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Description of key information

Although no information is available for the substance Imazalil sulphate, two oral carcinogenicity studies were considered reliable regarding to the assessment of the carcinogenic potential of the related substance Imazalil.

- Verstraeten et al. (1993): 50 male and 50 female Swiss Albino Mice were dietary administered for a period of 23 months at the following concentrations: 0, 50, 200 and 600 ppm (corresponding to 0, 8.1/9.9, 33/42 and 105/131 mg/kg in males/females respectively). Imazalil treatment resulted in an increase in the frequency of hepatocytic neoplasms and of neoplastic nodules (adenomas) at 200 and 600 ppm in males and at 600 ppm in females. In male mice, incidences of hepatic carcinoma were also increased, at 600 ppm.

- Van Deun et al. (1999): 50 male and 50 female Wistar rats were dietary administered for a period of 24 months at the following concentrations: 0, 50, 200, 1200 and 2400 ppm (corresponding to 0, 2.5/3.5, 10/14, 60/80 and 120/160 mg/kg bw/d in M/F, respectively). Imazalil treatment resulted in a statistically significantly higher incidence of thyroid follicular cell neoplasias (adenomas and carcinomas combined) at 1200 and 2400 ppm in male rats, together with swelling, increased thyroid weight and cystic follicular hyperplasia. Statistically significantly increased incidences of liver adenomas were seen in male rats at 2400 ppm only.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Endpoint conclusion
Endpoint conclusion:
adverse effect observed

Carcinogenicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Carcinogenicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

Summary on mechanistic data presetented in the RAC opinion on harmonised classification of Imazalil:

The mechanistic data seem to indicate that oxidative stress and peroxisome proliferation are unlikely to be involved in the development of the liver tumours following Imazalil treatment, and that there is also little evidence for cytotoxicity and (in rats) apoptosis. The mechanism is however non-genotoxic (Imazalil tested negative in a battery of mutagenicity studies), and most likely involves enzyme induction (with a practical threshold) as in several mechanistic studies Imazalil appeared to be a mixed type of microsomal enzyme inducer (indicative of CYP2B and CYP3A activity) in both rats and mice. The fact that in most studies Imazalil, similar to phenobarbital, further caused increases in liver weight and in hepatocellular hypertrophy and vacuolisation, and the up-regulation of several phenobarbital response signature genes, could point to a phenobarbital-like mode of action through PXR/CAR activation. Cell proliferation is however an additional essential step in the development of hepatocellular tumours by phenobarbital. IND argued that for phenobarbital it has been shown in vitro that there is a difference in ability between rodent and human hepatocytes in producing cell proliferation through CAR activation, by referring to Hirose et al. (2009). In this latter study, phenobarbital was able to induce CYP2b forms in both rat and human hepatocytes, but cell proliferation only in rat hepatocytes. Apparently a similar result has been observed for mouse versus human hepatocytes, given the results reported for phenobarbital in the Elcombe (2012b) study.

With reference to Ross et al. (2010), IND further argued that in vivo studies with humanised PXR/CAR mice exposed to phenobarbital confirmed the absence of cell proliferation, reason why phenobarbital-induced liver tumours in rodents are not considered relevant to human health (supported by the absence of an increased liver tumour risk in humans receiving phenobarbital for many years). Indeed, in the Ross et al. (2010) study, cell proliferation was only observed in wild type mice and not in hPXR/hCAR or knockout PXR/CAR mice following intraperitoneal injection of 80 mg/kg bw/d phenobarbital for 4 days. The RAC noted however that in the Elcombe (2012a) study, phenobarbital at a dietary dose equivalent to 127.8-155.3 mg/kg bw/d did induce cell proliferation in wild type and hPXR/hCAR mice. Apparently there is a threshold for phenobarbital-induced cell proliferation somewhere between 80 and 120 mg/kg bw/d. For Imazalil the mechanistic data on cell proliferation are equivocal: in (male) rats, no cell proliferation was observed, whereas in (male) mice cell proliferation was shown after relatively short exposure (4-7 days) but not after longer exposure. The recent experiments with Imazalil by Elcombe showed an absence of replicative DNA synthesis in human hepatocytes, but an increase in cell proliferation (albeit not dose-related) in humanised PXR/CAR mice.

Full References: Not available in the publicly available RAC reports.

Justification for classification or non-classification

Based on the available in vivo data on Imazalil and the RAC opinion published in 2013 on the harmonised classification and labelling for this substance. Based on the read-across approach described in section 13, the same conclusion applies for its salt Imazalil sulphate.

Imazalil sulphate should therefore be classified as Carc. cat 2 according to the criteria laid out in (EC) No 1272/2008, the Classification, Labelling and Packaging (CLP) Regulation.

Additional information

No reliable carcinogenicity study is available for Imazalil sulphate. Therefore, reliable data from the supporting substance Imazalil is used to cover this endpoint. Justification for this read across approach is included in IUCLID section 13.

2 key studies have been identified to evaluate the carcinogenic potential of Imazalil.

