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Repeated dose toxicity: Oral

A repeated-dose oral toxicity studywas performed to assess the toxic nature of the test chemical ofthe test chemicalupon repeated exposurein Sprague-Dawley rats. The test chemical was administered by gavage at the dose levels of 0, 250, 500 and 1000 mg/kg/body weight for 28 days.All animals were examined for clinical signs and general appearance daily throughout the study. Detailed clinical observations were conducted weekly. Body weight gain and food consumption values were recorded weekly.Motor activity of each animal was recorded, and stereotypic activity was calculated. After completion of the study period, animals were sacrificed and blood samples for haematology and clinical biochemistry were collected. All rats from each group were subjected to necropsy and gross lesions were noted. Histopathological examination was carried out on samples of control and 1000 mg/kg-treated animals.All the animals from control and treated dose groups survived throughout the dosing period of 28 days. No changes in body weight gain and food intake due to test chemical administration were noted. No clinical signs and differences in general appearance were seen in any of the treated animals. No dose-dependent and toxicologically relevant alterations in blood parameters were recorded. Although some significant change in organ weights (including liver, kidney and adrenals) were observed in animals from different dose groups, no related gross pathological or histological changes were seen, and findings were not dose-dependent and hence considered to be of no toxicological importance. Gross pathological examination of rats of all dose groups during necropsy did not reveal any abnormality attributable to the treatment. Similarly, histopathological examination conducted in rats of control and high dose groups did not reveal any abnormality attributable to the treatment. Motor activity values observed in male animals for control and different dose groups were comparable. In female animals, higher values for motor activity were observed from 500 mg/kg dose group but this change was within laboratory range and it was considered to be of no toxicological importance. Based on the observations made in the study, the repeated oral exposure ofthe test chemicalexerted no toxicologically significant changes in the morphology and function of a tissue/organ or have produced serious changes to the biochemistry or haematology of the organism. NOAEL was considered as 1000 mg/kg/day when male and female Sprague Dawley rats were orally treated with the test chemical for 28 days.

Repeated dose toxicity: Dermal

The acute dermal toxicity value for the given test chemical (as provided in section 7.2.3) is >2000 mg/kg body weight. Considering this, the end point for repeated dermal toxicity is considered as waiver.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from study report
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Principles of method if other than guideline:
Repeated dose oral toxicity study was performed to determine the toxic nature of the test chemical.
GLP compliance:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: National Institute of Biosciences, Pune.

- Age at study initiation: 6 to 8 weeks old.
- Weight at study initiation:
Male 168.62 g
Female 140.81 g

- Fasting period before study: No data available

- Housing: Animal was housed in polycarbonate cages. Three rats of same sex were housed together in each cage of size 39 cm X 28 cm X 14 cm. Paddy husk was used as bedding material.

- Diet (e.g. ad libitum): Rodent feed supplied by the Nutrivet Life Sciences, Pune, was provided ad libitum from individual feeders on cage top.

- Water (e.g. ad libitum): Water was provided ad libitum from individual bottles attached to the cages. Water was from a local source and passed through the reverse osmosis membrane before use.

- Acclimation period: 5 days prior to dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C (actual range: 19.7 °C to 24.1 °C)

- Humidity (%):30% to 70% (actual range: 52.8% to 59.7%).

- Air changes (per hr): Ten air changes per hour of 100% fresh air that has been passed through the HEPA filters.

- Photoperiod (hrs dark / hrs light): An artificial light and dark cycle of 12 hours each were provided.


Route of administration:
oral: gavage
Vehicle:
propylene glycol
Remarks:
Polyethylene Glycol-400
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: 4,4’-Methylenebis (2,6 dimethylphenol) was dissolved in Polyethylene Glycol-400 for preparation of dosing solution(s).

DIET PREPARATION
- Rate of preparation of diet (frequency):
No data available

- Mixing appropriate amounts with (Type of food): No data available

- Storage temperature of food: No data available

VEHICLE
- Justification for use and choice of vehicle (if other than water): No data available

Concentration in vehicle: The solution(s) were prepared at concentrations of 0, 25, 50 and 100 mg/ml such that dosage of 0 (vehicle), 250, 500 and 1000 mg/kg body weight.
- Amount of vehicle (if gavage): 0.00 mg/ml/day, 25.46 mg/ml/day, 49.02 mg/ml/ day and 101.23 mg/ml/day.

