1,3,5-tris[[4-tert-butyl-3-hydroxy-2,6-xylyl]methyl]-1,3,5-triazine-2,4,6(1H,3H,5H)-trione

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1977

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Doses of test material, homogeneous distribution, and stability in the diet were not confirmed analytically. More hematological, clinical chemistry and urinalysis parameters are measured in guideline studies. A high enough dose to produce a significant toxicological effect was not used.. GLP was not formally followed and no reference was made to following a specific guideline.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1976

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Doses of test material, homogeneous distribution, and stability in the diet were not confirmed analytically. Clinical chemistry, urinalysis and hematological parameters were not measured. GLP was not formally followed and no reference was made to following a specific guideline.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Test method was a standard multidose study for the time period, with a control group.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories
- Age at study initiation: =< 4 weeks
- Weight at study initiation: =< 100g
- Housing: individually in stock cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

Route of administration:

oral: feed

Vehicle:

not specified

Details on oral exposure:

Details about how the diets were prepared were not present.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

30 consecutive days

Frequency of treatment:

Daily in diet

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control, both males and females

Dose / conc.:

600 mg/kg bw/day (nominal)

Remarks:

males

Dose / conc.:

1 200 mg/kg bw/day (nominal)

Remarks:

males

Dose / conc.:

2 380 mg/kg bw/day (nominal)

Remarks:

males

Dose / conc.:

610 mg/kg bw/day (nominal)

Remarks:

females

Dose / conc.:

1 190 mg/kg bw/day (nominal)

Remarks:

females

Dose / conc.:

2 480 mg/kg bw/day (nominal)

Remarks:

females

Remarks:

Doses / Concentrations:
0 % (control), 0.5, 1.0 or 2.0 % test material
nominal in diet

No. of animals per sex per dose:

5

Control animals:

yes, plain diet

Details on study design:

Rats were randomly allocated to 4 groups of 5/sex.

Positive control:

No positive control reported

Observations and examinations performed and frequency:

All animals were observed daily to detect possible signs of toxicity. Food intake and body weight were measured weekly

Sacrifice and pathology:

Necropsies were performed on all animals at study termination. At necropsy, all animals received a thorough examination including all body surfaces, both internal and external, subcutaneous tissues, and the following organs: adrenal, aorta, urinary bladder, bone, bone marrow, cerebellum, cerebrum, colon, esophagus, eye, heart, ileum, jejunum, kidney, liver, lung, lymph node, mammary gland, skeletal muscle, sciatic nerve, ovary, pancreas, parathyroid, pituitary, prostate, salivary gland, seminal vesicle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid, tongue, trachea and uterus. The liver and kidneys (combined) were weighed. The animal carcass also was examined for lesions and other irregularities. The aforementioned organs were preserved in buffered 10% formalin and the livers of 2 high dose male and female rats were examined histologically.

Statistics:

Data were analyzed using a parametric analysis of variance (randomized block design) and Student's t tests. The critical level of significance was p < 0.05.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Rats receiving test material exhibited diarrhea (low and high-dose animals) and irritation of the face, ears and stomach (mid and high dose animals). This irritation of the face, ears and stomach is occassionally seen in this strain of rats and is not due to the test material administration.

Mortality:

no mortality observed

Description (incidence):

None of the animals died.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The statistically significant decrease in terminal body weight in the high dose females is thought to be of no biological significance beacause it is not present in males nor is it seen in week 4 body weights in these same rats, weighed 2 days earlier.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There was a trend towards increased food consumption in all treated groups, with the value for the high-dose females significantly different from control at week 4.

Food efficiency:

not specified

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

not specified

Haematological findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There was no effect of treatment on absolute organ weights. However, in females, average relative liver weights were increased.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Discoloration of the liver of 2 high dose males and 2 high dose females.

