Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Combined chronic toxicity and carcinogenicity study, oral (OECD 453), rat:

carcinogenicity: NOEL = 214 mg/kg bw/day (males) and 296 mg/kg bw/day (females)

systemic toxicity (24 month): NOAEL = 0.08 mg/kg bw/day (males) and 0.11 mg/kg bw/day (females)

Key value for chemical safety assessment

Carcinogenicity: via oral route

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Study duration:
chronic
Species:
rat
Quality of whole database:
The available information comprises an adequate and reliable study (Klimisch score 1), and is thus sufficient to fulfil the standard information requirements set out in Annex X, 8.5, of Regulation (EC) No 1907/2006.

Carcinogenicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Carcinogenicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The available data on carcinogenicity do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.

Additional information

The oncogenic potential and the chronic toxicity of the test substance were assessed in a study performed according to OECD Guideline 453 and in compliance with GLP (2006). Groups of 75 male and 75 female Wistar Rj:WI (IOPS HAN) rats were fed a diet containing 0, 2, 50, 1500 and 5000 ppm of the test substance for at least 104 weeks. Seventy-five rats per sex were used in the control and high dose groups (10 animals/sex designated for the interim sacrifice after 52 weeks, 15 animals/sex for the recovery group sacrifice after 68 weeks and 50 animals/sex for the final sacrifice after 104 weeks). Sixty rats per sex were used in the intermediate groups (10 animals/sex for the interim sacrifice and 50 animals/sex for the final sacrifice). Observations and examinations included mortality and clinical signs, detailed physical examinations including palpation for masses, body weight, food consumption, ophthalmological examinations, test substance analysis in the blood, hematology, clinical chemistry determinations and urinalysis. At the scheduled chronic, recovery and carcinogenicity phase sacrifice, selected organs were weighed and designated tissues sampled and examined microscopically. The mean achieved dose levels of the test substance received by the animals over the 12-month period were approximately 0.09, 2.33, 72.0 and 245 mg/kg bw/day in males and 0.13, 3.21, 99.6 and 337 mg/kg bw/day in females. The mean achieved dose levels of test substance received by the animals over the 24-month period were approximately 0.08, 2.03, 62.4 and 214 mg/kg bw/day in males and 0.11, 2.83, 88.6 and 296 mg/kg bw/day.

Over the first 12 months of the study, the mortality rate was low (1-5 per dose group) at most dose levels for both sexes with no evidence of a treatment or dose-related increase. In the male high dose group 16/75 animals were found dead or were sacrificed prematurely for humane reasons, principally within the first 6 weeks of the study. The main clinical signs in the early decedent males consisted of ocular or nasal discharge, soiled nose, soiled fur or anogenital region, wasted appearance, together with the usual signs associated with morbidity. Macroscopic examination indicated a hemorrhagic lesion as the cause of premature death in at least 8 of these males. During the second year of the study, no treatment-related effect was noted on the mortality incidence. After 2 years, 14-40 animals per dose group of 50 animals were found dead or sacrificed prematurely.

High dose group - 5000 ppm

Treatment-related clinical signs observed in a significant number of animals consisted of an increased incidence of white area on eyes (neovascularization as found in ophthalmologic examination, see below), localized soiled fur, soiled anogenital region, hair loss, reduced motor activity, cold appearance, general pallor, wasted appearance, and dental abnormality during the first year of the study in males and/or females. During the second year, the treatment-related clinical signs observed in a significant number of animals were: white area on eyes, skin lesions, reduced motor activity and focal swelling (principally of hindlimb) in males, and of white area on eyes, chromodacryorrhea and soiled anogenital region in females. At the end of the recovery phase, all clinical signs were found to be reversible, with the exception of white area on eyes that was observed in a single male only, and were therefore not considered to be adverse effects.

