Imazalil is used as a human and veterinary pharmaceutical, under the name ‘enilconazole’. Imazalil was evaluated by the 1977 JMPR, when a temporary ADI of 0-0.01 mg/kg bw was allocated (Annex 1, reference 28). An ADI of 0–0.01 mg/kg bw was allocated in 1986 on the basis of the NOAEL in a 2-year study in dogs (Annex 1, reference 47). The compound was re-evaluated in 1991, when an ADI of 0-0.03 mg/kg bw was established on the basis of the NOAEL in a new study in dogs and a safety factor of 100 (Annex 1, reference 62). The 2000 JMPR determined that an acute RfD was unnecessary and affirmed the ADI of 0–0.03 mg/kg bw; however, the Meeting was made aware of the existence of a new long-term study in rats (Annex 1, reference 89). The report of this study was supplied, together with those of studies on the mechanism by which imazalil affects the thyroid and liver, to the present Meeting.

Groups of 10 male and 10 female specific pathogen-free Wistar Hannover rats were given diets containing technical-grade imazalil (purity, 50%) at a concentration of 0, 800, 1600, 2400 or 3200 ppm, equal to 0, 64, 130, 180 and 250 mg/kg bw per day for males and 0, 79, 150, 240 and 330 mg/kg bw per day for females. The rats were observed daily, and body weights and food consumption were measured weekly. Deaths were recorded. Haematological and clinical chemical measurements were performed on blood samples from all surviving animals before the terminal sacrifice, and the concentration of imazalil was determined by gas chromatography with mass spectrometry (Sterkins, 1996). Urine was analysed before terminal sacrifice. Thyroid-stimulating hormone (TSH), triiodothyronine (T3), thyroxine (T4) and testosterone concentrations were measured in serum from blood taken at sacrifice. At autopsy, the animals were examined and weighed, and any gross pathological changes were noted. The liver was examined histopathologically, as were other organs adjudged to be grossly abnormal. Electron microscopy was carried on the livers of two male and two female control animals and two male and two female animals fed the highest dietary concentration of imazalil. Liver microsomes from four rats of each sex per group were assayed for protein and cytochrome P450 content, and the activities of UDP-glucuronyltransferase and several monooxygenases were determined (Vermeir, 1996).

A significantly lower body weight than that of concurrent controls was found from week 2 of the study in males at the two higher concentrations and from time to time in those at 1600 ppm. The females showed decreased body weight from week 1 at the two higher concentrations and from week 2 at 1600 ppm. The findings for weight gain were similar, except that decreased weight gain by comparison with controls was found in animals of each sex at 2400 and 3200 ppm throughout the study. Sporadic decreases in food consumption were seen in all groups of treated males, particularly frequently at 2400 ppm. Among females, decreased food consumption was observed at concentrations > 1600 ppm from time to time.

Sporadic changes in haematological parameters were seen, most of which were not dose-related and did not therefore appear to be related to treatment. However, a decrease in monocyte count was seen in males at dietary concentrations > 1600 ppm. Furthermore, increased erythrocyte volume fraction, haemoglobin and red cell count were seen, with decreases in mean cell volume and increases in mean cell haemoglobin concentration in females at most concentrations, including the lowest. Decreased aspartate aminotransferase activity was seen at all doses in both sexes, and decreased alanine aminotransferase activity and blood urea nitrogen concentration were seen at concentrations > 1600 ppm in males: such changes are not generally considered to be adverse. Decreased serum albumin was seen in females at the two higher dietary concentrations. A decrease in serum calcium and an increase in phosphate concentration was seen in females at 1600, 2400 and 3200 ppm. Decreased triglyceride and phospholipid concentrations were observed in males at all dietary concentrations, and a decreased triglyceride concentration was seen in females at the highest dietary concentration. No treatment-related inter-group differences were seen on urinary end-points. The TSH, T3, T4 and testosterone concentrations in serum showed no clear differences between groups.

At termination, the body weights of males at concentrations > 1600 ppm were decreased. Decreased absolute lung weights were seen at 2400 and 3200 ppm, and increases in the relative weight of the liver at all concentrations. Other changes in organs weights of males were either not dose-related or reflected changes in total body weight. Similar findings were made in females, which showed a decrease in body weight at dietary concentrations > 1600 ppm. A decrease in absolute lung weight was seen at these concentrations. At examination post mortem, gross changes that appeared to be related to treatment were seen only in the liver. The livers of males at 2400 and 3200 ppm were dark, and more pronounced lobulation was seen at dietary concentrations > 1600 ppm. Dark livers were also found in females at 2400 and 3200 ppm and more pronounced lobulation only at 3200 ppm. Histological examination revealed hepatocellular hypertrophy in males at all dietary concentrations, which was particularly pronounced in centrilobular zones. An increased frequency of hepatocytes with small vacuoles was seen at 2400 and 3200 ppm and an increased frequency of fine Oil Red O-positive material at all concentrations. Similar changes were seen in females: they had hepatocellular hypertrophy, which was particularly pronounced in centrilobular zones, at all dietary concentrations. Further, they showed an increased frequency of hepatocytes with small vacuoles at all concentrations and large vacuoles at > 2400 ppm. An increased frequency of fine Oil Red O-positive material was seen at concentrations > 1600 ppm. Histological examination of the thyroid gland revealed no significant intergroup differences. On electron microscopy, the livers of all animals at the four higher dietary concentrations, but not the controls, showed small and/or large lipid droplets in both centrilobular and perilobular areas. Additionally, whorls of smooth endoplasmic reticulum were seen, this change not being present in controls.