- Verstraeten et al. (1993): 50 male and 50 female Swiss Albino Mice were dietary administered for a period of 23 months at the following concentrations: 0, 50, 200 and 600 ppm (corresponding to 0, 8.1/9.9, 33/42 and 105/131 mg/kg in males/females respectively). Imazalil treatment resulted in increased liver weight in both sexes at 600 ppm. Macro and microscopic liver changes (non-neoplastic) were seen in male mice at 200 and 600 pm and consisted of foci, vacuolisation, sinusoidal cell pigmentation and swelling. A trend towards similar lesions was reported to be seen in female mice at 600 ppm, but no data were shown. Neoplastic changes (no data on statistical significance reported) consisted of increased incidences of hepatocytic neoplasms (i.e. combined hepatocellular adenoma/carcinoma) and neoplastic nodules (i.e. hepatocellular adenoma) at 200 and 600 ppm in males and at 600 ppm in females. In male mice, the incidence of hepatocellular carcinoma at 600 ppm was also increased. Other effects included a reduced body weight (by 5-10%) and body weight gain (by 15-20%) in males at 600 ppm. Haematological parameters were only affected in females (increased haemoglobin, haematocrit and RBC at 200 and 600 ppm), but only after 1-yr of dosing, not at the end.

Based on additional data presented by one industry representative (IND) to the Committee for Risk Assessment (RAC) on statistical significance for the liver tumors, as well as historical control data for hepatocellular tumours from 9 mouse carcinogenicity studies performed in the same test laboratory, starting within the same period of time and using the same strain of mice, it appears that the only tumour findings that reached statistical significance were the increases in adenoma and combined adenoma/carcinoma in males at 200 and 600 ppm and the increase in combined adenoma/carcinoma in females at 600 ppm. The incidences for combined adenoma/carcinoma were outside the historical control ranges for both males and females, the incidences for adenomas in males were at and above the upper level of the historical control range.

- Van Deun et al., (1999): 50 male and 50 female Wistar rats were dietary administered with for a period of 24 months doses of Imazalil at the following concentrations: 0, 50, 200, 1200 and 2400 ppm (corresponding to 0, 2.5/3.5, 10/14, 60/80 and 120/160 mg/kg bw/d in males and females, respectively). Imazalil treatment resulted in increased liver weight in both sexes at 1200 and 2400 ppm. In male rats, gross and microscopic liver changes (non-neoplastic) at these dose levels included (eosinophilic) foci, centriacinar hypertrophy, vacuolisation, focal cystic degeneration and pigment laden hepatocytes. An increase in this latter finding was already observed at 200 ppm in female rats, and this was accompanied by centriacinar and periacinar hypertrophy at higher dose levels where livers were dark and showed more pronounced lobulation. The only neoplastic finding in the liver was a statistically significantly increased incidence in adenomas in male rats at 2400 ppm.

Other effects included reductions in body weight and body weight gain in both sexes at 1200 and 2400 ppm. Food consumption was reduced in females at 1200 ppm and in both sexes at 2400 ppm. From 200 ppm, in female rats, haemoglobin values and red blood cell counts (RBC) were increased, while e.g. mean corpuscular volume, plasma potassium, urea nitrogen, ALT and AST were decreased compared to controls. At higher doses, the adverse effects on the blood and serum parameters were enhanced and included also males.

In addition, it is worth noting that in male rats, a statistically significantly higher incidence of thyroid follicular cell neoplasias (adenomas and carcinomas combined) was seen at 1200 and 2400 ppm together with swelling, increased thyroid weight and cystic follicular hyperplasia. The increase was mostly due to an increase in adenomas. However, mechanistic studies with Imazalil are available which indicate that that the observed thyroid tumours are not a primary effect of Imazalil, but are likely to be secondary to increased hepatic microsomal enzyme induction.

Given also that the thyroid tumours were mainly benign in nature and only occurred in males, that the thyroid gland related carcinogenicity is of low potency (with a T25 > 100 mg/kg bw/d), and that the mechanism behind these thyroid tumours was not genotoxic (Imazalil tested negative in a battery of mutagenicity studies), the RAC concluded that the thyroid tumours in rats do not warrant classification and that the thyroid appears to be a second target organ in rats.

Based on additional data presented by IND to the RAC on historical control data for hepatocellular adenoma and carcinoma in male rats from 8 rat carcinogenicity studies (with 10 control groups in total) performed in the same test laboratory, starting within the same period of time and using the same strain of rats. From this it appears that the incidence of hepatocellular adenomas in male rats at 2400 ppm was greater than the historical control range. IND however commented that the increase in this type of tumour only occurred at a dose level that was far beyond the MTD as a result of bad nutritional status due to dietary aversion (resulting in a decrease in body weight gain of 19%), and that therefore they should not be taken into account. Indeed, food wastage was observed in male rats dosed at 2400 ppm (and to an even greater extent in female rats dosed at 1200 and 2400 ppm), apparently due to lack of palatability of the treated food. Whether this dose can be considered ‘far beyond the MTD’ in males is questionable, as the poor nutritional status was not associated with overt clinical signs of toxicity, an increase in mortality, or severely altered serum biochemistry parameters. Besides, the reduction in body weight gain in female rats was even greater, and they showed no increased tumour incidence.

