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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1961
Reliability:
2 (reliable with restrictions)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1961
Report Date:
1961

Materials and methods

Test guideline
Qualifier:
no guideline available
Guideline:
other:
Principles of method if other than guideline:
A 90 day repeated dose study with test article incorporated in feed and a negative control group and positive control group (BHT) was conducted. Focused hematological examination was conducted to assure that the test article did not produce effects on clotting parameters similar to Vitamin K deficiency that had been seen with some other alkylated phenols under similar conditions. Body weights, food consumption, organ weights and histopathology of the viscera and endocrine glands were included in the study design.
GLP compliance:
no
Remarks:
Conducted prior to GLP regulations
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): "Ethyl" Antioxidant 702
- Chemical name: 4,4'-methylenebis (2,6-di-tert-butylphenol)
- Molecular formula (if other than submission substance): C29H44O2
- Physical state: Finely powdered pale yellow solid
- Analytical purity: 98.5%
- Physical Properties of Test Article:
Melting Point: 155 degrees C
Set Point: 154.7 degrees C
Boiling Poing: 290 degrees C at 40 mm Hg; 250 degrees C at 10 mm Hg
LD50 rats: > 24 grams/kg body weight

- Lot/batch No.: Nos. 0245 and 0729

Test animals

Species:
rat
Strain:
other: CF albino
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Carsworth Farms
- Age at study initiation: 10 week old weanlings
- Weight at study initiation:
Control Group: 296 +/- 5 grams
0.125% Test Article Group: 292 +/- 7.2 grams
0.50% Test Article Group: 295 +/- 7.8 grams
1.25% Test Article Group: 305 +/- 6.7 grams
2.50% Test Article Group: 288 +/- 9.2 grams
1.25% BHT Control Group: 304 +/- 7.1 grams
- Fasting period before study: none
- Housing: 5 males or females per cage,
- Diet (e.g. ad libitum): Purina Laboratory Chow meal basic diet, ad libitum A purified cellulose (alphacel) was added to the varous dients to keep non-nutritive bulk uniform at 5%
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 30 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
- Humidity (%):
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light):

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): every 2 weeks
- Mixing appropriate amounts with (Type of food): Test article and cellulose mixtures incorprated into dry feed by being stirred by a mixer for 15 minutes


Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
90 days of feeding
Frequency of treatment:
Daily in feed
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0.125%
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0.5%
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
1.25%
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
2.5%
Basis:
nominal in diet
No. of animals per sex per dose:
15 male/15 female per group (control group, 3 treated groups, positive control group (BHT)
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: High dose group was to be equal to the BHT positive control group, a level in the BHT group that was expected to cause toxicity. Maximum tolerance of BHT in human diet at the time of the study was 0.005%
Positive control:
Butylated hydroxytoluene (BHT); 2,6-di-tert-butyl-4-methylphenol. C15H24O (purity 99%)

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: initial, weekly, terminal

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each cage determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- 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

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: initial, and at 30 and 90 days for control, 0.125%, 0.50%, and 1.25% groups; initial, 30 days, 60 days and 90 days for 2.50% test group and the positive control (BHT 1.25%) groups
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes / No / No data
- How many animals: 5 males, 5 females per group
- Parameters examined: Total WBC, Polyleucocytes, Lymphocytes, Hemoblobin, Hematocrit, Prothrombin time (determined by modified micro-Kato method using whole blood)

CLINICAL CHEMISTRY: No data

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Other examinations:
Organ weights: brain, heart, lungs, liver, gonads, kidneys, spleen, adrenal, thyroid
Statistics:
Difference of mean values tested by analysis of variance for 4 and 70 degrees of freedom

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No difference in clinical signs compared to negative control; one female death in 0.5% group at 2 weeks probably not test article related
Mortality:
no mortality observed
Description (incidence):
No difference in clinical signs compared to negative control; one female death in 0.5% group at 2 weeks probably not test article related
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No statistical differences from negative control values
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No statistical differences from negative control values
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No differences compared to negative control for total WBC, differential, HB, Hct, or prothrombin time
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No specific effects that could be related to test article; differences in female groups related to differences in initial and terminal weight increases compared to control
Gross pathological findings:
no effects observed
Description (incidence and severity):
No differences in treated animals compared to negative control
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At high dose, 6 male and 3 female rats had slight degree of liver degeneration
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY: One rat in the 0.5% treatment group died after the second week, and had sustained progressive loss of weight, and respiratory difficulties prior to death. No other signs of illness were noted in the test article treatment animals. No significant effects on growth, food consumption or mortality compared to control were seen. In the positive control group (BHT), marked decrease in growth, in association with reduced food consumption, predominantly in males, was seen. The effectes in the BHT group were evident in the first week, and progressed to approximately the tenth week. Five of the male BHT rats and one female rat of that group died during weeks 3 to 10. Signs in the BHT rats included general weakness, which in some animals, became an acute illness with marked signs of pallor and listlessness, with large hematomas seen in some animals.

BODY WEIGHT AND WEIGHT GAIN: No effects seen in treated animals versus control animals. BHT animals showed decrease in weight gain beginning in the first week.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No differences in food consumption were seen in the treated animals compared to control.

