3-(2,2-dimethyl-3-hydroxypropyl)toluene

Endpoint summary

Description of key information

In a key subacute oral repeated dose toxicity study in rats according to OECD guideline 407, the NOAEL was 317.5 mg/kg bw/day (males) or  310 mg/kg bw/day (females).

In a supporting 14-day repeated dose palatibility study in rats similar to OECD guideline 407, the NOEL was found to be 373.9 mg/kg bw/day (males) or 737.2 (females) mg/kg bw/day.

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

Study period:

1987-11-10

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

1981

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: yes
- Age at study initiation: ca. 4 weeks
- Fasting period before study: not specified
- Housing: 5 per sex per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 + /-2 °C
- Humidity: at least 40 %
- Air changes: ca. 10 per hr
- Photoperiod: 12 / 12 hrs dark / hrs light

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was added to the stock diet to provide the concentrations wanted. The intended concentrations of the test substance in the diets were 0 (control), 800, 3000 or 10000 ppm. The diet with the highest concentration was prepared first by adding the appropriate amount of the test substance (liquidized at ambient temperature) to the stock diet. The diets with the lower concentrations were obtained by diluting the top-dose diet with stock diet. Homogeneity of the diets was achieved by mixing for 2 minutes in a mechanical blender (Stephan cutter). One batch of 6 kg of each of the diets was prepared on the starting day of the study. The diets were stored at -20°C until use. Twice a week the diets in the feeders were refreshed with portions of the test- or control diets (thawed immediately before use). Immediately after preparation of the diets, samples were taken and subsequently stored at -20°C.

DIET PREPARATION
- Rate of preparation of diet (frequency): once
- Storage temperature of food: -20 °C

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Immediately after preparation of the batch of diets, Samples were taken and subsequently stored at -20°C. The content of test item in each diet was determined in triplicate. The stability of the test item in the test diets was established by reanalyzing samples after storage for 2, 4 and 7 days in an open Container at room temperature. For the low-dose diet a liniar regression line was calculated. From this line, the mean actual daily concentrations during the 3 and 4 day feeding periods in a week, were calculated as well as the overall actual dietary concentration in the low-dose group. The analyses were carried out by TNO, Institute CIVO-Analysis according to an HPLC method provided by the sponsor (HPLC column: stainless steel, 120 x 4.6 mm i.d., packed with 5 um Hypersil ODS).

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily, 7 days per week

Dose / conc.:

0 ppm

Remarks:

control group

Dose / conc.:

800 ppm

Dose / conc.:

3 000 ppm

Dose / conc.:

10 000 ppm

No. of animals per sex per dose:

Male: 5 animals at 0 mg/kg bw/day
Male: 5 animals at 86.4 mg/kg bw/day
Male: 5 animals at 317.5 mg/kg bw/day
Male: 5 animals at 1102 mg/kg bw/day

Female: 5 animals at 0 mg/kg bw/day
Female: 5 animals at 82.5 mg/kg bw/day
Female: 5 animals at 310 mg/kg bw/day
Female: 5 animals at 1074.25 mg/kg bw/day

Control animals:

yes, plain diet

Details on study design:

- Rationale for animal assignment: random
- Fasting period before blood sampling for clinical biochemistry: not specified

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: initially and ond day 7, 14, 21, 28

FOOD CONSUMPTION AND FOOD EFFICIENCY:
Food intake was measured per cage (5 animals) weekly, and the efficiency of food utilization was calculated and expressed as gram weight gain per gram food consumed.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
Water intake was measured on a cage basis (5 animals) daily, during the first and the third week of the study.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 23
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: all
- Parameters checked: haemoglobin; packed cell volume; red blood cells; white blood cells; differential white blood cell count; thrombocytes; mean corpuscular volume (MCV); mean corpuscular haemoglobin (MCH); mean corpuscular haemoglobin concentration (MCHC)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 25 (glucose), day 28 (other parameters indicated below)
- Animals fasted: yes
- How many animals: all
- Parameters checked: glucose (day 25); alkaline phosphatase (ALP); glutamic-axalacetic transaminase (GOT)/ aspartate amino transferase (ASAT); glutamic-pyruvic transaminase (GPT)/alanine amino transferase (ALAT); gamma glutamyl transpeptidase; total protein; albumin; urea; ceatinine; bilirubin total; sodium (Na); potassium (K); Calcium (Ca); Chloride (Cl); inorganic phosphate

