Registration Dossier

Administrative data

Description of key information

NOAEL = 1000 mg/kg bw/day, i.e. 1005 or 980 mg/kg bw/day for males or females, respectively (OECD 407, GLP, K, rel.1)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included in Iuclid Section 13.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that the source and target substances have similar physico-chemical and toxicological properties because of their structural similarity.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target and source substances both contain the same two isomers, in a different ratio: the target substance is a multi-constituent, while the source substance is defined as a mono-constituent, the second constituent being an impurity.

3. ANALOGUE APPROACH JUSTIFICATION
The source substance and the target substance are isomers. Based on acute toxicity data, the source substance is expected to be a worst-case compared to the target substance considering the acute toxicological property.
The study design (OECD 407, GLP) is adequate and reliable for the purpose of the prediction based on read-across. The test material used represents the source substance as described in the hypothesis in terms of purity and impurities. The results of the studies are adequate for the purpose of classification and labelling.
Therefore, based on the considerations above, it can be concluded that the results of the sub-acute toxicity studies conducted in both the rat and the mouse with the source substance are likely to predict the properties of the target substance and are considered as adequate to fulfil the information requirement of Annex VIII, 8.6.1.

4. DATA MATRIX
Cf. Iuclid Section 13. 
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Other examinations:
None
Clinical signs:
no effects observed
Description (incidence and severity):
From Day 22 to the end of the study, pale faeces were noted for all groups treated with the test item and this continued throughout the remaining dosing period. Similar observations were also noted for Control animals from Day 26 of the study.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths in the study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Males given 1000 mg/kg/day showed moderate to marked reductions in weekly body weight gains with a terminal mean body weight 17 % lower than Controls (p<0.001). Group mean weekly body weight gains for males receiving 100 or 300 mg/kg/day were also slightly lower than Controls resulting in 8 % lower terminal body weights than Controls (p<0.05). Females given 1000 mg/kg/day had slightly lower overall mean weight gain which resulted in their terminal body weight being 7 % lower than Control (NS). There was no effect of treatment with the test item on body weight for females receiving 100 or 300 mg/kg/day.
=> The retardation of weight gain, most apparent in males given 1000 mg/kg/day, was linked to reduced food intake most notably in the first week of treatment, which is suggestive of unpalatability of the treatment diet. This secondary effect on body weight was therefore considered to be nonadverse.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Over the first week of the study, males given 1000 mg/kg/day showed a sharp decline in food intake (29 % lower than Controls), suggestive of possible unpalatability of the treatment diet. Thereafter, food consumption for these males improved but remained lower (9 % to 23 % lower) than Controls. Lesser reductions (up to 15 % vs. Controls) with no dose relationship were shown by males given 100 or 300 mg/kg/day. Females given 1000 mg/kg/day had a spuriously high apparent food intake over the first week (excluded from the data) which made any confirmation of unpalatability in females impossible. In remaining comparisons, where reductions occurred they were less than 14 % relative to Controls.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At all dose levels, males given the test item showed a dose-related lowering of total leucocyte counts, which was mainly due to decreases in absolute lymphocyte counts; reductions in both counts were statistically significant at 300 or 1000 mg/kg/day (p<0.05). Total leucocyte and lymphocyte counts in treated females were also lower than Controls but the only statistically significant reduction for leucocyte counts was seen in females given 1000 mg/kg/day (p<0.05).
=> The changes from Controls were only moderate with individual values largely within the background Control data ranges and this finding may have been linked to the reduced food intake for these animals.
Males receiving the test item also showed an apparent dose-related lowering in reticulocytes which achieved statistical significance at 300 and 1000 mg/kg/day (p<0.05). No such findings were apparent in females and, as individual values were within the background Control data ranges, this finding was considered not to be toxicologically significant.
Any other isolated statistically significant inter-group differences were considered not to be treatment-related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
At all dose levels, males given the test item showed statistically significantly higher levels of plasma creatinine than Controls (p<0.001). Most individual values exceeded the expected upper limit but in the absence of any changes in urea or electrolyte levels or any histopathology findings, this finding was considered not to be of toxicological significance.
