicity Mice Groups of 25 female Jcl:ICR mice were given doses of 0, 25, 100, or 200 mg/kg bw per day thiabendazole (purity, 99.8%) by gavage in olive oil on gestation days 6-15. Physical signs and body weight were recorded daily on gestation days 6-18. All dams were killed on gestation day 18 and examined grossly. Fetuses were examined externally and for visceral and skeletal abnormalities. The study was conducted in accordance with the GLP guidelines of the Japanese Ministry of Health and Welfare (No. 313) and the US FDA (21 CFR Part 58). No treatment-related physical signs, mortality, or abortions occurred. In animals at 100 and 200 mg/kg bw per day, food intake, body-weight gain, the numbers of implantations, and the weights of live fetuses were decreased. External malformations seen in treated groups included exencephaly, open eyelid, cleft palate, tail malformations, and clubbed hind foot. The low incidence of these malformations and the general lack of a dose-response relationship suggested they were unrelated to drug treatment. The incidences of visceral and skeletal malformations were not significantly increased. The incidence of incomplete ossification of the talus calcaneus was slightly increased in all treated groups; however, it was not dose-related, and the number of affected litters was similar in all groups. The NOEL was 25 mg/kg bw per day on the basis of reduced numbers of implantations (Nakatsuka et al., 1995). 2.1.5 Special study of thyroid function Rats Groups of 35 male Sprague-Dawley Crl:CD(SD)BR rats were fed thiabendazole (purity, 99.8%) in the diet at concentrations adjusted to achieve target doses of 0, 10, 90, or 270 mg/kg bw per day, for three months. Fifteen rats from each group were killed after 91 days of treatment; five were used to test for thyroxine clearance on day 94; and the remaining 15 animals were killed after a further three-month period of observation. Clinical signs were observed daily, and body weight and food consumption were recorded weekly. Serum levels of thyroid stimulating hormone (TSH), triiodothyronine (T3), and thyroxine (T4) were determined before treatment, during weeks 2, 4, 8, and 13 of treatment, and during recovery weeks 6 and 13. T4 clearance was measured during week 14 in five rats from each group 8, 22, 34, 48, and 72 h after an intravenous injection of radiolabelled thyroxine. The livers and thyroids from animals killed at the end of treatment and the end of recovery were weighed and subjected to pathological examination. The study was conducted in accordance with US EPA GLP guidelines (40 CFR Part 160). No clinical signs or deaths were attributable to treatment, but, during the treatment, body-weight gain was reduced in rats at 90 and 270 mg/kg bw per day and food intake in those at 270 mg/kg bw per day. These effects were no longer seen after cessation of treatment. The serum level of T4 was not significantly affected at any dose, whereas that of T3 was slightly decreased and that of TSH increased during treatment with 90 or 270 mg/kg bw per day. A subsequent decrease in TSH levels occurred mid-way through the recovery period only, but the T4 and T3 concentrations were similar to those of the controls. In the rats tested for thyroxine clearance, the volume of distribution of T4 was increased at 90 and 270 mg/kg bw per day but there were concomitant increases in the plasma half-lives. The net effect was increased clearance of T4 at 270 mg/kg bw per day with no significant changes at lower doses. Rats at 90 and 270 mg/kg bw per day killed at the end of the treatment had increased liver and thyroid weights, centrilobular hypertrophy in the liver, and diffuse hyperplasia of thyroid follicular cells. No evidence of such changes was seen after the recovery period. The NOEL was 10 mg/kg bw per day (Lankas et al., 1995). 3. COMMENTS New information considered by the Committee since the previous evaluation included data on short-term and long-term toxicity, genotoxicity, and reproductive toxicity. All of the studies were carried out according to appropriate standards for study protocol and conduct. In a one-year study, dogs were given thiabendazole at oral doses of 0, 10, 40, or 160 mg/kg bw per day. Anaemia, increased bone-marrow erythropoiesis, and thyroid follicular-cell hypertrophy were noted at the highest dose. At 40 and 160 mg/kg bw per day, bile-duct epithelial-cell vacuolation, renal tubular-cell vacuolation, cytoplasmic inclusions in the urinary bladder epithelium, and increased concentrations of haemosiderin and erythropoiesis in the spleen were observed. The incidences of both cytoplasmic vacuolation of the gall-bladder epithelium and inspissated (thickened) bile in the villi of the gall-bladder were increased in all treated groups. Similar observations have been reported previously both in dogs treated with thiabendazole and in untreated animals, including concurrent controls, which casts doubt on the toxicological significance of these effects. The NOEL was 10 mg/kg bw per day on the basis of changes indicative of haemolytic anaemia. Rats were given thiabendazole in the diet for two years at doses of 0, 10, 30, or 90 mg/kg bw per day. The incidences of centrilobular hypertrophy in the livers of males and of renal pelvic epithelial hyperplasia were increased in rats at the two highest doses. In the thyroid, follicular-cell hypertrophy and hyperplasia were