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

Endpoint summary

Description of key information

Repeated Dose oral toxicity

Study 1

A rat NOAEL of 1000 mg/kg bw/day was obtained in a study performed according to OECD 407 with the substance.

Study 2

Rat NOAELs of 750 mg/kg bw/day (females) and 1000 mg/kg bw/day (males) were obtained in a study performed according to OECD 408 with the substance.

Repeated dose inhalation toxicity

A short term repeated dose toxicity study doesnot need to be conducted as the exposure of humans via inhalation during production and/or use is highly unlikely based on the provided thorough and rigorous risk assessment. The test substance has very low vapor pressure (2,56 x 10-6 Pa at 25°C). Also, the particle size distribution of the substance was found to vary between 55-250 µm thus the potential for inhalable dust formation is low. Moreover, the normal conditions of use of this substance will not result in aerosols, particles or droplets of an inhalable size, thus exposure to humans via the inhalation route will be unlikely to occur, and therefore acute toxicity by inhalation route was considered to be waived.

Repeated dose dermal toxicity

A short term repeated dose toxicity study doesnot need to be conducted as the exposure of humans via dermal during production and/or use is highly unlikely based on the provided thorough and rigorous risk assessment.The acute dermal toxicity value for (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

Reference 1

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

Limit test:

no

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 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

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

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

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

Key result

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 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.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

1998

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo, Frederick, MD
- Females (if applicable) nulliparous and non-pregnant: [yes/no] : yes
- Age at study initiation: treatment began at 8 weeks of age.
- Housing: Animals were housed in Smithers Avanza Toxicology Services testing facility (Maryland, US). Animals were housed individually in clear polysulfone plastic cages with SaniChip certified hardwood bedding
- Diet (e.g. ad libitum): Animals were fed Teklad Certified Rodent Diet 2018C (Envigo Teklad Diets, Madison, WI; batch No. 2018CM-102116MA)

ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): a light cycle of 12-h light/12-h dark

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The diet preparation and formulation were conducted by Smithers Viscient Caroline Research Center.

Duration of treatment / exposure:

90 days

Frequency of treatment:

Daily by diet

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Control group

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Low-dose male and females

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Mid-dose male and females

Dose / conc.:

750 mg/kg bw/day (actual dose received)

Remarks:

High-dose females

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

High-dose males

No. of animals per sex per dose:

Main study: 10 rats per sex per dose.
Recovery groups: 5 rats per sex per dose treated at 0 (plain diet), 750 (females) and 1000 mg/kg bw/day (males).

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale:The dose levels were selected based on dose range-finding study
- Rationale for animal assignment (if not random): The test animals were randomly assigned to the control and treatment groups
- Fasting period before blood sampling for clinical biochemistry:
- Rationale for selecting satellite groups:
- Post-exposure recovery period in satellite groups: 28 days
- Section schedule rationale (if not random):
- Other:

Positive control:

None.

Observations and examinations performed and frequency:

Physical examinations, cage-side observations, body weight, food intake, ophthalmologic observations, functional observatory battery, grip strength, locomotor activity, clinical chemistry, hematology, coagulation, and urinalysis. See Table 1 for more details.

Sacrifice and pathology:

Not specified.

Statistics:

Prior to the ANOVA analysis, untransformed data were tested to determine if the data are normally distributed and have homogeneous variances among all groups using Shapiro-Wilk followed by the Levene’s test. The Dunnett’s t-test was used to determine which groups differed from the control group. Group comparisons were evaluated at the 0.05 (two-tailed) probability level. Statistical significance was considered at p ≤ 0.05

