[3R-(3α,3aβ,6α,7β,8aα)]-octahydro-6-methoxy-3,6,8,8-tetramethyl-1H-3a,7-methanoazulene

Endpoint summary

Description of key information

Repeated dosetoxicity: NOAEL: ≥ 330 mg/kg bw/day corresponding to ≥ 6000 mg/kg diet (OECD TG 422)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 November 2015 to 21 December 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approximately 9 weeks (males) or 8 weeks (females) of age
- Weight at study initiation: Mean body weight one day before the start of treatment was 299 grams for males and 185 grams for females.
- Housing: The rats were housed in makrolon cages with a bedding of wood shavings (Lignocel, Rettenmaier & Söhne GmbH & Co, Rosenberg, Germany) and strips of paper (Enviro-dri, Shepherd Specialty Papers, Michigan, USA) and a wooden block (ABEDD, Vienna, Austria) as environmental enrichment. During the premating period the animals were housed four rats to a cage (separated by sex). For mating, one male and one female were housed together. Mated females were housed individually.
- Diet: the rats receive a cereal-based (closed formula) rodent diet (VRF1 (FG)) from a commercial supplier (SDS Special Diets Services, Witham, England)., ad libitum.
- Water: Tap-water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 45-65%
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

The diets were mixed in a mechanical blender. Fresh batches of the experimental diets were prepared on 29 October 2015 and on 27 November 2015) and stored in a freezer (<-18°C) in plastic bags in portions sufficient for three to four days. The diets were provided as a powder in stainless steel cans, covered by a perforated stainless steel plate to prevent spillage. The food in the cans was replaced twice per week with fresh portions from the freezer.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

From both batches of diets prepared in the study (29 October 2015 and on 27 November 2015) samples were taken and stored in a freezer (≤ -18°C). The content of the test substance at each dietary level was determined in the batches prepared on 29 October 2015 and 27 November 2015. The homogeneity (and content) of the test substance in the experimental diets was assessed in the first batch (29 October 2015), by analysing five samples (taken at different locations in the feed container) of each test diet in duplicate. One sample of the control diet was analysed. To demonstrate the stability of the test substance under experimental conditions, samples of the batch of test diets prepared on 29 October 2015 (low-dose diet, mid-dose diet and high-dose diet) were analyzed at t=0 and analyzed after storage in the animal room (in an open container) for 4 days, and after storage in a freezer (<-18 °C) for at least 5 weeks.

The concentration of the test substance in diet was determined using the following procedure: The test substance was extracted from diet using extraction solvent (150 μl undecane in 2.5 liter dichloromethane) under shaking and ultrasonic treatment. Subsequently the extracts were 50-fold diluted with extraction solvent and centrifuged. The method of analysis was Gas Chromatography – Mass Spectrometry (GC-MS). Quantification was obtained by comparison of the peak area of the test substance in the extracts with those in calibration samples containing known amounts of the test substance and internal standard (undecane).

Before analysis of study samples, the analytical method was validated by analyzing three spiked samples per dose level, to conform to the following criteria:
- Linearity: the correlation coefficient of the calibration curve should be greater than or equal to 0.996;
- Recovery: the mean recovery of the test substance from the diet should be between 85% and 115% at each of the dose levels of the study;
- Repeatability: the relative standard deviation in the percentage recovery, when the recovery test is performed three times at each of the dose levels to be used in the toxicity study, should be less than 10%.
With respect to specificity: the signal obtained for samples should be corrected for the signal obtained with blank samples in case the signal obtained for blank samples is ≥ 5% of the signal obtained for low-dose samples.

Duration of treatment / exposure:

Males 28 days. Females received substance during a premating period of 2 weeks and during mating (1 week), gestation and lactation until 4-6 days after delivery.

Frequency of treatment:

Continuously

Dose / conc.:

0 mg/kg diet

Dose / conc.:

1 500 mg/kg diet

Remarks:

Males: ca. 79 mg/kg bw/day; Females: ca. 99 mg/kg bw/day

Dose / conc.:

3 000 mg/kg diet

Remarks:

Males: ca. 166 mg/kg bw/day; Females: ca. 203 mg/kg bw/day

Dose / conc.:

6 000 mg/kg diet

Remarks:

Males: ca. 330 mg/kg bw/day; Females: ca. 406 mg/kg bw/day

No. of animals per sex per dose:

12

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dose levels were selected in consultation with the sponsor and are based on the results of a 2-weeks dose-range finding study with the test substance in rats.