- Lot/batch no. (if required): No data available

- Purity: No data available

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis for concentration and stability of 4,4’-Methylenebis (2,6 dimethylphenol) were conducted at Subcontracted Laboratory. Test item formulation samples prepared day 1 (pre-dosing) were sent to Subcontracted Laboratory.
Duration of treatment / exposure:
28 days consecutively
Frequency of treatment:
Once daily
Remarks:
Doses / Concentrations:
0 (vehicle), 250, 500 and 1000 mg/kg body weight/day.
Basis:
actual ingested
No. of animals per sex per dose:
Control: 6 male, 6 female
250 mg/kg bw/day: 6 male, 6 female
500 mg/kg bw/day: 6 male, 6 female
100 0mg/kg bw/day: 6 male, 6 female
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on results from a preliminary 14-day study there was no chenge in the survivel, body weight, Daily clinical observations and Gross pathological examination of 1000 mg/kg/bw/day group. Based on these results, the 28 day study dose levels were finalized as 0 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg bw/day.

- Rationale for animal assignment (if not random): Animals were randomized by body weight.

- Rationale for selecting satellite groups: No data available

- Post-exposure recovery period in satellite groups: No data available

- Section schedule rationale (if not random): No data available
Positive control:
No data
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes

- Time schedule: No data available

- Cage side observations checked in table [No.?] were included. : Rats were observed for Behavior, Alterations, Vocalizations, Respiration and Palpebral closure.

DETAILED CLINICAL OBSERVATIONS: Yes

- Time schedule: Once before the start of dose administration and at least once a week thereafter until scheduled sacrifice.

BODY WEIGHT: Yes

- Time schedule for examinations: On the day of randomization, first day of dosing, weekly thereafter and a fasting body weight at scheduled sacrifice on day 29.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes

- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data available

- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data available

FOOD EFFICIENCY: No data available
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data available

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data available

- Time schedule for examinations: No data available

OPHTHALMOSCOPIC EXAMINATION: Yes

- Time schedule for examinations:
Once a day

- Dose groups that were examined: 0 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg bw/day.

HAEMATOLOGY: Yes, By using Beckman Coulter haematology analyzer.
- Time schedule for collection of blood: At termination.

- Anaesthetic used for blood collection: No data available

- Animals fasted: Yes, overnight fasted prior to sampling.

- How many animals: Blood collected from all rats of 0 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg bw/day group at termination.

- Parameters checked in table [No.?] were examined. : Hemoglobin, Red Blood Corpuscles, Hematocrit, Mean Corpuscular Volume, Mean Corpuscular Hemoglobin, Mean Corpuscular Hemoglobin Concentration, Platelets, White Blood Corpuscles, Neutrophils, Lymphocytes, Eosinophils, Monocytes, Basophil and Prothrombin time were checked, Table No.H; Appendix No.VII.

CLINICAL CHEMISTRY: Yes, By using Dimension XpandPlus and Acculyte 5P.

- Time schedule for collection of blood: At termination.

- Animals fasted: Yes, overnight fasted prior to sampling.

- How many animals: Blood collected from all rats of 0 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg bw/day group at termination.

- Parameters checked in table [No.?] were examined. : Total Protein, Blood Urea Nitrogen, Urea Nitrogen, Alanine Aminotransferase, Aspartate Aminotransferase, Alkaline Phosphatase, Gamma Glutamyl Transferase, Glucose, Calcium, Phosphorous, Albumin, Total Bilirubin, Creatinine , Total Cholesterol, Triglycerides, Globulin Calculated
Sodium, Potassium, Chloride were checked, Table No.I; Appendix No.VIII .

URINALYSIS: No data available
- Time schedule for collection of urine: No data available

- Metabolism cages used for collection of urine: No data available

- Animals fasted: No data available

- Parameters checked in table [No.?] were examined. : No data available

NEUROBEHAVIOURAL EXAMINATION: - No data available

Time schedule for examinations: No data available

- Dose groups that were examined: No data available

- Battery of functions tested: sensory activity / grip strength / motor activity / other: Yes

OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All the animals of 0 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg/bw/day group were sacrificed and gross lesions were noted.

