Methyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1969

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: very old study, non GLP and non guideline study, very young rats were used, only limited hematology parameters analyzed, limited clinical chemistry, no urinalysis, no details on compound intake, no individual results reported.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1969

Report date:

1969

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

The test article was administered orally over 90 days at dosing levels of 0.04, 0.2 and 1 % in the diet to young albino Wistar rats. The following parameters were analyzed: body weight, food consumption and food efficiency, hematology parameters (hemoglobin content, packed cell volume, red blood cell counts and total and differential white blood cell counts), clinical chemistry (serum glutamic-pyruvic transaminase (SGPT), serum glutamic-oxaloacetic transaminase (SGOT), serum alkaline phospahatase (SAP), serum ornithine carbamyl transferase (SOCT) and total serum proteins (TSP)). In addition, gross pathology was performed and the following organs were weighed: heart, liver, kidney, spleen, brain, testicle/ovary, thymus, pituitary, thyroid and adrenal. Detailed microscopic examination was performed on all surviving male and female rats of the highest dose group and of all control rats.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

Methyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate

EC Number:

228-985-4

EC Name:

Methyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate

Cas Number:

6386-38-5

Molecular formula:

C18H28O3

IUPAC Name:

methyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propanoate

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CIVO-colony
- Age at study initiation: weanling rats
- Weight at study initiation: males: 57.5 g, females: 52 g (average values)
- Housing: in screen bottom cages (five to a cage)
- Diet: stock diet consisting of: 28% yellow maize, 26% whole wheat, 10% rolled oats, 10% soybean oil meal, 8% fish meal, 4% meat scraps, 2.7% dried whey, 3% soybean oil, 3% grass meal, 1.5% minerals, 0.5 % sodium chloride, 3.3% vitamin preparations, ad libitum
- Water: tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test compound was thoroughly mixed into the diet by means of a mechanical blender (type Lodige, Paderborn).

DIET PREPARATION
- Rate of preparation of diet (frequency): every 2 weeks

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

90 days

Frequency of treatment:

continuously in food

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 0.04, 0.2 and 1.0 %
Basis:
nominal in diet

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dose levels were based on a range finder where feeding of dietary levels of 1.0 and 5.0 % resulted in deaths, decreased
growth rate, diminished food intake, increased water intake, increased relative weight of liver and damage of liver and kidney. At 0.2 % the only effect was an increase in liver weight (report nr. R 276U).
- Rationale for animal assignment (if not random): by weight

Examinations

Observations and examinations performed and frequency:

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Time schedule for examinations: during the first four weeks and in week 11 and 12.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption: Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in the 12th week
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- Parameters checked: haemoglobin content, packed cell volume, red blood cell counts and total and differential white blood cell counts

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the study
- Animals fasted: No data
- Parameters checked: serum glutamic-pyruvic transaminase (SGPT), serum glutamic-oxaloacetic transaminase (SGOT), serum alkaline phosphatase (SAP), serum ornithine carbamyl transferase (SOCT) and total serum proteins (TSP)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
In the 14th week all rats were killed by decapitation and examined macroscopically for pathological changes. The following organs were weighed: heart, liver, kidney, spleen, brain, testicle/ovary, thymus, pituitary, thyroid and adrenal. Samples of these organs and of a wide range of other organs were fixed in a 4 % neutralized formaldehyde solution.

HISTOPATHOLOGY: Yes
Detailed microscopic examination was performed on all surviving male and female rats of the highest dose group and of all control rats. Haematoxylin-eosin stained paraffin sections of the organs weighed and also of the following organs were examined: lung, salivary glands (sublingual, submaxillary and parotid gland) esophagus, Harderian gland, stomach, duodenum, ileum, caecum, colon, trachea, skeletal muscle, aorta, exorbital lacrimal gland, auxillary and mesenteric lymph nodes, pancreas, skin, urinary bladder, bone marrow (sternum), prostate, epididymis, coagulating gland, seminal vesicle, preputial gland, uterus, spinal cord and femoral nerve. Microscopic examination of rats at lower dosage levels (0.04 and 0.2 %) was restricted to liver, kidney, thyroid, thymus, stomach, testicle, submaxillary gland, ovary, uterus, adrenal and heart. A number of frozen kidney sections was stained with Sudan III. Selected paraffin liver sections were stained with PAS, Luxol fast blue, and according to Perl's method for iron. The latter procedure was also applied to a few kidney sections. In addition, a number of selected paraffin sections of the adrenals was stained according to Van Gieson's method for connective tissue.

Results and discussion

Results of examinations

Details on results:

CLINICAL SIGNS AND MORTALITY
Eight male and six female rats fed on 1 % test substance died during the experimental period. Most deaths occurred during the second and third
week of the experiment. Otherwise no abnormalities in condition or behavior were observed.

BODY WEIGHT AND WEIGHT GAIN
Considerable growth depression occurred at 1 % in either sex. At the 0.04 and 0.2 % the body weight figures were comparable to those of the
controls.

FOOD CONSUMPTION AND FOOD EFFICIENCY
The food intake of the 1 % group was distinctly diminished in either sex. At lower levels food consumption as well as food efficiency figures were not distinctly different from those of the controls.

HAEMATOLOGY
Haemoglobin content and number of white blood cells were significantly decreased at 1 % in either sex. At lower dosage levels the haematoglogical data were comparable to those of the controls.