observed at 90 mg/kg bw per day, and the incidences of thyroid follicular-cell adenomas were increased at 30 and 90 mg/kg bw per day. The NOEL was 10 mg/kg bw per day on the basis of pathological changes in the liver, thyroid, and kidney. In a study of thyroid function, rats were fed thiabendazole in the diet for three months at doses of 0, 10, 90, or 270 mg/kg bw per day. Treatment at 90 or 270 mg/kg bw per day was associated with increased clearance of thyroxine, decreased levels of triiodothyronine, and a concomitant increase in the level of thyroid stimulating hormone in serum. The Committee concluded that these findings are consistent with an indirect mechanism for stimulation of the thyroid gland by thiabendazole, leading to proliferative changes and subsequently to the formation of thyroid tumours in rats. Thiabendazole induced numerical chromosomal aberrations in a variety of assays in vitro. This effect is thought to be due to inhibition of tubulin polymerization, which is a characteristic effect of many benzimidazoles. Tests for mutations in bacteria and for chromosomal aberrations in mouse bone marrow gave negative results. In a two-generation study of reproductive toxicity, rats were given doses of 0, 10, 30, or 90 mg/kg bw per day in the diet. Reduced food intake and body-weight gain were observed in adult animals at 30 and 90 mg/kg bw per day and in offspring at the highest dose. Reproductive performance was not affected at any dose. The NOEL was 10 mg/kg bw per day on the basis of depressed maternal weight gain. No fetotoxicity was observed after oral administration of thiabendazole to pregnant mice at doses of 0, 25, 100, or 200 mg/kg bw per day. The numbers of live fetuses at the two highest doses were decreased due to reduced implantation, and these doses also decreased the food intake and body-weight gain of the dams. The NOEL was 25 mg/kg bw per day on the basis of reduced implantation. In a previously evaluated study in mice, fetal malformations were observed at doses of 240 mg/kg bw per day and above. 4. EVALUATION The results of these supplementary studies allowed the Committee to confirm the earlier evaluation. The NOELs in the 12-month study in dogs, the two-year study of toxicity in rats, and the two-generation study of reproductive toxicity in rats were all 10 mg/kg bw per day, identical to the NOEL identified previously that served as the basis for the ADI. The Committee applied a safety factor of 100 and confirmed the ADI of 0-100 µg/kg bw established at the fortieth meeting. 5. REFERENCES Galloway, S.M. & Lankas, G.R. (1994) Thiabendazole; assay for chromosomal aberrations in mouse bone marrow. Unpublished report No. TT94-8603 from Merck Research Laboratories, West Point, PA, USA. Submitted to WHO by Merck & Co., Three Bridges, NJ, USA. Lankas, G.R. & Wise, L.D. (1992) Thiabendazole: Two-generation dietary reproduction study in rats. Unpublished report No. TT90-733-0 from Merck Research Laboratories, West Point, PA, USA. Submitted to WHO by Merck & Co., Three Bridges, NJ, USA. Lankas, G.R., Morrissey, R.E. & Stabinski, L.G. (1993) Thiabendazole: Fifty-three-week oral toxicity study in dogs. Unpublished report No. TT91-068-0 from Merck, Sharp & Dohme Research Laboratories, USA. Submitted to WHO by Merck & Co., Three Bridges, NJ, USA. Lankas, G.R., Hubert, M.F. & Majka, J.A. (1995) Thiabendazole: Fourteen-week dietary thryroxine clearance study in rats with a 14-week recovery period. Unpublished report No. TT94-024-0 from Merck Research Laboratories, West Point, PA, USA. Submitted to WHO by Merck & Co., Three Bridges, NJ, USA. Nakatsuka, T., Ban, Y. & Fujimaki, Y. (1995) Thiabendazole: Oral developmental toxicity study in mice. Unpublished report No. TT94-9818 from Development Research Laboratories, Banyu Pharmaceutical Co., Ltd, Japan. Submitted to WHO by Merck & Co., Three Bridges, NJ, USA. Natarajan, A.T., Duivenvoorden, W.C.M., Meijers, M. & Zwanenburg, T.S.B. (1993) Induction of mitotic aneuploidy using Chinese hamster primary embryonic cells. Test results of 10 chemicals. Mutat. Res., 287, 47-56. Parry, J.M. (1993) An evaluation of the use of in vitro tubulin polymerization, fungal and wheat assays to detect the activity of potential chemical aneugens. Mutat. Res., 287, 23-28. Sina, J.F. & Lankas, G.R. (1992) Thiabendazole: Microbial mutagenesis assay. Unpublished reports Nos TT92-8074 and TT92-8079 from Merck Research Laboratories, West Point, PA, USA. Submitted to WHO by Merck & Co., Three Bridges, NJ, USA. Squibb, R.E., Wolfe, G.W. & Lankas, G.R. (1993) Thiabendazole: 106-week dietary toxicity/carcinogenicity study in rats. Unpublished report No. TT90-9009 from Hazleton Washington, USA, and Merck Research Laboratories, West Point, PA, USA. Submitted to WHO by Merck & Co., Three Bridges, NJ, USA. Warr, T.J., Parry, E.M. & Parry, J.M. (1993) A comparison of two in vitro mammalian cell cytogenetic assays for the detection of mitotic aneuploidy using 10 know or suspected aneugens. Mutat. Res., 287, 29-46.
See Also: Toxicological Abbreviations Thiabendazole (AGP:1970/M/12/1) Thiabendazole (WHO Pesticide Residues Series 1) Thiabendazole (WHO Pesticide Residues Series 2) Thiabendazole (WHO Pesticide Residues Series 5) Thiabendazole (Pesticide residues in food: 1977 evaluations) Thiabendazole (Pesticide residues in food: 1979 evaluations) Thiabendazole (Pesticide residues in food: 1981 evaluations)