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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable well-documented study report which meets basic scientific principles: non-GLP.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1987

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
other: rat: Sprague-Dawley and dog: beagle
Strain:
other: rat: Sprague-Dawley and dog: beagle
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS (rats)
- Source: Charles River
- Age at study initiation: young adult
- Weight at study initiation:

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
RAT EXPOSURE: TMCH was preblended with standard rat ration (Purina Rat Chow) at levels of 0, 3000, 10000, and 30000ppm (approx. 300, 1000, and 3000 mg/kg/day, respectively) and introduced to groups of 15 male and 15 female rats. Each rat was weighed on the first day of testing and at weekly intervals thereafter. Food consumption data were collected individually from five rats/sex/group on a weekly basis. Observations for deaths or overt signs of toxicity were recorded daily. Blood and urine samples were collected from five rats/sex from the control and high dose group after 45 and 84 days from study beginning and were analyzed for the following: hematology, blood chemistry, and urine analysis (pH, spec. gravity, microscopic elements, and glucose and albumin concentrations. Surviving rats were killed at study termination and received a complete gross necropsy. Absolute weights of the brain, gonads, heart, kidneys, liver, and spleen were recorded for each rat. Weight ratios (brain and body) were subsequently derived. Microscopic examination was conducted on representative sections of the following tissues from 10 rats/sex following preparation by conventional histological and staining techniques: adrenal glands, aorta, brain (three sections), caecum, colon, esophagus, eyes, gonads, heart, kidneys, liver, lungs, lymph nodes (cervical/mesenteric), muscle, optic nerve, pancreas, parathyroid glands, pituitary gland, prostate gland, salivary gland, sciatic nerve, small intestine (three sections), spinal cord, spleen, sternum, stomach (three sections), trachea, thyroid gland, urinary bladder and uterus. Additional kidney sections were processed and evaluated for 10 male and 10 female rats from the 3000 and 10 000 ppm TMCH-treated groups.

DOG EXPOSURE:
Groups of eight (4 male:4 female, approximately 5 months of age) purebred beagle dogs obtained from an in-house breeding colony were randomly assigned to each of 4 test groups (Table 1) and administered dietary levels of 0, 100, 300 or 1000 ppm TMCH (approximately 2.5, 7.5 and 25 mg/kg/day, respectively) for 90 days. Diet was introduced 6 hours/day and ad libitum feeding ensued. Each dog was examined daily and untoward signs of toxicity were recorded. The following determinations were made upon each dog just prior to study inception and after 42 and 84 days of testing: hematology (total red and white cell count, differential leukocytes, hemoglobin and hematocrit), blood chemistry (serum glucose, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase and blood urea nitrogen) and urine analysis (pH, specific gravity, microscopic elements, glucose and albumin). At the study conclusion, all animals were killed and given a thorough gross necropsy. Weights of the adrenal glands, brain, gonads, heart, kidneys, liver, pituitary gland, spleen and thyroid gland were recorded. Representative samples of the following tissues and organs from each dog on test were fixed in formalin, embedded and stained with hematoxylin and eosin for subsequent evaluation by light microscopy: adrenal glands, aorta, bone marrow (sternum), brain (three sections), caecum, colon esophagus, gall bladder, gonads, heart, kidneys, liver, lungs, lymph nodes (cervical/ mesenteric), muscle, pancreas, pituitary gland, prostate gland, salivary gland, sciatic nerve, small intestine (three sections), spinal cord, spleen, stomach (three sections), trachea, thyroid gland, uterus and urinary bladder.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
90 day exposure

RAT EXPOSURE:
TMCH was preblended with standard rat ration (Purina Rat Chow) at levels of 0, 3000, 10000, and 30000ppm (approx. 300, 1000, and 3000 mg/kg/day, respectively) and introduced to groups of 15 male and 15 female rats. Each rat was weighed on the first day of testing and at weekly intervals thereafter. Food consumption data were collected individually from five rats/sex/group on a weekly basis.