Statistically significantly higher mean plasma concentrations of bile acids were observed in animals of both sexes given 1000 mg/kg/day and males receiving 300 mg/kg/day in comparison with the respective Controls; individual animals in most groups showed noticeably higher values than the rest of the group. These findings might be due to the fact that these animals were not fasted prior to blood sampling.
Any other findings were either isolated, not in the expected direction for toxicological significance or in the opposite direction for the sexes, and therefore, were considered not to be related to treatment with the test item.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
There were no clear effects of treatment observed in standard arena observations conducted throughout the study or a functional observation battery. Males given 300 or 1000 mg/kg/day showed statistically significantly higher hind limb strength relative to Controls but because of high inter-animal variability and the absence of a dose-relationship this was considered unlikely to be treatment-related especially as any concern would be for a finding of a reduced grip strength.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Both sexes given 300 or 1000 mg/kg/day showed statistically significant increases in body weight-related liver weights relative to Controls. These increases were dose related and greater in males than in females with the largest increase being 22 % seen in males given 1000 mg/kg/day. Group mean body weight-related kidney weights for the treated males were also statistically significantly higher (7 % to 17 %) than Controls, but there was no such increase in females.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related macroscopic findings in rats given test item. The spectrum of incidental macroscopic findings was consistent with those normally encountered in rats of this age kept under laboratory conditions.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Histopathology was conducted on tissues from Controls and high dose animals only and treatment-related microscopic findings were seen in the livers of males and females given test item at 1000 mg/kg/day and in the kidneys and thyroid glands of males given test item at 1000 mg/kg/day.
Centrilobular hepatocyte hypertrophy (minimal) was found in some males and most females given test item at 1000 mg/kg/day. Increased hyaline droplets in the proximal tubules were found in all males given test item at 1000 mg/kg/day and there was also an increased incidence/severity of basophilic cortical tubules in males from this treatment group. These findings were not observed in the kidneys in females. Treatment-related minimal follicular epithelial hypertrophy was present in 2 males given test item at 1000 mg/kg/day but not in females.
The spectrum of other microscopic findings was consistent with those normally encountered in rats of this age kept under laboratory conditions.
=> At 300 or 1000 mg/kg/day, both sexes showed statistically significantly higher bodyweight-related liver weights relative to Controls. At 1000 mg/kg/day, the centrilobular hepatocyte hypertrophy found in the liver was considered to be an adaptive change indicative of hepatic enzyme induction which would account for the increase in body weight-related liver weights. Follicular epithelial hypertrophy in the thyroid, a rodent specific finding, seen in males given
1000 mg/kg/day is also associated with hepatic enzyme induction, secondary to increased metabolism of thyroid hormones as a result of induction of their metabolic pathways in hepatocytes.
Males given 1000 mg/kg/day also had statistically significantly higher body weight-related kidney weights than Controls. The histopathological changes seen in the male kidney at this dose level were considered to be typical of light hydrocarbon nephropathy (α2u-globulin nephropathy) in male rats. α2u-globulin nephropathy, a syndrome unique to male rats (and as such not relevant to human), is characterised by the accumulation of α2u-containing protein.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
1 005 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no adverse effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
980 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no adverse effects observed
Key result
Critical effects observed:
no

None

Conclusions:
Under the test conditions, the NOAEL of the source substance (and by analogy of the target substance) should be regarded as the nominal dose level of 1000 mg/kg bw/day, i.e 1005 or 980 mg/kg bw/day for males or females, respectively.
Executive summary:

In a sub-acute toxicity study performed according to the OECD test guideline No. 407 and in compliance with GLP, test item was administered by dietary admixture to Crl:WI(Han) rats (5/sex/group) at 100, 300 and 1000 mg/kg bw/day for four weeks (nominal), corresponding to achieved doses of 97, 299 and 1005 mg/kg bw/day for males and to 105, 296 and 980 mg/kg bw/day for females. A control group received the untreated diet (LabDiet 5KB3 EURodent Maintenance Diet Meal).

