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WHO FOOD ADDITIVES SERIES: 49

Toxicological evaluation of certain
veterinary drug residues in food

TIABENDAZOLE (THIABENDAZOLE)
(addendum)

First draft prepared by M.E.J. Pronk and G.J. Schefferlie
Centre for Substances and Risk Assessment
National Institute for Public Health and the Environment
Bilthoven, The Netherlands

Explanation

Biological Data

Biochemical aspects: Absorption, distribution and excretion

Toxicological studies

Acute toxicity

Short-term studies of toxicity

Reproductive toxicity

Multigeneration studies

Developmental toxicity

Observations in humans

Selection Of Relevant End-Points

Mechanism of action

Clinical signs

Toxicity to specific organs

Effects on the kidney

Effects on the haematopoietic system

Developmental toxicity

Comments

Evaluation

References

 

1. EXPLANATION

Tiabendazole (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 by the Committee at its fortieth meeting (Annex 1, reference 104). An ADI of 0–100 µg/kg bw was established on the basis of reduced body-weight gain in a 2-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. At its forty-eighth meeting (Annex 1, reference 128), the Committee reviewed the results of supplementary studies that allowed it to confirm its earlier evaluation. The NOELs in the 12-month study in dogs, the 2-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 that had 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 its fortieth meeting.

As tiabendazole is also used as a fungicide in plant protection, its toxicity was also evaluated by the 1970 and 1977 Joint FAO/WHO Meeting on Pesticide Residues (JMPR) (FAO/WHO, 1971, 1978). At the 2000 JMPR (FAO/WHO, 2001), at which the residue and analytical aspects of tiabendazole were evaluated, the Meeting concluded that the toxicological profile of tiabendazole included effects of concern that might indicate a need for an acute reference dose (RfD). The Meeting recommended that tiabendazole be considered further by JECFA, which had conducted the most recent toxicological assessment of this chemical.

The Committee did not receive new data for establishing an acute RfD for tiabendazole. All the data considered had been evaluated and summarized previously by the Committee, at its fortieth and forty-eighth meetings. Those data were re-evaluated by the Committee at its present meeting, when it focused on aspects relevant for establishment of an acute RfD. In addition, the Committee consulted the literature for recently published information on the toxicity of tiabendazole and considered those relevant for this evaluation.

2. BIOLOGICAL DATA

2.1 Biochemical aspects: Absorption, distribution and excretion

After oral administration to mice, rats, dogs and humans, tiabendazole is rapidly absorbed (peak plasma concentrations within 3 h), followed by rapid elimination, which is essentially complete within 1 day for humans and rats and 3 days for dogs and mice. The half-time in humans is < 2 h. In humans, rats and mice, urinary excretion predominated over faecal excretion. In dogs, the amount excreted in faeces was somewhat greater than that in urine (Annex 1, reference 105; Tocco et al., 1966). Given these toxicokinetics, accumulation in the body is not expected.

2.2 Toxicological studies

2.2.1 Acute toxicity

The acute toxicity of thiadibendazole is summarized in Table 1.

Table 1. Acute toxicity of thiabendazole

Species

Sex

Route

LD50
(mg/kg bw)

Reference

GLP/QA

Mouse

Female

Oral

3800

Robinson (1964)

No

Rat

Male

Oral

3300

Robinson (1964)

No

Rat

Male and female

Oral

4700-5100

Lankas (1981)

Yes

Rat

Male and female

Inhalationa

> 400 mg/m3

Gurman et al. (1981)

Yes

Rabbit

Not specified

Oral

3800

Robinson (1964)

No

Rabbit

Male and female

Dermal

> 2000

Blaszcak & Auletta (1987)

No

GLP, good laboratory oractice; QA, quality assurance

a Gravimetric concentration; 4 h exposure

 

The study by Lankas (1981) was not evaluated previously by the Committee. At doses ranging from 2200 to 11 000 mg/kg bw, the signs noted were decreased activity, bradypnoea, ptosis and loss of righting reflex.

In the studies of Robinson (1964), the doses used were not specified. The toxic signs were described as lethargy and stupor. No further details were given.

In the study with rabbits treated dermally (Blaszcak & Auletta, 1987), no toxic signs were noted.

The rats treated by inhalation had squinted eyes, polypnoea and body-weight loss (Gurman et al., 1981).