Spots and sores were observed on the head of the rat, but this is occassionally observed in this strain of rats and are not due to test material administration.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No histopathological changes were observed in 3/4 of these rats. One high dose male exhibited minimal intralobular scattered foci of extra medullary hematopoesis and mononuclear infiltrates. Infrequent degenerating individual hepatocytes were associated with the mononuclear infiltrates. This is not considered treatment-related.

Histopathological findings: neoplastic:

not specified

Details on results:

None of the animals died. Rats receiving test material exhibited diarrhea (low and high-dose animals) and irritation of the face, ears and stomach (mid and high dose animals). Because similar irritation is occasionally observed in Sprague-Dawley rats. it was not attributed to administration of test material. Alopecia was noted in controls as well as treated animals.

A significant effect of treatment on food consumption at week 4 was noted in females. There was a trend towards increased food consumption in all treated groups, with the value for the high-dose females significantly different from control at week 4. The average values for food consumption of females treated with 0, 0.5, 1.0 and 2.0% test material during week 4 were 118, 133, 131 and 138 g, respectively. The authors concluded that the increase in food consumption was not related to administration of test material.

Although the authors stated there was no effect on body weight, there was a trend towards decreased terminal body weight in females at the two highest doses, with the average value for the high dose group significantly different from control. The average values for terminal body weight of females treated with 0, 0.5, 1.0 and 2.0% test material were 199, 208, 173 and 159 g, respectively. The significant decrease in body weights is thought of no biological significance because it is not present in males nor is it seen in week 4 body weights in these same rats, weighed 2 days earlier.

There was no effect of treatment on absolute organ weights. However, in females, average relative liver weights (expressed as % of body weight) of animals treated with 0.5% (4.98)1.0% (5.97) and 2.0% (6.98) and average relative kidney weights of animals treated with 2.0% test material (1.207) were significantly different from control values for liver (4.67) and kidney (0.831).

The only significant gross observation seen at necropsy was discoloration of the liver of 2 high dose males and 2 high dose females. No histopathological changes were observed in 3/4 of these rats. One high dose male exhibited minimal intralobular scattered foci of extra medullary hematopoesis and mononuclear infiltrates. Infrequent degenerating individual hepatocytes were associated with the mononuclear infiltrates. It was concluded that the effects on the liver were not due to test material.

Dose descriptor:

NOAEL

Effect level:

1 190 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: Corresponds to 1% in diet

Dose descriptor:

LOAEL

Effect level:

2 480 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: Corresponds to 2% in diet

Dose descriptor:

NOAEL

Effect level:

2 380 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Remarks on result:

other: No adverse effects observed

Remarks:

Corresponds to 2% in diet

Conclusions:

Administration of the test material at 0, 0.5 and 1% in the diet for 30 days did not have significantly adverse effects.
Some minor effects on body weight and food consumption were observed when the rats were administered 2% in the diet.

Therefore the NOAEL was considered to be 1% (1190 mg/kg bw) for females and 2% (2380 mg/kg bw) for males.

Executive summary:

This report presents the results of a very minimal, "range-finding" study that was undertaken to provide a basis for establishing dietary levels for a full-scale, 90-day study.

The test material was administered daily for 30 days to rats by dietary admixture at concentrations of 0.5, 1.0 and 2.0%. There ware no deaths in any of the control or treatment groups. Food consumption, body weight gain, and organ weight (liver and kidneys) were slighlty affected by the treatment.

The only significant observation seen at necropsy was discoloration of the liver. Histopathological examination of liver sections from 4 high dose rats whoed no effects that could be attributed to the administration of the test substance. Thus, this liver discoloration is not treatment-related.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

reproductive toxicity, other

Remarks:

Examination of reproductive organs from an oral 90-day repeat dose study

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1977

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Pre GLP, pre guideline study. Doses of test material, homogeneous distribution, and stability in the diet were not confirmed analytically. The effects on mating and offspring were not tested. 90-day repeat dose study examining male testes.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Typical 90 day oral repeat dose study of the period, with examiniation of the reproductive organs.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories Incorporated, Massachusetts
- Age at study initiation: 4 weeks
- Weight at study initiation: 69-100g (males); 64-95g (females)
- Housing: Individually housed in suspended stainless ssteel cages with wire mesh bottoms.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-24
- Humidity (%): 40-50
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