The animals were checked twice daily for mortality and morbidity and daily for clinical signs of toxicity. Neurotoxicity was assessed by a functional observation battery which was performed on all animals in the final week of the treatment period. Body weight was recorded before the beginning of the study and then once a week, food consumption was recorded once a week during the study. The achieved dosages were calculated. Haematological and blood biochemical parameters were determined on Day 29. At scheduled necropsy, the animals were sacrificed; designated organs and tissues were weighed and preserved. A macroscopic post-mortem examination was performed on all animals. A microscopic examination was carried out for animals of the control and high-dose groups.

There were no deaths or treatment-related clinical signs at any dose level. Males given the test item at all dose levels and females given 1000 mg/kg bw/day showed slight to moderate decreases in group mean body weights, which were associated with slight reductions in food consumption that were more apparent in males. A reduction of 17 % in terminal body weight for males receiving 1000 mg/kg bw/day was considered to be a secondary response due to unpalatability of the diet causing reduced food intake and hence was considered not to be adverse.

At a dose level of 300 or 1000 mg/kg bw/day, males and females showed statistically significant increases in body weight-related liver weights when compared with the respective Controls. Centrilobular hepatocyte hypertrophy seen in the livers of both sexes given test item at 1000 mg/kg bw/day was considered to be an adaptive change, indicative of hepatic enzyme induction. Follicular epithelial hypertrophy seen in the thyroid glands of males given the same dose level was also considered to be associated with hepatic enzyme induction.

Group mean body weight-related kidney weights for the treated males were also statistically significantly higher than Controls. Histopathology findings in male kidneys included slightly increased hyaline droplets in the proximal tubules and increased incidence/severity of basophilic cortical tubules. These findings were considered to be typical of α2u-globulin nephropathy, a syndrome unique to male rats and as such not relevant to human and therefore it was considered not to be adverse.

Therefore, under the test conditions, the NOAEL of the source substance (and by analogy of the target substance) should be regarded as the nominal dose level of 1000 mg/kg bw/day, i.e 1005 or 980 mg/kg bw/day for males or females, respectively.

This sub-acute toxicity study is acceptable and satisfies the requirement for repeated dose toxicity endpoint. The source and the target substances only differ in the ratio of the isomers, therefore the analogue approach is justified (see §13 for read-across justification).

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 15 February to 22 May 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD test guideline No. 407 and in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Limit test:
yes
Species:
rat
Strain:
other: Crl:WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, CT9 4LT, England.
- Age at study initiation: 5 to 6 weeks.
- Weight at study initiation: Males: 162-196g; Females: 135-163g
- Housing: in groups of 5, in grid-floor cages suspended over paper-lined trays.
- Diet (e.g. ad libitum): powdered diet, LabDiet 5KB3 EURodent Maintenance Diet Meal (manufactured by PMI Nutrition International) supplied by International Product Supplies, ad libitum.
- Water (e.g. ad libitum): main tap water ad libitum.
- Acclimation period: from 8 days before the start of the treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C to 23°C (target range 19 to 23°C).
- Humidity (%): 34% to 57 (target range 55 % ± 15 %)
- Air changes (per hr): not reported.
- Photoperiod (hrs dark / hrs light): 12/12.
Route of administration:
oral: feed
Details on route of administration:
The rat is a suitable rodent species for toxicity testing, acceptable to regulatory authorities and for which extensive background data are available.
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with: LabDiet 5KB3 EURodent Maintenance Diet Meal (manufactured by PMI Nutrition International) supplied by International Product Supplies.
The concentrations of the test item in the diet will be adjusted according to body weight and food consumption to achieve the required dose levels. The following calculation will be used:
Concentration required (parts per million) = dose level (mg/kg/day) x Estimated mid-week body weight (g) / Estimated daily food intake (g).
Initially, a premix will be made at a fixed concentration. This premix will be mixed for at least 15 minutes in a double cone rotary mixer unless the premix is less than 1.5 kg when it will be mixed for at least 5 minutes using a desktop laboratory mixer. For each dietary concentration, a weighed amount of the premix will be mixed with the required amount of diet for at least 15 minutes using a double cone rotary mixer unless it is less than 1.5 kg when it may be mixed for at least 5 minutes using a desktop laboratory mixer.
- Storage temperature of food: no data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples from diets, including the Control and premix, used during Week 1 were analysed to assess homogeneity (where applicable) and achieved concentration. Samples were analysed using a GC-FID validated method DFATM-0006-01/C.
Dietary formulations containing the test item, at concentrations between 500 ppm and 20000 ppm, have been shown to be stable for up to 8 days when stored under ambient conditions.
Test item formulations used to dose animals during Week 1 of the study were considered accurate and homogeneous. The average measured concentrations of test item ranged from 91 % to 97 % with coefficients of variation ranging between 2 % to 5 % which fulfilled the acceptance criteria (± 20 % and ≤ 15 % for accuracy and homogeneity, respectively).
Duration of treatment / exposure:
28 days
Frequency of treatment:
Continuous
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Acutal: 97 (M) / 107 (F) mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Acutal: 299 (M) / 296 (F) mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Acutal: 1005 (M) / 980 (F) mg/kg bw/day
No. of animals per sex per dose:
5
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The dose levels were selected in consultation with the Sponsor, based on the results of a dose range finding study in the rat (Sequani Study Number: FCH0026).
- Rationale for animal assignment: random.
Positive control:
No
Observations and examinations performed and frequency:
MORTALITY / MORBIDITY: Yes
- Time schedule: twice daily

CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: on Days -1, 7, 14, 21, 28 and on the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE: YES
- amount of food consumed by each cage of animals recorded weekly during the treatment period.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on Day 29 of the study.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: No
- How many animals: all
- Parameters checked in table 7.5.1/1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on Day 29 of the study.
- Animals fasted: No
- How many animals: all
- Parameters checked in table 7.5.1/1 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: On one occasion in the final week of the treatment period.
- Dose groups that were examined: all animals.
- Battery of functions tested: sensorimotor responses to visual, acoustic, tactile or proprioceptive stimuli, grip strength and motor activity.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 7.5.1/2)
HISTOPATHOLOGY: Yes (see table 7.5.1/2)
Other examinations:
None
Statistics:
Depending on the nature of the data set to be analysed, appropriate tests will be applied as indicated in table 7.5.1/3. Where parametric tests may be appropriate they will be preceded by a check for homogeneity of variance using the Levene test and, where available, the Shapiro-Wilks test for normality. If either of these two assumptions fails a log transformation will be applied before retesting. If the transformation fails, appropriate nonparametric tests will be applied.
Clinical signs:
no effects observed
Description (incidence and severity):
From Day 22 to the end of the study, pale faeces were noted for all groups treated with the test item and this continued throughout the remaining dosing period. Similar observations were also noted for Control animals from Day 26 of the study.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths in the study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Males given 1000 mg/kg/day showed moderate to marked reductions in weekly body weight gains with a terminal mean body weight 17 % lower than Controls (p<0.001). Group mean weekly body weight gains for males receiving 100 or 300 mg/kg/day were also slightly lower than Controls resulting in 8 % lower terminal body weights than Controls (p<0.05). Females given 1000 mg/kg/day had slightly lower overall mean weight gain which resulted in their terminal body weight being 7 % lower than Control (NS). There was no effect of treatment with the test item on body weight for females receiving 100 or 300 mg/kg/day.
=> The retardation of weight gain, most apparent in males given 1000 mg/kg/day, was linked to reduced food intake most notably in the first week of treatment, which is suggestive of unpalatability of the treatment diet. This secondary effect on body weight was therefore considered to be nonadverse.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Over the first week of the study, males given 1000 mg/kg/day showed a sharp decline in food intake (29 % lower than Controls), suggestive of possible unpalatability of the treatment diet. Thereafter, food consumption for these males improved but remained lower (9 % to 23 % lower) than Controls. Lesser reductions (up to 15 % vs. Controls) with no dose relationship were shown by males given 100 or 300 mg/kg/day. Females given 1000 mg/kg/day had a spuriously high apparent food intake over the first week (excluded from the data) which made any confirmation of unpalatability in females impossible. In remaining comparisons, where reductions occurred they were less than 14 % relative to Controls.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At all dose levels, males given the test item showed a dose-related lowering of total leucocyte counts, which was mainly due to decreases in absolute lymphocyte counts; reductions in both counts were statistically significant at 300 or 1000 mg/kg/day (p<0.05). Total leucocyte and lymphocyte counts in treated females were also lower than Controls but the only statistically significant reduction for leucocyte counts was seen in females given 1000 mg/kg/day (p<0.05).
=> The changes from Controls were only moderate with individual values largely within the background Control data ranges and this finding may have been linked to the reduced food intake for these animals.
Males receiving the test item also showed an apparent dose-related lowering in reticulocytes which achieved statistical significance at 300 and 1000 mg/kg/day (p<0.05). No such findings were apparent in females and, as individual values were within the background Control data ranges, this finding was considered not to be toxicologically significant.
Any other isolated statistically significant inter-group differences were considered not to be treatment-related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