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 examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant changes in clinical chemistry included mild decreases in serum creatinine in females (≥100 mg/kg) and males (1000 mg/kg); mild increases in serum cholesterol in males (≥100 mg/kg) and females (≥300 mg/kg); minimal increases in serum total protein and globulin in males (1000 mg/kg); and minimal increases in serum total protein and albumin in females (750 mg/kg). Activated partial thromboplastin times were slightly increased in females (≥100 mg/kg) and males (≥300 mg/kg) however, no associated clinical findings or microscopic observations were made. At the end of the 28-day recovery period, the following statistically significant changes were observed in high-dosed animals: a minimal decrease in serum creatinine in males; a minimal increase in serum cholesterol in females; a minimal increase in serum albumin in females; and a mild decrease in serum alkaline phosphatase in both sexes. None of the statistical changes in clinical chemistry were considered adverse effect of treatment since they were either within normal biological variation, lacked dose-dependency, or lacked associated microscopic findings.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Organ weight changes at study day 91 included increased absolute and relative (body and brain) liver weights in males and females (≥100 mg/kg-bw/d), correlating microscopically with hepatocellular hypertrophy. There was also an increased absolute and relative kidney weight in females (750 mg/kg-bw/d) and males (1000 mg/kg-bw/d), correlating microscopically with tubular dilation and hypertrophy. Absolute and relative kidney weights for males given 1000 mg/kg-bw/d were calculated excluding one animal which had a large renal tumor and the magnitude of increase was still statistically significant. After the recovery period, organ weight changes remained in the liver and kidney of males given 1000 mg/kg-bw/d. Mean absolute and relative kidney weights were increased, correlating microscopically with tubular dilation, while mean absolute and relative liver weights were increased but had no microscopic correlates. Kidney weight changes were similar to study day 91, while the magnitude of change in liver weights for 1000 mg/kg-bw/d was less (up to 28%) than at study day 91, suggesting ongoing recovery.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

One male given 1000 mg/kg-bw/d was observed with a firm, red, tan mass in the left kidney, correlating microscopically with renal tubule carcinoma. As hyperplasia and adenomas were not observed in other high-dose animals and there were no tumors observed in recovery animals, it is unlikely that a single occurrence of carcinoma was test chemical-related in a 90-day study.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The liver weight changes were accompanied by hepatocellular hypertrophy (minimal/mild) whereas the kidney weight changes were accompanied by tubular dilatation and hypertrophy (minimal/mild). Additional histopathological findings included thymic epithelial proliferation in females treated at ≥300 mg/kg (minimal/mild) and ovarian follicular cysts at 750 mg/kg (minimal/mild). Females at 0 and 750 mg/kg appeared to be cycling normally as oestrogen stages were recorded in all of these animals. At the end of the recovery period, only microscopic evidence of tubular dilatation in both sexes remained.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopically, there were no findings attributable to administration of the test chemical. One male given 1000 mg/kg-bw/d was observed with a firm, red, tan mass in the left kidney, correlating microscopically with renal tubule carcinoma. As hyperplasia and adenomas were not observed in other high-dose animals and there were no tumors observed in recovery animals, it is unlikely that a single occurrence of carcinoma was test chemical-related in a 90-day study.

Other effects:

not specified

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

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: neoplastic

histopathology: non-neoplastic

mortality

ophthalmological examination

organ weights and organ / body weight ratios

urinalysis

Dose descriptor:

NOAEL

Effect level:

750 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

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: neoplastic

histopathology: non-neoplastic

mortality

ophthalmological examination

organ weights and organ / body weight ratios

urinalysis

Critical effects observed:

no

Table 1.Measurements at the end of the 90-day dietary treatment in TMBPF animals compared to controls. Statistically significant findings are marked with*