DOG EXPOSURE:
Groups of eight (4 male:4 female, approximately 5 months of age) purebred beagle dogs obtained from an in-house breeding colony were randomly assigned to each of 4 test groups (Table 1) and administered dietary levels of 0, 100, 300 or 1000 ppm TMCH (approximately 2.5, 7.5 and 25 mg/kg/day, respectively) for 90 days. Diet was introduced 6 hours/day and ad libitum feeding ensued.
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 3000, 10000, and 30000ppm (approx. 300, 1000, and 3000 mg/kg/day)
Basis:
other: nominal in diet: RATS
Remarks:
Doses / Concentrations:
0, 100, 300 or 1000 ppm TMCH (approximately 2.5, 7.5 and 25 mg/kg/day)
Basis:
other: nominal in diet: DOGS
No. of animals per sex per dose:
RATS: 15 male and 15 female rats per dose
DOGS: 4 male and 4 female dogs per dose
Details on study design:
RAT EXPOSURE: TMCH was preblended with standard rat ration (Purina Rat Chow) at levels of 0, 3000, 10000, and 30000ppm (approx. 300, 1000, and 3000 mg/kg/day, respectively) and introduced to groups of 15 male and 15 female rats. Each rat was weighed on the first day of testing and at weekly intervals thereafter. Food consumption data were collected individually from five rats/sex/group on a weekly basis. Observations for deaths or overt signs of toxicity were recorded daily. Blood and urine samples were collected from five rats/sex from the control and high dose group after 45 and 84 days from study beginning and were analyzed for the following: hematology, blood chemistry, and urine analysis (pH, spec. gravity, microscopic elements, and glucose and albumin concentrations. Surviving rats were killed at study termination and received a complete gross necropsy. Absolute weights of the brain, gonads, heart, kidneys, liver, and spleen were recorded for each rat. Weight ratios (brain and body) were subsequently derived. Microscopic examination was conducted on representative sections of the following tissues from 10 rats/sex following preparation by conventional histological and staining techniques: adrenal glands, aorta, brain (three sections), caecum, colon, esophagus, eyes, gonads, heart, kidneys, liver, lungs, lymph nodes (cervical/mesenteric), muscle, optic nerve, pancreas, parathyroid glands, pituitary gland, prostate gland, salivary gland, sciatic nerve, small intestine (three sections), spinal cord, spleen, sternum, stomach (three sections), trachea, thyroid gland, urinary bladder and uterus. Additional kidney sections were processed and evaluated for 10 male and 10 female rats from the 3000 and 10 000 ppm TMCH-treated groups.

DOG EXPOSURE: Groups of eight (4 male:4 female, approximately 5 months of age) purebred beagle dogs obtained from an in-house breeding colony were randomly assigned to each of 4 test groups (Table 1) and administered dietary levels of 0, 100, 300 or 1000 ppm TMCH (approximately 2.5, 7.5 and 25 mg/kg/day, respectively) for 90 days. Diet was introduced 6 hours/day and ad libitum feeding ensued. Each dog was examined daily and untoward signs of toxicity were recorded. The following determinations were made upon each dog just prior to study inception and after 42 and 84 days of testing: hematology (total red and white cell count, differential leukocytes, hemoglobin and hematocrit), blood chemistry (serum glucose, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase and blood urea nitrogen) and urine analysis (pH, specific gravity, microscopic elements, glucose and albumin). At the study conclusion, all animals were killed and given a thorough gross necropsy. Weights of the adrenal glands, brain, gonads, heart, kidneys, liver, pituitary gland, spleen and thyroid gland were recorded. Representative samples of the following tissues and organs from each dog on test were fixed in formalin, embedded and stained with hematoxylin and eosin for subsequent evaluation by light microscopy: adrenal glands, aorta, bone marrow (sternum), brain (three sections), caecum, colon esophagus, gall bladder, gonads, heart, kidneys, liver, lungs, lymph nodes (cervical/ mesenteric), muscle, pancreas, pituitary gland, prostate gland, salivary gland, sciatic nerve, small intestine (three sections), spinal cord, spleen, stomach (three sections), trachea, thyroid gland, uterus and urinary bladder.