At all dose levels, males given the test item showed statistically significantly higher levels of plasma creatinine than Controls (p<0.001). Most individual values exceeded the expected upper limit but in the absence of any changes in urea or electrolyte levels or any histopathology findings, this finding was considered not to be of toxicological significance.
Statistically significantly higher mean plasma concentrations of bile acids were observed in animals of both sexes given 1000 mg/kg/day and males receiving 300 mg/kg/day in comparison with the respective Controls; individual animals in most groups showed noticeably higher values than the rest of the group. These findings might be due to the fact that these animals were not fasted prior to blood sampling.
Any other findings were either isolated, not in the expected direction for toxicological significance or in the opposite direction for the sexes, and therefore, were considered not to be related to treatment with the test item.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
There were no clear effects of treatment observed in standard arena observations conducted throughout the study or a functional observation battery. Males given 300 or 1000 mg/kg/day showed statistically significantly higher hind limb strength relative to Controls but because of high inter-animal variability and the absence of a dose-relationship this was considered unlikely to be treatment-related especially as any concern would be for a finding of a reduced grip strength.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Both sexes given 300 or 1000 mg/kg/day showed statistically significant increases in body weight-related liver weights relative to Controls. These increases were dose related and greater in males than in females with the largest increase being 22 % seen in males given 1000 mg/kg/day. Group mean body weight-related kidney weights for the treated males were also statistically significantly higher (7 % to 17 %) than Controls, but there was no such increase in females.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related macroscopic findings in rats given test item. The spectrum of incidental macroscopic findings was consistent with those normally encountered in rats of this age kept under laboratory conditions.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Histopathology was conducted on tissues from Controls and high dose animals only and treatment-related microscopic findings were seen in the livers of males and females given test item at 1000 mg/kg/day and in the kidneys and thyroid glands of males given test item at 1000 mg/kg/day.
Centrilobular hepatocyte hypertrophy (minimal) was found in some males and most females given test item at 1000 mg/kg/day. Increased hyaline droplets in the proximal tubules were found in all males given test item at 1000 mg/kg/day and there was also an increased incidence/severity of basophilic cortical tubules in males from this treatment group. These findings were not observed in the kidneys in females. Treatment-related minimal follicular epithelial hypertrophy was present in 2 males given test item at 1000 mg/kg/day but not in females.
The spectrum of other microscopic findings was consistent with those normally encountered in rats of this age kept under laboratory conditions.
=> At 300 or 1000 mg/kg/day, both sexes showed statistically significantly higher bodyweight-related liver weights relative to Controls. At 1000 mg/kg/day, the centrilobular hepatocyte hypertrophy found in the liver was considered to be an adaptive change indicative of hepatic enzyme induction which would account for the increase in body weight-related liver weights. Follicular epithelial hypertrophy in the thyroid, a rodent specific finding, seen in males given
1000 mg/kg/day is also associated with hepatic enzyme induction, secondary to increased metabolism of thyroid hormones as a result of induction of their metabolic pathways in hepatocytes.
Males given 1000 mg/kg/day also had statistically significantly higher body weight-related kidney weights than Controls. The histopathological changes seen in the male kidney at this dose level were considered to be typical of light hydrocarbon nephropathy (α2u-globulin nephropathy) in male rats. α2u-globulin nephropathy, a syndrome unique to male rats (and as such not relevant to human), is characterised by the accumulation of α2u-containing protein.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
1 005 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no adverse effect observed
Key result
Dose descriptor:
NOAEL
Effect level:
980 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no adverse effect observed
Key result
Critical effects observed:
no