Endpoint	Mean ± SD (N = 10 animals/group/sex)
	Control (M)	TMBPF 100 (M)	TMBPF 300 (M)	TMBPF 1000 (M)	Control (F)	TMBPF 100 (F)	TMBPF 300 (F)	TMBPF 750 (F)
Serum creatinine (mg/dL)	0.59 ± 0.07	0.57 ± 0.07	0.58 ± 0.06	0.49 ±0.07*	0.76 ± 0.07	0.64 ± 0.17*	0.59 ± 0.07*	0.56 ± 0.08*
Cholesterol esters (mg/dL)	117.4 ± 10.2	148.4 ± 20.4*	163.0 ± 14.8*	195.7 ± 18.4*	113.3 ± 9.9	113.3 ± 9.10	145.2 ± 9.5*	177.0 ± 13.8*
Triglycerides (mg/dL)	42.2 ± 12.0	64.0 ± 20.8*	46.6 ± 22.2	51.2 ± 13.2	40.7 ± 18.6	36.7 ± 14.0	36.5 ± 8.6	42.4 ± 10.4
Total protein (g/dL)	6.28 ± 0.23	6.46 ± 0.20	6.54 ± 0.30	6.70 ± 0.32*	6.71 ± 0.32	6.69 ± 0.33	6.92 ± 0.42	7.14± 0.30*
Albumin (g/dL)	2.12 ± 0.13	2.20 ± 0.11	2.28 ± 0.19	2.29 ± 0.20	2.51 ± 0.20	2.60 ± 0.21	2.64 ± 0.24	2.73 ± 0.18*
Globulin (g/dL)	4.16 ± 0.19	4.26 ± 0.16	4.26 ± 0.26	4.41 ± 0.25*	4.20 ± 0.17	4.09 ± 0.20	4.28 ± 0.29	4.41 ± 0.25*
Hemoglobin (g/dL)	16.48 ± 0.39	16.05 ± 0.72	16.19 ± 0.59	16.05 ± 0.60	15.43 ± 0.46	15.36 ± 0.49	15.00 ± 0.41	14.93 ± 0.40*
Red cell distribution width (%)	12.47 ± 0.16	12.62 ± 0.29	12.68 ± 0.27	12.82 ± 0.20*	11.32 ± 0.27	11.33 ± 0.21	11.16 ± 0.24	11.31 ± 0.19
Activated partial thromboplastin time (sec)	16.63 ± 1.10	19.04 ± 3.82	22.93 ± 7.48*	20.03 ± 3.19*	17.04 ± 2.57	19.99 ± 3.0*	18.86 ± 1.29	23.44 ± 2.98*
Heart weight, body weight relative (g)	0.37 ± 0.02	0.35 ± 0.02	0.36 ± 0.04	0.38 ± 0.02	0.38 ± 0.02	0.42 ± 0.04^1	0.40 ± 0.03	0.42 ± 0.04*
Heart weight, brain weight relative (g)	0.83 ± 0.04	0.82 ± 0.06	0.80 ± 0.08	0.81 ± 0.07	0.54 ± 0.04	0.60 ± 0.06*	0.55 ± 0.03	0.59 ± 0.07
Kidney weight, absolute (g)	2.52 ± 0.19	2.65 ± 0.24	2.56 ± 0.17	2.82 ± 0.24*	1.51 ± 0.12	1.60 ± 0.13	1.60 ± 0.11	1.66 ± 0.11*
Kidney weight, body weight relative (g)	0.58 ± 0.03	0.61 ± 0.05	0.60 ± 0.03	0.64 ± 0.04*	0.58 ± 0.03	0.62 ± 0.05	0.64 ± 0.05*	0.66 ± 0.03*
Kidney weight, brain relative (g)	1.31 ± 0.09	1.41 ± 0.11	1.35 ± 0.11	1.47 ± 0.09*	0.83 ± 0.07	0.90 ± 0.07	0.89 ± 0.06	0.92 ± 0.07*
Liver weight, absolute (g)	11.03 ± 0.99	13.62 ± 1.45*	14.36 ± 1.77*	16.83 ± 0.94*	5.99 ± 0.47	6.59 ± 0.56*	7.06 ± 0.56*	8.61 ± 0.56*
Liver weight, body weight relative (g)	2.53 ± 0.13	3.14 ± 0.25*	3.36 ± 0.28*	3.85 ± 0.16*	2.32 ± 0.15	2.56 ± 0.14*	2.83 ± 0.17*	3.40 ± 0.16*
Liver weight, brain relative (g)	5.74 ± 0.49	7.23 ± 0.69*	7.60 ± 0.94*	8.73 ± 0.37*	3.28 ± 0.29	3.69 ± 0.34*	3.91 ± 0.31*	4.76 ± 0.40*
^*Statistical significance was considered at a p ≤ 0.05. M: male; F: Female ^1The not-rounded value is 0.41983 ± 0.04385 and it is not statistically significant compared to control.
Organ Histopathology (Incidence)
Liver
Hepatocellular hypertrophy
Minimal	FNO	4	5	1	FNO	FNO	1	6
Mild	FNO	FNO	FNO	6	FNO	FNO	FNO	3
Kidney
Tubular dilation
Minimal	8	5	5	4	3	4	4	7
Mild	FNO	3	3	5	FNO	2	FNO	2
Tubular hypertrophy
Minimal	FNO	3	4	5	FNO	1	FNO	3
Mild	FNO	FNO	FNO	1	FNO	FNO	FNO	FNO
Thymus
Epithelial proliferation
Minimal	1	NE	NE	1	FNO	2	4	5
Mild	FNO	NE	NE	FNO	FNO	FNO	FNO	1
Ovary
Follicular cyst(s)
Minimal	FNO	FNO	FNO	FNO	2	2	3	6
Mild	FNO	FNO	FNO	FNO	FNO	FNO	FNO	1
Incidence expressed as number of animals with findings; 10 males/female per group FNO: finding was not observed; NE: not examined

 

Table 2.List of findings in endocrine-related organs at the end of the exposure period and 28-day recovery period