Examinations

Observations and examinations performed and frequency:
RATS: Observations for deaths or overt signs of toxicity were recorded daily. Blood and urine samples were collected from five rats/sex from the control and high dose group after 45 and 84 days from study beginning and were analyzed for the following: hematology, blood chemistry, and urine analysis (pH, spec. gravity, microscopic elements, and glucose and albumin concentrations.

DOGS: Each dog was examined daily and untoward signs of toxicity were recorded. The following determinations were made upon each dog just prior to study inception and after 42 and 84 days of testing: hematology (total red and white cell count, differential leukocytes, hemoglobin and hematocrit), blood chemistry (serum glucose, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase and blood urea nitrogen) and urine analysis (pH, specific gravity, microscopic elements, glucose and albumin).
Sacrifice and pathology:
RATS: Surviving rats were killed at study termination and received a complete gross necropsy. Absolute weights of the brain, gonads, heart, kidneys, liver, and spleen were recorded for each rat. Weight ratios (brain and body) were subsequently derived. Microscopic examination was conducted on representative sections of the following tissues from 10 rats/sex following preparation by conventional histological and staining techniques: adrenal glands, aorta, brain (three sections), caecum, colon, esophagus, eyes, gonads, heart, kidneys, liver, lungs, lymph nodes (cervical/mesenteric), muscle, optic nerve, pancreas, parathyroid glands, pituitary gland, prostate gland, salivary gland, sciatic nerve, small intestine (three sections), spinal cord, spleen, sternum, stomach (three sections), trachea, thyroid gland, urinary bladder and uterus. Additional kidney sections were processed and evaluated for 10 male and 10 female rats from the 3000 and 10 000 ppm TMCH-treated groups.

DOGS: At the study conclusion, all animals were killed and given a thorough gross necropsy. Weights of the adrenal glands, brain, gonads, heart, kidneys, liver, pituitary gland, spleen and thyroid gland were recorded. Representative samples of the following tissues and organs from each dog on test were fixed in formalin, embedded and stained with hematoxylin and eosin for subsequent evaluation by light microscopy: adrenal glands, aorta, bone marrow (sternum), brain (three sections), caecum, colon esophagus, gall bladder, gonads, heart, kidneys, liver, lungs, lymph nodes (cervical/ mesenteric), muscle, pancreas, pituitary gland, prostate gland, salivary gland, sciatic nerve, small intestine (three sections), spinal cord, spleen, stomach (three sections), trachea, thyroid gland, uterus and urinary bladder.
Other examinations:
HEMATOLOGY
-erythrocyte count
-hematocrit
-hemoglobin
-leukocyte count (total and differential)
-mean corpuscular volume
- hemoglobin.

SERUM CHEMISTRY
-blood urea nitrogen
-glucose
-total protein
-triglycerides
-phosphorous
-alkaline phosphatase
-serum alanine aminotransferase
Statistics:
In the subchronic rat study, body weights, food consumption, absolute organ weights, hematological data and blood chemistry data were evaluated first by analysis of variance (t-test). Any significant effects disclosed were further analyzed by an appropriate method for comparison (Dunnett’s) of multiple groups. Organ weight ratios were analyzed by the Kruskal—Wallis statistical test or the multiple comparison test. In all cases, a minimum level of significance of p 0.05 was used.

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):
no effects observed
Food efficiency:
not examined
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:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
Administration of up to 30000 ppm TMCH to male and female rats produced no discernible effects on survival, demeanor, weight gain, food consumption, hematology, blood chemistry or urinalysis throughout the study period.