The protein content of microsomes was increased in males at concentrations > 1600 ppm and in females at > 2400 ppm. The activities of N-ethylmorphine N-demethylase, 7-ethoxyresorufin deethylase, 7-pentoxyresorufin- and 7-ethoxycoumarin O-dealkylase were significantly induced in animals of each sex at nearly all dietary concentrations. An increase in lauric acid hydroxylase activity was seen more frequently in the livers of males than in those of females but was significant only in males at 2400 ppm. Aniline hydroxylase activity was induced in females at all dietary concentrations. UDP-glucuronyltransferase activity was induced at the two higher dietary concentrations in females. At 800 ppm, the plasma concentration of imazalil was at or below the limit of detection of the assay, which was 1 ng/ml, in males and slightly above the limit of detection in females at 1.3 ng/ml. In the males, the concentrations of imazalil were 3.4. 4.4 and 11 ng/ml at 1600, 2400 and 3200 ppm, respectively, and the equivalent figures for females were 9.4, 6.8 and 17 ng/ml. These values suggest that detoxication pathways may have been saturated at dietary concentrations > 2400 ppm. It was concluded that imazalil is a nonspecific inducer of hepatic enzymes and that it caused morphological changes in the livers of rats. No effect was seen at 3 months either in the concentration of thyroid hormones or the histological appearance of the thyroid. No NOAEL could be identified, as increased liver weight and histopathological changes in that organ were seen at all dietary concentrations. Futhermore, liver enzyme induction, an adaptive change, was seen at all concentrations (van Deun et al., 1996a; Sterkins, 1996; Vermeir, 1996; van Deun et al., 1999).

Groups of 20 specific pathogen-free Wistar rats were given diets containing imazalil (technical grade) at a concentration of 0, 200, 400 or 800 ppm for 3 months. Over the initial month, these dietary concentrations resulted in intakes of 0, 21, 42 and 82 mg/kg bw per day for males and 0, 22, 45 and 90 mg/kg bw per day for females; however, over the entire 3 months of the study, the intakes of imazalil were lower, at 0, 16, 32 and 64 mg/kg bw per day for males and 0, 19, 38 and 76 mg/kg bw per day for females. Ten animals of each sex at each dietary concentration were killed after 1 month. The rats were observed clinically and for morbidity and mortality at least daily. Ophthalmological examinations were performed at the start of the study and before the interim and terminal sacrifices in the first 10 animals in the control group and those at the highest dietary concentration. Body weight was determined weekly, as was food consumption. Blood was taken at the interim and terminal sacrifice for haematological and clinical chemical investigations; urine was analysed at the same times. Autopsies were performed at both the interim and terminal sacrifices. Selected organs were weighed in each case, and any gross changes were noted. Pieces of liver from four rats of each sex per dietary concentration were taken at both the 1-month and 3-month sacrifices. Liver microsomes were assayed for protein and cytochrome P450 content and for the activities of certain microsomal enzymes, namely aniline hydroxylation, N-demethylation of N-ethylmorphine, 7-ethoxyresorufin O-deethylation, 7-pentoxyresorufin O-dealkylation, lauric acid hydroxylation, 7-ethoxycoumarin O-deethylation and UDP-glucuronosyltransferase activity towards 4-nitrophenol. The plasma concentrations of imazalil were measured.

No deaths were seen. The clinical effects were similar in the various groups, and no ophthalmological abnormalities were found. Body weight was reduced in females at 800 ppm by comparison with concurrent controls at weeks 7 and 9. Body-weight gain was reduced in males at 800 ppm at weeks 1–3 and in females at 400 and 800 ppm in week 1 and at 800 ppm in week 9. No effect was observed on food consumption, except in week 1, when there was decreased consumption by rats of each sex at the highest dietary concentration. At the 1-month sacrifice, the total leukocyte and lymphocyte counts were decreased at dietary concentrations of 400 and 800 ppm, and the neutrophil and basophil counts were decreased in males at 800 ppm. In females, the total leukocyte and lymphocyte counts were decreased at 200, 400 and 800 ppm, the monocyte count at 800 ppm and the mean cell haemoglobin concentration at 200 ppm. At 3 months, males showed a decrease in basophil count at 400 ppm and in erythrocyte volume fraction at 200, 400 and 800 ppm. The haemoglobin concentration was decreased at 400 ppm, the mean cell volume at 800 ppm and the mean cell haemoglobin concentration at 200 and 800 ppm. Females had a decrease in mean cell volume at 800 ppm and an increase in mean cell haemoglobin concentration at 200 and 800 ppm. The decrease in mean cell volume in animals of each sex at 800 ppm at 3 months may have been treatment-related. Otherwise, these changes were considered minor and were mostly not dose-related or fell within the reference range for the strain of rats.

Some minor changes were seen at lower doses in some clinical chemical parameters, but no treatment-related effects on clinical chemistry or urine were seen at concentrations < 800 ppm. At that concentration, males had increased the serum calcium, phosphate, albumin, cholesterol, triglyceride and blood urea nitrogen concentrations at 1 month, whereas females had decreased calcium, glucose and triglycerides; the total protein was increased. At 3 months, serum calcium concentration was decreased in males and females. The decreases in bilirubin concentration and aminotransferase activities that were observed in the study were considered not adverse, and the inconsistent nature of the other clinical chemical changes casts doubt on their clinical significance. Treatment-related changes were not seen on urinary parameters.

