6,6'-di-tert-butyl-4,4'-thiodi-m-cresol

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

> 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Unfortunately, GLP conditions or Guidelines for testing are not mentioned, and certain key data i.e. the study period and attachments with raw data are missing.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): 4,4'-Thiobis(6-tert-butyl-3-cresol)
- Substance type: white, crystalline powder, insoluble in water, soluble in acetone and methanol
- Physical state: solid
- Analytical purity: > 98%
- Purity test date: not mentioned
- Lot/batch No.: not mentioned
- Expiration date of the lot/batch: not mentioned
-Stability under test conditions: stable during animal test period according to authors of report; data not shown
- Storage condition of test material: room temperature, protected from light

Test animals

Species:

rat

Strain:

other: SPF

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc.
- Age at study initiation: 6
- Weight at study initiation: males 193-222 g, females 148-171 g
- Fasting period before study: not mentioned
- Housing: individually in metal mesh cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 -26
- Humidity (%): 30 - 70
- Air changes (per hr): 11-13
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: arabic gum

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: once weekly

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Data in attached documents 1, 2, 3 are missing

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 16, 60, 250 mg/kg bw
Basis:
nominal in diet

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based upon 15 days toxicity study

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 3 times a day

DETAILED CLINICAL OBSERVATIONS: YES

BODY WEIGHT: Yes
- Time schedule for examinations: twice a week

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal 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

Haematology: Yes
- Time schedule for collection of blood: at begin and end of study, and start of recovery period
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes
- How many animals: all
- Parameters checked: red blood cells, hemoglobin, hematocrit, mean corpuscular volume, reticulocyte rate, platelet count, leucocytes, prothrombin time, APTT

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at begin and end of study, and start of recovery period
- Animals fasted: Yes
- How many animals: all
- Parameters checked: GOT, GPT, LDH, gamma-GPT, total cholesterol, triglycerides, phospholipids, bilirubin, blood glucose, urea nitrogen, creatinine, sodium, potassium, chlorine, calcium, phosphor, total protein, albumin, A/G ratio.

URINALYSIS: Yes
- Time schedule for collection of urine: week 4 and 6
- Parameters checked: pH, protein, ketone, glucose, occult blood, bilirubin, urobilinogen, color, urinary sediment, urinary output, gravity, water intake.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes: organ weight plus examination
HISTOPATHOLOGY: Yes: macroscopic examination of various tissues

Statistics:

For the results that were digitalized from among the examination parameters, tests for homogeneity of variance in each group were first performed using Bartlett's method. As a result, analysis of variance was performed by the one-way layout method if the variance was homogeneous, and if significant differences were observed between groups, tests were performed on the mean differences between the control group and each treatment group using Dunnett's method when the numbers of cases in each group were equal and Scheffe's method when the numbers of cases in each group differed. If the variance was not homogeneous, the Kruskal-Wallis rank sum test was performed, and if the results were significant, tests were performed on the mean rank differences between the control group and each treatment group using Dunnett's method (when the numbers of cases in each group were equal) and Scheffe's method (when the numbers of cases in each group differed). All tests are performed on both sides with significance levels of 5 and 1%

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

minor

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
No deaths were observed throughout the study period.

BODY WEIGHT AND WEIGHT GAIN
No changes caused by the administration of the test substance were observed in general signs or body weight profiles.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Although the food consumption decreased in the males and females of the 250 mg/kg treatment group on day 4 after administration was begun, the food consumption of males slightly exceeded that of the control group thereafter. The food consumption of the males of the 60 mg/kg treatment group slightly exceeded that of the control group. Since the body weights in these treatment groups were roughly the same as those in the control group, it was assessed that the dietary efficiency was diminished, and it was inferred that the damage to the intestinal tract was a contributing factor.

HAEMATOLOGY
In hematology, increases in platelet counts, decreases in lymphocyte ratios, and increases in segmented neutrophil ratios were observed primarily in the females of the 250 mg/kg treatment group. When the actual numbers of lymphocytes and segmented neutrophils of the females of the 250 mg/kg treatment group were found from the white blood cell count and the differential ratio, the lymphocyte count was approximately 20% greater than the mean value of the control group, and the segmented neutrophil count was approximately 160% greater than the mean value of the control group. Therefore, changes in the differential leukocyte counts in this study are caused by increases in segmented neutrophils, which suggests a relationship with the cellular infiltration into large intestine mucosa.

CLINICAL CHEMISTRY
In blood biochemistry, increases in total cholesterol and phospholipids were observed in the 250 mg/kg treatment group in addition to the fluctuations in blood sugar, urea nitrogen, and inorganic phosphorus described above, which suggests effects on lipid metabolism.

URINALYSIS
In urinalysis, decreases in urinary pH were observed in the groups treated at 60 mg/kg or higher, and increases in urinary protein and ketone bodies were observed primarily in females. At the same time, in blood biochemistry, increases in urea nitrogen and inorganic phosphorus were observed in females, which yields the possibility that the increase in urinary protein is an effect of this test substance on the kidneys, but no histological changes were observed in the kidneys. Moreover, since the blood sugar levels of the females of the 250 mg/kg treatment group were reduced, it was inferred that the increases in urinary ketone bodies were due to increases in lipid requirements as an energy source in step with the decreases in blood sugar levels.

ORGAN WEIGHTS
Significant increases in the relative weight of the liver were observed in the males and females of the 250 mg/kg treatment group at the end of the administration period. Significant increases in the relative weight of the heart were observed in the males of each treatment group at the end of the recovery period.

GROSS PATHOLOGY
Dilatation of the cecum was observed in 5 males and 4 females of the 250 mg/kg treatment group, and thickening of the small intestine wall was observed in 4 males and 4 females of the 250 mg/kg treatment group at the end of the administration period.

HISTOPATHOLOGY: NON-NEOPLASTIC
In the small intestine, macroscopic thickening of the wall was observed in the 250 mg/kg treatment group, and villus hypertrophy was observed histologically in the ileum. As described above, reduced dietary efficiency was suggested in the groups treated at 60 mg/kg or higher, but the morphological changes in the small intestine are considered to be adaptive biological reactions to decreases in digestion/absorption ability. Moreover, in the large intestine, the macroscopic dilatation of the cecum was observed in the 250 mg/kg treatment group, and histologically, and the vacuolization of absorptive epithelial cells and cellular infiltration of the mucosa were observed in the cecum and the colon in the groups treated at 60 mg/kg or higher, which suggests damage to the large intestine. However, the animals demonstrating vacuolization of absorptive epithelial cells did not necessarily coincide with the animals demonstrating cellular infiltration, so the relationship between the two was not clear. In mesenteric lymph nodes, "tingible body macrophages" in the paracortical region were observed somewhat frequently in the 250 mg/kg treatment group. Since this change was not observed in other lymphoid organs such as the cervical lymph nodes, thymus, or spleen, it is possible that this is a finding related to intestinal tract damage and not a direct effect on lymphoid organs.

Effect levels

Key result

Dose descriptor:

NOEL

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

none

Applicant's summary and conclusion

Conclusions:

Male and female SD rats were treated with 0, 15, 60, 250 mg/kg 4,4'-thiobis(6-tert-butyl-3-cresol) once daily during 28 days. 4,4'-thiobis(6-tert-butyl-3-cresol) induced primarily changes in the large intestine in the groups at 60 mg/kg or higher and in the small intestine and the liver in the 250 mg/kg treatment group. On the other hand, no changes were observed in the 15 mg/kg treatment group. Based on the above results, the NOEL of 4,4'-thiobis(6-tert-butyl-m-cresol) in this study was estimated to be 15 mg/kg/day.