Observations and examinations performed and frequency:

- General clinical observations: Each animal was observed daily in the morning hours by cage-side observations. On working days, all cages were checked again in the afternoon for dead or moribund animals to minimize loss of animals from the study. All abnormalities, signs of ill health or reactions to treatment were recorded.

- Neurobehavioral testing (FOB and spontaneous motor activity): In addition to the above daily general clinical observations, detailed clinical examinations outside the home cage were performed on all rats of all groups prior to the first exposure and then once weekly throughout the study. In the last week of the study the detailed clinical examinations were part of the Functional Observational Battery tests (FOB) in the animals concerned. Signs noted included but were not limited to changes in skin and fur, piloerection, changes in the eyes, gait (including posture), and presence of clonic or tonic movements, stereotypies and bizarre behaviour. In the week prior to sacrifice, FOB tests and spontaneous motor activity were performed in 5 males/group on day 24 of the study and in 5 females with a litter/group on PN day 4. For females both tests were performed after sacrifice of their pups on PN day 4. During neurobehavioural testing, the observer was unaware of the treatment of the animals.

- Body weight: Body weights of male and female animals were recorded shortly before the start of the treatment (to enable randomization) and at the start of the study (day 0). Subsequently males were weighed weekly until sacrifice. Females were weighed once per week during the premating and mating period. Mated females were weighed on days 0, 7, 14 and 20 during presumed gestation and on day 0 and 4 of lactation. The animals were weighed on their scheduled necropsy date in order to calculate the organ to body weight ratios.

- Food consumption: Food consumption was measured per cage for the same periods as the body weight are measured, except during the mating period when food intake was not registered. The results are expressed in g per animal per day. Food was refreshed twice weekly.

- Intake of test substance: The intake of the test substance per kg body weight per day was calculated from the nominal dietary concentrations, the food consumption and the body weight of each study phase period for males and females separately.

- Hematology: Hematology was conducted in samples collected prior to the end of the premating period (on day 13) in 5 rats/sex/group. The rats were fasted overnight (water was freely available). Blood was taken by orbita punction whilst under CO2/O2 anesthesia. EDTA was used as anticoagulant. In each sample the following determinations were carried out: Hemoglobin, packed cell volume, red blood cell count, reticulocytes, total white blood cell count, differential white blood cell counts (neutrophils, lymphocytes, eosinophils, basophils, monocytes), prothrombin time, thrombocyte count, mean corpuscular volume (MCV; calculated), mean corpuscular hemoglobin (MCV; calculated), mean corpuscular hemoglobin concentration (MCHC; calculated)

- Clinical chemistry: Clinical chemistry was conducted in the same rats as indicated above (hematology). Blood was collected in heparinized plastic tubes and plasma was prepared by centrifugation. In each plasma sample the following determinations were carried out: Alkaline phosphatase activity (ALP) , aspartate aminotransferase activity (ASAT), alanine aminotransferase activity (ALAT), gamma glutamyl transferase activity (GGT), total protein, albumin, ratio albumin to globulin (calculated), urea, creatinine, glucose (fasting), bilirubin (total), cholesterol (total), triglycerides, phospholipids, calcium (Ca), sodium (Na), potassium (K), chloride (Cl), inorganic phosphate (PO4).