2.2.2 Short-term studies of toxicity

Rats

In a 13-week study of toxicity reviewed by the Committee at its fortieth meeting (Annex 1, reference 105), groups of 10 male and 10 female Sprague-Dawley Crl:CD BR rats received diets containing tiabendazole at concentrations intended to provide a daily dose of 0, 10, 40, 160 or 320 mg/kg bw. The calculated mean intakes of tiabendazole were 0, 9, 37, 150 and 300 mg/kg bw per day, respectively. Clinical signs were recorded weekly, and haematological end-points were measured in samples taken at weeks 6 and 13. The following observations may be relevant for the acute toxicity of this compound. No effects were seen in controls or in animals at the lowest dose. At 37 mg/kg bw per day, females showed an increased platelet count at both weeks 6 and 13, and both sexes showed an increased incidence of erythroid hyperplasia in the bone marrow. At the two higher doses, the following dose-related changes were seen: an increase in the incidence of alopecia; a decrease in erythrocyte count, haemoglobin concentration and erythrocyte volume fraction at weeks 6 and 13; an increased frequency of abnormal erythrocyte morphology at both times; an increase in platelet count at both times (only at week 13 in males at 150 mg/kg bw per day); and increased incidences of erythroid hyperplasia in bone marrow and haemoglobin pigment in spleen.

The Committee at its previous meeting identified a NOEL of 9 mg/kg bw per day on the basis of decreased body-weight gain and food intake in males, increased liver weight and centrilobular hypertrophy, increased thyroid weight, follicular cell hypertrophy and bone-marrow erythroid hyperplasia at doses of 37 mg/kg bw per day and above. The effects on the haematopoietic system at both sampling times (6 and 13 weeks), together with the erythroid hyperplasia in bone marrow, were indicative of anaemia and were considered potentially relevant for acute exposure. The NOEL for these effects was 9 mg/kg bw per day (Myers & Lankas, 1990).

Dogs

In a 14-week study of toxicity reviewed by the Committee at its fortieth meeting (Annex 1, reference 105), groups of four male and four female beagle dogs received tiabendazole orally in gelatin capsules at a daily dose of 0, 35, 75 or 150 mg/kg bw. Clinical signs were recorded twice daily, and haematological end-points were measured at weeks 4, 8 and 12. The following observations may be relevant for acute toxicity. No changes were seen in controls or those at the lowest dose. Animals at the two higher doses showed dose-related increases in salivation and emesis, the latter effect mainly during the first few weeks of the study. At the highest dose, decreased erythrocyte count, haemoglobin and erythrocyte volume fraction were seen, the effects being more frequent at weeks 4 and 8 than at week 12.

The Committee at its previous meeting identified a NOEL of 35 mg/kg bw per day on the basis of histopathological changes in the gall-bladder. This effect was, however, considered not relevant for assessing the effects of acute exposure. The haematological findings at the first two sampling times may be indicative of anaemia and were considered potentially relevant for acute exposure. The NOEL for these effects was 75 mg/kg bw per day (Batham & Lankas, 1990).

Groups of four male and four female beagle dogs received tiabendazole orally in gelatin capsules at a daily dose of 0, 10, 40 or 160 mg/kg bw for 1 year in a study of toxicity reviewed by the Committee at its fortieth meeting (Annex 1, reference 105). Clinical signs were recorded daily and haematological end-points were measured in samples taken at weeks 4, 12, 26 and 52. The following observations may be relevant to the acute toxicity of this compound. No effects were seen in controls or animals at the lowest dose. Animals at the highest dose showed emesis, especially during the first 3 weeks and in females throughout the first half of the study. Animals at the highest dose showed decreased erythrocyte count, haemoglobin concentration and erythrocyte volume fraction and increased activated partial thromboplastin time and platelet count throughout the study (weeks 4–52). Histological examination showed a dose-related increase in the frequency of distal tubule vacuolation in the kidney of females; a dose-related increase in haemosiderin deposition in the spleen of females, with a very slight, not statistically significant effect in males at all doses with no dose–response relationship; and a dose-related increase in splenic erythropoiesis in animals at the two higher doses. Increased bone-marrow erythropoiesis was seen only in females at the highest dose.

The Committee at its previous meeting identified a NOEL of 10 mg/kg bw per day on the basis of (histopathological) changes indicative of haemolytic anaemia. As haematological changes were observed throughout the study, all changes to the haematopoietic system, including the histopathological changes, were considered potentially relevant to acute intake as well. It was noted that the effects in spleen and bone marrow seen after 53 weeks were not found in the 14-week study in dogs (Lankas, 1993).

2.2.3 Reproductive toxicity

(a) Multigeneration studies

Rats

In a two-generation study of reproductive toxicity, groups of 33 male and 33 female Sprague-Dawley Crl:CD BR rats received diets containing tiabendazole (purity, > 99%) at concentrations providing a dose of 0, 10, 30 or 90 mg/kg bw per day (Wise & Lankas, 1992). This study was summarized by the Committee at its forty-eighth meeting (Annex 1, reference 129). The only treatment-related findings were effects on food consumption and body-weight gain, for which the Committee previously identified a NOEL of 10 mg/kg bw per day. These effects were considered irrelevant for acute exposure.

(b) Developmental toxicity

Mice

Three studies of developmental toxicity were undertaken in pregnant Jcl:ICR mice. Tiabendazole (purity, 98.5%) was given orally as a suspension in olive oil by gastric intubation. The animals were killed on day 18 of gestation. These studies were summarized by the Committee at its fortieth meeting (Annex 1, reference 105).