After 1 month, the liver microsomal protein content was increased in males at all concentrations and in females at the highest concentration. The liver cytochrome P450 content was increased animals of each sex at the highest concentration. The activities of 7-ethoxyresorufin O-deethylase and 7-pentoxyresorufin O-dealkylase were increased in males at the two higher dietary concentrations, while the activity of 7-ethoxycoumarin O-deethylase was increased at 200 and 800 ppm; UDP-glucuronyltransferase activity was increased at the highest concentration. At 1 month in females, increased activity was seen for N-ethylmorphine N-demethylase, 7-ethoxyresorufin O-deethylase, 7-pentoxyresorufin O-dealkylase and 7-ethoxycoumarin O-deethylase at all dietary concentrations and for UDP-glucuronyltransferase activity at the highest concentration. Additional induction was not observed in males at 3 months. and increased activity of 7-ethoxyresorufin O-deethylase was observed at dietary concentrations of 200 and 800 ppm and of 7-pentoxyresorufin O-dealkylase and 7-ethoxycoumarin O-deethylase only at the highest concentration. However, in females, the cytochrome P450 content was further increased. Increased aniline hydroxylation was seen at all dietary concentrations, N-ethylmorphine N-demethylase activity at the two higher concentrations, 7-ethoxyresorufin O-deethylation and 7-pentoxyresorufin O-dealkylase activities at all dietary concentrations and lauric acid hydroxylation at 200 and 800 ppm. Females at 3 months also showed increased activity of 7-ethoxycoumarin O-deethylase at 400 and 800 ppm and of UDP-glucuronosyltransferase at 800 ppm. After 1 month, the plasma concentrations of imazalil ranged from < 2 to 4.4 ng/ml in males and from < 2 to 3.8 ng/ml in females at the three dietary concentrations. At 3 months, the corresponding concentrations were 1.1–2.2 ng/ml in males and 1.0–4.0 ng/ml in females.

Increased absolute and relative liver weights were observed after 1 month in males and females at 400 and 800 ppm. Increased relative liver weights were seen in males and increased absolute liver weights in females at 200 ppm. Increased relative thyroid weights were observed in males and increased absolute adrenal weights in females, both at 800 ppm. Decreased thymus weights, both absolute and relative, were seen in males at the highest dietary concentration. At 3 months, males at 800 ppm had increased relative liver and kidney weights, while females had increased relative weight of the kidneys at 800 ppm and increased absolute and relative weights of the adrenals at 400 and 800 ppm. No gross pathological changes were observed that appeared to be related to treatment. Hepatic changes were seen histologically at dietary concentrations > 400 ppm at 1 month; these comprised hepatocyte swelling in males and large vacuoles in females. Adrenal changes (cortical-cell swelling) were observed at 3 months in 2/10 females at 800 ppm and 1/10 females at 400 ppm; the study authors considered that this finding corresponded to the increase in adrenal weights observed. No histopathological differences between groups were seen in the liver at 3 months. At neither 1 nor 3 months were histopathological differences observed in the thyroid (Vermeir, 1995; van Deun et al., 1996b).

In a study to investigate the mechanisms underlying the effects of imazalil on the thyroid gland, groups of 50 specific pathogen-free, male Wistar (Hannover substrain) rats were given diets contaning imazalil (purity, 98.8%) at a concentration of 0, 400, 1200 or 3200 ppm for 4 weeks; a further group received a diet containing phenobarbital at 1200 ppm. The dietary concentrations of imazalil resulted in mean intakes of 0, 41, 120 and 340 mg/kg bw per day, while the dose of phenobarbital was 130 mg base equivalent per kg bw. Interim sacrifices were carried out after 1, 2 and 4 weeks of treatment and after a 4-week (week 8) or 9-week (week 13) recovery period, so that the size of the group at each dietary concentration at the time of sacrifice was 10. The rats were observed daily, and body weights and food consumption were measured weekly. Blood was taken at sacrifice for measurement of TSH, T3 and T4 and other clinical chemical end-points, including bilirubin, total protein and alkaline phosphatase and alanine and aspartate aminotransferase activities. The animals were killed at the times described above, five animals from each group being injected 6 h before death with bromodeoxyuridine (BrdU). At sacrifice, a full post mortem was carried out, and macroscopic abnormalities were noted. The thyroid (including parathyroids) and liver were weighed. The thyroid and liver were examined histologically after haematoxylin and eosin staining, and the thyroid, liver and sections of the jejunum from BrdU-treated rats were stained immunohistochemically for BrdU-labelled cells. Liver and thyroid microsomes were prepared from liver pieces and the left thyroid glands (Vermeir, 2000). Induction or inhibition of the hepatic microsomal cytochrome P450 enzymes aniline hydroxylase, N-ethylmorphine N-demethylase, 7-pentoxyresorufin O-dealkylase, 5’-monodeiodinase and T4 glucuronyltransferase was assessed in tissue from the five animals from each group not injected with BrdU; microsomal protein and thyroid peroxidase activity in the thyroid were also measured.