3 additional supporting studies were considered in the evaluation of the carcinogenic potential of Imazalil.

- Lina et al (1984): In this 18 -month oral toxicity study, groups of 20 male and 20 female rats were treated with Imazalil at dose levels of 0, 25, 100, or 400 ppm in the diet (equivalent to about 1.25, 5 and 20 mg/kg bw/day. The no-toxic-effect level of Imazalil for rats over a period of 18 months was placed at 100 ppm in the diet based on the effects observed at the top dose (slight decrease in mean bodyweight in females, decrease in plasma albumin concentration in males, treatment related changes in liver). This study was however not considered reliable due to deficiencies in dose selection.

- In a lifespan oral carcinogenicity study in rats, Til et al. (1985) administered 50 male and 50 female rats with Imazalil at levels of 0, 25, 100 or 400 ppm in the diet. This study is the follow-up study to the previous 6- and 18 -month studies directed by Lina et al. The long term oral administration of Imazalil to rats did not reveal tumorigenic properties of the test substance.

- The long-term study in Beagle dogs (Verstraeten et al., 1989; 1 year, daily capsule administration of 0, 1.25, 2.5 and 20 mg/kg bw/d, similar to OECD TG 452) was considered less relevant for carcinogenicity due to the limited exposure and observation duration (1-year exposure, no post-exposure observation period) and the limited number of animals (4M+4F/exposure dose).

To conclude, the liver tumours observed form a borderline case for classification for carcinogenicity. In male rats the increase only involved adenomas and was limited to the highest dose, with no dose-response at lower doses. This is considered ‘limited evidence’ for carcinogenicity. The increase in liver tumours in male mice was observed against relatively high background incidences (26% for combined adenoma/carcinoma, 16% for adenoma, 10% for carcinoma) and was statistically significant for adenomas and combined adenoma/carcinoma only, with no dose-response at 200 and 600 ppm. The increase in liver tumours in female mice was limited to the highest dose and reached statistical significance only by combining adenomas (that were increased, but not statistically significantly, and without dose-response at lower doses) and carcinoma. The evidence for carcinogenic effects in mice is therefore also considered ‘limited’.

Given the limited evidence in both rats and mice, there are insufficient grounds for a category 1B classification for carcinogenicity.

The evidence presented on Imazalil-induced cell proliferation is not sufficient to allow the conclusion that this will not be operative in humans.

As the relevance to humans of the mechanism behind Imazalil-induced liver tumour formation in rodents cannot be convincingly excluded, Imazalil is classified as Carcinogenic cat 2.

The same conclusion applies to Imazalil Sulphate.

Source: Committee for Risk Assessment RAC. Opinion proposing harmonised classification and labelling at EU level of Imazalil (ISO). CLH-O-0000002720-08-03/F. Adopted 4 June 2013.

References

• Lina, B., Til, H.P., van Nesselrooij, J.H.J., Beems, R.B. & Falke, H.E. (1984) Eighteen-month oral toxicity study with imazalil base-R 23979 in rats. Unpublished report V 84.140/220555 from Netherlands Organization for Applied Scientific Research, Division for Nutrition and Food Research, TNO, Zeist, Netherlands. Submitted to WHO by Janssen Pharmaceutica NV.

• Til, H.P., Lina, B.A.R., van Nesselrooij, J.H.J., Beems, R.B. & Falke, H.E. (1985) Lifespan oral carcinogenicity study with imazalil base-R 23979 in rats. Unpublished report V 85.153/220555 from Netherland Organization for Applied Scientific Research, Division for Nutrition and Food Research, TNO, Zeist, Netherlands. Submitted to WHO by Janssen Pharmaceutica NV.

• Van Deun, K. (1999a) Combined oral chronic toxicity/carcinogenicity study in the SPF Wistar rat. Unpublished study No. R023979 dated 8 June 1999 from Department of Toxicology, Janssen Pharmaceutica NV, Turnhoutseweg, Beerse, Belgium. Submitted to WHO by Janssen Pharmaceutica NV, Beerse, Belgium.

• Verstraeten, A., Teuns, G., Vandenberghe, J., Coussement, W., van Cauteren, H. & Marsboom, R. (1989) Imazalil base: R 23979 technical. Chronic toxicity study in beagle dogs subjected to repeated dosage for 12 months by oral administration. Unpublished report No. 1899 from Department of Toxicology, Janssen Research Foundation, Turnhoutsweg, Beerse, Belgium. Submitted to WHO by Janssen Pharmaceutica NV

• Verstraeten, A., Vandenberghe, J., Lampo, A., Van Deun, K., Coussement, W. & van Cauteren, H. (1993) Imazalil base: carcinogenicity study in Swiss mice administration through the diet for 2 years. Unpublished report No. 2194 from Department of Toxicology, Janssen Research Foundation, Turnhoutsweg, Beerse, Belgium. Submitted to WHO by Janssen Pharmaceutica NV