URINALYSIS: Yes
- Time schedule for collection of urine: day 25
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: volume; density

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
adrenals; spleen; heart; testes; kidneys; thymus; liver; thyroid (with parathyroids); mesenteric lymph nodes; urinary bladder; ovaries; gross lesions

HISTOPATHOLOGY: Yes
Tissue samples required for microscopic examination were processed and embedded in paraffin wax. Sections were cut at 5 µm, stained with haematoxylin and eosin and then examined macroscopically. Histopathological examination was carried out on the liver, kidneys, heart,
adrenals, spleen and mesenteric lymph node of all animals of the control group and of the top-dose group, and on organs showing gross alterations at autopsy.

Statistics:

Data on body weights were evaluated by one-way analysis of co-variance followed by Dunnett's multiple comparison test. Data an red blood cell variables, clinical chemistry values, volume and density of the urine, and organ weights were evaluated by one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. Differential white blood cell counts were analyzed by the Mann Whitney U-test.,The histopathological changes were examined by Fisher's exact probability test.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no noticeable differences in appearance or behaviour between the treated rats and the controls.

Mortality:

no mortality observed

Description (incidence):

None of the rats died in the course of the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights were relatively low in males of the mid- and top-dose group and in females of all treatment groups. The differences with the controls did, however, not reach the level of statistical significance.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food intake was relatively low in males of the mid- and top-dose group and in females of all treatment groups.

Food efficiency:

no effects observed

Description (incidence and severity):

Food conversion efficiency was generally slightly decreased in the top-dose group in both sexes.

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

no effects observed

Description (incidence and severity):

There were no outstanding differences in water intake among the various groups, but in the top-dose group the figures were generally higher than in the other groups.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

A slight, though statistically significant decrease in MCV and MCH was observed in males of the top-dose group. Total white blood cell count was decreased in males of the top-dose group, and in females of the mid-dose group. The latter change was considered to be incidental. The differential white blood cell count did not reveal significant changes in the distribution of the individual cells.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Plasma sodium concentration showed a dose-related increase in females of the mid- and top-dose group. There were no other statistically significant changes in clinical chemistry parameters, except for an incidental decrease in glucose concentration in males of the low-dose group.

Urinalysis findings:

no effects observed

Description (incidence and severity):

The urine concentration test did not indicate treatment-related changes in renal function.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The relative weight of the liver was statistically significantly increased in males of the mid- and top-dose group and in females of the top-dose group. The relative weight of the kidneys was statistically significantly increased in females of the mid- and top-dose group. There were
no statistically significant changes in absolute organ weights.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Gross examination at autopsy did not reveal any treatment related changes, either in males or in females.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Upon microscopic examination, special attention was paid to the liver and kidneys, in view of their increased relative weight. However, neither in these organs nor in any of the other organs examined, histopathological changes were observed that could be related to treatment. Most changes observed are common findings in the strain of rats used. Moreover, the lesions were about equally distributed between the top-dose rats and the controls, or they occurred in a single animal only.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

haematology

organ weights and organ / body weight ratios

other: corresponding to 317.5 mg/kg bw (m) and 310 mg/kg bw/d (f)

Key result

Dose descriptor:

NOEL

Effect level:

800 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: corresponding to 86.4 mg/kg bw (m) and 82.5 mg/kg bw/d (f)

Key result

Critical effects observed:

no

Conclusions:

Under the conditions of this study, the 28 d NOAEL in male and female rats was determined to be 317.5 or 310 mg/kg bw/d, respectively, based on body weight and food consumption decrease as well as liver and kidney organ weight changes and haematological changes.