During the first week of treatment, mean body weight was lower by 5 and 4% and mean body weight gain by 21 and 29%, in males and females, respectively, compared to controls. This trend was observed throughout the course of the study, leading to a final body weight reduction on Day 708 of 5 and 15% in males and females, respectively, and to a body weight gain reduced by 6 and 23% in males and females, respectively, when compared to controls. No relevant changes in body weight were noted during the recovery phase; indicating that these effects were reversible. The food consumption was slightly reduced by up to 6% during the first week of treatment in both sexes, but was similar to controls thereafter. No significant changes in food consumption were noted during the recovery phase. The ophthalmological examinations revealed a high incidence of corneal opacity, neovascularization and oedema of the cornea and snow flake-like corneal opacity in both sexes throughout the study. After 13 weeks of recovery, the corneal opacity, oedema of the cornea and snow flake-like corneal opacity were completely reversible, whereas neovascularization of the cornea was still observed in some high dose  animals from both sexes. The type of corneal lesions observed at 5000, 1500 and 50 ppm in the rat (together with the eye keratitis noted at the microscopic examination), are characteristics of a compound such as the test substance that inhibits 4-hydroxyphenylpyruvic acid dioxygenase (4-HPPDase), an enzyme of the tyrosine catabolic pathway. These lesions are related to an increase in plasma tyrosine level caused by a blockade of the 4-HPPDase enzyme in the rat. However, the rat is a species particularly sensitive to inhibition of the 4-HPPDase enzyme and is atypical in its susceptibility to develop tyrosine-related eye lesions. Therefore, although these lesions (corneal lesions and eye keratitis) were treatment-related, they were considered not to be toxicologically relevant to humans.

The clinical chemistry assessments showed a higher mean total cholesterol concentration up to Month 18 of the study in both sexes, together with a higher mean triglycerides concentration up to Month 12 in males and up to Month 3 in females, in comparison to controls. All changes noted were reversible after the 3-month recovery period. In the absence of clear changes in the histopathological examination, they were considered to be treatment-related, but not adverse, effects.

Urinalysis showed higher ketone level throughout the study in both sexes, and lower pH values during the first year in both sexes and during the second year in males, when compared to controls. In addition, higher protein level and a lower amount of crystals were observed in males up to Month 18, compared with controls. After 13 weeks of recovery, only the tendency towards a lower pH value was observed in females, while the rest of the above-mentioned changes were reversible. The observed effects on urinary parameters were considered not to be adverse, due to the reversibility and lack of corresponding histopathological effects.

At the end of the chronic phase (12 months) the mean absolute and/or relative kidney weights were higher in males at 5000 ppm (statistically significant) in comparison to controls. These changes were considered to be treatment-related.  Mean absolute and relative liver weights were higher in males at 5000 ppm (statistically significant), in comparison to controls. These changes were considered to be treatment-related. However, in view of the limited changes noted at the microscopic observation, they were considered to have no toxicological relevance. Mean absolute brain weight was decreased by 6% (p<0.05) in males at 5000 ppm, in comparison to controls. This change was considered not to be toxicologically relevant, since it was observed for this parameter only and was not associated with any change at the macroscopic and microscopic examinations.  All organ weight changes detected at the end of the chronic phase were found to be reversible. The few changes noted after the 3-month recovery period were considered to be incidental in view of their individual variation.