HISTOPATHOLOGY: Yes
Control and treated at the highest dose level of 1000 mg/kg were subjected to sacrifice.
Organs examined: Stomach, Seminal Vesicles with Coagulation Gland Testes, Thymus, Thyroid, Trachea, Urinary Bladder, Adrenals, Aorta Brain, Caecum Cervix Colon Duodenum Eyes Heart Ileum Jejunum Kidneys, Liver, Lungs and Mesenteric Lymphnodes of 1000 mg/kg/bw/day group.
Statistics:
Data were analyzed for reporting group means and standard deviations with significance between the controls and treated groups, using in-house developed and validated MS-Excel 2003 based statistical software. All the parameters characterized by continuous data such as body weight, per cent body weight change, feed consumption, organ weight, relative organ weight, haematological and clinical chemistry data were subjected to Bartlett’s test to meet the homogeneity of variance before conducting Analyses of Variance (ANOVA) and Dunnett’s t -test. Where the data did not meet the homogeneity of variance, Student’s t -test was performed to calculate significance.

Significance was calculated at 1% as well as 5% level and indicated in the summary tables as follows:

* = Significant than control at 95% level of confidence (p≤ 0.05).
** = Significant than control at 99% level of confidence (p≤ 0.01).
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Details on results:
Clinical signs and mortality
Clinical signs :
Daily clinical observations did not reveal any signs of toxicity in male and female animals from of 0 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg bw/day dose groups during the dosing period of 28 days.

Mortality:
No mortality were observed in any of the traeted groups of 0 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg bw/day.

Body weight and weight gain
All the of 0 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg bw/day dose groups exhibited normal body weight gain at the end of the study period of 28 days.

Food consumption and compound intake
Food consumption :
Food intake of animals from control and 250 mg/kg, 500 mg/kg and 1000 mg/kg bw/day dose groups were found to be normale throughout the study period of 28 days.

Food efficiency
No data available

Water consumption and compound intake
No data available

Opthalmoscopic examination
No data available

Haematology
Statistically significant increase in the values of MCHC of male rats dosed at 250 mg/kg/bw/day and 1000 mg/kg/bw/day. In addition statistically significant decrease was observed in the values of Hb and HCT of male rats dosed at 1000 mg/kg. The increase / decrease in the values of different parameters were marginal and within the normal laboratory limits.
No significant changes were observed in the values of different parameters studied in female animals from different dose groups when compared with that of controls.

Clinical chemistry

Increase level of Calcium and Sodium in male rats dosed at 250 mg/kg, 500 mg/kg and 1000 mg/kg of 4,4’-Methylenebis (2,6-dimethylphenol).

Significant increase level of Cholesterol levels in male rats dosed at 250 mg/kg of 4,4’-Methylenebis (2,6-dimethylphenol).

Decrease level of Phosphorous in male rats dosed at 1000 mg/kg of 4,4’-Methylenebis (2,6-dimethylphenol) were observed.

Decrease level of Chloride in male rats dosed at 250 mg/kg, 500 mg/kg and 1000 mg/kg.

Decrease level of Glucose in female rats dosed at 500 mg/kg of 4,4’-Methylenebis (2,6-dimethylphenol) was observed.

Although there was an increase/decrease in the values of various biochemical parameters are within the normal range of limits.

Urinanalysis
No data available

Neurobehaviour
No data available

Organ weights
Female animals revealed increased relative weights of liver of animals from 250 mg/kg and 500 mg/kg dose groups, increased relative weights of kidneys of animals from 250 mg/kg dose group and decreased relative weights of spleen of animals from 1000 mg/kg dose group.

Although significant change in organ weights were observed in animals from different dose groups, but the effect was not due to 4,4’-Methylenebis (2,6-dimethylphenol).

Gross pathology
In male and female animals from control and 250 mg/kg/bw/day, 500 mg/kg/bw/day and 1000 mg/kg/bw/day of test item. All the treatment groups did not reveal any abnormality.