CLINICAL CHEMISTRY
The SGOT-activities at 0.2 and 1 % were lower than those of the controls in either sex. In females the differences were statistically significant. Alkaline phosphatase activities were increased in male and female rats at the highest level. The slightly higher SAP value in males at 0.04 % did not suggest any relationship with the level of the test substance. Total serum protein, SGPT and SOCT values were comparable in all groups in both sexes.

ORGAN WEIGHTS
At the feeding level of 1.0 % several organ weights differed considerably from those of the controls. This may partly be ascribed to the very low body weights in this group. At the 0.2 % level liver weights and thyroid weights were increased in both sexes. Thyroid weights were also slightly increased in males at the 0.04 % level.

GROSS PATHOLOGY
The surviving animals of the highest dose-group were small and emaciated. All of them had enlarged, dark-brown livers and large thyroids. In addition, one of the two male survivors in this group showed severe pulmonary emphysema and atrophy of both testicles, prostate and seminal vesicle.
At lower dosage levels the gross post mortem examination did not reveal treatment-related pathological changes. Other lesions noted at necropsy, in the test groups and in the control group, such as early signs of murine pneumonia and proteinaceous plugs in the urinary bladder, are regularly seen in the strain of rats used. They may, therefore, not be ascribed to the ingestion of the test article.

HISTOPATHOLOGY
At microscopic examination the most outstanding treatment-related pathological changes were found in liver and thyroid.
- LIVER
Greatly enlarged hepatocytes with homogeneous cytoplasm often containing large, eosinophilic droplets which had a laminary structure, variations in size and chromatin pattern of hepatocytic nuclei, an increased number of binucleated cells, necrosis of individual parenchymal cells, and slight proliferation of bile ducts ("oval cells") were regularly seen in the livers of the surviving animals at the 1 % level of feeding. At 0.2 % the hepatic change only consisted of slightly homogeneous cytoplasm and nuclei which varied slightly in size. At the lowest dosage level the livers were morphologically indistinguishable from those of the controls.
-THYROID
An activated appearance of the thyroid gland was seen in most of the male and female rats of the control group and of the various test groups. Degree and/or frequency of this "morphological activation" increased, however, with increasing levels of the test substance. Moreover, in a few rats of the two highest dose-groups papillary infoldings of the follicular epithelium occurred, which resulted in a thyroid picture similar to that in rats given thyroid-stimulating hormone (TSH). This finding shows that the test substance has a goitrogenic effect in rats. Signs of this effect were visible already at the lowest dosage level of 0.04 %.

Less important than the above mentioned changes in liver and thyroid, but (at least indirectly) compound-related abnormalities were observed in kidney (fatty infiltration, pigmentation, cystic tubules), male and female genital organs (atrophic changes), lung (edema and emphysema), stomach (hyperkeratosis), adrenal (fatty "vacuoles", hyperaemia and distended sinusoids), bone marrow (hypoplasia) and submaxillary salivary gland (increased ratio ducts: acini). The abnormeilities in these organs were seen only at the highest dose level and they may partly or completely be ascribed to inanition, except for the degenerative changes in the gonads which occurred also in a few males at 0.2 %. Additional histopathological changes occurred only in a single animal or were about equally distributed between the control group and the various test groups. Most of these lesions are, moreover, quite common findings in the strain of rats used. They may, therefore, not be attributed to the ingestion of the test material.
From gross and microscopic pathological examination it appears that 1 % of the test substance in the diet induced distinct lesions in a range of organs. Feeding of 0.2 % of the test article resulted in slight changes in liver, thyroid and testicle, whereas at the lowest dietary level of 0.04 % the only relevant finding was a slightly more activated appearance of the thyroid as compared with controls.

Effect levels

Dose descriptor:

NOAEL

Effect level:

0.04 other: % in food

Sex:

male/female

Basis for effect level:

other: morphological thyroid activation, hepatocellular changes and testicular alterations in the intermediate dose group

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In a 90 day oral feeding study with rats, the NOEL is below 0.04 % per day. However, since the thyroid effect is known to be a rat-specific response to inductive processes in the liver, the NOAEL can be set at 0.04%.

Executive summary:

In a 90 day oral feeding study, the test article was administered continuously at dosing levels of 0.04, 0.2 and 1 % in the diet to young albino Wistar rats. At a dietary level of 1%, serious ill effects as shown by mortality, considerable growth retardation and increased SAP levels were induced. Microscopically, hepatocellular hypertrophy accompanied by eosinophilic droplets in the cytoplasm, variations in the size of nuclei and increased numbers of mitotic figures as well as single cell necrosis and proliferation of bile ducts were observed in high dose rats. Additional histopathological findings in high dose rats were: fatty infiltration and pigmentation in renal epithelial cells, hyperkeratosis in the stomach, adrenal fatty changes, bone marrow hypoplasia and an increased ratio of ducts versus acini in the submaxillary gland and atrophic changes in the male and female reproductive organs. At a level of 0.2 % the major abnormalities were increased relative weights of livers and thyroids which were attended with histological changes. Testicular alterations were also observed in the intermediate group. At the lowest feeding level of 0.04 % slight differences with the control group were revealed only by the thyroid which showed an increased relative weight in males and histological differences with the controls in both sexes. The increased weight of this organ in males might be an incidental finding because the thyroid weight of male controls was low in comparison with that of control males in other sub-chronic studies. However, microscopically the thyroids of the 0.04 % group showed a slightly more activated appearance than did the control thyroids in both sexes. The NOEL is therefore below 0.04 %. However, since the thyroid effect is known to be a rat-specific response to inductive processes in the liver, the NOAEL can be set at 0.04%.