At the end of the carcinogenicity phase (24 months), the mean absolute and relative liver weights were increased by 25 and to 31%, respectively, in males, while the relative liver weight was increased by 17% in females. These changes were considered to be a treatment-related, adaptive response to the test substance exposure. Mean absolute and relative kidney weights were increased by 16 and 22%, respectively, in males, in comparison to controls. During the macroscopic examination, toxicologically relevant changes were noted in the kidneys. The incidence of irregular surface on kidney was increased in females (3), in comparison to controls (0). In the kidney, the incidence of minimal to severe chronic progressive nephropathy was statistically significantly higher in males (38), compared with controls (30). However, this change is a common age-related and spontaneously occurring disease in the rat and was observed with a high incidence in control males (30/50). This change is not considered to be toxicologically relevant. The histopathological examination showed toxicologically relevant changes in the pancreas and thyroid gland, in comparison to controls. In the pancreas, the incidence of acinar atrophy/fibrosis was higher in both sexes (36 for males, 31 for females vs. 18 for males, 13 for females in control), whilst in the thyroid gland the incidence of follicular cell hypertrophy was elevated in both sexes (27 for males, 19 for females vs. 3 for males, 1 for females in control). Other changes noted during the histopathological examination were considered not to be toxicologically relevant. The overall incidence of neoplastic findings observed after the chronic phase was very low and did not indicate any treatment related relationship. No neoplastic finding was observed after the recovery phase on the organs examined. The overall incidence of neoplastic findings observed in animals from the carcinogenicity phase was within expectations for a two-year study and did not indicate any treatment-relationship.

1500 ppm

The treatment-related clinical signs consisted of a significantly increased incidence of white area on eyes in both sexes, together with soiled anogenital region in 4/60 females during the first year. During the 2nd year, a significantly increased number of males had white area on eyes, reduced motor activity and focal swelling (principally of hindlimb), compared with controls; while an increased number of females had a white area on eyes, compared with control.

The mean body weight was reduced by up to 6% during the first year of treatment in both sexes, in comparison to controls, while the mean body weight gain was reduced by up to 11 and 18% throughout the first year in males and females, respectively. The effect persisted in both sexes, as the final body weight on Day 708 was reduced by 9 and 15% in males and females, respectively, when compared to controls. The body weight gain was reduced by 14 and 21% in males and females, respectively, when compared to controls. The food consumption was unaffected by treatment.

The ophthalmological examinations revealed a high incidence of corneal opacity, neovascularization and oedema of the cornea and snow flake-like corneal opacity in both sexes throughout the study. These observations are considered not to be relevant to humans, as described above.

At the end of the chronic phase (12 months) the clinical chemistry evaluation showed higher mean total cholesterol and/or triglycerides concentrations were noted in both sexes (statistically significant on most occasions), in comparison to controls. Lower mean alkaline phosphatase activity was noted in males at Months 3 and 6 (statistically significant). These changes were considered to be treatment-related. However, since the decrease was observed only transiently and in the absence of any changes at the histopathological examination, they were considered not to be toxicologically relevant. Slight variations were also observed in males in total protein and albumin concentrations and consequently in globulin concentrations and albumin/globulin ratio (calculated parameters), principally at Month 3. However, the magnitude of the variations relative to the controls was less pronounced throughout the sampling periods. Therefore, these differences were considered to be meaningless and not to be toxicologically relevant. After the carcinogenicity phase no changes were observed.

The urinalysis showed higher ketone level on most occasions during the study in both sexes, compared with control. Lower mean pH values compared to controls were observed in both sexes throughout the first year and only in males throughout the second year, though no clear dose related effect was noted in males, a lower amount of crystals was observed up to Month 18, together with a higher protein level at Months 6, 12 and 18.

At the end of the chronic phase (12 months), the mean absolute and relative liver weights were higher in males (statistically significant only for the relative liver weight), in comparison to controls. These changes were considered to be treatment-related. However, in view of the limited changes noted at the microscopic observation, they were considered to have no toxicological relevance. Mean absolute and/or relative kidney weights were higher in males (statistically significant for the relative weight parameter), in comparison to controls. These changes were considered to be treatment-related. The mean absolute brain weight was decreased by 7% (p<0.01) in males at 1500 ppm, in comparison to controls. This change was considered not to be toxicologically relevant, since it was observed for this parameter only and was not associated with any change at the macroscopic and microscopic examinations.

