2,4-di-tert-butylphenyl 3,5-di-tert-butyl-4-hydroxybenzoate

Repeated dose toxicity: oral

Administrative data

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:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1975

Report date:

1975

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)

Version / remarks:

1998

Deviations:

no

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,4-di-tert-butylphenyl 3,5-di-tert-butyl-4-hydroxybenzoate

EC Number:

224-166-0

EC Name:

2,4-di-tert-butylphenyl 3,5-di-tert-butyl-4-hydroxybenzoate

Cas Number:

4221-80-1

Molecular formula:

C29H42O3

IUPAC Name:

2,4-di-tert-butylphenyl 3,5-di-tert-butyl-4-hydroxybenzoate

Test animals

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Olac (Western) Limited
- Age at study initiation: 30 weeks (mean)
- Weight at study initiation: 9805 g (mean)
- Housing: All dogs were housed singly in kennels at Alconbury.
- Diet: The dogs were fed throughout with a complete dry diet (Spratt's Dog Diet). The diet is normally obtained in expanded pellet form, but as the test compound had to be introduced into the diet, a finely ground form of meal was obtained, made to the same standard as the pelleted diet. Each dog was offered 400 grams daily. When new food was offered in the morning, the residue from the previous day was removed and weighed. From these residual weights food intake was calculated. It was, therefore, possible for each animal to consume a maximum of 2800 grams of dry diet per week. Supplements of milk (approximately 200 mL) were offered to each animal on weekdays only throughout the experiment.
- Water: Water was freely available in the kennels at all times

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: ground diet

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: 920 grams of test material were weighed out weekly and thoroughly mixed with 3680 grams of powdered diet to give 4.6 kg of premix containing 200000 ppm. The diets for the three dosage groups were obtained by serial dilution of this premix with more ground diet.

DIET PREPARATION
- Mixing appropriate amounts with: Dry diet (Spratt's Dog Diet)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the diets fed to the dogs have been analysed for their content of the test item by spectrophotometry. The relative reproducibility of the method is +/- 10%. Within the limits of error of the sampling technique and of the analytical method there was good correspondence between the concentrations found in the diets and the nominal values.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

5 000 ppm

Remarks:

corresponding to 171 mg/kg and 187 mg/kg in males and fermales, respectively

Doses / concentrations 2

Dose / conc.:

10 000 ppm

Remarks:

corresponding to 340 mg/kg and 399 mg/kg in males and fermales, respectively

Doses / concentrations 3

Dose / conc.:

20 000 ppm

Remarks:

corresponding to 690 mg/kg and 704 mg/kg in males and fermales, respectively

No. of animals per sex per dose:

Group 4 (High dose level): 5 males and 5 females
Group 2+3 (Intermediate dose level): Each group 4 males and 4 females
Group 1 (Controls): 6 males and 6 females

Control animals:

yes, concurrent no treatment

Details on study design:

- Post-exposure recovery period: 4 weeks (1 male and 1 female - High dose level, 2 males and 2 females - Control)

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE :
- Food consumption for each animal determined and mean daily diet consumption in mg/kg/day: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: 5 days

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Once before dietary intake and after 4, 8 and 12 weeks of dietary intake

HAEMATOLOGY: Yes
- Time schedule for collection of blood: once before dietary intake commenced
after 4 weeks of dietary intake
after 8 weeks of dietary Intake
after 12 weeks of dietary intake
- Investigations were performed on all dogs. Samples of blood were obtained prior to feeding on the day of examination with the animals in a fasting
condition, food having been removed from the kennels approximately 16 hours previously.
- Following parameters were checked: Erythrocyte sedimentation rate, packed cell volume, haemoglobin, red cell count, reticulocyte count, Absolute indices: Mean corpuscular haemoglobin concentration, total white cell count, differential count, platelet count - direct visual count, prothrombin index

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: once before dietary intake commenced, after 4 weeks of dietary intake, after 8 weeks of dietary Intake, after 12 weeks of dietary intake
- Fasting: Yes: food have been removed from the kennels approximately 16 hours previously.
- The following parameters were checked: Plasma urea, plasma Glucose, Total serum proteins, serum protein electrophoresis and AG ratio, Serum alkaline phosphatase, Serum glutamic - pyruvic transaminase, serum glutamic oxaloacetic transaminase, Serum Bilirubin, Serum cholesterol, Electrolytes: Sodium and Potassium