Test diets were prepared by mixing the appropriate amount of test material with equal amounts of ground Purina Lab Chow in a high speed blender (blending time was not noted). After blending, an additional 2 parts of ground lab chow was mixed by hand into the blended material. After this step, an additional amount of ground lab chow was added to give the appropriate dose level (25 mg/kg bw, 100 mg/kg bw or 400 mg/kg bw). The diet was then placed in a twin-shell mixer and mixed until the test material was distributed throughout the diet. The formula used to determine the amount of material added to feed was as follows: mean body weight x dietary level x 7 days/mean weekly food consumption = mg test material/kg feed. Fresh diets were prepared at least once per week. Doses were adjusted weekly on the basis of the weight and food consumption for the preceding week. Doses were not adjusted for purity of the material.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

90 consecutive days

Frequency of treatment:

continuous

Dose / conc.:

25 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

400 mg/kg bw/day (nominal)

No. of animals per sex per dose:

20

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Based on a 30d range-finding study (see other study record available)
- Fasting period before blood sampling for clinical biochemistry: overnight

Positive control:

none

Parental animals: Observations and examinations:

Food consumption and body weights were measured weekly. Animals selected for clinical chemistry, hematology and urinalysis (5 per sex per group) were determined by protocol and a table of randomization. Clinical chemistry (gamma-glutamyl transpeptidase, glucose, glutamic-oxaloacetic transaminase, glutamic pyruvic transaminase, and blood urea nitrogen), hematology (erythrocyte count, hematocrit, hemoglobin, differential and total leukocyte count, and platelet count) and urinalyses (appearance, glucose, microscopic examination of sediment, pH, protein and specific gravity) were performed on the same animals at appropriate time intervals (day 90 for clinical chemistries, days 45 and 90 for hematologies). Animals were fasted overnight before blood was collected from the orbital sinus on day 45 and from an unknown site on day 90. Urine also was collected on days 45 and 90.

Oestrous cyclicity (parental animals):

N/A

Sperm parameters (parental animals):

N/A

Litter observations:

N/A

Postmortem examinations (parental animals):

At study termination, all animals were weighed. They were then euthanized. The urogenital orifaces, tail, each pinna, eyes and external auditory meatus were examined visually and by palpatation for distortions. All subcutaneous tissues were examined including regional lymph nodes, mammary and salivary glands. Abdominal contents, and the brain, pituitary gland and cranial nerves also were examined grossly. The following tissues were taken and preserved for possible histologic evaluation: adrenal, aorta, urinary bladder, bone, bone marrow, cerebellum, cerebrum, pancreas, pituitary, skin , stomach, thyroid, colon, esophagus, eye, heart, ileum, jejunum, kidney, prostate, salivary gland, spleen, testes, tongue, uterus, liver, lung, lymph node, mammary gland, muscle (skeletal), sciatic nerve, ovary, parathyroid, seminal vesicle, spinal cord, thymus and trachea. Any organ or tissue exhibiting a lesion was noted and the lesion was taken for histopathological examination. Complete histopathology was performed on 10 animals/sex/group from the control and high dose groups. These animals were selected randomly from a table. Microscopic examination of the heart, liver, lungs and kidneys was performed on all additional animals that survived to termination.

Postmortem examinations (offspring):

N/A

Statistics:

Data for food intake, weight gain, clinical chemistries, hematologies, urinalyses, and organ weights were analyzed using a blocked 2-way analysis of variance that compared data between and within groups. If F values were significant, data were compared using Dunnett's t-test. The level of significance is p < 0.05.