At the end of the carcinogenicity phase (24 months), the mean absolute and relative liver weights were higher in males (25% and 36%, respectively, p<0.01), when compared to controls. Mean relative weight was also higher in females (12%, p<0.05), in comparison to controls. There were no toxicologically relevant findings during the macroscopic examination. The histopathological examination showed toxicologically relevant changes in the pancreas and thyroid gland, in comparison to controls. In the pancreas, the incidence of acinar atrophy/fibrosis was higher in both sexes (33 in males and 32 in females vs. 18 in males and 13 in females in controls), whilst in the thyroid gland, the incidence of follicular cell hypertrophy was elevated in both sexes (19 in males and 20 in females vs. 3 in males and 1 in females in controls). Other changes noted during the histopathological examination were considered not to be toxicologically relevant.

The overall incidence of neoplastic findings observed after the chronic phase was very low and did not indicate any treatment related relationship. No neoplastic finding was observed after the recovery phase on the organs examined. The overall incidence of neoplastic findings observed in animals from the carcinogenicity phase was within expectations for a two-year study and did not indicate any treatment-relationship.

50 ppm

Treatment-related clinical signs were limited to a significantly increased incidence of white area on eyes in both sexes throughout the study, together with a significant increase in the incidence of reduced motor activity and focal swelling (principally of hindlimb) in males during the second year. The Body weight parameters were comparable to control values in both sexes during the first year of treatment.

Throughout the second year, the mean body weight was reduced by up to 11 and 8% and the mean body weight gain by up to 17 and 12% in males and females, respectively, compared to controls. The food consumption was unaffected by treatment.

The ophthalmological examinations showed a high incidence of corneal opacity, neovascularization and oedema of the cornea and snow flake-like corneal opacity in both sexes throughout the study. These observations are considered not to be relevant to humans, as described above.

The clinical chemistry evaluation showed a significantly higher mean total cholesterol level up to Month 12 of the study in males and a significantly higher mean triglycerides concentration on one or two sampling periods in both sexes, in comparison to controls.

The urinalysis showed higher ketone level on most occasions during the study in both sexes and lower pH values during the first year in both sexes and also during the second year in males, when compared to controls. In addition in males, a lower amount of crystals was observed up to Month 18, together with a higher protein level at Months 6, 12 and 18.

At the end of the chronic phase (12 months), the mean kidney weights were increased by between 17 to 22% in males, in comparison to controls. There were no toxicologically relevant findings during the macroscopic examination. The few changes noted during the histopathological examination were considered to be incidental.

At the end of the carcinogenicity phase (24 months), the mean relative liver and kidney weights were significantly increased by 20% each in males, when compared to controls. There were no toxicologically relevant findings during the macroscopic examination. The histopathological examination revealed toxicologically relevant changes in the pancreas and thyroid gland, in comparison to controls. In the pancreas, the incidence of acinar atrophy/fibrosis was higher in females (28 vs. 13 in controls), whilst in the thyroid gland the incidence of follicular cell hypertrophy was elevated in both sexes (16 in males and 22 in females vs. 3 in males and 1 in females in controls). Other changes noted during the histopathological examination were considered to be incidental.

The overall incidence of neoplastic findings observed after the chronic phase was very low and did not indicate any treatment related relationship. No neoplastic finding was observed after the recovery phase on the organs examined. The overall incidence of neoplastic findings observed in animals from the carcinogenicity phase was within expectations for a two-year study and did not indicate any treatment-relationship.

2 ppm

No toxicologically relevant changes were noted throughout the course of the study in either sex for any of the parameters evaluated.

 

In conclusion, no treatment-related neoplastic changes were observed at any dose level tested in either sex. Thus, under the conditions of this study the NOEL in terms of carcinogenic potential was 5000 ppm for males and female rats (equivalent to 214 and 296 mg/kg bw/day, respectively). Due to adverse effects on pancreas and thyroid gland the NOAEL for males and females in terms for systemic toxicity over a 24-month period of dietary administration with the test substance to the Wistar rat was 2 ppm (equivalent to 0.08 and 0.11 mg/kg bw/day in males and females, respectively).