Sacrifice and pathology:

- Gross necropsy and histology of parental animals: All male and female parent animals were sacrificed by exsanguination from the abdominal aorta whilst under CO2/O2 anesthesia at necropsy and then examined grossly for pathological changes. All males were sacrificed after the mating period on 2 December 2015 (after 28 days of treatment). The dams were sacrificed on day 4 of lactation or one day thereafter (those selected for FOB/motor activity testing. High-dose female 89 was sacrificed at day 6 of lactation. Pups were sacrificed by gradual CO2 anesthesia, followed by irreversible hypothermia. The dams and the various lots were sacrificed between 14 – 21 December 2015. Samples of the following tissues and organs of all parent animals were preserved in a neutral aqueous phosphate buffered 4% solution of formaldehyde; except for the testes and the epididymides which were preserved in Bouin's fixative. Organs with asterisk were weighed (paired organs together) as soon as possible after dissection to avoid drying. Testes*, epididymides*, prostate, all gross lesions, ovaries (after counting the corpora lutea), uterus (after counting of the implantation sites), seminal vesicles (with coagulating glands). In addition, the following organs of five parent animals/sex/group (males with the lowest identification numbers in each cage; females with a litter were selected) were preserved. Of all males the kidney was preserved. The organs with an asterisk were weighed (paired organs together) (five animals/sex/group) as soon as possible after dissection to avoid drying: Adrenals*, heart*, kidneys*, lungs, spleen*, stomach, thymus*, thyroid, urinary bladder, brain* (including sections of cerebrum, cerebellum, medulla/pons), bone marrow (femur), liver*, mesenterial and axillary lymph nodes, peripheral nerve (sciatic or tibial), small and large intestines (including Peyer’s patches), spinal cord (cervical, mid-thoracic, and lumbar), trachea.

Tissues for microscopic examination were embedded in paraffin wax, sectioned at 5 μm, and stained with hematoxylin and eosin, except for sections of the testes which were stained with PAS hematoxylin. Microscopic examination was performed on the preserved organs of all animals of the control (group 1) and high-dose group (group 4). The kidneys from all dose groups of all males were stained with hematoxylin and eosin and examined for alpha2-globulin accumulation using immunohistochemistry. Furthermore, of rats of all dose groups organs showing gross lesions were microscopically examined.

Other examinations:

- Epididymal sperm motility, count and morphology: At scheduled necropsy on 02 December 2015, epididymal sperm was derived from the left cauda epididymis of 5 males of each dose group. For this purpose, the cauda epididymis was dissected, weighed and then minced in M199 medium containing 0.5% Bovine Serum Albumin. Sperm motility and, after sonification and DNA-staining, the cauda epididymal sperm reserves (sperm count) were measured for the selected males, using the Hamilton Thorne Integrated Visual Optical System (IVOS). In addition, a smear of the sperm solution was prepared and stained for males of all groups, but only the smears of the control and the high-dose males were examined microscopically for morphology. No treatment-related changes were observed in the high-dose group, therefore the examination of the sperm morphology was not extended to the intermediate-dose groups.

- Testicular sperm count: At scheduled necropsy on 02 December 2015, the left testis of the same males as used for epididymal sperm analysis were placed on dry ice and subsequently stored in a freezer (<-70°C) for later determination of the number of homogenization-resistant spermatids. The testes to be analyzed was thawed just before further processing. Following removal of the tunica albuginea, the testicular parenchyma was weighed, minced and homogenized in Saline
Triton X-100 solution. Following DNA-staining, the homogenization-resistant sperm heads were enumerated using the IVOS. The daily sperm production was calculated. No treatment-related changes were observed in the high-dose group, therefore the examination of the sperm morphology was not extended to the intermediate-dose groups.

Statistics:

The data were analysed using appropriate methods. Tests were generally performed as two-sided tests with results taken as significant where the probability of the results is p<0.05 (*) or p<0.01 (**). Continuous data were subjected to the “Decision tree for continuous data” and dichotomous data were subjected to the “Decision tree for dichotomous data”

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

One male rat (number 18) in the control group had encrustations of the skin in the neck area(s) during the mating and post-mating period. One female (number 47) in the low-dose group had encrustations of the skin in the neck region from premating until gestation. These observations and common observations for this strain and therefore not considered to be treatment related. During the end of the gestation period and the start of the lactation period, a few rats in the low, mid and high dose had sparsely haired area(s) and one rat in the high dose had ventral encrustations during lactation (female number 89). This observation is common for this strain and therefore not considered to be treatment related. Around the time of delivery piloerection was observed in one female rat in the high dose and two in the mid dose. This was due to delivery and not considered related to the treatment.

