Registration Dossier

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity 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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 4,4’-Methylenebis (2,6 dimethylphenol (4,4'-methylenedi-2,6-xylenol)
- Molecular formula (if other than submission substance): C17H20O2
- Molecular weight (if other than submission substance): 256.3395 g/mole
- Substance type: Organic
- Physical state: Solid
- Impurities (identity and concentrations): 00.47 %

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals 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.


Administration / exposure

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
Doses / concentrations
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

Examinations

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).

Results and discussion

Results of examinations

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.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on Clinical biochemistry, Organ weight, Gross pathological and Histopathology.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The no observed adverse effect level (NOAEL) was considered to be 1000 mg/kg bw/day when Sprague-Dawley rats exposed to the test chemical orally for 28 days.
Executive summary:

A subacute study was conducted to evaluate the toxic effects of repeated administration of the test chemical in male and female Sprague-Dawley rats by gavage. The test chemical was administered to 6 animals/sex/species in Polyethylene Glycol-400 at doses of 0, 250, 500 or 1000 mg/kg/bw/day for 28 days.Blood samples for Clinical Biochemistry and Haematology were collected. No abnormalities occurred that could be directly attributed to treatment. Although significant change in organ weights were observed in animals from different dose groups, no related gross pathological or histological changes were seen and findings were not treatment dependent and hence considered to be of no toxicological importance. All the rats of 0, 250, 500 and 1000 mg/kg/bw/day dose group survived and no mortality was observed.

Therefore, the no observed adverse effect level (NOAEL) for repeated dose toxicity study was considered to be 1000 mg/kg/bw/day in male and female Sprague-Dawley rats when exposed to to the test chemical by oral route for 28 days.