None

Conclusions:
Under the test conditions, the NOAEL should be regarded as the nominal dose level of 1000 mg/kg bw/day, i.e 1005 or 980 mg/kg bw/day for males or females, respectively.
Executive summary:

In a sub-acute toxicity study performed according to the OECD test guideline No. 407 and in compliance with GLP, test item was administered by dietary admixture to Crl:WI(Han) rats (5/sex/group) at 100, 300 and 1000 mg/kg bw/day for four weeks (nominal), corresponding to achieved doses of 97, 299 and 1005 mg/kg bw/day for males and to 105, 296 and 980 mg/kg bw/day for females. A control group received the untreated diet (LabDiet 5KB3 EURodent Maintenance Diet Meal).

The animals were checked twice daily for mortality and morbidity and daily for clinical signs of toxicity. Neurotoxicity was assessed by a functional observation battery which was performed on all animals in the final week of the treatment period. Body weight was recorded before the beginning of the study and then once a week, food consumption was recorded once a week during the study. The achieved dosages were calculated. Haematological and blood biochemical parameters were determined on Day 29. At scheduled necropsy, the animals were sacrificed; designated organs and tissues were weighed and preserved. A macroscopic post-mortem examination was performed on all animals. A microscopic examination was carried out for animals of the control and high-dose groups.

There were no deaths or treatment-related clinical signs at any dose level. Males given the test item at all dose levels and females given 1000 mg/kg bw/day showed slight to moderate decreases in group mean body weights, which were associated with slight reductions in food consumption that were more apparent in males. A reduction of 17 % in terminal body weight for males receiving 1000 mg/kg bw/day was considered to be a secondary response due to unpalatability of the diet causing reduced food intake and hence was considered not to be adverse.

At a dose level of 300 or 1000 mg/kg bw/day, males and females showed statistically significant increases in body weight-related liver weights when compared with the respective Controls. Centrilobular hepatocyte hypertrophy seen in the livers of both sexes given test item at 1000 mg/kg bw/day was considered to be an adaptive change, indicative of hepatic enzyme induction. Follicular epithelial hypertrophy seen in the thyroid glands of males given the same dose level was also considered to be associated with hepatic enzyme induction.

Group mean body weight-related kidney weights for the treated males were also statistically significantly higher than Controls. Histopathology findings in male kidneys included slightly increased hyaline droplets in the proximal tubules and increased incidence/severity of basophilic cortical tubules. These findings were considered to be typical of α2u-globulin nephropathy, a syndrome unique to male rats and as such not relevant to human and therefore it was considered not to be adverse.

Therefore, under the test conditions, the NOAEL should be regarded as the nominal dose level of 1000 mg/kg bw/day, i.e 1005 or 980 mg/kg bw/day for males or females, respectively.

This sub-acute toxicity study is acceptable and satisfies the requirement for repeated dose toxicity endpoint.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
980 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The key study is GLP-compliant and of high quality (Klimisch score = 2).

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:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In a sub-acute toxicity study performed according to the OECD test guideline No. 407 and in compliance with GLP (Sequani, 2014), 4-tert-butylcyclohexyl acetate diluted in corn oil was administered via gavage to Crl:WI(Han) rats (5/sex/group) at 0, 300, 500 and 1000/750 mg/kg bw/day for four weeks (nominal). Satellite animals (5/sex) were included in the control and high-dose group to assess the reversibility of any observed effects over a 14 day treatment-free period.

The oral (gavage) administration of 4-tert-butylcyclohexyl acetate was not particularly well tolerated with six animals either dying or being sent to necropsy during the treatment period. These deaths appeared to be the result of the tremors and convulsions that occurred primarily in females. These transient effects were noted clinically as increased activity, twitching and convulsions. In general, the prevalence of these findings increased with study duration and were eventually seen at all dose levels tested. At the low dose (300 mg/kg bw/day) only very minor effects were seen early in the study but began to increase after about Day 22. There were no effects seen in the functional observation battery and no histopathological changes seen in the nervous tissue which provides more evidence that the test item only caused transient effects and no permanent damage to the nervous tissues.

