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.