Mortality:

no mortality observed

Description (incidence):

No mortality and morbidity was observed during premating, mating, post-mating (males) or gestation and lactation (females).

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

In females no changes were observed in body weight and body weight gain in any of the doses groups in the premating period, gestation period and lactation period. In males no changes were observed in the body weight in any of the doses groups in the premating period and in the post-mating period. In addition, no changes were observed in the total body weight gain during the pre-mating period. In males in the high-dose group a slight, but statistically significant decrease in body gain was observed from day 7 to day 14 in the premating period and in the post-mating period for males in the high-dose group. As this slight effect on body weight change did not result in effects on body weight, it is considered treatment-related but not an adverse effect

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

In males and females no changes were observed in food consumption in any of the dose groups.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

In male and female rats no changes were observed in red blood cells and coagulation parameters in any of the dose groups. Also no changes were observed in total and differential white blood cell counts.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In females in the high-dose a statistically significant decrease in bilirubin was observed. In females that received the low-dose ALP concentrations were statistically significantly decreased. In males of the low-dose group triglycerides concentration were statistically significantly increased. Because both statistically significant findings were not dose-dependent and observed in only one sex, these finding are considered chance findings. In males of the mid-dose group the concentration potassium was statistically significantly increased. Because this effect was not observed in the high-dose group and not in female rats, this finding is considered a change finding. In females in the high-dose group, phospholipids concentration and phosphate concentration were statistically significantly increased. Since these findings were slightly outside the historical control range, these effects were considered slight and not adverse. Male rats in the high-dose group had a statistically significant increase in creatinine concentrations, which might be related to the alpha2-microglobuline nephropathy in kidneys observed in this group.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Clinical observations, Functional Observation Battery (FOB) and motor activity assessment (MAA) did not indicate any neurotoxic potential of the test substance in rats.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No effects were observed on all organ weights of males and females of all groups. A statistically significantly decrease in absolute and relative thymus weight was observed in male rats in the high-dose group. The relative thymus weight was also statistically significantly decreased in males of the low-dose group. In absence of a dose-response relation this was considered a chance finding.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At necropsy no treatment related macroscopic changes were observed. The few gross changes observed represented background pathology in rats of this strain and age and occurred only incidentally or at random incidence in the different groups.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic evaluation revealed a treatment-related increase of accumulation of hyalin droplets in tubular epithelial cells in the outer cortex of the kidneys of 11/11 high-dose and 6/12 mid-dose males. This was accompanied by degenerative changes of the epithelial cells involved. In most cases these kidneys also showed scattered tubular dilatation in the corticomedullary area, containing granular material, representing accumulation of sloughed degenerated epithelial cells. Further, a treatment-related increased incidence of basophilic tubules was observed. The characteristics of the findings in the kidneys, along with the observation that they occurred in males only, were highly suggestive for alpha 2-microglobulin (alpha 2u-) nephropathy. To further specify these changes the kidneys were immunohistochemically stained with a monoclonal antibody against alpha 2-microglobulin. The kidneys of all males showed positive staining, which was expected, because alpha 2-microglobulins are normal for male rats. In general, the overall positivity (i.e. intensity of the immuno-staining) of the kidneys was not very convincingly higher in the treated groups when compared to the controls. However, detailed examination of the outer cortex revealed that the structures identified as hyalin droplets in the HE stained slides showed a positive reaction in the immunostaining indicated as ‘alpha 2u-positive droplets'. Unfortunately the kidneys of animal no.96 (high-dose group) was not properly fixed which precluded evaluation of the HE slide, though the immunostaining still demonstrated some positive reaction. The other organs and tissues did not reveal treatment related histopathological changes. The histopathological changes observed were about equally distributed amongst the different treatment groups or occurred in one or a few animals only. They are common findings in rats of this strain and age or occurred as individual chance findings. Therefore, they were not considered to be related to treatment.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

Fertility: In each group 12 females were placed with males for mating. Within one week, all females were mated. All females were pregnant (12 females for each dose group). There were no relevant differences in pre-coital time, male and female mating indices, male or female fertility indices and the duration of gestation between the groups. No effects were found on the mean number of corpora lutea and implantation sites between the group. Pre-implantation loss was not affected by the treatment.
Reproductive performance: All pregnant rats survived until scheduled necropsy. The mean number of live born pups was comparable in all groups. No litters in control, low- mid- high-dose groups contained still born pups. Prenatal loss was comparable in all groups.
Epididymal sperm motility, count and morphology: No treatment related effects were observed on epididymal sperm motility, epididymal sperm count, and epididymal sperm morphology.
Testicular sperm count: No treatment related effect was observed in the testicular sperm count between all groups.