In the first experiment, mice were given a dose of 0, 700, 1300 or 2400 mg/kg bw on days 7–15 of gestation. Maternal body-weight gain was decreased in a dose-related fashion at all doses, and the mortality rate increased with increasing dose, being 0/39 controls, 0/39 at the lowest dose, 5/39 at the intermediate dose and 24/39 at the highest dose. The weights of the liver, kidney, heart and spleen were increased at all three doses. A dose-related increase in the frequency of resorptions was seen at the two higher doses, and a dose-related decrease in fetal body weight occurred at all doses. In the offspring, the incidence of cleft palate was increased in a dose-related fashion at all doses, and the incidence of fusion of vertebral arches and vertebral bodies was increased in offspring of dams at the two lower doses.

In the second experiment, animals were given a dose of 2400 mg/kg bw on a single day between days 6 and 15 of gestation. The maternal mortality rates were 2/7, 2/12, 1/12, 2/11, 2/11, 6/11, 2/11, 1/11, 4/11 and 6/11 on days 6–15, respectively. The rate of resorptions was increased and the fetal body weight decreased after dosing on any day. Increased frequencies were seen of microencephaly and exencephaly after treatment on day 6, 7 or 8; short or absent tail and anal atresia after dosing on day 9; open eyelids after treatment on day 7, 8, 10, 13 or 14; reduction deformity of the limbs after dosing on day 9, 10, 11 or 12; cleft palate after dosing on day 8, 9, 10, 11, 12 or 13; fusion of vertebral arches and vertebral bodies after treatment on day 7, 8, 9, 10 or 13; and fusion of the ribs after dosing on day 7, 8 or 9.

In the third experiment, groups of 21–31 mice were given tiabendazole at a single dose of 0, 30, 60, 62, 120, 130, 240, 270, 480, 560, 670, 800, 960, 1200, 1400, 1700, 2000 or 2400 mg/kg bw on day 9 of gestation. Maternal weight gain was decreased at 1200 mg/kg bw, the maternal mortality rate was increased at 1700 mg/kg bw, and the weights of the liver, heart and kidney weight were decreased at 1400 mg/kg bw. The resorption rate was increased at 1700 mg/kg bw, and the fetal body weight was decreased at 60 mg/kg bw. The incidence of reduction deformity of the limbs was increased at 480 mg/kg bw, and that of fusion of vertebral arches and vertebral bodies and of ribs was increased at 240 mg/kg bw (Ogata et al., 1984).

In a study of developmental toxicity, groups of 25 pregnant Jcl:ICR mice were given tiabendazole (purity, 99.8%) in olive oil by oral gavage at a dose of 0, 25, 100 or 200 mg/kg bw per day on days 6–15 of gestation. The animals were killed on day 18 of gestation. This study was evaluated by the Committee at its forty-eighth meeting (Annex 1, reference 129). Dams at the two higher doses showed dose-related decreases in food consumption and weight gain, and there were dose-related decreases in the number of implantations and fetal body weight at these doses. An increased incidence of delayed ossification was seen at all doses, but this was not dose-related, and the number of affected litters was similar in all groups, including controls. The NOEL was 25 mg/kg bw per day on the basis of the reduced number of implantations at a maternally toxic dose (Nakatsuka et al., 1995).

Rats

In a study of developmental toxicity, groups of 25 pregnant Sprague-Dawley Crl:CD BR rats received tiabendazole (purity, 98.9%) in 0.5% methylcellulose by gavage at a daily dose of 0, 10, 40 or 80 mg/kg bw on days 6–17 of gestation. The animals were killed on day 20 of gestation. This study was evaluated by the Committee at its fortieth meeting (Annex 1, reference 105). The food consumption and weight gain of the dams at the two higher doses were decreased in a dose-related manner, and dams at the highest dose showed ptosis and regurgitation. A dose-related decrease in fetal body weight was seen at the two higher doses. The NOEL was 10 mg/kg bw per day (Wise, 1990).

Rabbits

In a study of developmental toxicity, groups of 18 pregnant New Zealand white rabbits received tiabendazole (purity, 98.9%) in 0.5% methylcellulose by gavage at a dose of 0, 24, 120 or 600 mg/kg bw on days 6–18 of gestation. The animals were killed on day 29 of gestation. This study was evaluated by the Committee at its fortieth meeting (Annex 1, reference 105). Dose-related decreases in food consumption and weight gain were seen at the two higher doses, with a loss of body weight at the highest dose. Four of 18 dams at the highest dose aborted. The mortality rates were 2/18 controls (due to intubation errors), 0/18 at the lower dose, 0/18 at the intermediate dose and 1/18 at the highest dose. A dose-related increase in the rate of early and late resorptions was seen at the two higher doses, and fetuses at these doses had dose-related increased incidences of domed head, hydrocephalus and marked enlargement of the fontanelle. The NOEL was 24 mg/kg bw per day (Hoberman, 1989).

