4,4'-methylenedi-2,6-xylenol

Repeated dose toxicity: oral

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

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

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-methylenedi-2,6-xylenol

EC Number:

226-378-9

EC Name:

4,4'-methylenedi-2,6-xylenol

Cas Number:

5384-21-4

Molecular formula:

C17H20O2

IUPAC Name:

4-[(4-hydroxy-3,5-dimethylphenyl)methyl]-2,6-dimethylphenol

Test material form:

solid

Details on test material:

- Name of test material (as cited in study report):4,4'-methylenedi-2,6-xylenol
- Molecular formula :C17H20O2
- Molecular weight :256.34 g/mol
- Substance type:Organic
- Physical state:White Solid (Powder)
- Analytical purity:99.79%
- Lot/batch No.:Lot 1/03
- Storage condition of test material:Stored in cool, dry place. Kept container closed when not in use.
- Other:
Handling and Disposal
Safety precautions: Avoided excessive heat and light. Did not breath dust. Aprons, caps, gloves and goggles were used to ensure the health and safety of the Personnel.
Disposal:The remaining unused test item formulations were disposed as per internal SOPs of sa-FORD.

Test animals

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.

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 the test chemical 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

Description (incidence and severity):

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 observed

Description (incidence):

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 changes:

no effects observed

Description (incidence and severity):

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 (if feeding study):

no effects observed

Description (incidence and severity):

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:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Description (incidence and severity):

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 biochemistry findings:

no effects observed

Description (incidence and severity):

Increase level of Calcium and Sodium in male rats dosed at 250 mg/kg, 500 mg/kg and 1000 mg/kg of the test chemical. Significant increase level of Cholesterol levels in male rats dosed at 250 mg/kg of the test chemical. Decrease level of Phosphorous in male rats dosed at 1000 mg/kg of the test chemical 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 the test chemical was observed. Although there was an increase/decrease in the values of various biochemical parameters are within the normal range of limits.

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

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 the test chemical.

Gross pathological findings:

no effects observed

Description (incidence and severity):

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.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

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 the test chemical were observed.

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Effect levels

Key result

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

Target system / organ toxicity

Key result

Critical effects observed:

not specified

Applicant's summary and conclusion

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 study was performed to assess the toxic nature of the test chemical of the test chemical upon repeated exposure in 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.