	TMBPF Dose level (mg/kg-bw/day
	90-days dietary exposure				90-days plus 28-days recovery
	0	100	300	1000	0	1000
MALES
Absolute and relative organ weights
Testes + epididymides (g)	5.2840 ± 0.4550	5.0792 ± 0.7636	5.1579 ± 0.8963	5.5039 ± 0.3516	5.5446 ± 0.3184	5.8558 ± 0.3130
Testes + epididymides:BdW	1.21242 ± 0.06231	1.17388 ± 0.18480	1.21367 ± 0.22182	1.26020 ± 0.06276	1.29478 ± 0.15283	1.28292 ± 0.13141
Testes + epididymides:BrW	2.74828 ± 0.20426	2.69644 ± 0.38493	2.72460 ± 0.45800	2.85557 ± 0.14398	2.82936 ± 0.15144	2.91722 ± 0.17377
Adrenal glands (g)	0.04958 ± 0.00543	0.05079 ± 0.00831	0.04948 ± 0.00599	0.05692 ± 0.01185	0.05156 ± 0.00648	0.05280 ± 0.00720
Adrenal glands:BdW	0.01139 ± 0.00128	0.01176 ± 0.00225	0.01162 ± 0.00158	0.01305 ± 0.00272	0.01196 ± 0.00104	0.01146 ± 0.00080
Adrenal glands:BrW	0.02581 ± 0.00274	0.02700 ± 0.00459	0.02622 ± 0.00372	0.02956 ± 0.00608	0.02628 ± 0.00274	0.02622 ± 0.00273
Thyroid gland + parathyroids (g)	0.02175 ± 0.00472	0.02086 ± 0.00722	0.02302 ± 0.00478	0.02352 ± 0.00242	0.02162 ± 0.00449	0.02696 ± 0.00563
Thyroid gland + parathyroids:BdW	0.00499 ± 0.00101	0.00479 ± 0.00148	0.00539 ± 0.00113	0.00540 ± 0.00068	0.00498 ± 0.00076	0.00598 ± 0.00165
Thyroid gland + parathyroids:BrW	0.01132 ± 0.00245	0.01105 ± 0.00366	0.01219 ± 0.00260	0.01222 ± 0.00137	0.01100 ± 0.00199	0.01356 ± 0.00353
Organ histopathology
Thyroid gland	GO: NVL; HP: WNL	GO: NVL	GO: NVL	GO: NVL; HP: WNL	GO: NVL	GO: NVL
	90-days dietary exposure				90-days plus 28-days recovery
	0	100	300	1000	0	1000
Parathyroid gland	GO: NVL; HP: not examined, not present (2)	GO: NVL	GO: NVL	GO: NVL; HP: not examined, not present (1)	GO: NVL	GO: NVL
Adrenal gland	GO: NVL; HP: WNL	GO: NVL	GO: NVL	GO: NVL; HP: WNL	GO: NVL	GO: NVL
Pituitary gland	GO: NVL; HP: WNL	GO: NVL	GO: NVL	GO: NVL; HP: WNL	GO: NVL	GO: NVL
Testis	GO: NVL; HP: minimal hypospermatogenesis (1)	GO: NVL	GO: NVL	GO: NVL; HP: WNL	GO: NVL	GO: NVL
Epididymides	GO: NVL; HP: WNL	GO: NVL	GO: NVL	GO: NVL; HP: minimal mononuclear cell infiltrate (2)	GO: NVL	GO: NVL
Prostate	GO: NVL; HP: minimal mononuclear cell infiltrate (2)	GO: NVL	GO: NVL	GO: NVL; HP: minimal mononuclear cell infiltrate (5)	GO: NVL	GO: NVL
Seminal vesicles	GO: NVL; HP: WNL	GO: NVL	GO: NVL	GO: NVL; HP: WNL	GO: NVL	GO: NVL
FEMALES
Absolute and relative organ weights
Uterus + cervix (g)	0.7352 ± 0.4447	0.6084 ± 0.1524	0.6436 ± 0.1768	0.5082 ± 0.1239	0.7054 ± 0.0858	0.6132 ± 0.0407
Uterus + cervix:BdW	0.28813 ± 0.18735	0.23692 ± 0.05814	0.25822 ± 0.06718	0.20295 ± 0.05974	0.26728 ± 0.03310	0.24268 ± 0.02747
Uterus + cervix:BrW	0.39892 ± 0.22758	0.34062 ± 0.08429	0.35660 ± 0.09568	0.28104 ± 0.06968	0.38648 ± 0.05899	0.33780 ± 0.02962
