Ethylene bis[3,3-bis(3-tert-butyl-4-hydroxyphenyl)butyrate]

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1977

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well performed and reported study with design equivalent to OECD 408/415.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1977

Report date:

1977

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

test animals were exposed to substance in utero before study start, ophthalmology and FOB not performed

Principles of method if other than guideline:

The toxicity of DTB-glycolester was examined in a sub-chronic (16-week) study in which the test substance was fed to groups of 50 male and 50 female weanling rats each at levels of 0 (control), 0.1, 0.4 or 1.6 % in stock diet. The rats were derived from parents that had been fed the test diets for 31 days before mating and during gestation and lactation.

GLP compliance:

no

Remarks:

study was performed before GLP implementation

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Bis-(3,3-di-(4-oxy-3-tert. Butyl-phenyl)butane acid)-gylcolester

IUPAC Name:

Bis-(3,3-di-(4-oxy-3-tert. Butyl-phenyl)butane acid)-gylcolester

Constituent 2

Reference substance name:

DTB-glycolester

IUPAC Name:

DTB-glycolester

Test material form:

solid: crystalline

Details on test material:

white crystalline material, pure substance

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: : the rats were selected from the first litter of parents which had been fed diets containing the test item at levels of 0, 0.1, 0.4 and 1.6% for 31 days before mating and during gestation and lactation; these parent rats were obtained from the Central Institute for the Breeding of Laboratory Animals TNO, Zeist, The Netherlands
- Age at study initiation: weanling age (parent animals were also treated with the test substance while reproduction phase)
- Weight at study initiation: 37.1 - 46.5 g (males), 37.7 - 46.2 g (females)
- Housing: in suspended, screen-bottomed, stainless steel cages (5 per cage)
- Diet (e.g. ad libitum): CIVO stock diet by means of a mechanical blender (Lödige type) at levels of 0, 0.1, 0.4 or 1.6 % test substance, ad libitum
- Water (e.g. ad libitum): water, ad libitum
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24-26°C
- Humidity (%): 50%
- Photoperiod (hrs dark / hrs light): 12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: CIVO stock diet

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): The test material was thoroughly mixed into CIVO stock diet by means of a mechanical blender (Lödige type) at levels of 0 (control), 0.1, 0.4 or 1.6%. Batches of 40 kg of each diet were freshly prepared every three to four weeks and stored in an unheated room at ambient temperature.

VEHICLE: CIVO stock diet

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

16 weeks via diet (test animals were pretreated in utero via parent animals and lactation)

Frequency of treatment:

continuously

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0.1 %
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0.4 %
Basis:
nominal in diet

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1.6 %
Basis:
nominal in diet

No. of animals per sex per dose:

50

Control animals:

yes, plain diet

Details on study design:

The toxicity of DTB-glyoclester was examined in a sub-chronic (16-week) study in which the test substance was fed to groups of 50 male and 50 female weanling rats each at levels of 0 (control), 0.1, 0.4 or 1.6% in stock diet. The rats were derived from parents which had been fed the test diets for 31 days before mating and during gestation and lactation. Observations were made on general appearance and behavior, growth, food intake and food efficiency, haematology, blood biochemistry and urine composition. At week 17 all rats were killed and examined grossly for pathological changes. The major organs were weighed and extensive histopathological examinations were carried out on 15 males and 15 females of each group.

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
The animals were observed at intervals for signs of illness and or abnormal behavior.

BODY WEIGHT: Yes
Individual body weights were recorded at the end of weeks 0, 1, 2, 4, 6, 8, 10, 12 and 16.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
The food intake of 20 males and 20 females of each group was measured during the first four weeks and in weeks 12 and 13.

FOOD EFFICIENCY:
Food efficiencies were calculated from the gain in body weight and the total food consumption over the first four weeks.