Details on results:

Test substance analysis:
- The concentration of the test substance was close to intended (85-115%) for the diets prepared on 29 October 2015. The measured concentrations in the diets prepared on 27 November 2015 were slightly lower than intended (-11%, -22%, -19% as compared to the nominal concentration for the low-, mid-, and high-dose respectively).
- The test substance was considered to be homogeneously distributed in the diets.
- After storage in an open container in the animal room for 4 days, the test substance was considered stable (relative differences upon storage were -2%, -11% and -15% for the low dose, mid-dose and high-dose groups, respectively). Re-analysis at 7 December 2015 of the high-dose samples (after subsequent storage in the freezer for 5 weeks) showed a relative difference of -20%. After storage in a closed container freezer for at < -18 °C for 5 weeks the low-dose and mid dose were considered stable after storage. The high-dose was slightly lower than intended (-21%). These concentrations were accepted and no corrections with respect to the analysis were made in the test item intake calculations.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 330 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: corresponding to NOAEL ≥ 6000 mg test substance/kg diet

Dose descriptor:

NOAEL

Effect level:

>= 406 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: corresponding to NOAEL ≥ 6000 mg test substance/kg diet

Key result

Critical effects observed:

no

	Mean (range) substance intake (mg Cedramber/kg body weight/day
	Low dose	Mid dose	High dose
Males during the study	79.85 – 95.29	166.42 – 195.49	330.43 – 384.97
Premating females	99.27 - 106.75	203.81 - 214.43	406.65 - 407.57
Gestation females	99.43 - 108.38	204.06 - 222.34	411.74 – 418.74
Lactation females	173.74	366.26	759.56

Conclusions:

Based on the absence of adverse effects in male and female rats the NOAEL for parental toxicity was stablished at ≥ 6000 mg test substance per kg diet (corresponding to a dose of 330 mg/kg body weight for males and 406 mg/kg body weight for females).

Executive summary:

The possible effects of the test substance on general toxicity, reproductive performance and development of pups was examined according to OECD 422 and in compliance with GLP.Groups of 12 male and female Wistar rats were exposed via the diet to levels of 0, 1500, 3000 and 6000 mg/kg diet during a premating period of 2 weeks and during mating, gestation until day 4 of lactation. Female rats and pups were sacrificed at or shortly after day 4 of lactation. Male rats were sacrificed after the mating period. The content, homogeneity and stability of the test substance in the carrier were accepted. Stability in a freezer resulted in slightly lower concentrations. No mortality and morbidity was observed in this study. No treatment related clinical signs were noted in male and female rats. Neurobehavioural observations and motor activity assessment did not indicate specific neurotoxic effects of the test substance. In females no treatment related effects were observed in body weight and body weight change. In males no effects were observed in body weight. In males in the high-dose group a slight, but significant decrease in body weight gain was observed in the last week of the premating period and the post-mating period. In absence of effects on body weight this was not considered to be adverse. No treatment related changes were observed in food consumption in any of the dose groups. No treatment-related effects were observed on hematology and clinical chemistry parameters. No treatment-related effects were observed on organ weight. In the kidneys of 11/11 high-dose and 6/12 mid-dose male animals clear treatment related histopathological changes were observed, which were confirmed to be hyaline droplets by immunohistochemical staining. Alpha 2u-nephropathy is considered species-specific toxicity and is therefore not considered relevant for humans. Based on the absence of adverse effects in male and female rats the NOAEL for parental toxicity was stablished at ≥ 6000 mg test substance per kg diet (corresponding to a dose of 330 mg/kg body weight for males and 406 mg/kg body weight for females).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

330 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

OECD 422 guideline study in compliance with GLP, available as unpublished report, no restrictions, fully adequate for assessment