Reproductive indices:

N/A

Offspring viability indices:

N/A

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Grossly observable deviation from normal was seen in all groups. These deviations included alopecia around the nares and front paws, diarrhea, watery eyes and encrustment around nares. The alopecia appeared and disappeared in all groups throughout the study and was attributed to mechanical irritation due to movement in and out of the feeding container. The diarrhea was seen in an occasional individual in all groups at various periods, and was not considered to be related to the test material. No animal had diarrhea for two consecutive days. Watery eyes appeared primarily immediately after orbital bleeding of some animals for hematology and clinical chemistry determination and was considered to be the result of bleeding. Encrustment around nares is common to animals fed compound mixed in feed. There were no clinical signs manifested in this study attributable to the test material up to and including 400 mg/kg

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One animal in the mid-dose group (100 mg/kg) died after 12 days of treatment. Since no additional deaths occurred, this death was considered artifactual.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no significant differences in weight changes among treated groups when compared to control throughout the entire study.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

During first week on test, females in the 25 mg/kg treated group ate significantly less food than did the other female groups during that week. Males in the 25 mg/kg treated group ate less food than the other males during the third week on test. No other effets of the test material were seen upon food intake throughout the 13 weeks of the study. Therefore, it was concluded that dose levels up to 400 mg/kg bw of feed did not affect food intake in rats.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

After 45 days on study there was no significant difference in erythrocyte count in either males or females.
At 90 days, there was a trend toward increased red blood cells in low (8.27 x 10E6/mm3) and mid-dose animals [(8.63 x 10E6/mm3, value significantly different from study control (7.99 x 10E6/mm3) and historical controls]; however, counts in high dose animals (8.04 x 10E6/mm3) were similar to controls. Since the increase was not dose-related, this observation was thought to be artifactual in nature and not related to administration of test material.

There were no changes in hematocrit, hemoglobin, leukocyte count, platelet estimate and erythrocyte morphology.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Gamma-glutamyl transpeptidase (GGTP) values in males treated with 400 mg/kg were higher than control at 90 days (4.1 vs. 2.6 IU/l), but were within historical limits. The elevation was due to 2/5 males that had GGTP values that were twice the value of the other males. This effect was not seen in females. Since this elevation of GGTP was not dose related in males and since females did not have a change in GGTP values, it was concluded that this observation was not treatment-related.
Glucose values in females treated with 100 and 400 mg/kg (163 and 162 mg/dl, respectively) were lower than controls (209 mg/dl) at 90 days but were within the range of historical values (90-284 mg/dl). This effect was not seen in males. The significant differences in glucose between high and mid dose females and controls were considered to be a result of some unusually high values in the controls and were similar to the historical means. Therefore this effect was not considered to be related to test material.

Glutamic-oxaloacetic transaminase, Glutamic-pyruvic transaminase and urea were unaffected by the treatment.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no differences in the color, appearance, pH, specific gravity, milligram protein, milligram sugar, RBC see under a high power field, and WBC seen under a high power field in rats fed the test material for 90 days.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

The type and incidence of lesions detected by the histopathological examination were considered to represent spontaneous change in albino rats and unrelated to the administration of the test material.

There was no effect of treatment on any of the reproductive organs examined.


One animal in the mid-dose group (100 mg/kg) died after 12 days of treatment. Study personnel did not attribute this death to administration of test material. Animals were generally healthy during the study, but exhibited alopecia around the nares and paws, diarrhea, watery eyes (animals that were bled, only) and encrustment around the nares. Study personnel attributed these signs to ingestion of a powdered food and did not consider them to be related to test material. Other than week one (where females treated with 25 mg/kg test material ate less food than females in other groups) and week 3 (when males in the 25 mg/kg group ate less food than males in the other groups), there was no difference between food intake of treated and control animals. There was no effect of treatment on weight or weight gain.