No treatment-related deaths were seen, and the only treatment-related clinical effects were seen at the dietary concentration of 3200 ppm, as food wastage. Sedation was noted in the phenobarbital-treated animals. Body weight and body-weight gain were decreased from week 1 to week 8 in animals at 1200 and 3200 ppm, to a greater extent in the latter. Animals given phenobarbital showed decreased weight gain during weeks 5–8. Food consumption was decreased sporadically in animals at 400 ppm, but this was considered not biologically significant. At 1200 ppm, food consumption was decreased during dosing and for the first 3 weeks of the recovery period, while at 3200 food consumption was lower than in the controls during treatment and for 1 week afterwards. In the group given phenobarbital, food consumption was increased during week 2 of dosing. At 400 ppm, no difference from controls in clinical chemical end-points was seen.

At 1200 ppm, slight decreases in alkaline phosphatase activity were seen during week 2 and in aspartate aminotransferase activity during weeks 1 and 2. At 3200 ppm, aspartate aminotransferase activity was decreased during weeks 1, 2 and 4. animals given phenobarbital showed an increase in total protein content during dosing. TSH concentrations were increased in the phenobarbital-treated animals and in all imazalil-treated animals at 8 weeks. No statistically significant increases in TSH concentration were seen at other times; nevertheless, the increases at 1 and 2 weeks may have been biologically significant. The T4 concentrations were lower in all imazalil-treated groups after 1 week. After 2 weeks, no differences in T4 concentration were seen, while at 4 weeks the concentrations were increased in rats at 1200 and 3200 ppm, and in those given phenobarbital. At week 8 (i.e. during the recovery period), an increased T4 concentration was seen in the group at 1200 ppm and in that given phenobarbital. An increase was also seen in the group given 3200 ppm at week 13. The T3 concentration was decreased in the group given 3200 ppm at week 4 and in that given phenobarbital at weeks 1, 2 and 4. Increased hepatic activities of cytochrome P450, aniline hydroxylase, N-ethylmorphine N-demethylase and 7-pentoxyresorufin O-dealkylase were seen in both the imazalil-treated and the phenobarbital-treated groups at weeks 1, 2 and 4. Induction was often found at the lowest dietary concentration. Enzyme induction was largely absent during week 8, indicating that it was a reversible phenomenon. The activity of 5’-monodeiodinase was decreased in the group given 3200 ppm of imazalil, and T4 glucuronyltransferase activity was induced in all imazalil-treated groups after 1 week of treatment and also at week 4 in the group given 3200 ppm. Thyroid peroxidase activity was increased in the group at 3200 ppm after weeks 1 and 4 and reduced after 2 weeks of treatment; however, the changes were neither statistically significant nor clearly dose-related.

Increases were seen in the relative weight of the thyroid of animals at 3200 ppm at week 2 only, while phenobarbital increased both the relative and absolute weights at this time. At week 2, a positive trend in the relative thyroid weight with dose of imazalil was found. At week 1, the relative and absolute weights of the liver were increased at 400 and 3200 ppm, and the relative weight only at 1200 ppm. At week 2, the relative liver weight was increased at 1200 and 3200 ppm, while at week 4 the relative and absolute weights were increased at 400 and 1200 ppm. The relative liver weight was increased at week 4 at the highest dietary concentration. At 8 weeks, a decrease in absolute but not relative liver weight was observed at the two higher dietary concentrations of imazalil. Phenobarbital increased the absolute and relative weights of the liver at 1, 2 and 4 weeks, but this effect had disappeared by 8 weeks. At the terminal kill at 13 weeks, the highest concentration of imazalil had produced a decrease in the absolute but not the in the relative weight of the liver, while phenobarbital had no effect.

The only gross findings of interest were in the liver and consisted of swelling in a few animals at 400 and 1200 ppm at week 2 and 4, respectively, and swelling and increased lobulation at 3200 ppm from 1 week onwards. Histologically, hepatic centrilobular hypertrophy was found at concentrations > 400 ppm, and the occurrence of this change appeared to be dose-related. Periportal hypertrophy was found at 1200 and 3200 ppm, while hepatic vacuolation was seen at the highest dietary concentration. All three changes in the liver were also seen with phenobarbital. These changes were not observed 8 and 13 weeks after the start of the study (i.e. during the recovery period). Significant increases in the frequency of thyroid follicular hypertrophy were seen only with phenobarbital. This lesion disappeared during the recovery period. Immunohistochemistry for BrdU-labelled cells did not reveal intergroup differences. The authors concluded that imazalil alters thyroid status by affecting hepatic and thyroid enzymes involved in the synthesis, metabolism and excretion of T4. As histopathological changes were present in the liver at all concentrations, no NOAEL could be identified (Piccirillo, 2000; Verbeek et al., 2000; Vermeir, 2000). The USA’s Environmental Protection Agency concluded that these findings may explain the mechanism whereby imazalil induces thyroid metabolism (Khasawinah, 2000).

Qualititatively, these studies show that imazalil causes hypertrophy of the liver and, at high doses, some interference with thyroid function. The quantitative differences between the studies may be related to their duration. The results for induction of specific hepatic enzymes were not easy to interpret, as they tended to be inconsistent and dose–effect relationships were not always clear. However, the Meeting concluded that imazalil is a relatively nonspecific enzyme inducer in the liver and its enzyme-inducing effects are reversible.