Histopathology
Focal to multifocal periportal mononuclear cell infiltration in the liver; minimal interstitial haemorrhages and/or tubular dilatation in the kidneys; minimal alveolar haemorrhages and/or alveolar histiocytosis in the lungs; minimal multifocal haemosiderosis and/or diffused congestion in spleen; minimal eosinophilic infiltration and/or luminal dilatation and/or endometrial gland dilatation in uterus; minimal dilatation of zona reticularis and/or presence of accessory adrenocortical tissue in adrenals; minimal multifocal haemorrhages in thymus; presence of ultimobranchial cysts in thyroid; in male or female animals from control and high dose group. All the changes observed in the control and 1000 mg/kg/bw/day dose group animals were similar and no toxic effect of 4,4’-Methylenebis (2,6-dimethylphenol) were observed.

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
gross pathology
haematology
histopathology: non-neoplastic
mortality
organ weights and organ / body weight ratios
urinalysis
other: No adverse effect was seen at any dose tested.
Critical effects observed:
not specified
Conclusions:
The repeated oral exposure to the test chemical exerted no toxic effects at the doses tested in male and female Sprague-Dawley rats. The No Observed Adverse Effect Level (NOAEL) was considered to be 1000 mg/kg bw/day when SD rats were exposed to the test chemical for 28 days.
Executive summary:

A repeated-dose oral toxicity studywas performed to assess the toxic nature of the test chemical ofthe test chemicalupon repeated exposurein Sprague-Dawley rats. The test chemical was administered by gavage at the dose levels of 0, 250, 500 and 1000 mg/kg/body weight for 28 days.All animals were examined for clinical signs and general appearance daily throughout the study. Detailed clinical observations were conducted weekly. Body weight gain and food consumption values were recorded weekly.Motor activity of each animal was recorded, and stereotypic activity was calculated. After completion of the study period, animals were sacrificed and blood samples for haematology and clinical biochemistry were collected. All rats from each group were subjected to necropsy and gross lesions were noted. Histopathological examination was carried out on samples of control and 1000 mg/kg-treated animals.All the animals from control and treated dose groups survived throughout the dosing period of 28 days. No changes in body weight gain and food intake due to test chemical administration were noted. No clinical signs and differences in general appearance were seen in any of the treated animals. No dose-dependent and toxicologically relevant alterations in blood parameters were recorded. Although some significant change in organ weights (including liver, kidney and adrenals) were observed in animals from different dose groups, no related gross pathological or histological changes were seen, and findings were not dose-dependent and hence considered to be of no toxicological importance. Gross pathological examination of rats of all dose groups during necropsy did not reveal any abnormality attributable to the treatment. Similarly, histopathological examination conducted in rats of control and high dose groups did not reveal any abnormality attributable to the treatment. Motor activity values observed in male animals for control and different dose groups were comparable. In female animals, higher values for motor activity were observed from 500 mg/kg dose group but this change was within laboratory range and it was considered to be of no toxicological importance. Based on the observations made in the study, the repeated oral exposure ofthe test chemicalexerted no toxicologically significant changes in the morphology and function of a tissue/organ or have produced serious changes to the biochemistry or haematology of the organism. NOAEL was considered as 1000 mg/kg/day when male and female Sprague Dawley rats were orally treated with the test chemical for 28 days.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Data is from study report.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
a short-term toxicity study does not need to be conducted because exposure of humans via inhalation in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available
Quality of whole database:
Waiver

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
other justification
Justification for data waiving:
a short-term toxicity study does not need to be conducted because exposure of humans via the dermal route in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment
Endpoint conclusion
Endpoint conclusion:
no study available
Quality of whole database:
Waiver

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Data available for the test chemical was reviewed to determine the toxic nature of the test chemical. The studies are as mentioned below:

Repeated dose toxicity: Oral

Study 1: A repeated dose 28 -day oral toxicity studywas conducted to evaluate the toxic nature of4,4’-Methylenebis(2,6-dimethylphenol) in male and female Sprague-Dawley rats. The test chemical was administered by gavage to 6 animals/sex/group in Polyethylene glycol-400 at doses of 0, 250, 500 or 1000 mg/kg/bw/day.The control animals were administered with vehicle only. All animals were examined for clinical signs and general appearance daily throughout the study. Detailed clinical observations were conducted weekly. Body weight gain and food consumption values were recorded every week.Motor activity of each animal was recorded, and stereotypic activity was calculated. After completion of the study period animals were sacrificed and blood samples for hematology and clinical biochemistry were collected. All rats from each group were subjected to necropsy and gross lesions were noted. Histopathological examination was carried out on samples of control and 1000 mg/kg-treated animals. All the rats of 0, 250, 500 and 1000 mg/kg/bw/day dose group survived the administration period. No abnormalities in blood parameters occurred that could be directly attributed to treatment. Some significant changes in organ weights were observed in animals from different dose groups, but no related gross pathological or histological changes were seen, and findings were not treatment dependent and hence considered to be of no toxicological importance. Therefore, the NOAEL for repeated dose toxicity was considered to be 1000 mg/kg/bw/day in male and female Sprague-Dawley rats when orally exposed to the test chemical for 28 days.