Gamma-glutamyl transpeptidase (GGTP) values in males treated with 400 mg/kg were higher than control at 90 days (4.1 vs. 2.6 IU/l), but were within historical limits. The elevation was due to 2/5 males that had GGTP values that were twice the value of the other males. This effect was not seen in females. Since this elevation of GGTP was not dose related in males and since females did not have a change in GGTP values, it was concluded that this observation was not treatment-related. Glucose values in females treated with 100 and 400 mg/kg (163 and 162 mg/dl, respectively) were lower than controls (209 mg/dl) at 90 days but were within the range of historical values (90-284 mg/dl). The significant differences in glucose between high and mid dose females and controls were considered to be a result of some unusually high values in the controls and were not considered to be related to test material.

At 90 days, there was a trend toward increased red blood cells in low (8.27 x 10E6/mm3) and mid-dose animals [(8.63 x 10E6/mm3, value significantly different from study control (7.99 x 10E6/mm3) and historical controls]; however, counts in high dose animals (8.04 x 10E6/mm3) were similar to controls. Since the increase was not dose-related, this observation was thought to be artifactual in nature and not related to administration of test material.

There was no effect of treatment on urinalysis, organ weights, or gross pathology. One of the males with a high GGTP value had focal perivascular and periductal mononuclear leukocyte infiltration in the liver, but the other animal did not demonstrate any liver pathology. Livers of other males in the high dose group had histopathology similar to that of controls. Other mild inflammatory lesions characterized by leukocytic infiltration also occurred in the lungs, liver and kidneys of a few animals in each group. All changes were considered to be spontaneous and not related to administration of test material.

Dose descriptor:

NOEL

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reproductive organs: No effects observed

Conclusions:

Test material was not toxic to reproductive organs at the highest dose tested.

Executive summary:

Sprague-Dawley rats were used in this study. 20 males and 20 females were assigned to control, and to each of the test groups (25, 100 and 400 mg/kg). The length of the treatment period was 90 days.

There were no specific effects noted on the reproductive organs of the rats. Therefore it is concluded that the test material did not affect adversely those organs after 90 days of administration at the highest dose tested. The NOEL is 400 mg/kg bw.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1977

Report date:

1977

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Test method was a standard multidose study for the time period, with a control group.

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1,3,5-tris[[4-tert-butyl-3-hydroxy-2,6-xylyl]methyl]-1,3,5-triazine-2,4,6(1H,3H,5H)-trione

EC Number:

254-996-9

EC Name:

1,3,5-tris[[4-tert-butyl-3-hydroxy-2,6-xylyl]methyl]-1,3,5-triazine-2,4,6(1H,3H,5H)-trione

Cas Number:

40601-76-1

Molecular formula:

C42H57N3O6

IUPAC Name:

tris[(4-tert-butyl-3-hydroxy-2,6-dimethylphenyl)methyl]-1,3,5-triazinane-2,4,6-trione

Constituent 2

Reference substance name:

Cyanox (TM) 1790 Antioxidant

IUPAC Name:

Cyanox (TM) 1790 Antioxidant

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories Incorporated, Massachusetts
- Age at study initiation: 4 weeks
- Weight at study initiation: 69-100g (males); 64-95g (females)
- Housing: Individually housed in suspended stainless ssteel cages with wire mesh bottoms.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-24
- Humidity (%): 40-50
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

not specified

Details on oral exposure:

Test diets were prepared by mixing the appropriate amount of test material with equal amounts of ground Purina Lab Chow in a high speed blender (blending time was not noted). After blending, an additional 2 parts of ground lab chow was mixed by hand into the blended material. After this step, an additional amount of ground lab chow was added to give the appropriate dose level (25 mg/kg bw, 100 mg/kg bw or 400 mg/kg bw). The diet was then placed in a twin-shell mixer and mixed until the test material was distributed throughout the diet. The formula used to determine the amount of material added to feed was as follows: mean body weight x dietary level x 7 days/mean weekly food consumption = mg test material/kg feed. Fresh diets were prepared at least once per week. Doses were adjusted weekly on the basis of the weight and food consumption for the preceding week. Doses were not adjusted for purity of the material.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