Groups of 50 specific pathogen-free Wistar (Hannover substrain) rats of each sex received diets containing a preparation called ‘imazalil 50 premix’, which nominally contained 50% imazalil (in fact 49–50%) at a concentration of 0, 50, 200, 1200 or 2400 ppm, equal to intakes of 0, 2.4, 9.7, 58 and 120 mg/kg bw per day for males and 0, 3.3, 14, 79 and 160 mg/kg bw per day for females. The animals were observed at least daily. Ophthalmic examinations were carried out in controls and in animals at the highest dose before dosing and towards the end of the treatment. Body weight was measured on the first day of dosing, thence weekly and at the end of dosing and additionally in any animal killed in extremis. Food consumption was measured weekly. Haematological and clinical chemical variables were measured in blood samples from all groups at 6, 12 and 18 months and near the end of dosing. At the same time, urine was analysed. Immediately before the terminal kill at 24 months, each rat was examined. A full necropsy was undertaken on all animals, and macroscopic abnormalities were noted. Selected organs were removed and weighed, and samples of tissue were taken and processed for histopathological examination. Blood samples were taken at autopsy for measurement of testosterone, luteinizing hormone, TSH, T4 and T3 by enzyme immunoassay, the assays for TSH and luteinizing hormone being rat-specific. Toxicokinetics was determined for the first four rats at each dietary concentration other than the controls, in blood samples taken on days 180, 362 and 726 of the study. Imazalil was measured in these blood samples.

The mortality rate was not affected. Increased food wastage was observed for males at the highest dietary concentration and for females at the two higher concentrations. Females at the highest concentration showed a decrease in subcutaneous tissue mass and alopecia. No significant difference was seen between animals at the highest concentration and controls in the ophthalmic examination. The body weights of males at 50 ppm were lower than those of controls at weeks 96, 99, 101 and 102, and their weight gain was decreased during weeks 90–94 and weeks 96–104. An increase in weight gain was seen in males at 200 ppm in week 1, but no other effect was seen on body weight or weight gain. Decreased body weight and weight gain were seen in males at 1200 ppm, consistently from week 1 to week 26 and less so thereafter. In males at 2400 ppm, body weight and weight gain were decreased throughout the study (by 15–25%). The body weights of females at 50 ppm were lower than those of controls at weeks 82, 84, 85, 87, 88, 100 and 101, and weight gain was decreased in weeks 54, 73, 78, 81–89 and 100–104. A decrease in weight gain was seen in females at 200 ppm in week 34, with no other significant change in weight or weight gain at any other time. Decreases in body weight were seen in females at 1200 ppm from the third week of the study and in weight gain from the first week. In females at 2400 ppm, body weight and weight gain were decreased throughout the study (by 25–35%). The food consumption of males was decreased in weeks 23, 52, 93 and 96 for those at 50 ppm and in weeks 53, 90, 93 and 95 for those at 200 ppm. Decreased food consumption was seen at 1200 ppm intermittently throughout the study and at 2400 almost throughout the study. Total food intake of males throughout the study was decreased at the two higher dietary concentrations. Females at 50 ppm had increased food consumption in week 27 and decreased food consumption at weeks 52, 83 and 88. At 200 ppm, increased food consumption was seen in weeks 10, 11 and 35–37 and decreased food consumption in weeks 51, 83 and 93. The food consumption of females at 1200 and 2400 ppm was decreased almost throughout the study. As for males, total food intake was decreased at the two higher dietary concentrations, and food conversion was increased by 10 and 12% at 1200 ppm and 2400 ppm, respectively.

In males at the two higher dietary concentrations, decreased erythrocyte volume fraction, mean cell volume and mean cell haemoglobin were found consistently, with decreased haemoglobin concentration at weeks 78–79 and 104–105. Minor changes, including decreased mean cell volumes at weeks 25 and 52, were seen in males at 200 ppm. The haematological picture in females was more complex: increases were observed in haemoglobin concentration at weeks 25–26 and 78–79 and increases in erythrocyte count at weeks 25–26, 52–53 and 78–79 in animals at dietary concentrations of 200, 1200 and 2400 ppm, respectively. Additionally, an increase in erythrocyte count was seen at 104–105 weeks in rats at the highest dietary concentration. Decreased mean cell volume and mean cell haemoglobin concentration were seen at all times in females at 1200 and 2400 ppm and at weeks 25–26 and 52–53 in those at 200 ppm. Thus, imazalil at a dietary concentration of 50 ppm did not affect haematological parameters, minor changes were seen at 200 ppm, and definite changes were seen at 1200 and 2400 ppm.

A number of differences between groups were observed in clinical chemical parameters, many of which were transient or inconsistent. The more consistent changes included decreased activity of alkaline phosphatase and aspartate aminotransferase in animals of each sex at all dietary concentrations. Of greater clinical significance were decreases in calcium and total protein concentrations observed at most times in animals at 1200 and 2400 ppm. Increased glucose concentrations were observed at all times in males and females at the highest dietary concentration; at weeks 52–53 and 104–105 in males and at weeks 52–53, 78–79 and 104–105 in females at 1200 ppm; and at all four times in females at 200 ppm. Triglyceride and phospholipid concentrations were decreased in males at 2400 ppm and in females at the two higher dietary concentrations at all times. The blood urea nitrogen concentration was decreased in males at the two higher dietary concentrations at all four times and in females at concentrations > 200 ppm. Decreases in alkaline phosphatase and aminotransferase activity and blood urea nitrogen concentration are generally considered not to be adverse. However, the changes in calcium and total protein, which were observed at most times in animals at 1200 and 2400 ppm, may be clinically significant. Furthermore, the increases in blood glucose concentration seen at 2400 ppm in both sexes, at 200 ppm in females at all times, and from time to time in both sexes at 1200 ppm are likely to be clinically significant.