Study 2: A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was carried out to evaluate the toxic potential of the test chemical on reproductive performance and the development of offspring in male and female Sprague-Dawley rats. The test chemical was administered by gavage daily at the concentrations of 12.5, 50, 200 and 800 mg/kg/ bw/day. Males were dosed for the total of 50-52 days (14 days before mating and 36 to 38 days after mating), while for females, the total administration period was 40 to 48 days (14 days before mating, 14 days of mating, 21 days pregnancy and 3 days of lactation). The test chemical was solved in 5% aqueous gum Arabic solution and employed as a vehicle in controls. The purity and the stability of the test chemical were confirmed, and it was stored in refrigerator under light-proof conditions.During the administration period, ratswere observed for general condition, body weight alteration and food consumption. After the last administration day, a complete necropsy of reproductive organs was carried out. The sperm parameters including sperm motion, morphology, viability and survivability were recorded in males. Detailed histopathological examination of testis and epididymis were performed, as well. In females the weight of ovaries, and uterus parameters including number of corpora lutea, implantation, gestation length and number of live pups born were recorded. Newborn pups were observed for general conditions, neonatal survival and body weights. In male rats, no difference in body weights and food intake values were observed during the experiment. However, in the 200 and 800 mg/kg groups the relative and absolute weights of testis and epididymis were significantly decreased.The results of sperm analysesshowedsignificantlyreduced ofsperm motility, viability, survivability and the number of sperms in the left epididymis in the group treated with 50 mg/kg or more of the test chemical compared with controls. The percentage of sperms with abnormal morphology significantly increased in 50, 200 and 800 mg/kg groups in comparison to control. The histopathological examination of the gonads revealed atrophy of seminiferous tubule of testis and the epididymis in the two highest dose groups (200 and 800 mg/kg). Giant cell formation in the testis, which is usually accompanies germinal celldegeneration/atrophy was observed at the dose of 50 mg/kg and above. No death and alterations in general conditions were observed among the female rats during the test chemical administration. However, the body weight gain was significantly decreased in the high-dose group between 0-21 day of pregnancy and during the lactation period in 200 and 800 mg/kg treated group compared to controls. Similarly, the food consumption in female rats is dropped significantly on day 3-6 of pre-mating period, on day 2 of pregnancy and day 4 of lactation in the two highest dose groups. No abnormalities were seen by necropsy and the weights of ovaries showed no significant alterations between any treated and control groups. The histopathological analyses showed no alterations of uterus parameters. Similarly, no changes in fertility and mating performance between any of the treated and control groups were observed. However, the number of pups born showed a significant and dose-dependent decrease in the 200 and 800 mg/kg group compared with controls. The number of live pups born and the number of live pups on lactation day 4 significantly dropped in the two high-dose groups. The necropsy of newborns revealed no abnormalities in any of the treated-group, although the body weight of pups dropped significantly in the two highest-dose group. In summary, the present study provides clear evidence that the repeated oral exposure ofthe test chemical exertedstrong adverse effect of testicular spermatogenesis and promotes the atrophy of male gonads in male Sprague-Dawley rats. The reproductive NOAEL (No Observed Adverse Effect Level) for male SD rats was 12,5 mg/kg/day based on findings of necropsy, organ weight measurement,sperm testfindingsand histopathology. The repeated exposure of the test chemical during pre-mating and mating period had no effect on female gonad function, fertility, mating behaviour and conception as it was evidenced by the unchanged fertility and copulation index and by the lack of histopathological findings. The reproductive NOAEL for maternal generation was 800 mg/kg/day. The number of pups born, number of live pups born and the number of live pups on lactation day 4, as well as the delivery index were significantly decreased at 200 mg/kg and remained reduced, but not significantly at 800 mg/kg, which was still considered important from toxicological point of view, when female SD rats were administeredthe test chemicalduring the period of pre-mating mating, pregnancy and early lactation. Hence, the developmental NOAEL was estimated to be 50 mg/kg/day for both maternal and F1 generations. The NOAEL for systemic maternal toxicity was considered as 50 mg/kg/day based on reduced body weight and feed consumption at the two highest dose. The NOAEL for general toxicity for males was 800 mg/kg/day as no alteration of bodyweight and food consumption was seen.