90 consecutive days

Frequency of treatment:

Daily in diet

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

25 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

400 mg/kg bw/day (nominal)

No. of animals per sex per dose:

20

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Based on a 30d range-finding study (see other study record available)
- Fasting period before blood sampling for clinical biochemistry: overnight

Positive control:

No positive control reported

Examinations

Observations and examinations performed and frequency:

Food consumption and body weights were measured weekly. Animals selected for clinical chemistry, hematology and urinalysis (5 per sex per group) were determined by protocol and a table of randomization. Clinical chemistry (gamma-glutamyl transpeptidase, glucose, glutamic-oxaloacetic transaminase, glutamic pyruvic transaminase, and blood urea nitrogen), hematology (erythrocyte count, hematocrit, hemoglobin, differential and total leukocyte count, and platelet count) and urinalyses (appearance, glucose, microscopic examination of sediment, pH, protein and specific gravity) were performed on the same animals at appropriate time intervals (day 90 for clinical chemistries, days 45 and 90 for hematologies). Animals were fasted overnight before blood was collected from the orbital sinus on day 45 and from an unknown site on day 90. Urine also was collected on days 45 and 90.

Sacrifice and pathology:

At study termination, all animals were weighed. They were then euthanized. The urogenital orifaces, tail, each pinna, eyes and external auditory meatus were examined visually and by palpatation for distortions. All subcutaneous tissues were examined including regional lymph nodes, mammary and salivary glands. Abdominal contents, and the brain, pituitary gland and cranial nerves also were examined grossly. The following tissues were taken and preserved for possible histologic evaluation: adrenal, aorta, urinary bladder, bone, bone marrow, cerebellum, cerebrum, pancreas, pituitary, skin , stomach, thyroid, colon, esophagus, eye, heart, ileum, jejunum, kidney, prostate, salivary gland, spleen, testes, tongue, uterus, liver, lung, lymph node, mammary gland, muscle (skeletal), sciatic nerve, ovary, parathyroid, seminal vesicle, spinal cord, thymus and trachea. Any organ or tissue exhibiting a lesion was noted and the lesion was taken for histopathological examination. Complete histopathology was performed on 10 animals/sex/group from the control and high dose groups. These animals were selected randomly from a table. Microscopic examination of the heart, liver, lungs and kidneys was performed on all additional animals that survived to termination.

Statistics:

Data for food intake, weight gain, clinical chemistries, hematologies, urinalyses, and organ weights were analyzed using a blocked 2-way analysis of variance that compared data between and within groups. If F values were significant, data were compared using Dunnett's t-test. The level of significance is p < 0.05.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Grossly observable deviation from normal was seen in all groups. These deviations included alopecia around the nares and front paws, diarrhea, watery eyes and encrustment around nares. The alopecia appeared and disappeared in all groups throughout the study and was attributed to mechanical irritation due to movement in and out of the feeding container. The diarrhea was seen in an occasional individual in all groups at various periods, and was not considered to be related to the test material. No animal had diarrhea for two consecutive days. Watery eyes appeared primarily immediately after orbital bleeding of some animals for hematology and clinical chemistry determination and was considered to be the result of bleeding. Encrustment around nares is common to animals fed compound mixed in feed. There were no clinical signs manifested in this study attributable to the test material up to and including 400 mg/kg

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One animal in the mid-dose group (100 mg/kg) died after 12 days of treatment. Since no additional deaths occurred, this death was considered artifactual.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no significant differences in weight changes among treated groups when compared to control throughout the entire study.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

During first week on test, females in the 25 mg/kg treated group ate significantly less food than did the other female groups during that week. Males in the 25 mg/kg treated group ate less food than the other males during the third week on test. No other effets of the test material were seen upon food intake throughout the 13 weeks of the study. Therefore, it was concluded that dose levels up to 400 mg/kg bw of feed did not affect food intake in rats.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

After 45 days on study there was no significant difference in erythrocyte count in either males or females.
At 90 days, there was a trend toward increased red blood cells in low (8.27 x 10E6/mm3) and mid-dose animals [(8.63 x 10E6/mm3, value significantly different from study control (7.99 x 10E6/mm3) and historical controls]; however, counts in high dose animals (8.04 x 10E6/mm3) were similar to controls. Since the increase was not dose-related, this observation was thought to be artifactual in nature and not related to administration of test material.