In another study of developmental toxicity, evaluated by the Committee at its fortieth meeting (Annex 1, reference 105), groups of 18 pregnant New Zealand white rabbits received tiabendazole (purity, 98.6%) in 0.5% methylcellulose by gavage at a dose of 0, 50, 150 or 600 mg/kg bw per day on days 6–18 of gestation. The animals were killed on day 28 of gestation. Effects were seen only at the highest dose; they comprised decreased maternal food consumption and weight gain, increased rates of early and late resorptions, decreased fetal body weight and increased incidences of variation in lung lobation and incompletely ossified metacarpals. No evidence for compound-related fetal hydrocephaly was found. The NOEL was 150 mg/kg bw per day (Lankas & Wise, 1991/1993).

2.3 Observations in humans

In a study with volunteers, 50 men aged 20–57 years received capsules containing placebo and 50 received 125 mg of tiabendazole twice daily for 24 weeks. Neither the study subjects nor the investigators were aware of who had received placebo. In total, 36 men receiving tiabendazole and 41 receiving placebo completed the study. One man was removed from the study at his own request because of daytime sedation and markedly decreased energy. The other withdrawals were clearly unrelated to the treatment. Weekly interviews were conducted to record side-effects. General physical examinations and laboratory examinations (haematology, measurement of cholesterol, glucose, urea, alkaline phosphatase, thymol turbidity, bilirubin in serum and urine analysis) were carried out before the test and after 4, 12 and 24 weeks. The haematological parameters measured were reported only as ‘CBC’ and haematocrit. Protein-bound iodine in serum and electrocardiographic traces were evaluated only at the beginning and after 24 weeks of the study.

This study was previously summarized by the 1977 JMPR (FAO/WHO, 1978), which noted that, under the conditions of the study, tiabendazole was well tolerated, and no effect on any of the parameters could be clearly ascribed to treatment. The JMPR identified a NOEL of 3–4 mg/kg bw per day, which was confirmed by the Committee at its fortieth meeting (Annex 1, reference 105). The following observations may be relevant for the acute toxicity of tiabendazole, although the time of onset of clinical signs was not specified. The men reported the following possible side-effects (treated versus placebo): increased appetite (26/50 vs 30/50), flatulence (6/50 vs 3/50), nausea (4/50 vs 2/50), increased urinary frequency (3/50 vs 3/50) and sedation (7/50 vs 5/50) (Colmore, 1965).

In a review of studies on the efficacy of tiabendazole against parasites in humans, the standard therapeutic oral dose was 25 mg/kg bw twice daily for 1–4 days, although higher doses were used in some studies. The incidences of minor transient side-effects were generally 25–30% with the standard dose and higher with higher doses. The effects comprised anorexia, nausea, vomiting and dizziness. Serious side-effects were rare and comprised numbness, collapse, tinnitus, abnormal sensation in the eyes, xanthopsia, enuresis, decreased pulse rate and systolic blood pressure and transient rises in the frequency of cephalin flocculation and in aspartate aminotransferase activity (Campbell & Cuckler, 1969).

Side-effects in humans after therapeutic oral doses (not specified) of thiabendazole were reported in another literature review. Common effects were dizziness (the frequency ranging from < 5% to 80%, depending on dosage) and nausea and vomiting (5–15%). Rarely observed side-effects included anorexia, abdominal pain, headache, drowsiness, weariness, heartburn, diarrhoea or constipation, flatulence, blurring of vision, xanthopsia, skin eruption, malodorous urine and vomiting of live Ascaris. The extent to which these frequencies differed from those in untreated subjects is unknown, although in two placebo-controlled studies dizziness was reported to be approximately twice as common in tiabendazole-treated subjects as in placebo-treated subjects (Cuckler & Mezey, 1966).

In a clinical case report, the following side-effects were reported in 14 of 23 patients with trichinosis who had received tiabendazole orally at a dose of 50 mg/kg bw in two daily doses for 10 days: nausea (11/23), retching (11/23), vomiting (11/23), aversion to tablets (3/23), exanthema (3/23), impotence (2/23), diarrhoea (1/23), liver damage (1/23), fever (1/23) and dizziness (1/23) (Hennekeuser et al., 1969). Again, the incidence of side-effects in untreated patients was not reported, and the extent to which these effects might have been influenced by the underlying condition was not reported.

3. SELECTION OF RELEVANT END-POINTS

The end-points relevant to acute intake are identified below, on the basis of guidance given by the Commission of the European Union (2001), the 2000 JMPR (FAO/WHO, 2001) and van Raaij (2001).

3.1 Mechanism of action

The mechanism of action of tiabendazole is not clear, but it might involve inhibition of the fumarate reductase system of worms, thereby interfering with their source of energy (Parfitt, 1999). As fumarate is a link in the citric acid cycle, which is a common pathway in organisms, this possible mode of action of tiabendazole may also be relevant in assessing the risks of humans after acute exposure. An indication of the toxicity resulting from this mode of action may be found in the mitochondrial dysfunction observed in the renal cortex of mice given a single oral dose of 1000 or 2000 mg/kg bw (see section 3.3.1).