The urine of females showed decreased specific gravity at some times, with increased pH and increased urinary volume at the two higher dietary concentrations.

The testosterone and luteinizing hormone concentrations were little altered in male rats, a significant increase in testosterone being observed only at 1200 ppm. The TSH concentrations tended to be higher at 200 and 1200 ppm, and the T4 concentrations were definitely lower in males at 1200 and 2400 ppm, the T3 concentrations being similar in all groups. In females, the T3 concentration was decreased at the two higher dietary concentrations, T4 was decreased at 1200 ppm only, while TSH was not consistently affected. Despite these inconsistent results, it is seems likely that imazalil affected the pituitary–thyroid axis when given at a dietary concentration of 1200 or 2400 ppm.

The organ weights of animals killed at term after receiving the higher dietary concentration showed some differences from controls, but these were considered to be a reflection of changes in total body weight, with the following exceptions. The relative weight of the liver was increased in males at the two higher dietary concentrations, with no effect on absolute liver weight. The absolute liver weight was decreased at 2400 ppm, and the relative liver weight was increased at concentrations > 200 ppm in females. At termination, the absolute thyroid weight was increased in males at 1200 and 2400 ppm and the relative thyroid weight at all dietary concentrations, while in females the absolute thyroid weight was decreased at 1200 and 2400 ppm, and the relative thyroid weight was unaffected (Table 1). The sponsor argued that it was appropriate to include decedents in the analysis of organ weights, as the effects on the thyroid were unlikely to be fatal (Tables 2 and 3). The study authors concluded that the NOAEL for changes in organ weights was 200 ppm, presumably on the grounds that the increased relative liver weight observed at 200 ppm in females, unaccompanied as it was by a decrease in absolute body weight, was an incidental finding. Further, the increase in relative thyroid weight seen at 50 and 200 ppm in males, in the absence of absolute thyroid weight changes, is not likely to be biologically significant.

The pathological changes seen grossly at terminal sacrifice included an increased frequency of liver foci and swollen thyroid glands in males at 1200 and 2400 ppm and decreased adiposity in males at 2400 ppm. The changes seen in females included decreased adiposity and decreased inspissated secretions in the mammary glands at 1200 and 2400 ppm. There was evidence of decreased mammary gland stimulation at all dietary concentrations.

An increased prevalence of hepatocellular adenoma was seen in males at the highest dietary concentration, in those killed at termination of the study and when all animals were included in the analysis. Males at 1200 and 2400 ppm also had an increased incidence of follicular-cell neoplasia of the thyroid. In males at 2400 ppm that were killed at termination, the incidences of both follicular-cell adenoma and total follicular-cell neoplasia were increased (Table 4). Addition of animals that died before the end of the study (Table 5) had little effect on the total incidence of the tumours (Table 6). As these tumours are likely to be incidental findings, and not the cause of death, the most appropriate tabulation is probably that in Table 6, showing a clear LOAEL for total follicular-cell neoplasia at 1200 ppm and a NOAEL at 200 ppm. The decreased incidences of pituitary and mammary gland hyperplasia were probably related to the reduced body weights of animals at the highest concentration in the diet. No differences were found between groups in neoplastic findings.

The non-neoplastic findings included decreased incidences of haemorrhagic degeneration of the adrenals in female rats at the highest dietary concentration, in all animals and in the survivors to termination when the results for these animals were analysed separately. The incidence of transitional-cell hyperplasia was increased in all treated males, but with no dose–response relationship; furthermore, this finding was significant only in rats at 50, 200 and 1200 ppm when the decedents were included in the analysis. Females at the highest dietary concentration that survived to termination and survivors and decedents taken together had an increased frequency of mineralization of the transitional epithelium of the renal pelvis. In the livers of animals that survived to termination and in all animals, an increased frequency of centriacinar hepatocyte hypertrophy was seen at the two higher concentrations. An increase in the frequency of periacinar hepatocyte hypertrophy was seen in females at the two higher dietary concentrations when the results for all animals were analysed together and at 1200 ppm in survivors only. Changes in the portal tract were discerned in males at the highest concentration, only when all animals were included in the analysis. Clear-cell foci were observed in females at the highest concentration that survived to termination. Decreased frequencies of basophilic foci and foci of hepatocellular alteration were observed in the livers of surviving females at 200 and 2400 ppm and in survivors and decedents together at 2400 ppm. Eosinophilic foci and foci of hepatocellular alteration were seen in surviving males at the two higher concentrations and in all animals at the highest dietary concentration when the results for all animals were analysed together. Fatty vacuolation was found in the livers of surviving males and in survivors and decedents taken together at the two higher concentrations and in female survivors at 1200 ppm at termination. Focal cystic degeneration of the liver was seen in males at 2400 ppm (both survivors separately and all animals), while pigment-laden hepatocytes were seen in females at the three higher dietary concentrations, whether or not the decedents were included in the analysis. In the thyroid gland, cystic focal follicular hyperplasia was seen in male survivors at the two higher concentrations and in those at the highest concentration only when all animals were included in the analysis. Few differences were found between groups with respect to pathological changes in decedents, when the findings were analysed separately; none appeared to be clearly treatment-related, with the possible exception of an increased incidence of transitional-cell hyperplasia in the kidney in males and periacinar hepatocyte hypertrophy and pigment-laden hepatocytes in females at the highest dietary concentration.