There were no changes in hematocrit, hemoglobin, leukocyte count, platelet estimate and erythrocyte morphology.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Gamma-glutamyl transpeptidase (GGTP) values in males treated with 400 mg/kg were higher than control at 90 days (4.1 vs. 2.6 IU/l), but were within historical limits. The elevation was due to 2/5 males that had GGTP values that were twice the value of the other males. This effect was not seen in females. Since this elevation of GGTP was not dose related in males and since females did not have a change in GGTP values, it was concluded that this observation was not treatment-related.
Glucose values in females treated with 100 and 400 mg/kg (163 and 162 mg/dl, respectively) were lower than controls (209 mg/dl) at 90 days but were within the range of historical values (90-284 mg/dl). This effect was not seen in males. The significant differences in glucose between high and mid dose females and controls were considered to be a result of some unusually high values in the controls and were similar to the historical means. Therefore this effect was not considered to be related to test material.

Glutamic-oxaloacetic transaminase, Glutamic-pyruvic transaminase and urea were unaffected by the treatment.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no differences in the color, appearance, pH, specific gravity, milligram protein, milligram sugar, RBC see under a high power field, and WBC seen under a high power field in rats fed the test material for 90 days.

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No differences in weight of hearts, livers, kidneys, gonads or brain.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no descernable changes in most rats in each group. The few changes noted were considered unrelated by type or incidence to the administration of the test material. The gross pathological changes were not unlike those with spontaneous lesions to the laboratory rat of this age group. The lesions included artifacts of necropsy procedures (discoloration of tissue due to congestion); physiological changes (alteration in size of endocrine gland) and spontaneous inflammatory processes (pneumonia, arthritis, etc.). Each tissue with a gross change was examined microscopically to determine if histological correlation could be detected. No related histopathological correlation could be detected. Therefore, it was concluded that alterations seen at necropsy are unrelated to the treatment and have no apparent biological significance.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

The type and incidence of lesions detected by the histopathological examination were considered to represent spontaneous change in albino rats and unrelated to the administration of the test material.

Histopathological findings: neoplastic:

not specified

Details on results:

One animal in the mid-dose group (100 mg/kg) died after 12 days of treatment. Study personnel did not attribute this death to administration of test material. Animals were generally healthy during the study, but exhibited alopecia around the nares and paws, diarrhea, watery eyes (animals that were bled, only) and encrustment around the nares. Study personnel attributed these signs to ingestion of a powdered food and did not consider them to be related to test material. Other than week one (where females treated with 25 mg/kg test material ate less food than females in other groups) and week 3 (when males in the 25 mg/kg group ate less food than males in the other groups), there was no difference between food intake of treated and control animals. There was no effect of treatment on weight or weight gain.

Gamma-glutamyl transpeptidase (GGTP) values in males treated with 400 mg/kg were higher than control at 90 days (4.1 vs. 2.6 IU/l), but were within historical limits. The elevation was due to 2/5 males that had GGTP values that were twice the value of the other males. This effect was not seen in females. Since this elevation of GGTP was not dose related in males and since females did not have a change in GGTP values, it was concluded that this observation was not treatment-related. Glucose values in females treated with 100 and 400 mg/kg (163 and 162 mg/dl, respectively) were lower than controls (209 mg/dl) at 90 days but were within the range of historical values (90-284 mg/dl). The significant differences in glucose between high and mid dose females and controls were considered to be a result of some unusually high values in the controls and were not considered to be related to test material.