HAEMATOLOGY: Yes
Blood samples from the tip of the tail of ten rats/sex/group were taken at week 15 for measurements of the following parameters: Haemoglobin, Packed cells volume as microhaematocrit, red cell count, white cell count, differential white cell count (Lymphocytes, Neutrophils, Monocytes and Eosinophils)

CLINICAL CHEMISTRY: Yes
At necropsy, blood was collected from 15 rats/sex/group by punction of the aorta abdominalis. In sera, which were obtained by centrifugation the following determinations were carried out: Serum alkaline phosphatase (SAP), Serum glutamic-oxalacetic transaminase (SGOT), Serum glutamic-pyruvic transaminase (SGPT), total Serum protein (TSP), Albumin

URINALYSIS: Yes
Individual urine samples were collected at week 15 from 10 rats/sex/group and at week 16 from 10 rats/group in females only. Collections were made during the last 16 hours of a 14-hour period of deprivation of food and water. The following measurements were made of individual urine samples: volume, urine glutamic-oxalacetic transaminase, specific gravity. The following qualitative tests were made in pooled samples from 10 rats/sex/group: pH, protein, sugar, occult blood, ketones and microscopy of spun deposit (Erythrocytes, Leucocytes, epithelial cells, amorph deposits, crystalline deposit, casts, bacteria, worm eggs and sperm cells.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
At week 17, 15 animals/sex/group were ether-anaesthetized, bled to death by puncture of the aorta abdominalis and examined grossly for pathological changes.

HISTOPATHOLOGY: Yes
Detailed microscopic examination was performed on all of the 15 male and 15 female rats of the highest dose group and of the control group. Haematoxylin-eosin stained paraffin sections of the organs weighed and also of the following organs were examined: lung, trachea, salivary glands, prostate, seminal vesicle, coagulating gland, epididymis, uterus, urinary bladder, skeletal muscle, thoracic aorta, oesophagus, gastro-intestinal tract, pancreas and axillary and mesenteric lymph nodes.
The remaining 35 animals/sex/group were killed by decapitation. A wide variety of organs was collected and preserved in a 4% neutralized Formaldehyde solution for possible, additional histological examination in the future.

Other examinations:

Organ weight:
The following organs were weighed: heart, kidneys, liver, spleen, brain, testicles/ovaries, thymus, thyroid and adrenals.

Statistics:

All data were analysed statistically by means of Student’s t-test.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

one male, cause of death could not be detected

Mortality:

mortality observed, treatment-related

Description (incidence):

one male, cause of death could not be detected

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

decreased body weight gain in males

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

Clinical signs:
No abnormalities of condition or behavior were observed.

Mortality:
One male rat of the control group died in week 16 of the study. No cause of death could be detected.

Body weights/food efficiency:
Already at the start of the study, body weights were statistically significant decreased at the highest dose level (1.6%) as compared to controls. The low initial body weight was caused by growth depression during lactation period. In males, the differences with the controls increased during the course of the study, but this did not happen in females. Body weights in the medium-dose (0.4%) were slightly higher than in controls, especially in males; in the low-dose group (0.1%) body weights were similar to those of the controls.
Food intake was distinctly decreased in the group fed 1.6% DTB-glycolester during the first two weeks of the study. Thereafter, the difference with the controls diminished and at weeks 12 and 13 the figures for males were similar and for females even higher than those of the controls. The food intake in the medium and low dose groups was generally comparable to that of the controls.
Food efficiency figures were not adversely affected by feeding DTB-glycolester.

Hematology:
There were no changes in the parameters examined which could be attributed to treatment. The only statistically significant difference with the controls consisted of a decrease in the percentage of lymphocytes in males at 0.1%. Since at the higher dose levels no such difference occurred, the low value at 0.1% is considered to be incidental.

Blood chemistry:
The activities of the serum enzymes Glutamic-oxalacetic transaminase, Glutamic-pyruvic transaminase and Alkaline phosphatase did not show any dose-related difference amongst the various groups.
Total serum protein values showed a slight though statistically significant decrease in males of the top-dose group. Since the serum Albumin content of this group was equal to that of the controls, the decrease in total serum protein is probably caused by a relatively low Globulin level. The other statistically significant decreases in total serum protein and serum Albumin occurred in the medium and low-dose groups and are, therefore, considered to be incidental findings.

