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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:
Not specified
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study performed in compliance wiith GLP and analytical verification performed, however no reference guidelines detailed in the report.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report date:
1988

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
3 month feeding study to albino rats at three dose levels plus control. Homogeneity and dose level verification conducted by GC. The results of this study include: measurements of body weight, once a week, food and water consumption and detailed observations (haematology, clinical biochemistry and urinalysis), as well as gross necropsy and histopathology.
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
6,6'-di-tert-butyl-4,4'-butylidenedi-m-cresol
EC Number:
201-618-5
EC Name:
6,6'-di-tert-butyl-4,4'-butylidenedi-m-cresol
Cas Number:
85-60-9
Molecular formula:
C26H38O2
IUPAC Name:
6,6'-di-tert-butyl-4,4'-butylidenedi-m-cresol
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Identification: SANTOWHITE powder antioxidant
EHL Substance Identification Code: T870041
Lot Number: N7E009
Stated Purity: 95+ %
Date Received: June 10, 1987
Description at Receipt: White powder

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
Species: Albino rat
Strain: Sprague-Dawley
Source: Charles River Breeding Laboratory, Portage MI
Date of Arrival at EHL: September 29, 1987
Number Used in Study: 120 (60 male, 60 female)
Test Group Size: 15/ sex
Method of Assignment: Computer randomization by weight
Method of Identification: Individual ear tag, bar-coded cage card
Age at Study Start: Approximately seven weeks
Weight Range at Study Start: Males- 218.5 to 245.6 grams
Females- 146.0 to 172.9 grams
Type of Housing: Individual stainless steel cages
Water Availability: Free choice (St. Louis public water supply)
Food Availability: Free choice
Light Cycle: 12 hours daily (On at 6: 30 A.M.)
Animal Room Environmental Specifications: Temperature- 70 to 74 degrees F.; humidity- 35 to 60% relative; there were no excursions in animal room environmental conditions which had any obvious impact on the results of the study.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
not specified
Details on oral exposure:
Diet Formulation
Basal Diet: Purina Mills Certified RODENT CHOW #5002
Levels Prepared: T-3 (1,000 ppm) , T-2 (500 ppm), T-l (100 ppm)
Frequency of Preparation: Approximately weekly
Mixing Machine: HOBART HCM-450
Mixing Time: 10 minutes
Batch size: Premix (also used as T-3), 15 kilograms; other levels, 9 kilograms each
Mixing Procedure Number: DP-87188-1
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test Material Stability: Gas chromatography with flame ionization detector
Homogeneity of Diet Mixtures: Analysis of duplicate samples from top t middle t and bottom of mixer of lowest and highest dietary levels
Diet Mixture Stability: Analysis of samples kept frozen (closed container, 35 days) or at ambient temperature (open container, 21 days)
Dietary Level Verification: Extraction of test substance with acetonitrile, analysis by gas chromatography with flame ionization detector; all dietary levels for first five weeks, at least one level/week thereafter
Duration of treatment / exposure:
3 months
Frequency of treatment:
Daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 500, 1000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
Number Used in Study: 120 (60 male, 60 female)
Test Group Size: 15/ sex
Control animals:
not specified
Positive control:
Not specified

Examinations

Observations and examinations performed and frequency:
Checks for Mortality and Moribundity: Twice daily
Detailed Observations for Signs of Toxicity: Once weekly
Body Weight and Food Consumption Measurement: Once weekly