3.2 Clinical signs

The studies of acute toxicity gave oral LD50 values above 2000 mg/kg bw, raising no concern. The clinical signs described in these studies were relatively unspecific (e.g. decreased activity), and the correlation with doses was not reported. It remains unclear whether these signs were substance-specific or related to the dosing procedure. They were therefore not considered in establishment of an acute RfD.

In the studies with repeated doses, the only specific clinical sign in rats (13-week study) was alopecia, which was observed at the highest dose of 300 mg/kg bw per day, with a NOEL of 150 mg/kg bw per day. However, the onset of this sign was not earlier than after 5 or 6 weeks of treatment, and it is therefore not relevant for acute exposure. It was noted that this effect was also reported in a 28-day study in rats treated orally (summarized by the Committee at its fortieth meeting (Annex 1, reference 105) but not available in the present dossier) at the highest dose of 800 mg/kg bw per day, but not at lower doses up to 200 mg/kg bw per day. In dogs, the only treatment-related clinical signs were salivation and emesis. These effects, particularly emesis, are considered relevant for acute exposure, because vomiting is also a common side-effect in humans. The NOEL for emesis in dogs was 40 mg/kg bw per day.

Common side-effects reported in humans after oral doses > 25 mg/kg bw twice daily for 1–10 days comprised anorexia, nausea, vomiting and dizziness (data from 1966–69; no recent data available and often no control data for comparison). NOELs could not be identified. In a placebo-controlled study in volunteers, a dose of 125 mg of tiabendazole twice a day for 24 weeks (equivalent to 3.6 mg/kg bw per day for a 60-kg person) did not cause significant changes in subjective side-effects.

3.3 Toxicity in specific organs

3.3.1 Effects on the kidney

No effects on the kidney were found in the studies available to the Committee, after repeated doses in rats or in the studies on reproductive and developmental toxicity in rats and rabbits. In dogs, renal toxicity was observed in the 1-year study (NOEL, 10 mg/kg bw per day) but not in the 14-week study at comparable doses. In contrast, renal toxicity was observed after acute and short-term oral intake of tiabendazole in mice. This effect was described in earlier evaluations by the Committee, as well as in some published articles, but none of these studies was submitted in the dossier.

In a short-term study of toxicity, mice were given tiabendazole at a dose of 0, 250 or 500 mg/kg bw per day by gavage for 1, 3, 5 or 7 days. Dose-related renal toxicity was observed, which included tubule dilatation, degenerative desquamation, cell infiltration, fibrosis and regeneration of tubule epithelium. Electron microscopy showed evidence of glomerular damage, such as flattening of the foot processes of podocytes and oedematous changes in the mesangium in treated mice. No NOEL could be identified (Tada et al., 1989).

Effects on the kidneys were also observed in one 13-week study in rats and some of the long term (2-year) studies in rats that had been evaluated earlier by the JMPR or JECFA but which were not available in the present dossier.

In a published report not previously evaluated by the committee, male Crj:CD-1 (ICR) mice were given tiabendazole (purity, 98.5%) in olive oil as a single dose of 0, 250, 500 or 1000 mg/kg bw by oral gavage, and the kidneys were examined histologically 1, 3, 5 or 24 h after dosing. The histological findings were desquamation of degenerated cells in proximal tubules and production and release of apical cytoplasmic blebs from epithelial cells into the proximal tubule lumen of treated mice from 1 h onwards. Dilatation of proximal, distal and collecting tubules was apparent in treated mice at 3, 5 and 24 h. Pre-treatment with inducers of the microsomal mono-oxygenase system reduced the renal injury, while pre-treatment with inhibitors of that system enhanced the effects, suggesting that the toxicity was caused by the parent compound rather than by its metabolites. Pre-treatment with inhibitors of organic cation or anion transport indicated that tiabendazole is transported into tubule cells through the organic cation transport system for 1–5 h after dosing. no NOEL could be identified (Tada et al., 1992).