Additional information

14 -day dose range finding study:

A 14-day range-finding study was performed to select the dose levels for a subsequent oral (diet) reproduction toxicity screening study (OECD 422) with the test substance in rats. One control group and three test groups, each consisting of 4 male and 4 female rats, were administered different dose levels of the test substance in the diet for 14 subsequent days. Test diets with concentrations of 0, 2,300, 5,400 and 11,550 mg/kg diet, corresponding to ca 1000 mg/kg bw in the high dose. In-life parameters included mortality, clinical observations, body weight and food consumption. At sacrifice animals were examined for gross anatomical changes and terminal body weight and weight of liver and kidney were determined. In the low-, mid-, and high-dose groups of female rats and in the high-dose group of male rats a decrease in food consumption was observed. This was due to reduced palatability of the diet. In female rats, this decrease in food consumption was followed by a one-day increase in food consumption, which reflects compensation for the previous decrease in food consumption. The observed effects were transient.

In the low-, mid- and high-dose group of the males and the high-dose group of the females the (absolute) and relative liver weights of the liver of were increased. The relative liver weights were increased with 18 and 21%, in males and females, respectively. The effect was considered to be on the edge of tolerability. The absolute and relative weights of the kidney were increased in the mid- and high-dose group of male rats, reaching 7% in the high dose, considered to be on the edge of tolerability. The calculated actual concentration test substance in the diets showed that the concentrations were about 20% higher (low-dose 190 mg/kg; mid-dose 425 mg/kg; high-dose 925 mg/kg) than the intended concentrations (low-dose 150 mg/kg; mid dose 350 mg/kg; high-dose 750 mg/kg) for the low- mid- and high-dose diet. Based on the above findings and taking into account the duration of exposure, lower dose levels were selected for the subsequent combined oral repeated dose toxicity study and reproduction/ developmental toxicity screening test in rats with the test substance. Dose levels of 1500, 3000 and 6000 mg/kg diet were selected.

Main study:

The possible effects of the test substance on general toxicity, reproductive performance and development of pups was examined according to OECD 422 and in compliance with GLP. Groups of 12 male and female Wistar rats were exposed via the diet to levels of 0, 1500, 3000 and 6000 mg/kg diet during a premating period of 2 weeks and during mating, gestation until day 4 of lactation. Female rats and pups were sacrificed at or shortly after day 4 of lactation. Male rats were sacrificed after the mating period. The content, homogeneity and stability of the test substance in the carrier were accepted. Stability in a freezer resulted in slightly lower concentrations. No mortality and morbidity was observed in this study. No treatment related clinical signs were noted in male and female rats. Neurobehavioural observations and motor activity assessment did not indicate specific neurotoxic effects of the test substance. In females no treatment related effects were observed in body weight and body weight change. In males no effects were observed in body weight. In males in the high-dose group a slight, but significant decrease in body weight gain was observed in the last week of the premating period and the post-mating period. In absence of effects on body weight this was not considered to be adverse. No treatment related changes were observed in food consumption in any of the dose groups. No treatment-related effects were observed on hematology and clinical chemistry parameters. No treatment-related effects were observed on organ weight. In the kidneys of 11/11 high-dose and 6/12 mid-dose male animals clear treatment related histopathological changes were observed, which were confirmed to be hyaline droplets by immunohistochemical staining. Alpha 2u-nephropathy is considered species-specific toxicity and is therefore not considered relevant for humans. Based on the absence of adverse effects in male and female rats the NOAEL for parental toxicity was stablished at ≥ 6000 mg test substance per kg diet (corresponding to a dose of 330 mg/kg body weight for males and 406 mg/kg body weight for females).

In summary, the NOAEL of 330 mg/kg bw is selected including the relative liver weight increase in the dose-range finding at ca 1000 mg/kg bw. This approach is considered conservative as the effects in this dose range finding may well be due to adaptive effects.

Justification for classification or non-classification

The NOAEL of the substance is ≥ 330 mg/kg bw/day, therefore the substance does not have to be classified for repeated dose toxicity in accordance with CLP Regulation (EC) No. 1272/2008 and its amendments.