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Description of key information

subacute toxicity:

OECD 410, dermal, rat, 28 -days: NOAEL = 10 mg/kg bw/day

subchronic toxicity:

OECD 408, oral, rat, 90 -days: NOAEL = 259 mg/kg bw/day (males) and 302 mg/kg bw/day (females)

chronic toxicity:

OECD 453, oral, rat, 24 months: NOAEL = 0.08 mg/kg bw/day (males) and 0.11 mg/kg bw/day (females)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
0.08 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
The available information comprises adequate, reliable (Klimisch score 1-2) and consistent studies, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.
System:
endocrine system
Organ:
pancreas
thyroid gland

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
10 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available information comprises one adequate, reliable (Klimisch score 1) and consistent study, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.
System:
endocrine system
Organ:
pancreas

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
5.16 mg/cm²
Study duration:
subacute
Species:
rat
Quality of whole database:
The available information comprises one adequate, reliable (Klimisch score 1) and consistent study, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.

Additional information

Short-term, sub-chronic and chronic oral and a short-term dermal repeated dose toxicity studies performed with the test substance are available.

Oral:

Sub-acute

Two short-term oral repeated dose toxicity studies performed in rats and mice (2001) are available. Because the treatment duration lasted only 14 days, this is considered to be supporting data only. A short summary is provided below:

5 rats/sex were treated by gavage with 30, 150 and 600 mg/kg bw/day of test substance for 14 days. A satellite subgroup of 3 rats per sex was added to the high dose and control groups for interim sacrifice after 3 days of treatment to check only liver weight and hepatic cellular proliferation and histopathology. The following parameters in the main group were evaluated: detailed clinical observation, body weight, food consumption, ophthalmoscope examination, haematology, and clinical chemistry including cytochrome P-450 content, gross pathology and histopathology of liver, kidney, spleen, ovaries, testes and thyroid gland. In the 600 mg/kg bw/day dose group only higher liver weights and slight centrilobular hepatocellular hypertrophy in males were observed. Hepatocellular hypertrophy in combination with higher liver weights and without further histologic or clinical pathology alterations indicative of liver toxicity is considered an adaptive and a non-adverse reaction. Therefore, the NOAEL was determined to be 600 mg/kg bw/day, which is the highest dose tested.

5 mice/sex were treated by gavage with 100, 300 and 1000 mg/kg bw/day of test substance for 14 days. In the 1000 mg/kg bw/day dose group only higher liver weights and centrilobular hepatocellular hypertrophy in males and females were observed. Hepatocellular hypertrophy in combination with higher liver weights and without further histologic or clinical pathology alterations indicative of liver toxicity is considered an adaptive and a non-adverse reaction. Therefore, the NOAEL was determined to be 1000 mg/kg bw/day, which is the highest dose tested.

Sub-chronic

Three sub-chronic oral repeated dose toxicity studies are available with rats and mice; two 90-day repeated toxicity studies performed in the rat and the mouse, respectively, and one neurotoxicity study performed in rats.

Oral 90-day repeated dose toxicity study in rats (2003):

The key study was considered to be the oral repeated dose toxicity study in rats, conducted according to OECD TG 408 (2003). The test substance was administered continuously via the diet to separate groups of Wistar rats (10/sex/group) at concentrations of 1.25, 600, 4 000 and 12 000 ppm (equivalent to approximately 0.08, 39.0, 259 and 787 mg/kg bw/day in males and 0.09, 45.6, 302 and 902 mg/kg bw/day in females, respectively) for at least 90 days. A similarly constituted group of 10 males and 10 females received untreated diet and acted as a control.

12000 ppm:

There were two male mortalities at 12 000 ppm. One male was found dead on study Day 84, clinical signs prior to death consisted of pale eyes, cold to touch, general pallor, reduced motor activity and red soiled fur. A second male died during anaesthesia for blood sampling on study Day 86. This animal had lost weight during the previous week together with a corresponding reduction in food consumption. At necropsy, this animal had red soiling around the muzzle and a pale appearance. Treatment-related clinical signs in animals surviving to terminal kill consisted of white area on the eye of one male, anogenital soiling in one male and five females, hair loss in six females and wasted appearance in one female. Absolute body weight was reduced by between 9-12% in males and 6-10% in females during the whole treatment period, compared with the control groups (significant for males). The body weight gain was significantly reduced by 47% in males and 82% in females, after 1 week of treatment, with an overall reduction after 90 days of 19% (p>0.05) in males and 13% (p>0.05) in females, compared with the control group. Overall food consumption was significantly reduced by 6% in males, with the most marked effect during Week 1 when there was a 16% reduction compared with controls. In females the food consumption was reduced during Week 1 only, by 24% compared with the control females. Body weight changes are not considered to be adverse since they are significantly reduced over the whole period and are also in correlation with lower food consumption. At the ophthalmic examination, one male had a bilateral 'snow flake' corneal opacity together with neovascularisation of the cornea at Weeks 8 and 12, plus unilateral haemorrhaging in the iris at Week 3. A second male had unilateral haemorrhaging of the retina and bilateral pale retinal fundus at Week 12. This was considered to be attributable to the effects of administration of the dietary admixture. Diet residue may have been accidentally deposited in and around the eyes, causing irritation. Neovascularisation is a mechanism which promotes corneal healing and is a common pathological response following corneal insult and thus not considered to be an adverse effect. The haematology evaluation showed a 13% increase (non-significant) in the prothrombin time in males, compared with control males. In addition, the male which died during anaesthesia for blood sampling had a very low erythrocyte count and haemoglobin concentration, together with a high leucocyte and neutrophil count, plus the presence of immature cells. At the clinical chemistry assessment, total cholesterol concentration was significantly increased by 45% in males and 34% in females, triglycerides concentrations were significantly increased by 81% in males and inorganic phosphorus concentration was significantly increased by 16% in females, compared with the relevant control groups. These effects show no clear dose dependency or relation to histopathological findings and are therefore not considered to be adverse. Urinalysis revealed a significant higher urinary volume in females. Marginally lower pH values and slightly higher ketone levels in both sexes are not considered to be an adverse effect since no clear dose response effect was observed. Absolute liver weight and relative weights were increased by between 22-38% in the high dose males compared with the control group. Microscopic examination of the two decedent males revealed similar treatment-related changes within the liver, pancreas and thymus. Both animals had a diffuse centrilobular acute hepatocellular necrosis in the liver, moderate acute/subacute pancreatitis with necrosis in the pancreas and moderate to marked single cell necrosis, mild to moderate atrophy/involution and slight multifocal parenchymal haemorrhages in the thymus. Microscopic examination of animals surviving to termination revealed treatment-related changes in the liver and pancreas of 6/8 surviving males. These changes consisted of slight to mild diffuse hepatocellular hypertrophy, mainly in the centrilobular region of the liver. Without any critical changes found in liver enzymes and the related liver increase this is a common adaptive response of the liver in rats found after treatment therefore, this effects is not considered to adverse. In the pancreas, 4/8 males had pancreatic lesion of which one was a mild acute/subacute pancreatitis with necrosis, similar to the lesion observed in decedent males. Other lesions included slight to mild atypical periductular fibrosis, interstitial oedema and the presence of interstitial intracytoplasmic golden brown pigmentation.

Taken together male animals show adverse treatment-related effects when fed with 12000 ppm test substance. The effects found in females are not considered adverse since none died, and there was no correlation between the effects found in clinical chemistry and in histopathologic findings.

4000 ppm:

Treatment-related clinical signs consisted of white area on the eye in 2 males and anogenital soiling in 1 female. In females, absolute body weight was reduced by between 3-8%, with a reduction in body weight gain of 47% after 1 week of treatment and an overall reduction in body weight gain of 10% after 90 days, compared with the control group. There was a corresponding reduction in food consumption of 12% during the first week of treatment in females, compared with the control group. Therefore, this effect is not considered to be adverse. At the ophthalmic examination two males and one female had a unilateral 'snow flake' corneal opacity and neovascularisation of the cornea, which were present at Weeks 3, 8 and 12 in one male, Weeks 8 and 12 in the second male and Week 12 in the female. In addition, one female had unilateral haemorrhaging in the iris at Week 3. This was considered to be attributable to the effects of administration of the dietary admixture. Diet residue may have been accidentally deposited in and around the eyes, causing irritation. Neovascularisation is a mechanism which promotes corneal healing and is a common pathological response following corneal insult. At the haematology evaluation the prothrombin time was found to be increased by 25% in the males which is considered to be in single occurrence and thus not adverse. Clinical chemistry assessment revealed a 47% increase in total cholesterol concentration and a 46% increase in triglyceride concentrations in males, compared with the control males.  The effects were not clearly dose-related and for the triglycerides only a few animals were affected in each group. Therefore, these effects are not considered to be adverse. Urine analysis revealed marginally lower pH values and slightly higher ketone levels in both sexes compared with the control groups; these are not considered to be adverse because no clear dose response was observed. Absolute liver weight and relative weight were increased by between 30-41% in males, compared with the control males. At the microscopic examination, a slight to moderate diffuse hepatocellular hypertrophy, mainly in the centrilobular region, was observed in 10/10 males. Hepatocellular hypertrophy in combination with higher liver weights and without further histologic or clinical pathology alterations indicative of liver toxicity is considered to be an adaptive and a non-adverse reaction. An interstitial oedema within the pancreas associated with an interstitial mixed cell infiltrate was observed in 2/10 males. This is considered to be an isolated occurrence.