It might be argued that no NOAEL could be identified in this study, as effects on body weight, weight gain and food consumption were seen at some times in males and females at 50 ppm. However, the findings at 200 ppm suggest that the first two effects were not dose-related, and the LOAEL for body weight and weight gain was likely to be 1200 ppm. In females, increased food consumption was observed at some times at 50 ppm, whereas, in males, the situation was less clear, the decreases observed were small and occurred at only a few times. The NOAEL was therefore 50 ppm, equal to 2.4 mg/kg bw per day, on the basis of minor haematological changes in animals of each sex and increased blood glucose concentration and liver changes (increases in relative liver weight and increased incidence of pigmented hepatocytes) in females at 200 ppm. The NOAELs for tumours in males and females were 200 and 2400 ppm, equal to 9.7 and 160 mg/kg bw per day, respectively (van Deun, 1999).

All the mechanistic studies were carried out in rats and were conducted at dietary concentrations that resulted in daily intakes of up to approximately 350 mg/kg bw per day. These studies consisted of a 3-month dose range-finding study and a 3-month study of the toxicity of imazalil given orally, which included a 1-month interim sacrifice. A 1-month study was also undertaken with repeated oral doses of imazalil, with interim sacrifices at 1 and 2 weeks and recovery periods of 4 and 9 weeks. Hepatocellular hypertrophy and vacuolation were observed, and there was evidence of liver enzyme induction. Some effects were observed on thyroid weight and pituitary and thyroid hormones (increased TSH and decreased T4 concentrations), but the effects were weak and inconsistent, and clear dose–effect relationships were not always found. The Meeting concluded that imazalil is a relatively non-specific enzyme inducer in vivo and its enzyme-inducing effects are reversible. Thus, imazalil may alter thyroid status by affecting hepatic and thyroid enzymes involved in the synthesis, metabolism and excretion of T4.

In a long-term study of toxicity and carcinogenicity, rats received diets containing imazalil at a concentration of 0, 50, 200, 1200 or 2400 ppm, providing intakes of 0, 2.4, 9.7, 58 and 120 mg/kg bw per day for males and 0, 3.3, 14, 79 and 160 mg/kg bw per day for females. Consistent decreases in body weights, weight gain and food consumption were seen in males and females at the two higher concentrations. Changes in haematological parameters were seen at concentrations > 200 ppm in both sexes. Increased blood glucose concentrations were observed at the two higher dietary concentrations in animals of each sex and in females at 200 ppm at all times. The changes in thyroid hormone concentrations in serum were inconsistent: in males, the concentrations of TSH tended to be higher and those of T4 definitely lower at the two higher dietary concentrations; in females, the level of T3 was decreased at the two higher dietary concentrations, and that of T4 was decreased only at 1200 ppm. Males at the highest dietary concentration had an increased prevalence of hepatocellular adenoma, and an increased incidence of follicular-cell neoplasia of the thyroid was observed in males at 1200 and 2400 ppm. An increased frequency of pigment-laden hepatocytes was seen in females at the three higher dietary concentrations.

Effects on body weight, weight gain and food consumption, seen at some times in males and females at 50 ppm, were considered not to be dose-related because they were mild and no effects on body weight or body-weight gain were seen at 200 ppm. The overall NOAEL was 50 ppm, equal to 2.4 mg/kg bw per day, on the basis of minor haematological changes in animals of each sex and increased blood glucose concentrations and hepatic changes (increased relative liver weight and an increased frequency of pigment-laden hepatocytes) in females at 200 ppm. The NOAEL for tumours in males was 200 ppm, equal to 9.7 mg/kg bw per day, whereas that in females was 2400 ppm (the highest dose tested), equal to 160 mg/kg bw per day.

The Meeting concluded that the ADI of 0–0.03 mg/kg bw established by the 1991 Joint Meeting and reaffirmed by the 2000 Meeting is supported by the new data.

van Deun, K. (1999) Combined oral chronic toxicity/carcinogenicity study in the SPF Wistar rat. Unpublished study No. R023979 dated 8 June 1999. Submitted to WHO by Janssen Pharmaceutica NV, Beerse, Belgium. Guideline: USEPA guideline for chronic feeding/oncogenicity in the rat, subdivision F, Guideline ref. 83-5 (December 1999). GLP USEPA 40 CFR part 160.

van Deun, K., Lammens, L., Benze, J., Coussement, W., Vandenberghe, J., Lampo, A. & van Cauteren, H. (1996a) 3-month dose range finding and mechanistic toxicity study in SPF Wister rats. Unpublished study No. 3672 from Janssen Pharmaceutica NV, Department of Pharmacokinetics, Beerse, Belgium. Submitted to WHO by Janssen Pharmaceutica NV, Beerse, Belgium. GLP USEPA 40 CFR 160.