Adrenal glands (g)	0.06322 ± 0.00533	0.06317 ± 0.00562	0.06357 ± 0.01262	0.05939 ± 0.00545	0.06314 ± 0.01247	0.06192 ± 0.00729
Adrenal glands:BdW	0.02451 ± 0.00245	0.02459 ± 0.00196	0.02565 ± 0.00557	0.02358 ± 0.00302	0.02394 ± 0.00482	0.02440 ± 0.00240
Adrenal glands:BrW	0.03462 ± 0.00339	0.03541 ± 0.00367	0.03524 ± 0.00692	0.03280 ± 0.00319	0.03458 ± 0.00730	0.03402 ± 0.00364
Thyroid gland + parathyroids (g)	0.01875 ± 0.00581	0.02127 ± 0.00613	0.01824 ± 0.00154	0.01998 ± 0.00273	0.01738 ± 0.00365	0.01914 ± 0.00309
Thyroid gland + parathyroids:BdW	0.00732 ± 0.00241	0.00831 ± 0.00257	0.00736 ± 0.00074	0.00790 ± 0.00099	0.00660 ± 0.00141	0.00758 ± 0.00151
Thyroid gland + parathyroids:BrW	0.01024 ± 0.00312	0.01186 ± 0.00320	0.01013 ± 0.00103	0.01104 ± 0.00156	0.00948 ± 0.00186	0.01058 ± 0.00194
Organ histopathology
Thyroid gland	GO: NVL; HP: WNL	GO: NVL	GO: NVL	GO: NVL; HP: WNL	GO: NVL	GO: NVL
Parathyroid gland	GO: NVL; HP: WNL	GO: NVL	GO: NVL	GO: NVL; HP: not examined, not present (4)	GO: NVL	GO: NVL
Adrenal glands	GO: NVL; HP: accessory adrenocortical nodule present (2)	GO: NVL	GO: NVL	GO: NVL; HP: accessory adrenocortical nodule present (1)	GO: NVL	GO: NVL
Pituitary gland	GO: NVL; HP: WNL	GO: NVL	GO: NVL	GO: NVL; HP: WNL	GO: NVL	GO: NVL
Ovaries	GO: NVL; HP: minimal atrophy (1), minimal follicular cyst (2)	GO: NVL; HP: minimal follicular cyst (2), mild angiectasis (1)	GO: NVL; HP: mild atrophy (1), minimal follicular cyst (3),	GO: NVL; minimal follicular cyst (6), mild follicular cyst (1)	GO: NVL; HP: minimal atrophy (1), mild atrophy (1), minimal follicular cyst (2), mild follicular cyst (2)	GO: NVL; HP: minimal atrophy (1), mild atrophy (1), moderate atrophy (1), minimal follicular cyst (3), moderate follicular cyst (1)
Cervix	GO: NVL; HP: mild epithelium atrophy (1), mild epithelium mucification (1)	GO: NVL	GO: NVL	GO: NVL; HP: WNL	GO: NVL	GO: NVL
Vagina	GO: NVL; HP: mild epithelium atrophy (1), mild epithelium mucification (1)	GO: NVL	GO: NVL	GO: NVL; HP: WNL	GO: NVL	GO: NVL
Uterus	GO: distention, fluid-filled, bilateral (2); HP: mild lumen dilation (2), moderate lumen dilation (2)	GO: distention, fluid-filled, bilateral (1) ; HP: moderate lumen dilation (1)	GO: distention, fluid-filled, bilateral (1) ; HP: mild lumen dilation (1)	GO: NVL; HP: mild lumen dilation (2)	GO: NVL	GO: NVL
Mammary glands	GO: NVL; HP: WNL	GO: NVL; HP: WNL	GO: NVL; HP: WNL	GO: NVL; HP: WNL	GO: NVL; HP: WNL	GO: NVL; HP: WNL
SD, standard deviation; BrW, brain weight; BdW, body weight; GO, gross observations; NVL, no visible lesions; HP, histopathology observations; WNL: within normal limits