Study 3: In a subchronic study, Wistar rats were fed with diet the test chemical at the concentrations of 0, 0.12, 0.6 or 3.0 % for 12 weeks. The body weight and food consumption values were recorded weekly. During the entire course of the study, rats were daily observed. Haematological and serum biochemical examinations were conducted. All animals were studied for histopathological changes. Severe suppression of body weight gain was observed in both sexes of 0.6 and 3.0 % groups. Death accompanied by haemorrhage from nasal cavity was observed in 0.6 and 3.0 % males and 3.0 % females. Significant and dose-dependent decrease in the absolute and relative liver weight was noted in both sexes of the 0.6 and 3.0 % groups. The blood chemical test showed elevated parameters relevant for liver disfunction as well. Significant and dose-dependent decrease in absolute and relative testis weights were observed in 0.12, 0.6 and 3.0 % males. Similarly, the uterus weight dropped significantly and dose-dependently in the 0.6 and 3.0 % groups. Histopathological examination revealed testicular atrophy, the appearance of giant cells and decrease of spermatogenesis in a dose-and time-dependent manner in all treated males. Interstitial edema in the testis was also apparent in all treated rats at week 12. At weeks 4 and 12, epididymis atrophy and hypospermia in the 3 % males and the atrophy of the seminal vesicles and prostate in the 0.6 % and 3.0 % group were noted. In females, atrophy of the ovaries and uterus were apparent in the 0.6 and 3.0 % groups. In both sexes, thymus atrophy and bone marrow hyperplasia were noted in the 0.6 % and 3.0 % groups. No histopathological changes were observed in any other organs. In conclusion, oral administration ofthe test chemicalexerted toxicologically serious adverse effectson male and female reproductive organs. In males, the remarkable effectswerethe atrophy of testicular tubules, decrease of spermatogenesis and the significant reduction of testis weight at all doses applied. In females, significant decrease of absolute and relative ovaries weightwas observed, which was accompanied by the atrophy of ovaries and uterus at 0.6 and 3 %. Hence, the reproductive NOAEL for female rats was considered as 0.12 % (104 mg/kg body weight/day). No reproductive NOAEL for male rats was possible to determine as testicular atrophy and reduced spermatogenesis were observed at the lowest applied doseslevel. Hence, the reproductive LOAEL for male rats was considered as 0.12 % (88 mg/kg body weight/day). The NOAEL for systemic toxicity was 0,12 % for both males and females, based on mortality, clinical signs of toxicity, decreased body weight, increased liver weight, thymus hypertrophy and bone marrow hyperplasia, when male and female Wistar rats were fed the test chemicalfor 12 weeks.

Repeated dose toxicity: Inhalation

The test substance has very low vapor pressure (0.00000256 Pa at 25 degC). Also, the particle size distribution of the substance was found to vary in the size of 55-250 µm, so the potential for the generation of inhalable dust forms is low. Moreover the normal conditions of use of this substance will not result in aerosols, particles or droplets of an inhalable size, so exposure to humans via the inhalatory route will be unlikely to occur, and therefore acute toxicity by inhalation route was considered to be waived.

Repeated dose toxicity: Dermal

The acute dermal toxicity value for the given test chemical (as provided in section 7.2.3) is >2000 mg/kg body weight. Considering this, the end point for repeated dermal toxicity is considered as waiver.

Based on the data available, the test chemical is not likely to be toxic upon repeated exposure by oral, dermal and inhalation route of exposure and hence it is not likely to be a toxicant as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

According to the requirements of REACH regulations, a sub-chronic (90-day) toxicity study have to be performed to fulfil the criteria at this tonnage level. Currently, the sub-chronic study of the test chemical is lacking thus the classification of the test chemical according to the criteria of CLP regulations cannot be performed.