Recovery from the nephrotoxicity of tiabendazole was reported in mice after a single dose in a published report that had not previously been evaluated by the Committee. Male and female Crj:CD-1 (ICR) mice were given a single dose of tiabendazole (purity, 98.5%) suspended in olive oil at a dose of 0, 125, 250, 500, 1000 or 2000 mg/kg bw by oral gavage. The animals were necropsied 24 h thereafter. Additional groups of mice were given single oral doses of 1000 or 2000 mg/kg bw and necropsied 1, 2, 3, 5, 7 or 10 days after dosing in order to study recovery from the effects. Further groups of mice were given a single oral dose of 0 or 2000 mg/kg bw and were observed for 10 days to study the effect on the mortality rate. By 10 days after administration of 2000 mg/kg bw, 30% of the females and 60% of the males had died. mice given 1000 or 2000 mg/kg bw showed higher serum urea nitrogen and aspartate aminotransferase concentrations 24 h after dosing. At the same time, mice given 250 mg/kg bw or more showed dose-dependent renal toxicity. histological examination showed desquamation of degenerated cells in the proximal tubules and dilatation of proximal, distal and collecting tubules. Necrosis of proximal tubules was observed at doses of 500 mg/kg bw and higher. the changes at 250 mg/kg bw were slight, and the kidneys of mice treated at 125 mg/kg bw were not different from those of control mice. In the recovery experiment, tiabendazole-treated mice had higher urine volumes with higher concentrations of glucose, protein and sodium, in particular during the first few days after administration. The serum urea nitrogen concentrations returned to control levels within 3 and 7 days in mice given 1000 and 2000 mg/kg bw, respectively. The increased aspartate aminotransferase concentration in all treated mice recovered to normal within 3 days. The increased absolute and relative kidney weights had returned to control levels at 5 and 7 days, respectively, but pale foci and rough surface were still observed in the kidneys at the end of the study. The dilatation of tubules observed at both doses did not revert during the 10-day observation period. Mitochondrial swelling was seen in proximal tubule epithelium during the first 2 days after treatment. necrosis of proximal tubules and desquamation of degenerated cells in proximal tubules were not observed after 7 and 10 days of recovery, respectively. Tissue repair processes (cell infiltration, fibrosis, regeneration of tubules) started 3 days after exposure and were still apparent in all animals 10 days after exposure. The committee concluded that the tiabendazole-induced renal toxicity was most frequent 2 or 3 days after dosing and was followed by regeneration. The NOEL for renal toxicity was 125 mg/kg bw (Tada et al., 1994).

In a 13-week study of toxicity, groups of male and female Crj:CD-1 (ICR) mice received a diet containing 0, 0.8 or 1.6% tiabendazole (purity not reported). The report was not included in the dossier and had not previously been evaluated by the committee. The doses were equivalent to 0, 1200 and 2400 mg/kg bw per day, respectively. Anaemia and liver and kidney damage were the main effects at both doses (Tada et al., 1996). The study was considered to be of less value than others for determination of an acute RfD in view of its long duration and the high doses.

Another study of toxicity in mice was reported in a published paper that had not previously been evaluated by the committee. Male Crj:CD-1 (ICR) mice were given diets containing 0, 0.8, 1.2 or 1.6% tiabendazole (purity, > 99%) for 44 weeks. The doses were equivalent to 0, 1200, 1800 and 2400 mg/kg bw per day, respectively. Neither haematological nor urine analyses were performed. Liver, kidney and gall-bladder damage were the main effects at all doses (Tada et al., 2001). The study was considered to be of less value than others for determination of an acute RfD in view of its long duration and the high doses used.

The same group of investigators studied the structural alterations to mitochondria in renal proximal tubule cells to determine the mechanism of the acute renal tubule necrosis. The published paper had not previously been evaluated by the Committee. Groups of male CRj:CD-1(ICR) mice were given tiabendazole in olive oil by oral gavage at a single dose of 0, 1000 or 2000 mg/kg bw and were killed 3, 6 or 16 h after dosing. The following end-points were determined: mitochondrial respiration, the tiabendazole concentration in renal cortex and mitochondrial fraction (also at 0.5, 1 and 24 h), histochemical analysis of mitochondrial enzyme (NAD-linked isocitrate dehydrogenase) and the concentration of ATP in the renal cortex. Mitochondrial respiration was statistically significantly decreased in a dose-dependent manner 6 and 16 h after dosing but not 3 h after dosing. When additional groups of mice were treated with tiabendazole at 0, 250 or 500 mg/kg bw and killed 16 h later, no effects on mitochondrial respiration were observed. The concentration of tiabendazole in the renal cortex reached a maximum 1 h after dosing and declined thereafter. The concentration in the mitochondrial fractions followed the same pattern. Effects on the activity of NAD-linked isocitrate dehydrogenase (a marker enzyme of mitochondria) were not observed 3 or 6 h after dosing, whereas significant inhibition of this activity was observed 16 h after administration of 1000 or 2000 mg/kg bw. Dilatation of tubules, observed at all times after administration of 1000 or 2000 mg/kg bw, was observed mainly in the area where inhibition of NAD-linked isocitrate dehydrogenase was found. Necrosis of renal tubules was not observed. The ATP concentration in the renal cortex, determined only 16 h after a single oral dose of 0, 500, 1000 or 2000 mg/kg bw, was significantly decreased at 1000 and 2000 mg/kg bw but not at the lowest dose (Fujitani et al., 1998).