Mechanistically, hydrolysis of the acetate form of the test item to the alcohol seems a plausible cause of the toxicity seen in this study; this is consistent with the timing of the observations because rats have quite low stomach pH and so a degree of hydrolysis might be expected immediately after dosing. The later effects could correlate with absorbed test item being subject to the activity of liver hydrolases. These enzymes are certainly inducible, which may account for the increased effect with prolonged treatment and also correlate with the increased liver weights seen in the study. The presence of follicular epithelial hypertrophy in the thyroid and the increase in liver weight are indicative of the presence of induction of hepatic enzymes.

The clinical observations appear to be Cmax effects (occurring at the time of peak systemic exposure) and a function of the gavage administration used in the study. Given the likely method of human exposure the relevance of any Cmax effect is questionable in terms of normal human exposure. It was therefore decided to conduct another study, using dietary route of exposure, on the analogue substance, cis-4-tert-butylcyclohexyl acetate (>80% cis isomer, up to 20% trans isomer).

None of the previously reported adverse effects were observed in the dietary study, confirming the hypothesis of Cmax effect. Such bolus oral exposure is not considered relevant to longer-term or chronic exposure scenarios in humans. Therefore the results of Sequani were not used as the basis for classification.

The dietary study of Sequani (2013) conducted on cis-4-tert-butylcyclohexyl acetate was therefore selected as the key study. In this sub-acute toxicity study performed according to the OECD test guideline No. 407 and in compliance with GLP, cis-4 -tert-butylcyclohexyl acetate was administered by dietary admixture to Crl:WI(Han) rats (5/sex/group) at 100, 300 and 1000 mg/kg bw/day for four weeks (nominal). A control group received the untreated diet (LabDiet 5KB3 EURodent Maintenance Diet Meal).

Body weight was recorded before the beginning of the study and then once a week, food consumption was recorded once a week during the study. The achieved dosages were calculated. Haematological and blood biochemical parameters were determined on Day 29. At scheduled necropsy, the animals were sacrificed; designated organs and tissues were weighed and preserved. A macroscopic post-mortem examination was performed on all animals. A microscopic examination was carried out for animals of the control and high-dose groups. There were no deaths or treatment-related clinical signs at any dose level. Males given the test item at all dose levels and females given 1000 mg/kg bw/day showed slight to moderate decreases in group mean body weights, which were associated with slight reductions in food consumption that were more apparent in males. A reduction of 17 % in terminal body weight for males receiving 1000 mg/kg bw/day was considered to be a secondary response due to unpalatability of the diet causing reduced food intake and hence was considered not to be adverse. At a dose level of 300 or 1000 mg/kg bw/day, males and females showed statistically significant increases in body weight-related liver weights when compared with the respective Controls. Centrilobular hepatocyte hypertrophy seen in the livers of both sexes given the analogue at 1000 mg/kg bw/day was considered to be an adaptive change, indicative of hepatic enzyme induction. Follicular epithelial hypertrophy seen in the thyroid glands of males given the same dose level was also considered to be associated with hepatic enzyme induction. Group mean body weight-related kidney weights for the treated males were also statistically significantly higher than Controls. Histopathology findings in male kidneys included slightly increased hyaline droplets in the proximal tubules and increased incidence/severity of basophilic cortical tubules. These findings were considered to be typical of α2u-globulin nephropathy, a syndrome unique to male rats and as such not relevant to human and therefore it was considered not to be adverse. Therefore, under the test conditions, the NOAEL should be regarded as the nominal dose level of 1000 mg/kg bw/day, i.e 1005 or 980 mg/kg bw/day for males or females, respectively.

The source substance and the target substance being only different in their ratio of the isomers (both being present at more than 10%, i.e. above generic concentration limit triggering classification of a mixture as STOT-RE 1&2), the conclusion on cis-4 -tert-butylcyclohexyl acetate was extended to 4 -tert-butylcyclohexyl acetate. (see §13 for additional read-across justification)

Justification for classification or non-classification

Harmonised classification:

The substance has no harmonised classification according to the Regulation (EC) No. 1272/2008 (CLP).

Self-classification:

Based on the available information, no additional classification is proposed according to the CLP or GHS.