URINALYSIS: Yes
The urine specimens collected for routine studies include an assessment of concentrating power of the kidneys. Since urine passed overnight is more consistent in its constitution, samples collected over this period are considered to be more indicative of basic renal function than specimens passed during the daytime. For this reason, therefore, the procedure adopted in collecting urine specimens was to withdraw the supply of drinking water for the animals at midday, but delay the collection of urine until about 5 p.m. All urine passed between 5 p.m. and 9 a.m. the following morning was collected in suitable containers and the whole specimen was analysed.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

On completion of the dosing period or subsequent observation period each animal received on Intravenous injection of sodium pentobarbitone and, when deeply unconscious, was rapidly exsanguinated by incision of the carotid blood vessels. Immediately afterwards a full macroscopic examination of the tissues was performed during which the following organs were removed and weighed:

Brain
Pituitary
Heart
Lungs
Liver
Spleen
Pancreas
Thymus
Prostate/Uterus
Kidneys
Thyroids
Adrenals
Gonads (including epididymis)

Small portions of these tissues together with pieces of: aorta (arch and abdominal), trachea, lymph nodes (cervical and mesenteric), gall bladder, urinary bladder, salivary gland, tongue, oesophagus, stomach, ducxienum, jejunum, ileum, colon, skin, mammary gland, skeletal muscle, bone marrow, peripheral nerve, eye and optic nerve and spinal cord were then placed In fixative.

Statistics:

Whenever it was necessary to determine whether significant differences existed between mean values relating to test and control animals, the method used was analysis of variance followed by Student's ' t ' test, the results being expressed as the 'least significant difference' (LSD), that is, the least difference that had to exist between mean values for dosed and control animals for significance at specified levels of probability.
The only other statistical procedure adopted was that used to describe the range of results obtained during the pre-dosing investigation. These results have been described by the grand mean and the "95% range", the latter phrase being used to describe the mean - ('t' x standard deviation).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

One dog (20000 ppm) passed liquid faeces containing mucus, and possibly blood on day 29 of dietary Intake. However, a sample sent for examination gave a negative result to the test for occult blood (Modification of Ortho-toluidine technique). Another dog (20000 ppm) showed a sudden and complete loss of appetite on days 29 and 30 of dietary intake. Green, liquid faeces and vomitus were observed in the kennel on both days. On day 31 the animal passed red coloured urine and a slight purulent discharge from the vulva was noted. However, most of the food offered was consumed on this day. No other signs were observed during the dosing or observation periods.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Most dogs were gaining weight satisfactorily before dosing commenced. One dog lost weight during the final pre-dosing week and showed erratic weight changes subsequently.
Inspection of the group mean bodyweight shows that weight gain was similar for all groups. It may be concluded therefore, that there was no adverse effect on bodyweight that could be related to administration of the test compound. Bodyweight remained satisfactory during the subsequent observation period for all dogs.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

With the onset of dietary Intake and throughout the dosing period food consumption remained satisfactory for all dogs (except for a short period with one dog). It may be concluded that dietary intake of the test substance up to a level of 20000 ppm had no effect on appetite. Appetite remained satisfactory for those dogs maintained for observation study.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

During weeks 1-4 and 9-12 water consumption remained comparable for all groups, and it was concluded that dosage with the test compound produced no adverse effect on water consumption.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No abnormalities were detected that could be related to dietary intake of the test item.

Haematological findings:

no effects observed

Description (incidence and severity):

All finding were considered to be within normal limits.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

One dogs of the high dose group and one dog of the intermediate dose group had plasma glucose levels which just exceeded or equalled our accepted upper limit of 110 mg%. All finding were considered to be within normal limits.

Urinalysis findings:

no effects observed

Description (incidence and severity):

All results were within normal limits and all dogs were showing satisfactory urinary concentration.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The uterus weights for 4 dogs (20000 ppm) were considered normal for dogs undergoing oestral changes.
When expressed either In grams or as a percentage of bodyweight and brain weight, it was possible to show that the mean spleen weights for dogs receiving 10000 ppm or 20000 ppm were significantly greater than the control value. However, the individual values for these groups were well within normal limits for animals of this age, whilst the control values tended to be lower than usual.
All other organ weights were considered to be within normal limits.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No morphological change or variation from normal was seen In any of the tissues examined that was considered to be related to treatment.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

690 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No mortality or other symptoms occurred during the study.

2

Key result

Dose descriptor:

NOAEL

Effect level:

704 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No mortality or other symptoms occurred during the study.

Target system / organ toxicity

Key result

Critical effects observed:

no