HAEMATOLOGY: Treated animals showed no differences in total white blood counts, percent polyleucocytes, percent lymphocytes, hemaglobin, hematocrit, or prothrombin times. Animals fed BHT, especially the males, showed increased prothrombin times, and a decrease in hemoglobin content, with a shift in leucocyte distribution characterized by increase in polymorphonuclear cells and corresponding decrease in lymphocytes. These changes in the BHT animals were most pronounced in the 30 to 60 day blood samples.


ORGAN WEIGHTS: differences in average visceral weights among the female treated gropus appeared to be associated with different initial and terminal body weights. Weights of most of the viscera of animals given BHT were altered compared with control weights. Nearly all of the livers of animals in this group were acutely hyperemic and dark reddish brown.

GROSS PATHOLOGY: No alterations were seen in gross pathology in rats in control or test article added diets. One of the rats receiving BHT was euthanized on day 20 of exposure, and showed a urethral injury associated with urine retention. Three males given BHT died after 39 and 52 days of exposure, and showed extensive degeneration and necrosis of the livers, with marked pneumonitis, and small lung hemorrhages. Another male died after 63 days, but decomposition obscured lesions. One female rat given BHT died after 67 days of exposure, and showed extensive necrosis and liver degeneration.

HISTOPATHOLOGY: NON-NEOPLASTIC: Animals given 0.125, 0.5, or 1.25% test article showed no microscopic pathology. The livers of animals given 2.5% test article in 9/15 males and 12/15 females showed no pathologic changes. The remainder (6 males and 3 females) showed slight degree of liver degeneration as evidenced by clumping of cytoplasmic granules at edges of hepatocytes, cellular swelling, and either one large or two small nuclei, and multiple prominent nucleoli. The degenerated cells were found sporadically throughout the liver lobules. The other viscera and endocrine glands in these 6 males and 3 females of the high dose group were normal except for a slight to moderate degree of pneumonitis.
All male rats given BHT showed degeneration of hepatic cells ranging from modreate to severe, and varying from focal to diffuse in distribution. The lesions were similar, but more severe, compared to the animals given test article at 2.5% in the diet. Four of the 14 female rats that survived to 90 days of BHT in diet had normal viscera; The other 10 had moderate to severe degeneration of the liver. In addition, some of the male and female rats given BHT had a slight to moderate degree of pneumonitis.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
ca. 1.25 other: wt % in diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
LOAEL
Effect level:
2.5 other: wt. % in diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Weight Gain, Food intake, and Mortality over 90 days feeding

Dietary Additive Group

Wt. % added to diet

Mean wt. gain (per cent)

M            F

Mean daily food intake (grams)

M            F

Mortality (deaths/group)

M            F

Control

0

+41        +26

20.6         13.8

0/15        0/15

702

0.125

+36        +29

19.6         14.9

0/15        0/15

702

0.50

+35        +26

19.8         14.6

0/15        1/15

702

1.25

+38        +18

20.5         13.7

0/15        0/15

702

2.50

+42        +23

19.3         14.9

0/15        0/15

BHT

1.25

- 3          + 4

16.2         12.2

5/15        1/15

Applicant's summary and conclusion

Executive summary:

A 90-day repeated dose feeding study was conducted in male and female albino rats with antioxidant “Ethyl” 702 as the test substance, and Butylated hydroxytoluene (BHT) as the positive control substance. Test concentrations were control, 0.125, 0.5, 1.25 and 2.50 wt. percent of the diet; BHT was tested at a single concentration of 1.25 wt. percent of the diet. The purpose of the study was to examine the effects of the test article to some aspects of toxicity seen with other alkylated phenols. No grossly harmful effects were seen in the animals exposed to test substance. No significant differences were noted in the groups with respect to growth, consumption of food, or apparent physiological activity. The administration of BHT at a level of 1.25% of the diet caused death in 20% of the animals during the course of the study.

           Hematological examinations of the test animals carried out in intervals of 30 days disclosed no abnormalities in the numbers or types of leucocytes in peripheral blood, in hematocrit, hemoglobin, or prothrombin times. These findings of no effect was interpreted to mean that the test article did not cause the cause the anticoagulative effects seen in rats fed some other alkyl phenols in comparable diets. These hemorrhagic tendencies were thought to be associated with an increase in ‘prothrombin time” and with the metabolism of Vitamin K seen in rats fed some other alkyl phenols.

           At study determination, there were no adverse effects seen among the test article treated groups with respect to survival or gross pathological changes. There were significant effects in the final weights of females in the various groups (the test substance treated animals had heavier than control weights), and in the final weights of individual viscera. That is, the larger animals had heavier viscera, and vice versa, but without a uniform proportional relationship of body weight to weight of organs.

           The histopathologic examination of viscera and endocrine glands of these rats revealed no changes in the groups 0.125%, 0.5%, or 1.25% test article in the diet. Slight degeneration of the liver was observed at the dietary concentration of 2.5% test article in the diet in about 30% of the animals in the group. Comparatively, marked degeneration of the livers was found in almost all of the rats fed BHT at 1.25% in the diet.