The results of the studies summarized above suggest that, after single doses of tiabendazole to mice, the compound is taken up by tubule cells in the renal cortex through the organic cation transport system and subsequently inhibits mitochondrial respiration, leading to depletion of ATP and ultimately to necrosis of proximal tubule cells. The reduced mitochondrial respiration may be due to inhibition of the fumarate-reductase system, which is the putative mechanism of action of tiabendazole (Parfitt, 1999). Tada and colleagues have suggested that cell debris obstructs the tubules, leading to dilatation. This explanation is not entirely consistent, because dilatation was observed at lower doses than necrosis and lasted longer. Dilatation of proximal tubules was observed at single doses of 250 mg/kg bw and higher. The lowest dose of 125 mg/kg bw was therefore the NOEL for acute renal toxicity in mice.

3.3.2 Effects on the haematopoietic system

In both rats and dogs, a number of haematological parameters were changed after repeated oral intake of tiabendazole, sometimes with related histopathological changes in the spleen and bone marrow. Although the studies were of short duration (13 weeks in rats and 14 and 53 weeks in dogs), the interim analysis of blood samples (starting at week 4 or 6) showed haematological changes early in the study, which were occasionally more frequent at earlier times than at the end of the study. The related histopathological changes were also observed at doses at which haematological changes were not yet or no longer seen. The haematological and histopathological changes are indicative of anaemia, and, as these changes could have occurred after one or a few doses, they were considered relevant to acute intake. The NOELsfor this effect in rats and dogs were 9 and 10 mg/kg bw per day, respectively.

In addition to the studies summarized above, the Committee at its fortieth meeting reviewed three other studies of the same or shorter duration in rats treated by gavage. These studies, which were not available at the present meeting, comprised two 4-week studies with animals given tiabendazole at doses of 50–1600 mg/kg bw per day and one 13-week study with rats given doses of 25–400 mg/kg bw per day. The 4-week studies also showed haematological changes (NOEL, 100 mg/kg bw per day) and histopathological changes in the spleen and/or bone marrow (LOEL, 50 mg/kg bw per day). in the 13-week study, changes were observed in red blood cell parameters, and histopathological changes were seen in the spleen (NOEL for both effects, 25 mg/kg bw per day). as the studies could not be reviewed by the present committee, it could not verify whether the bone marrow was affected in the 13-week study.

It should be noted that in the study of Colmore (1965) with male volunteers, a dose of 125 mg twice a day for 24 weeks (equivalent to 3.6 mg/kg bw per day for a 60-kg person) did not affect haematological parameters after 4, 12 or 24 weeks of treatment. However, this study has a number of serious shortcomings: the haematological parameters (other than haematocrit and ‘CBC’) investigated were not specified; only one dose was tested, so that the dose–response relationship could not be investigated; and, more importantly, no histopathological examination was carried out, while, in animals, this appeared to be a more sensitive indicator of haematotoxicity than the haematological parameters.

3.4 Developmental toxicity

As short treatments are used in studies of developmental toxicity or teratogenicity and because, in particular, teratogenic effects and resorptions may be induced by a single dose within a certain (sensitive) period, effects on the fetus were considered relevant for setting an acute RfD.

With regard to teratogenic effects, domed heads, hydrocephalus and marked enlargement of fontanels were observed in one study in rabbits at doses of 120 mg/kg bw per day and higher. The NOEL was 24 mg/kg bw per day. In another study in rabbits, no such effects were observed at doses up to 600 mg/kg bw per day. The NOEL in this study was 150 mg/kg bw per day, on the basis of an increased incidence of malformations, which was seen relatively often in this species. In mice, the teratogenic effects after a single exposure on day 9 of gestation consisted of deformed limbs at doses of 480 mg/kg bw and higher (NOEL, 26 g/kg bw) and fusion of vertebral arches, bodies and ribs at doses of 240 mg/kg bw and higher (NOEL, 130 mg/kg bw). In rats, no teratogenic effects were observed.

Increased resorption rates were observed in mice and rabbits but not in rats. In mice, the NOELs for this effect were 700 mg/kg bw per day when the dams were exposed on days 7–15 of gestation and 1400 mg/kg bw when a single dose was given on day 9 of gestation. In rabbits, the resorption rate was increased at doses of 120 mg/kg bw per day and higher. The NOEL for this effect was 24 mg/kg bw.

Another developmental effect that was observed consistently in all the laboratory animal species tested was reduced fetal body weight. This effect is generally regarded as nonspecific and secondary to maternal toxicity, and this would appear to be the case for tiabendazole: decreased fetal body weight occurred at doses at which the dams had reduced food intake and/or reduced weight gain. The reduction in fetal body weight at doses that did not appear to induce concurrent maternal toxicity, as observed in mice given tiabendazole only on day 9 of gestation, might also have been secondary to maternal toxicity, as it is possible that the dams had normal weights after the single dose (day 10 until time of death) but that the fetuses did not recover their normal weights within that period.

On the basis of the effects considered relevant for acute intake, the overall NOEL for developmental toxicity, including teratogenicity, was 24 mg/kg bw per day.