At 90 days, there was a trend toward increased red blood cells in low (8.27 x 10E6/mm3) and mid-dose animals [(8.63 x 10E6/mm3, value significantly different from study control (7.99 x 10E6/mm3) and historical controls]; however, counts in high dose animals (8.04 x 10E6/mm3) were similar to controls. Since the increase was not dose-related, this observation was thought to be artifactual in nature and not related to administration of test material.

There was no effect of treatment on urinalysis, organ weights, or gross pathology. One of the males with a high GGTP value had focal perivascular and periductal mononuclear leukocyte infiltration in the liver, but the other animal did not demonstrate any liver pathology. Livers of other males in the high dose group had histopathology similar to that of controls. Other mild inflammatory lesions characterized by leukocytic infiltration also occurred in the lungs, liver and kidneys of a few animals in each group. All changes were considered to be spontaneous and not related to administration of test material.

Effect levels

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: No adverse effects noted at the highest dose tested

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

The changes that were observed (elevated gamma-glutamyl transpeptidase in high dose males, decreased glucose in females, and increased red blood cell counts in mid-dose males at 90 days) were not considered to be related to test material since they were not dose-dependent and were within normal limits. Therefore, the NOAEL was 400 mg/kg bw.

Executive summary:

Sprague-Dawley rats were used in this study. 20 males and 20 females were assigned to control, and to each of the test groups (25, 100 and 400 mg/kg). The length of the treatment period was 90 days.

One animal in the mid-dose group (100 mg/kg) died after 12 days of treatment but was an isolated case and so not related to administration of test material.  Animals were generally healthy during the study, but exhibited alopecia around the nares and paws, diarrhea, watery eyes (animals that were bled, only) and encrustment around the nares. These signs were attributed to ingestion of a powdered food and were not considered to be related to test material.  

Food intake was measured weekly, other than a random week where a particular test group ate less food than comparable test groups or control groups, there was no significant difference in food intake between control and test animals in any sex grouping. Animals were weighed initially and then weekly thereafter. Weight gains were essentially comparable in treated and control groups with only a sex difference noted; males gained more weight than females.

Gamma-glutamyl transpeptidase (GGTP), glutamix-oxaloacetic transpeptidase, glutamic-pyruvic transaminase and urea were measured at 90 days in 5 males and 5 females from each test group. GGTP values were variable within the different dose groups but were within the values usually observed in this strain of rat. Glucose levels of treated animals were lower than controls but were within the range of normal values reported for this strain of rat. Since there were not statistically significant differences between the means of treated animals, the significant difference between the high-dose females and their control was considered to be a result of some unusually high values among the controls and was not considered to be the result of administering the test material. Glutamic-oxaloacetic transaminase, Glutamic-pyruvic transaminase and urea were unaffected by the treatment.

At 45 days, no changes in erythrocyte counts were noted. At 90 days, there was a trend toward increased red blood cells in low and mid-dose animals; however, counts in high dose animals were similar to controls. Since the increase was not dose-related, this observation was thought to be artifactual in nature and not related to administration of test material. There were no changes in hematocrit, hemoglobin, leukocyte count, platelet estimate and erythrocyte morphology.

There was no effect of treatment on urinalysis, organ weights, or gross pathology.  One of the males with a high GGTP value had focal perivascular and periductal mononuclear leukocyte infiltration in the liver, but the other animal did not demonstrate any liver pathology.  Livers of other males in the high dose group had histopathology similar to that of controls.  Other mild inflammatory lesions characterized by leukocytic infiltration also occurred in the lungs, liver and kidneys of a few animals in each group.  All changes were considered to be spontaneous and not related to administration of test material.

Therefore it is concluded that the test material did not affect adversely the rats after 90 days of administration at the highest dose tested. The NOAEL is 400 mg/kg bw.