 

 

Table 3.Selected mean (±SD) hematology, coagulation, and clinical chemistry parameters for 90-day repeated dose dietary toxicity study in Sprague-Dawley rats, followed by a 28-day recovery.

	Dose level (mg/kg bw/day)
	0	1000
Males
Serum creatinine (mg/dL)	0.64 ± 0.05	0.56 ± 0.05*
Alanine aminotransferase (U/L)	41.8 ± 4.8	35.2 ± 3.0*
Alkaline phosphatase (U/L)	75.2 ± 6.8	55.0 ± 4.2*
Fibrinogen (mg/dL)	360.0 ± 48.50	431.6 ± 24.3*

SD, standard deviation. *p ≤ 0.05

 

Table 4.Selected mean (±SD) hematology, coagulation, and clinical chemistry parameters for 90-day repeated dose dietary toxicity study in Sprague-Dawley rats, followed by a 28-day recovery.

	Dose level (mg/kg bw/day)
	0	750
Females
Phosphorus (mg/dL)	8.60 ± 0.42	7.76 ± 0.25*
Cholesterol esters (mg/dL)	118.2 ± 10.10	152.2 ± 15.80*
Albumin (g/dL)	3.32 ± 0.13	3.58 ± 0.16*
Aspartate aminotransferase (U/L)	95.6 ± 18.20	73.2 ± 4.20*
Alkaline phosphatase (U/L)	49.2 ± 13.9	32.2 ± 6.50*
Mean platelet volume (fL)	7.48 ± 0.18	7.10 ± 0.19*

SD, standard deviation. *p ≤ 0.05

 

Table 5.Selected mean (±SD) absolute and relative organ weights for 90-day repeated dose dietary toxicity in male Sprague Dawley rats, followed by a 28-day recovery.

	Dose level (mg/kg bw/day)
	0	1000
Males
Kidney (g)	2.49 ± 0.19	2.81 ± 0.16*
Kidney:BdW	0.578 ± 0.03	0.613 ± 0.03
Kidney:BrW	1.27 ± 0.09	1.40 ± 0.06*
Liver (g)	9.93 ± 1.59	12.43 ± 1.11*
Liver:BdW	2.89 ± 0.16	2.71 ± 0.10*
Liver:BrW	5.06 ± 0.16	6.18 ± 0.37*

SD, standard deviation. *p ≤ 0.05

 

Tabular data on terminal body weights were not provided in the article, however these can be estimated by using tabular data for absolute and relative organ weight. That is, by dividing e.g. absolute liver weight with relative liver weight, approximate terminal body weights can be estimated, as presented in the tables below.

 

Table 6.Estimated male terminal body weight (g):

	Control	100 mg/kg	300 mg/kg	1000 mg/kg
Data set 1^†	435,97	433,76	427,38	437,14
Data set 2^‡	434,48	434,43	426,67	440,63
^†Based on absolute and relative liver weight. Calculated based on supplemental data using the following formula: (Liver weight absolute) / (Liver weight, body weight relative) × 100.   ^‡Based on absolute and relative kidney weights. Calculated based on supplemental data using the following formula: (Kidney weight absolute) / (Kidney weight, body weight relative) × 100.

 

Table 9.Estimated female terminal body weight (g):

	Control	100 mg/kg	300 mg/kg	750 mg/kg
Data set 1^†	258,19	257,42	249,47	253,23
Data set 2^‡	260,34	258,06	250,00	251,52
^†Based on absolute and relative liver weights. Calculated based on supplemental data using the following formula: (Liver weight absolute) / (Liver weight, body weight relative) × 100.   ^‡Based on absolute and relative kidney weight. Calculated based on supplemental data using the following formula: (Kidney weight absolute) / (Kidney weight, body weight relative) × 100.

Conclusions:

The study-derived NOAEL was considered to be 750 mg/kg/day (females) and 1000 mg/kg/day (males).

Executive summary:

Materials and Methods: The test item, with a purity of >98% of the Substance, i.e. 4,4'-methylenedi-2,6-xylenol (CAS: 5384-21-4; EC: 226-378-9), was tested for sub-chronic toxicity in Sprague Dawley rats according to OECD Test Guideline 408 (adopted in 1998) and GLP standards. Male and female Sprague Dawley rats (obtained from Envigo, Frederick, MD, USA) were randomly assigned to vehicle control and treatment groups. At the age of 8 weeks, the Sprague Dawley rats were exposed to the test item by diet. Male rats in the main study (10 per dose level) were treated at dose levels corresponding to 0 (basal diet), 100, 300 and 1000 mg/kg bw/day whereas female rats in the main study (10 per dose level) were treated at dose levels corresponding to 0 (basal diet), 100, 300 and 750 mg/kg bw/day. Recovery animals (5 rats/sex/dose) treated at 0 (males/females), 750 (females) and 1000 mg/kg/day (males) for 90 days were also included in the study and these animals were allowed to recover for 28 days after 90 days of treatment. The diet used is the study was of the brand Teklad Certified Rodent Diet (Envigo Teklad Diets, Medison, WI, USA; batch 2018CM-102116MA). The diet preparation and formulation were conducted by Smithers Viscient Caroline Research Center. Dose levels were selected based on the results of a dose range-finding study (details not available). Animals were housed in Smithers Avanza Toxicology Services testing facility (Maryland, USA). They were housed individually in clear polysulfone plastic cages with SaniChip certified hardwood bedding. A light cycle of 12-h light/12-h dark was used. The animals were euthanized by carbon dioxide inhalation followed by exsanguination prior to necropsy. Smithers Avanza’s Institutional Animal Care and Use Committee (IACUC) reviewed the study protocol for its compliance with provisions of the US Dept of Agricultural Animal Welfare Act of 1996, the Public Health Service Policy on Human Care and Use of Laboratory Animals and the US Interagency Research Animal Committee Principles for the Utilization and Care of Research Animals. Quantitative data (body weights, body weight changes, food consumption, clinical pathology, and organ weights) from the treated groups were compared statistically to the data of the control group using one-way Analysis of Variance (ANOVA) techniques; sexes were analyzed separately. Prior to the ANOVA analysis, untransformed data were tested to determine if the data were normally distributed and had homogeneous variances among all groups using Shapiro-Wilk followed by the Levene’s test. The Dunnett’s t-test was used to determine which groups differed from the control group. Group comparisons were evaluated at the 0.05 (two-tailed) probability level. Statistical significance was considered at p ≤ 0.05.Results and discussions: Main study: No remarkable effects on mortality, cage-side/physical observations, body weight, body weight changes, food intake, ophthalmology, functional observatory battery, grip strength, locomotor activity, or gross pathology were observed up to 750 mg/kg (females) or 1000 mg/kg (males). Statistically significant changes in clinical chemistry included mild decreases in serum creatinine in females (≥100 mg/kg) and males (1000 mg/kg); mild increases in serum cholesterol in males (≥100 mg/kg) and females (≥300 mg/kg); minimal increases in serum total protein and globulin in males (1000 mg/kg); and minimal increases in serum total protein and albumin in females (750 mg/kg). Activated partial thromboplastin times were slightly increased in females (≥100 mg/kg) and males (≥300 mg/kg) however, no associated clinical findings or microscopic observations were made. Statistically significant changes in organ weight included increased absolute and relative liver weights in male and females (≥100 mg/kg) and increased absolute and relative kidney weights in females (750 mg/kg) and males (1000 mg/kg). The liver weight changes were accompanied by hepatocellular hypertrophy (minimal/mild) whereas the kidney weight changes were accompanied by tubular dilatation and hypertrophy (minimal/mild). Additional histopathological findings included thymic epithelial proliferation in females treated at ≥300 mg/kg (minimal/mild) and ovarian follicular cysts at 750 mg/kg (minimal/mild). Females at 0 and 750 mg/kg appeared to be cycling normally as oestrogen stages were recorded in all of these animals.Recovery groups:At the end of the 28-day recovery period, the following statistically significant changes were observed in high-dosed animals: a minimal decrease in serum creatinine in males; a minimal increase in serum cholesterol in females; a minimal increase in serum albumin in females; a mild decrease in serum alkaline phosphatase in both sexes; increased absolute and relative liver weights in both sexes; increased absolute and relative kidney weights in both sexes; and microscopic evidence of tubular dilatation in both sexes. No other remarkable effects were observed at 750 mg/kg (females) or 1000 mg/kg (males).Discussion and conclusions: None of the statistical changes in clinical chemistry were considered adverse effect of treatment by the authors of the study since they were either within normal biological variation, lacked dose-dependency, or lacked associated microscopic findings. The kidney effects (minimal/mild in severity) were not considered adverse effects of treatment by the authors, but rather due to exacerbated chronic progressive nephropathy, which is a common feature in the kidneys of aging rats (1, 2). The liver effects were considered adaptive responses to treatment by the authors. As such, the study-derived NOAEL values were 750 mg/kg/day (females) and 1000 mg/kg/day (males).

References:

1. Palazzi et al.Characterizing "Adversity" of Pathology Findings in Nonclinical Toxicity Studies: Results from the 4th ESTP International Expert Workshop. Toxicol Pathol 2016; 44: 810-824.

2. Pandiri et al. Is It Adverse, Nonadverse, Adaptive, or Artifact? Toxicol Pathol 2017; 45: 238-247.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

750 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Klimisch 2

Repeated dose toxicity: inhalation - systemic effects

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

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

Repeated Dose oral toxicity

Two studies have been performed to evaluate the oral toxicity of the test chemical when dosed repeatedly for longer durations. The executive summaries are presented below:

Study 1

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 of the test chemical exerted 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.