Clinical Pathology
Frequency: Weeks 6/7 (interim) and 13/14 (scheduled termination)
Number of Animals: 5/sex/level at interim, 10/sex/level at scheduled termination
Fasting: Food withheld overnight prior to blood collection. Food and water withheld 4 hours prior to urine collection
Collection Method: Blood - Vena cava of anesthetized rat. Urine - Metabolism cages
Hematology Determinations: Total erythrocyte count (RBC), total leukocyte count (WEC), platelet count (PLT) , hematocrit (HCT) , level of haemoglobin (HGB), and red blood cell indices [mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC)]: Whole blood treated with anticoagulant (EDTA pre treated commercial tubes) processed on a COULTER S Plus II blood cell counter using the manufacturer's methods.
Leukocyte differential: Thin blood smears on labelled glass slides prepared, stained with Wright's stain, and examined microscopically
Reticulocyte count: A portion of the EDTA sample mixed with a vital stain (methylene blue), a slide prepared and examined mic roscopically
Blood Chemistry Determinations: Albumin, total protein, blood urea nitrogen (BUN), total
bilirubin, direct bilirubin, glucose, glutamic pyruvic transaminase (SGPT / ALT) , alkaline phosphatase, glutamic oxaloacetic transaminase (SGOT / AS!), gamma glutamyl transpeptidase (Gamma-GT), creatinine, cholesterol (CHOL), calcium, phosphorus, chloride, sodium, and potassium.
Serum harvested by centrifugation of samples submitted in commercial clot tubes assayed with a KDA clinical analyzer using the manufacturer's methods. Globulin determined by subtraction of albumin value from total protein value.
Urinalysis Determinations: Urine protein, pH, blood, glucose, ketone, bilirubin, and urobilinogen: Urine samples assayed with MULTISTIX reagent strips and a CLINI-TEK urinalysis strip reader using the manufacturer's methods.
Specific Gravity: Determined with an AO T.S. meter using the manufacturer’s methods
Sacrifice and pathology:
Gross Pathology
Animal Termination: C02 anesthesia followed by exsanguination
Animals Examined: All at scheduled termination (interim sacrifice animals discarded without examination)
Scheduled Sacrifice: Week 13/14
Extent of Examination: External and internal; internal cavities opened and organs examined in place and then removed; hollow organs opened and examined
Organs Weighed: Adrenals, brain, kidneys, liver, spleen, testes with epididymides
Tissues Retained (when present): Aorta, adrenals, bone and bone marrow, brain, colon, duodenum, esophagus, eyes, heart, ileum, jejunum, kidneys, lesions or abnormal masses, liver, lung (with mainstem bronchi), lymph node (mesenteric and submandibular), musc Ie (quadriceps femoris), ovaries, pancreas, pituitary, prostate, sciatic nerve, seminal vesicles, skin (with mammary tissue), spinal cord (thoracolumbar), spleen, stomach, submaxillary salivary gland, testes with epididymides, thymus, thyroid/parathyroid, trachea, uterus (corpus and cervix), urinary bladder
Histopathology
Tissues Examined: All retained tissues from all control and high dose level animals; target organs from two intermediate groups as defined by the highest dose level
Tissue Preparation: Fixed tissues washed, dehydrated, embedded in paraffin, sectioned at approximately 5 microns, and stained with hematoxylin and eosin
Examination: Light microscopy
Statistics:
The following statistical procedures were used to detect statistically significant differences between treated animals and their respective controls:
Dunnett' s Multiple Comparison Test (two-tailed) (1): Body weights and food consumption
Fisher's Exact Test with Bonferroni Inequality Procedure (2): Incidence of microscopic lesions
Terminal body weights, absolute organ weights, organ/body weight ratios and selected non categorical clinical pathology data were evaluated by decision-tree statistical analysis procedures which, depending on the results of tests for normality (3) and homogeneity of variances (Bartlett's Test), chose either the parametric (Dunnett's Test and Linear Regression) or nonparametric (Kruskal-Wallis, Jonckheere' sand/ or Mann-Whitney Tests) routines to detect group differences and analyzed for trend.
Other statistical routines used for some data were: Bartlett's Test (4) to evaluate homogeneity of variances, Analysis of Variance (6) to determine if the sample (group) means could be considered as an estimate of a common population, and Grubb's Test (5) to detect outliers.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
reduction in bw gain for high dose males only
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
high dose males only
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
Description (incidence and severity):
High dose only
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Increase in liver weights high and intermediate doses only
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
high and intermediate doses only
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
high dose animals only
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Details on results:
Analysis of Test Material and Diets: Results of analyses for test material stability conducted over a span of time approximately equal to the study length indicated the test material was stable. The homogeneity of the diet mixtures was determined to be adequate for study use. The stability of the test material/diet mixture was demonstrated for the low and high dose levels; stored in open containers at room temperature for 21 days, and stored in closed containers in a freezer for 35 days.
Mortality: There were no deaths during this study.
Body Weight: Slightly decreased weight gain for high dose group males was noted. All other treated males gained weight comparable to their control group. Although not statistically significant, prior to fasting for the terminal sacrifice the highest dose group females had slightly reduced body weights when compared to control. This effect was noted at approximately Week 6 and continued through the end of the study. All other female dose groups gained weight comparable to control.
Food Consumption: Reduced food consumption for high dose level males was statistically significant during the first two weeks of testing, but occurred only sporadically throughout the remainder of the study. High dose level females had slight, but consistently reduced food consumption throughout the study.
Clinical Signs: There were no clinical signs considered related to treatment. One high dose level female (F3002) had a palpable mass at Week 5 that remained through the rest of the study.