4. COMMENTS

The studies of the acute toxicity of tiabendazole given orally, which gave LD50 values > 2000 mg/kg bw, did not provide any indication of acute effects. The only substance-specific clinical sign relevant for acute exposure in studies with single or repeated doses was emesis in dogs (NOEL, 40 mg/kg bw per day). The common side-effects reported in humans receiving therapeutic doses (> 25 mg/kg bw twice daily for 1–10 days) included anorexia, nausea, vomiting and dizziness. However, these effects were poorly described and did not allow identification of a NOEL. In a study in volunteers, in which controls were given a placebo, a dose of 125 mg of tiabendazole twice a day for 24 weeks (equivalent to 3.6 mg/kg bw per day for a 60-kg person) did not cause significant changes in subjective side-effects.

In the report of its fortieth meeting, the Committee noted renal injury in mice given tiabendazole for 1–7 days. In a number of published papers, the renal toxicity of tiabendazole in mice was investigated after single or repeated oral administration. Although renal toxicity was observed in the studies with repeated doses, these studies were considered of limited value for establishing an acute RfD because of the high doses used (1200, 1800 or 2400 mg/kg bw per day in the diet) and their long duration (13–44 weeks). In the studies with single doses, mice received 0, 125, 250, 500, 1000 or 2000 mg/kg bw by gavage. Renal toxicity, mainly in the proximal tubules, was observed at doses of 250 mg/kg bw and higher and consisted of histopathological changes including mitochondrial swelling. The toxic effects were due to the parent compound and were most severe 2–3 days after dosing; after that time, tissue repair processes began. All the effects except tubule dilatation were either fully or partly reversed within 10 days of administration. These studies showed that tiabendazole is taken up by proximal tubule epithelial cells in the renal cortex and ultimately causes necrosis of those cells. The lowest dose of 125 mg/kg bw was the NOEL for acute renal toxicity in mice.

Haematotoxicity was observed in studies with repeated oral doses in rats and dogs, lasting 4 and 13 weeks in rats and 14 and 53 weeks in dogs. Analysis of blood samples from week 4 or 6 showed changes indicative of anaemia early in the studies, and these were occasionally seen more often earlier than at the end of the study. Related histopathological changes in the spleen and/or bone marrow were observed at the same and lower doses. As it cannot be excluded that histopathological changes indicative of anaemia could occur after one or a few doses, they were considered relevant for assessing acute exposure. The NOELs in rats and dogs were 9 and 10 mg/kg bw per day, respectively.

In a study with volunteers, 50 men received an oral dose of 125 mg of tiabendazole twice a day for 24 weeks (equivalent to 3.6 mg/kg bw per day for a 60-kg person), and 50 other men were given a placebo. Tiabendazole did not affect haematological parameters after 4, 12 or 24 weeks of treatment. However, owing to a number of shortcomings, no NOEL could be identified in this study. In particular, it was not possible to perform histopathological examinations, which in animals appeared to provide more sensitive indicators of haematotoxicity than the haematological parameters.

In a study of developmental toxicity in rabbits, changes related to hydrocephalus were observed after oral doses of tiabendazole of 120 mg/kg bw per day and higher (NOEL, 24 mg/kg bw per day). In another study with rabbits, no such effects were observed at oral doses of up to 600 mg/kg bw per day (NOEL, 150 mg/kg bw per day). In mice, teratogenic effects were observed after a single oral dose on day 9 of gestation. They consisted of deformed limbs at doses of 480 mg/kg bw and higher (NOEL, 270 mg/kg bw) and fusion of vertebrae and ribs at 240 mg/kg bw and higher (NOEL, 130 mg/kg bw). Tiabendazole was not teratogenic in rats in doses up to 80 mg/kg bw, the highest tested.

Increased resorption rates were observed in mice and rabbits but not in rats. In mice, the NOELs for this effect were an oral dose of 700 mg/kg bw per day when the animals were exposed on days 7–15 of gestation and 1400 mg/kg bw when they were given a single oral dose on day 9 of gestation. Rabbits showed increased resorption rates at oral doses of 120 mg/kg bw per day and higher, with a NOEL of 24 mg/kg bw.

The overall NOEL for developmental toxicity, including teratogenicity, was 24 mg/kg bw per day.

5. EVALUATION

Emesis and effects on the kidney, haematopoietic system and development were considered relevant end-points for establishing an acute RfD. The most sensitive effect was haematotoxicity, specifically histopathological changes in the spleen and bone marrow indicative of anaemia, for which almost identical NOELs were found in rats (9 mg/kg bw per day) and dogs (10 mg/kg bw per day). Using these NOELs and a safety factor of 100, the Committee established an acute RfD of 100 µg/kg bw, the same value as the ADI. In view of the lack of appropriate data for this effect after single doses, the acute RfD is based on data from studies of repeated administration and hence may be conservative. The results of a study designed specifically to generate data after a single dose might allow refinement of the estimated acute RfD.

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       Toxicological Abbreviations