Absolute kidney weights were statistically increased in all groups of male rats at termination of the study. Also statistically increased were the weights relative to brain in mid-dose and to body weight in high-dose males. No statistically significant changes were observed in any treated groups of female rats. Absolute liver weights were statistically (p < 0.01) lower than controls in the 30000 ppm group males but not females. However, the lack of any morphological changes observed microscopically in liver sections suggested that this observation was unrelated to TMCH treatment. No significant differences were observed in absolute or relative liver weights of any test group of female rats.

Treatment-related microscopic changes were confined to the kidney of male rats in all test groups. These effects were observed in all TMCH-treated male rats on study and were dose related in regards to the relative severity and degree of parenchymal involvement. The renal lesion observed consisted of protein resorption droplets within the cytoplasm of epithelial cells lining the proximal convoluted tubules in the cortex of both kidneys. Histologically, these droplets had an eosinophilic staining, were oval-shaped and of variable size. While this lesion is considered degenerative, no evidence of necrosis or inflammation was observed. In some sections, there were foci of tubules in the cortex which were lined with enlarged basophilic staining cells which appeared to represent regeneration of the tubular epithelium. The microscopic changes in the male kidneys are typical of a syndrome that occurs specifically in male rats and is unlikely to have a correlation to humans. The syndrome, alpha-2u-globulin nephropathy or Light Hydrocarbon Nephropathy is related to the accumulation of alpha-2u in the lysosomes of the kidney.
No treatment-related microscopic effects were observed in any tissue, including the kidney, of any female rat on test.

In the subchronic dog study, no effects related to treatment were noted in survival, average food consumption, or abnormal behavior up to 1000 ppm TMCH. A slight body weight suppression was observed in male dogs during the first week on test. Recovery to normal weight occurred within the next study week. No such effects were observed in groups of female beagles at any test level. Results of hematological and blood chemistry studies and urine analysis revealed no test material-related effects at any dose level tested. No changes in absolute organ weight, organ-to-body weight or organ-to-brain weight ratios were found which could be attributable to treatment with TMCH. There were no relevant gross autopsy or histopathological findings among animals tested in this study.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Remarks:
in Rats
Effect level:
>= 30 000 ppm
Sex:
male/female
Basis for effect level:
other: ~3000 mg/kg; No treatment-related mortality or significant adverse clinical effects occurred.
Dose descriptor:
NOAEL
Remarks:
in Dogs
Effect level:
>= 1 000 ppm
Sex:
male/female
Basis for effect level:
other: ~25 mg/kg; No treatment-related mortality or significant adverse clinical effects occurred.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, the NOAEL for rats was 3000 mg/kg/day which was the highest dose tested. The NOAEL for dogs was determined to be 1000ppm (25 mg/kg/day) which was the highest dose tested.
Executive summary:

Tetramethylcyclohexane (TMCH) was preblended with standard rat ration (Purina Rat Chow) at levels of 0, 3000, 10000, and 30000ppm (approx. 300, 1000, and 3000 mg/kg/day, respectively) and introduced to groups of 15 male and 15 female rats.  Each rat was weighed on the first day of testing and at weekly intervals thereafter.  Food consumption data were collected weekly and observations for deaths or overt signs of toxicity were recorded daily.  Blood and urine samples were collected and analyzed. Groups of eight (4 male/4 female) beagle dogs were administered dietary levels of 0, 100, 300 or 1000 ppm TMCH (approximately 2.5, 7.5 and 25 mg/kg/day, respectively) for 90 days. Diet was introduced 6 hours/day and ad libitum feeding ensued. Each dog was examined daily and untoward signs of toxicity were recorded. Results of subchronic exposure of TMCH to rats and dogs failed to show any treatment-related morphological or qualitative changes in the cellular elements of the peripheral blood picture. This result is consistent with a similar lack of effects noted after acute TMCH exposure.  The NOAEL for rats was 30000ppm.  The NOAEL for dogs was 1000ppm. In both instances, these were the highest levels tested.