PATHOLOGY
Clinical Pathology
Hematology: There were slight increases in platelet and absolute neutrophil counts in high dose males at Period 1 which were not considered to be treatment-related due to the small magnitude of change at Period 1, and because of the absence of the increase at Period 2. At Period 2, there were slight decreases in MCV and MCH values in males at the highest dose level. Likewise, there were slight decreases in HGB, HCT and MCV and slight increases in the reticulocyte values in high dose females. The biological significance of these changes, and their relationship to treatment, were equivocal due to their small magnitude. Other changes in hematologic parameters were sporadic and inconsistent, and therefore not considered related to treatment.
Clinical Chemistry: Relevant serum chemical changes which were apparently exposure related were primarily limited to increases in enzyme values. These included SGOT and SGPT values in males from the two highest dose groups at Periods 1 and 2, alkaline phosphatase values in high dose males at Period 1, and SGOT and SGPT in females from the highest dose group at Period 1 and from the two highest dose groups at Period 2.
The only other change which may have been treatment-related was a small increase in cholesterol in high dose females at Period 2. The statistically significant increases in chloride values for males from the two highest dose groups at Period 2 were not considered to be biologically relevant due to the small magnitude of change, which resulted in mean values well within the historical control range. Other changes in serum chemical parameters were sporadic, inconsistent and of small magnitude. Values remained within the normal historical control range, and therefore -were considered to be neither biologically relevant nor related to chemical exposure.
Urinalysis: Slightly increased urine specific gravity and ketones in high dose males at Period 1 were not present in females, or in either sex at Period 2. Therefore, these changes were not considered biologically significant or related to treatment.
Gross Pathology: There was a slight (although statistically significant) decrease in the mean terminal body weights of high dose group females. The only notable changes in organ weights were slight increases in absolute liver weights for males at the highest dose level, and for females at the two highest dose levels. Liver to body weight ratios were slightly increased for both sexes at the highest dose level. Other increases in organ to body weight ratios were attributed to low terminal body weights, rather than a direct treatment effect on the organs.
A slight increase in the occurrence of uterine dilatation at the highest dose level was observed at necropsy. Other changes observed at necropsy were considered to be of spontaneous origin and were not associated with chemical exposure.
Microscopic Pathology: Microscopic examination of a palpable mass from female F3002 revealed a single mammary gland adenoma. Microscopically, the occurrence of uterine dilatation was slightly higher than that observed at necropsy. Other microscopic changes which were apparently related to chemical administration occurred in the livers and mesenteric lymph nodes of both sexes. Centrilobular hepatocellular vacuolization occurred at a higher incidence in males at the two highest dose levels and in females at the highest dose level. Instances of hepatocellular hypertrophy, karyomegaly, multinucleated hepatocytes and increased mitoses were probably a result of chemical exposure. Macrophages with distended cytoplasm were present in sinusoids and portal areas of livers from both sexes at the highest dose level.
Likewise, accumulations of macrophages which apparently contained foreign material were prevalent in mesenteric lymph nodes of both sexes from the two highest dose levels. Other changes observed microscopically were considered to be of spontaneous origin and were un associated with chemical exposure.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
100 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Overall averages (uncorrected for quality control results)

 

TEST GROUPS

 

T-1

T-2

T-3

Target Exposure (ppm)

100

500

1000

Mean Analytical (ppm)

97

500

1000

Standard Deviation (ppm)

8.1

24

42

Mean % Difference

-3.0

0.0

0.0

 

Group mean body weight data

Group

Study Mean

% Difference from control (Study Mean)

% Difference from Control (Final Body Weight)

% Gain from Initial

MN

434.6

----

----

131.6

M1

433.7

-0.2

0.6

133.2

M2

436.1

0.4

2.1

136.9

M3

422.7

-2.7

-3.7

123.1

FN

249.1

----

----

85.1

F1

257.2

3.3

5.7

95.4

F2

255.3

2.5

5.0

93.9

F3

240.3

-3.5

-7.6

71.0

 

Overall group mean food consumption

 

FOOD CONSUMPTION GM/DAY

Group

Study Mean

% Difference from control (Study Mean)

MN

25.5

----

M1

25.1

-1.6

M2

25.4

-0.5

M3

23.9

-6.2

FN

18.5

----

F1

19.3

4.3

F2

19.8

6.8

F3

17.1

-7.5

 

Clinical Signs

There were no clinical signs considered related to treatment.