Executive summary:

The toxicity of the test item was examined in a sub-acute oral toxicity study with four groups of 5 male and 5 female Wistar rats, which received the test substance in the diet for a period of 4 weeks. The intended dietary levels of the test item were 0 (control), 800, 3000 or 10000 ppm, but the actual levels were lower due to disappearance of the test substance from the diet. General condition and behaviour were not adversely affected by the administration of the test item. None of the rats died in the course of the study. There were no statistically significant differences in body weights among the various groups, although body weights and food intake were relatively low in males of the mid- and top-dose group and in females of all dose groups. In males of the top-dose group decreases were observed in total white blood cell counts and in MCV and MCH. Plasma sodium concentration was increased in females of the mid- and top-dose group. There were no treatment-related changes in volume and density of the urine among the various groups. The relative weight of the liver was increased in males of the mid- and top-dose group and in females of the top-dose group. The relative weight of the kidneys was increased in females of the mid- and top-dose group. Gross examination at autopsy and histopathological examination did not reveal any treatment-related changes. From the present study it was concluded that certainly the low-dose level was a clear no-effect level. However, as the changes observed at the mid-dose level were not considered to be of obvious toxicological significance, even the mid-dose level was not a clear effect level. The NOAEL was thus considered to be 3000 ppm, corresponding to 317.5 or 310 mg/kg bw/d in males or females, respectively.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

310 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP and guideline study

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

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

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Subacute toxicity study, rats, (OECD 407)

The toxicity of the test item was examined in a sub-acute oral toxicity study with four groups of 5 male and 5 female Wistar rats, which received the test substance in the diet for a period of 4 weeks. The intended dietary levels of the test item were 0 (control), 800, 3000 or 10000 ppm, but the actual levels were lower due to disappearance of the test substance from the diet. General condition and behaviour were not adversely affected by the administration of the test item. None of the rats died in the course of the study. There were no statistically significant differences in body weights among the various groups, although body weights and food intake were relatively low in males of the mid- and top-dose group and in females of all dose groups. In males of the top-dose group decreases were observed in total white blood cell counts and in MCV and MCH. Plasma sodium concentration was increased in females of the mid- and top-dose group. There were no treatment-related changes in volume and density of the urine among the various groups. The relative weight of the liver was increased in males of the mid- and top-dose group and in females of the top-dose group. The relative weight of the kidneys was increased in females of the mid- and top-dose group. Gross examination at autopsy and histopathological examination did not reveal any treatment-related changes. From the present study it was concluded that certainly the low-dose level was a clear no-effect level. However, as the changes observed at the mid-dose level were not considered to be of obvious toxicological significance, even the mid-dose level was not a clear effect level. The NOAEL was thus considered to be 3000 ppm, corresponding to 317.5 or 310 mg/kg bw/d in males or females, respectively.

 

Subacute toxicity study, rats, (OECD 407 (two-week palatibility study)

In a two-week palatibility study similar to OECD guideline 407, the potential toxicity of the test item was evaluated to determine the dose levels for reproduction/developmental toxicity screening test when administered daily by orally (dietary) to Sprague-Dawley rats of both sexes for 2 weeks. Test groups consisted of dose groups which received 0.5, 1 and 2 % of the test substance in feed (corresponding to achieved concentrations of 373.9, 776.3 and 1572.8 mg/kg bw/day in males and 371.8, 737.2 and 1435.5 mg/kg bw/day in females) and a control group (10 mL of acetone for 1 kg of powder feed) with 5 animals of each sex per group. All animals were dosed daily for 2 weeks by orally (dietary). During the observation period, observation of clinical signs, measurement of body weights and food consumption were performed, and after the observation period, hematology and clinical chemistry examinations, organ weight measurement, gross post mortem examinations were performed. No abnormal clinical signs or mortality were observed during the duration of the study. There were no test substance-related differences in body weight, food consumption, hematology and clinical signs and necropsy. The absolute and/or relative weight of livers were significantly increased in male in the 1, 2 % and in female in the 2 % dosing group compared to the control group. Based on the results, the maximal high dose level for the OECD 421 study should be selected at 1.5 % to gain a daily test substance uptake of about 1000 mg/kg b.w./day.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on repeated dose toxicity, the test item does not require classification for STOT RE according to Regulation (EC) No 1272/2008 (CLP), as amended for seventeenth time in Regulation (EU) No 2021/849.