600 ppm:

One male and one female had a unilateral "snow flake" corneal opacity together with neovascularisation of the cornea at Weeks 8 and 12 in the male and Week 12 in the female, at the ophthalmic examination. This finding corresponded to the clinical sign of white area on the eye observed in these two animals. As described above this is considered to be due to dietary application and not considered adverse. Total cholesterol concentration and triglyceride concentrations were increased by 43% (p<0.01) and 41% (p>0.05) respectively, in males, compared with the control group. These effects are not considered to be adverse because no corresponding histological changes were observed. Urine analysis revealed marginally lower pH values and slightly higher ketone levels in both sexes which are not considered adverse since no dose response was observed. Absolute and relative liver weights were increased by between 25-32% in males, compared with the control group. Microscopically, a slight to moderate diffuse centrilobular hepatocellular hypertrophy, mainly in the centrilobular region, was observed in 4/10 males. This is considered to be an adaptive response to the metabolic load of the test substance on the liver.

1.25 ppm:

No treatment-related findings were observed at 1.25 ppm in either sex.

In conclusion, the No Observed Adverse Effect Level (NOAEL) in the male Wistar rats when administered the test substance in the diet over a 90-day period was 4000 ppm (equivalent to 259 mg/kg/day) as in the 12000 ppm dose group 2 animals died and surviving animals showed histopathologic effects in the pancreas. For female rats the NOAEL was set to 12000 ppm (equivalent to 902 mg/kg/day) since no adverse effects were observed at the highest dose level tested.

Oral 90-day repeated dose toxicity study in mice (2003):

This study is considered to be a supporting study since it was performed similar to OECD 408 and in the mouse. The test substance was administered continuously via the dietary administration to groups of C57BL/6 J@ Ico mice (10/sex/group) at concentrations of 700, 2 500 and 7 000 ppm (equivalent to approximately 115, 411, and 1251 mg/kg bw/day in males and 150, 559, and 1564 mg/kg bw/day in females, respectively) for at least 90 days. A similarly constituted group of 10 males and 10 females received untreated diet and acted as a control.

The test substance dietary administration for 90 days in C57BL/6 mice at dose level of 7 000, 2 500 and 700 ppm induced no treatment-related mortality.

At 7 000 ppm, treatment-related clinical signs consisted of coloured urine (intense yellow) observed from Day 6 to the end of the study in both sexes. A slight body weight loss (up to 4%) was observed during the first week of treatment in males and during the first two weeks in females of the 7000 ppm group. The rest of the treatment period, body weight changes were similar to the control values. As a consequence of the initial body weight loss, mean body weight was reduced throughout the study when compared to control values (up to -5% in males and -8% in females). The difference was statistically significant for half of the recorded Days throughout the treatment period. Food consumption was unaffected by treatment. As the reduction was less than 10%, these effects are considered to be not adverse. The clinical chemistry evaluation revealed a statistically significant decrease in mean total cholesterol concentration in males (-33%) and in mean alkaline phosphatase activity in females (-28%), when compared to control values. Since these effects were the only significant clinical chemistry changes observed and no correlated histopathological effects were seen, these findings were not considered adverse. At terminal sacrifice, mean terminal body weight was significantly lower in males (-6%) when compared to control values. Mean absolute and relative liver weights were significantly higher in females (+29 and +29%, respectively), when compared to control values. The histopathological examination revealed treatment-related changes in the liver: a slight centrilobular hepatocellular hypertrophy associated with a slight tendency toward a decrease in incidence and severity of the centrilobular hepatocellular vacuolation was observed in both sexes. These liver effects are considered as an adaptive response to the treatment and are therefore treatment-related, but not adverse effects.

At 2 500 and 700 ppm, treatment-related clinical signs consisted of coloured urine (intense yellow) observed from Day 6 to the end of the study in both sexes. No other treatment-related effects were observed. This effect is not considered to be adverse as it does not have any clinical importance.

In conclusion, since all found effects are not considered to be adverse the No Observed Adverse Effect Level (NOAEL) of the test substance is 7000 ppm in males and females (approximately 1251 and 1564 mg/kg body weight/day, respectively).

Chronic

Combined chronic toxicity (24-month) and carcinogenicity study in the rat

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.

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 lexicologically 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.

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.

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, due to adverse effects on the pancreas and thyroid gland in males and females the NOAEL over a 24-month period of dietary administration with the test substance was 2 ppm (equivalent to 0.08 and 0.11 mg/kg bw/day in males and females, respectively).

Taking together all results from oral repeated dose toxicity studies, the chronic toxicity study had the lowest NOAEL, based on with adverse effect on the pancreas and thyroid gland in rats. Therefore, the NOAEL of 0.08 mg/kg bw/day found in male and female rats, is used for classification and risk assessment.