One high dose level female (F3002) had a palpable mass at Week 5 that remained through the rest of the study.

Hematology:

There were slight decreases in MCV and MCH values in males at the highest dose level. Likewise, there were slight decreases in HGB, HCT and MCV and slight increases in the reticulocyte values in high dose females. The biological significance of these changes, and their relationship to treatment, were equivocal due to their small magnitude. Other changes in hematologic parameters were sporadic and inconsistent, and therefore not considered related to treatment.

Clinical Chemistry:

Relevant serum chemical changes which were apparently exposure related were primarily limited to increases in enzyme values. These included SGOT and SGPT values in males from the two highest dose groups, alkaline phosphatase values in high dose males and SGOT and SGPT in females from the two highest dose groups. The only other change which may have been treatment-related was a small increase in cholesterol in high dose females. The statistically significant increases in chloride values for males from the two highest dose groups were not considered to be biologically relevant due to the small magnitude of change, which resulted in mean values well within the historical control range. Other changes in serum chemical parameters were sporadic, inconsistent and of small magnitude. Values remained within the normal historical control range, and therefore were considered to be neither biologically relevant nor related to chemical exposure.

Urinalysis:

No biologically significant or related to treatment effects.

Gross Pathology:

There was a slight (although statistically significant) decrease in the mean terminal body weights of high dose group females. The only notable changes in organ weights were slight increases in absolute liver weights for males at the highest dose level, and for females at the two highest dose levels. Liver to body weight ratios were slightly increased for both sexes at the highest dose level. Other increases in organ to body weight ratios were attributed to low terminal body weights, rather than a direct treatment effect on the organs. A slight increase in the occurrence of uterine dilatation at the highest dose level was observed at necropsy. Other changes observed at necropsy were considered to be of spontaneous origin and were not associated with chemical exposure.

Microscopic Pathology

Microscopic examination of a palpable mass from one high dose female revealed a single mammary gland adenoma. Microscopically, the occurrence of uterine dilatation was slightly higher than that observed at necropsy. Other microscopic changes which were apparently related to chemical administration occurred in the livers and mesenteric lymph nodes of both sexes. Centrilobular hepatocellular vacuolization occurred at a higher incidence in males at the two highest dose levels and in females at the highest dose level. Instances of hepatocellular hypertrophy, karyomegaly, multinucleated hepatocytes and increased mitoses were probably a result of chemical exposure. Macrophages with distended cytoplasm were present in sinusoids and portal areas of livers from both sexes at the highest dose level. Likewise, accumulations of macrophages which apparently contained foreign material were prevalent in mesenteric lymph nodes of both sexes from the two highest dose levels. Other changes observed microscopically were considered to be of spontaneous origin and were unassociated with chemical exposure.

Applicant's summary and conclusion

Conclusions:
The no-observed-adverse-effect-level (NOAEL) in this study was considered to be 100 ppm in the diet of rats of both sexes for up to three months.
Executive summary:

Santowhite powder antioxidant was fed to Sprague-Dawley rats at targeted levels of 0, 100, 500 and 1,000 parts per million (ppm) for three months. Stability of the test material both neat and when mixed with the diet was confirmed. Diet homogeneity and verification of concentrations of the test material in the diet revealed satisfactory results.

There were no deaths during the study. Chemical exposure, at the highest exposure level for both sexes, resulted in slightly reduced body weights and food consumption, altered serum enzymes (including SGOT and SGPT), increased liver weights, and microscopic liver and lymph node changes. The mid-dose level produced similar changes in SGOT and SGPT, liver weights, and in liver and lymph node tissue. The liver appeared to be the target organ for this test compound. The no-observed-adverse-effectlevel (NOAEL) was considered to be 100 ppm in the diet of rats of both sexes for up to three months of exposure.

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