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    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 g/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 g/plate        +     Negativea     Sina & Lankas
                             TA97, TA98,                               -     Negativea     (1992)
                             TA100, TA1535

    Reverse mutation         E. coli WP2,       3-6000 g/plate        +     Negativea     Sina & Lankas
                             WP2uvrA,                                  -     Negativea     (1992)
                             WP2uvrA,
                             pKM101

    Inhibition of tubulin    Bovine tubulin     100 mol/litre               Positive      Parry (1993)
       polymerization

    Aneuploidy               A. nidulans        > 19.87 mol/litre           Positive      Parry (1993)
                             S. cerevisiae      > 49.7 mol/litre            Positive
                             Hexaploid wheat    > 114.2 mol/litre           Positive

    Aneuploidy               Chinese hamster    50 g/ml                     Positiveb     Natarajan et
                             embryonic                                                     al. (1993)
                             fibroblasts

    Cell-division            Chinese hamster    > 10 g/ml                   Positivec     Warr et al.
       aberration            cells; Don, LUC2                                              (1993)

    Chromosome               Chinese hamster    100 g/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 g/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)