Study 2:

The test chemical was given by diet to 10 rats per sex per dose for a total of 90 days. Males were treated at doses corresponding to 0, 100, 300 and 1000 mg/kg/day whereas female rats were treated at doses corresponding to 0, 100, 300 and 750 mg/kg/day. Recovery animals (5 rats/sex/dose) treated at 0 and 750 (females) and 1000 mg/kg/day (males) for 90 days were also included in the study and these animals were allowed to recover for 28 days after the final dose. Dose levels were selected based on the results of a dose range-finding study. The study followed OECD 408 (adopted in 1998) and was conducted in compliance with GLP standards as per information provided by the authors of the study report.Results (main study):No remarkable effects on mortality, cage-side/physical observations, body weight, body weight changes, food intake, ophthalmology, functional observatory battery, grip strength, locomotor activity, or gross pathology were observed up to 750 mg/kg (females) or 1000 mg/kg (males). Statistically significant changes in clinical chemistry included mild decreases in serum creatinine in females (≥100 mg/kg) and males (1000 mg/kg); mild increases in serum cholesterol in males (≥100 mg/kg) and females (≥300 mg/kg); minimal increases in serum total protein and globulin in males (1000 mg/kg); and minimal increases in serum total protein and albumin in females (750 mg/kg). Activated partial thromboplastin times were slightly increased in females (≥100 mg/kg) and males (≥300 mg/kg) however, no associated clinical findings or microscopic observations were made. Statistically significant changes in organ weight included increased absolute and relative liver weights in male and females (≥100 mg/kg) and increased absolute and relative kidney weights in females (750 mg/kg) and males (1000 mg/kg). The liver weight changes were accompanied by hepatocellular hypertrophy (minimal/mild) whereas the kidney weight changes were accompanied by tubular dilatation and hypertrophy (minimal/mild). Additional histopathological findings included thymic epithelial proliferation in females treated at ≥300 mg/kg (minimal/mild) and ovarian follicular cysts at 750 mg/kg (minimal/mild). Females at 0 and 750 mg/kg appeared to be cycling normally as oestrogen stages were recorded in all of these animals.Results (recovery groups):At the end of the 28-day recovery period, the following statistically significant changes were observed in high-dosed animals: a minimal decrease in serum creatinine in males; a minimal increase in serum cholesterol in females; a minimal increase in serum albumin in females; a mild decrease in serum alkaline phosphatase in both sexes; increased absolute and relative liver weights in both sexes; increased absolute and relative kidney weights in both sexes; and microscopic evidence of tubular dilatation in both sexes. No other remarkable effects were observed at 750 mg/kg (females) or 1000 mg/kg (males).Discussion:None of the statistical changes in clinical chemistry were considered adverse effect of treatment since they were either within normal biological variation, lacked dose-dependency, or lacked associated microscopic findings. The kidney effects (minimal/mild in severity) were not considered adverse effects of treatment but rather due to exacerbated chronic progressive nephropathy, which is a common feature in the kidneys of aging rats. The liver effects were considered adaptive responses to treatment.Conclusion:The study-derived NOAEL was considered to be 750 mg/kg/day (females) and 1000 mg/kg/day (males).

Repeated dose inhalation toxicity

A short term repeated dose toxicity study doesnot need to be conducted as the exposure of humans via inhalation during production and/or use is highly unlikely based on the provided thorough and rigorous risk assessment. The test substance has very low vapor pressure (2,56 x 10-6 Pa at 25°C). Also, the particle size distribution of the substance was found to vary between 55-250 µm thus the potential for inhalable dust formation is low. Moreover, the normal conditions of use of this substance will not result in aerosols, particles or droplets of an inhalable size, thus exposure to humans via the inhalation route will be unlikely to occur, and therefore acute toxicity by inhalation route was considered to be waived.

Repeated dose dermal toxicity

A short term repeated dose toxicity study doesnot need to be conducted as the exposure of humans via dermal during production and/or use is highly unlikely based on the provided thorough and rigorous risk assessment.The acute dermal toxicity value for (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.

Justification for classification or non-classification

The substance is regarded to be classified as Not Classified for STOT RE as per Regulation EC 1907/2006 based on a rat NOAEL of 1000 mg/kg bw/day in a study performed according to OECD 407, a female rat NOAEL of 750 mg/kg bw/day in a study performed according to OECD 408, and a male rat NOAEL of 1000 mg/kg bw/day in a study performed according to OECD 408.