van Deun, K., Lammens, L., Benze, J., Coussement, W., Vandenberghe, J., Lampo, A. & van Cauteren, H. (1996b) 3-month dose range finding and mechanistic toxicity study with one month interim sacrifice in SPF Wister rats. Unpublished study No. 3514 from Janssen Pharmaceutica NV, Department of Pharmacokinetics, Beerse, Belgium. Submitted to WHO by Janssen Pharmaceutica NV, Beerse, Belgium. GLP USEPA 40 CFR 160. Guidelines: USEPA FIFRA pesticide assessment guidelines, subdivision F, hazard evaluation (1984); US FDA toxicological principles for the safety assessment of direct food additives and color addtives in food (1982); Ministry of Health and Welfare, Japan; the rules governing medicinal products in the European Community, Volume III.

van Deun, K., Lammens, L., Vandenberghe, J., Lampo, A., Jansen, T. & Coussement, W. (1999) 3-month dose range finding and mechanistic toxicity study in SPF Wistar rats: Unpublished amendment No. N117794 to the final report (report No. 3672) dated 30 September 1999 from Janssen Pharmaceutica NV, Department of Pharmacokinetics, Beerse, Belgium. Submitted to WHO by Janssen Pharmaceutica NV, Beerse, Belgium. GLP USEPA 40 CFR 160. Guideline 21 CFR part 58 (US FDA).

Khasawinah, A. (2000) Memorandum to Mark Howard and Betty Shackleford dated 4 October on imazalil, mechnistic study to support the mode of action of thyroid tumors. USEPA.

Piccirillo, V.J. (2000) Imazalil: One-month repeated dose oral toxicity study with 1 and 2 week interim sacrifices and 4 and 9 week recovery periods to evaluate thyroid effects. Unpublished report no VJP 5452-00-1 from VJP Consulting Inc., Sterling, Virginia, USA. Submitted to WHO by Janssen Pharmaceutica NV, Beerse, Belgium. Guidelines: not applicable. GLP USEPA 40CFR 160.

Sterkins, P. (1996) The toxicokinetics of imazalil (R023979) in SPF Wistar rats at the end of a three-month dose-range-finding and mechanistic toxicity study. Unpublished addendum to study No. 3672 from Janssen Pharmaceutica NV, Department of Pharmacokinetics, Beerse, Belgium. Submitted to WHO by Janssen Pharmaceutica NV, Beerse, Belgium. GLP USEPA 40 CFR 160.

Verbeek, J., Verstyenen, B., Vandenberghe, J., Vinckier, A., de Coster, R., Lampo, A., Jansen, T. & Coussement, W. (2000) One-month repeated dose oral toxicity study in the Wistar rat with 1 and 2 week interim sacrifice and 4 and 9 weeks of recovery. Unpublished study No. 5009 from Janssen Pharmaceutica NV, Department of Pharmacokinetics, Beerse, Belgium. Submitted to WHO by Janssen Pharmaceutica NV, Beerse, Belgium. Guidelines: not applicable.

Vermeir, M. (1995) Study on the possible induction and/or inhibition of effects of hepatic drug metabolizing enzymes by imazalil in male and female SPF Wistar rats, after oral adminstration through the diet for one or three consective months at levels of 200, 400 and 800, 1600 ppm. Unpublished study No. FK 1960 from Janssen Pharmaceutica NV, Department of Pharmacokinetics, Beerse, Belgium. Submitted to WHO by Janssen Pharmaceutica NV, Beerse, Belgium. GLP: USEPA 40 CFR 160 and US FDA 21 CFR 58. Guidelines: not applicable.

Vermeir, M. (1996) Study on the possible induction and/or inhibition of effects of hepatic drug metabolising enzymes by imazalil in male and female SPF Wistar rats, after oral adminstration through the diet for three months at levels of 800, 1600 and 2400 ppm. Unpublished study No. FK 2060 from Janssen Pharmaceutica NV, Department of Pharmacokinetics, Beerse, Belgium. Submitted to WHO by Janssen Pharmaceutica NV, Beerse, Belgium. GLP: USEPA 40 CFR 160 and US FDA 21 CFR 58. Guidelines: not applicable.

Vermeir, M. (2000) Study on the possible induction and/or inhibition of effects of hepatic drug metabolising enzymes and of hepatic 5’-monodeiodinase and thryoid peroxidase activities in male SPF Wistar rats, after oral adminstration through the diet for one, two and four weeks at levels of 400, 1200 and 3200 ppm. Unpublished study No. FK 3378 from Janssen Pharmaceutica NV, Department of Pharmacokinetics, Beerse, Belgium. Submitted to WHO by Janssen Pharmaceutica NV, Beerse, Belgium. Guidelines: not applicable. GLP USEPA 40CFR 160 (not analysis of samples for 5’-monodeiodinase activity).

    See Also:
       Toxicological Abbreviations
       Imazalil (ICSC)
       Imazalil (Pesticide residues in food: 1977 evaluations)
       Imazalil (Pesticide residues in food: 1980 evaluations)
       Imazalil (Pesticide residues in food: 1984 evaluations)
       Imazalil (Pesticide residues in food: 1984 evaluations)
       Imazalil (Pesticide residues in food: 1985 evaluations Part II Toxicology)
       Imazalil (Pesticide residues in food: 1986 evaluations Part II Toxicology)
       Imazalil (Pesticide residues in food: 1991 evaluations Part II Toxicology)
       Imazalil (JMPR Evaluations 2000 Part II Toxicological)
       Imazalil (JMPR Evaluations 2005 Part II Toxicological)