Kidney function/Urinalysis:
There was a dose-related decrease in specific gravity, with a simultaneous increase in the volume of the urine in females fed 0.4 and 1.6% DTB-glycolester. These findings were, however, not confirmed by determinations, one week later, of the same parameters in the animals. Therefore, these changes are considered to be incidental. Glutamic-oxalacetic transaminase values of urine samples were comparable in the various groups.
There were no changes in the composition of the urine which could be ascribed to the feeding of DTB-glycolester. The occurrence of occult blood in females at week 15 was probably caused by a contamination of blood from the tail-tips, since the collection of blood and urine was done on the same day. Moreover, at week 16 no occult blood was found in the animals.

Relative Organ weights:
The relative organ weights of the liver were slightly increased in females of the high-dose group. In males of this group the liver weights also tended to be increased, but the difference with the controls was not statistically significant.
The relative weight of the kidneys only showed a statistically significant increase in the top-dose group males.
The increase in the relative weights of brain and testicles exhibited the well-known inverse relationship with body weight and are, therefore, not considered to be of toxicological significance.
The relative weights of ovaries showed a dose-related increase in the medium-dose and high-dose-groups.
The statistically significant changes in the weights of the spleen and thyroid in females were either not dose-related or only occurred in the intermediate-dose level. Therefore, they are not attributed to the feeding of the test substance.

Gross examination:
Gross examination at necropsy did not reveal pathological findings that could be ascribed to treatment.

Microscopy:
The degree and incidence of the changes observed were about equally distributed among the test groups and controls or they occurred only in a single animal. None of these abnormalities, which are common findings in the strain of rats used, could, therefore, be related to the feeding of the test compound. Although special attention was paid to the microscopic appearance of ovaries and testes, because of their increased relative weights, no abnormalities could be detected. The cause of death of the control animal, which died in week 16 of the experiment, could not be established.

Effect levels

Dose descriptor:

NOEL

Effect level:

1.6 other: % in diet

Sex:

male/female

Basis for effect level:

other: no deleterious effects of toxicological relevance observed up to 1.6% in diet

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Since no treatment-related, deleterious effect occurred in the medium-dose group it was concluded that the NOAEL of the compound was 1.6% in the diet, which is equivalent to 1600 mg/kg bw/d considering a diet conversion factor (ppm to mg/kg bw/d) of 10.

Executive summary:

The toxicity of DTB-glycolester was examined in a sub-chronic (16 week) study in which the test substance was fed to groups of 50 male and 50 female weanling rats each at levels of 0 (control), 0.1, 0.4 or 1.6% in stock diet. The rats were derived from parents which had been fed the diets for 31 days before mating and during gestation and lactation. Observations were made on general appearance and behavior, growth, food intake and food efficiency, hematology, blood biochemistry and urine composition. At week 17 all rats were killed and examined grossly for pathological changes. The major organs were weighed and extensive histopathological examinations were carried out on 15 males and 15 females of each group.
General condition and behavior were not adversely affected at any level of the test substance.
Gain in body weight was relatively low in males of the high-dose group. Food intake was decreased at 1.6% dose level in both sexes during the first two weeks. Food efficiencies were not adversely affected.
Haematological examinations did not reveal any treatment related differences amongst various groups.
There were no toxicologically significant differences amongst the groups with respect to enzyme activities and protein values of blood serum.
Kidney-function tests and urine composition did not exhibit any harmful effects of the test substance.
There were slight increases in the relative weights of the kidneys, liver and ovaries at the high-dose level. The weights of ovaries were also increased in the medium-dose group.
Gross and microscopic pathology failed to reveal changes that could be attributed to ingestion of the test substance.
It was concluded that the NOAEL of DTB-glycolester in the present study was 1.6% (1600 mg/kg bw/d) in the diet of rats for 16 weeks.