Dermal:

A 28-day dermal toxicity study according to OECD GL 410 was performed with the test substance in rats (2013). Each experimental group consisted of 10 Wistar rats/sex. The test substance was applied to the rat skin (shaved flank) and covered with a semi-occluded application at doses of 10, 100 and 1000 mg/kg bw/day, for 6 h. The test substance was held in place using special jackets. The test substance was gently wiped from the skin with lukewarm water after the 6-hour exposure period to prevent ingestion. Control animals were similarly treated with gauze patches and restrainers (Lomir jackets with inserts).

There was no unscheduled mortality during the study, and no clinical signs or skin irritation related to test substance administration were observed. No test substance-related effects were noted during the ophthalmoscopic examination or during the functional observation battery tests, landing food splay test or grip strength evaluation. There were no test substance-related or adverse effects on the animal body weights, body weight gain or animal food consumption during the study as well as no adverse effects on of the test substance on the haematology or urinalysis. Evaluation of the vaginal smears prior to necropsy showed the expected distribution of the oestrous cycle phases within the normal population of female Wistar rats. Changes that were considered to be related to test substance treatment were noted in the clinical chemistry parameters. Albumin (Alb.) concentration was slightly higher than control in all male dose groups, reaching statistical significance (p<0.01). Consequently Total protein (Tot.Prot) and the Albumin to Globulin ratio (A/G) were also higher, reaching statistical significance (p<0.05 or p<0.01). However, there were no histopathological or other changes associated with these differences at any dose level. As all values in the 10 mg/kg/day group fell within the normal historical control range, these changes were considered to be non-adverse at this dose level. There were no test substance-related changes at dose levels of 10, 100 and 1000 mg/kg bw at necropsy.

Statistically significant differences in male liver (ca. 10%) and kidney (ca. 10%) organ weight at 1000 mg/kg bw/day were noted when adjusted to body weight or brain weight. These differences could be related to treatment, as is indicated in the microscopic findings. There were no biologically significant differences among groups in the weights of other organs measured, related to body weight or brain (compared to control). Test substance-related microscopic findings were noted in the liver, kidneys and pancreas. The microscopic findings in the liver and kidneys were in correlation with organ weight changes in the males. In the liver, the centrilobular hepatocellular hypertrophy was only observed in males dosed at 1000 mg/kg bw/day. Numerous findings were seen in the kidneys including increased presence of eosinophilic droplets in the cortical/corticomedullary tubules, tubular proteinaceous casts, tubular basophilia or dilatation of renal pelvis. The changes seen were considered to represent an increased incidence of common background features. However, the casts were accompanied with minimal tubular degeneration in one male at the High dose. These renal changes were ascribed to treatment with test substance only in High dose males. Casts were also present in one High dose female but the incidence and severity could be within the normal range, so the observation cannot be unequivocally ascribed to treatment. An additional histopathological evaluation of the animals dosed at 100 mg/kg bw/day did not show any microscopic changes in the females. In the males, tubular cast was only present in one rat without accompanied alterations; therefore this change was not ascribed to treatment with the test substance. There were no significant differences between treated males and females in treatment-related findings of the pancreas. Increased acinar zymogen granules, necrosis of acinar cells or mixed cell inflammation were microscopically visualized. The severity of these lesions was decreased when comparing 1000 mg/kg bw/day-group (minimal to moderate) versus 100 mg/kg bw/day-group (minimal to mild). No test substance-related microscopic findings in the pancreas were recorded in rats dosed at 10 mg/kg bw/day.

Due to the dose-dependent effects found in pancreas at 100 and 1000 mg/kg bw/day in males and females, a dose level of 10 mg/kg bw/day was considered to be the overall NOAEL for both males and females. No skin irritation was observed up to the highest dose tested, therefore a NOAEL local of 1000 mg/kg bw/day (equivalent to 8 and 5.16 mg/cm²/day for males and females, respectively, based on a generic modification recommended by REACH Guidance R.8, p.112 as follows: NOAELtest*mean weight/44,5 = NOAELmodified where 44.5cm² is 10% of an average total body surface of a rat) was considered.

Justification for classification or non-classification

The available data on oral repeated dose toxicity with 2-{2-chloro-4-mesyl-3-[(tetrahydrofuran-2-ylmethoxy)methyl]benzoyl}cyclohexane-1,3-dione (CAS 473278-76-1) meets the criteria for classification according to Regulation (EC) No 1272/2008, and has therefore to be classified as STOT RE Cat. 1 with pancreas and thyroid gland as target organs.

The available data on dermal repeated dose toxicity with the test substance meets the criteria for classification according to Regulation (EC) No 1272/2008 and therefore has to be classified as STOT RE Cat. 2 with pancreas as target organ.