2,5-thiophenediylbis(5-tert-butyl-1,3-benzoxazole)

Endpoint summary

Description of key information

Several studies with repeated expoure are available in both rats and dogs. The most sensitive species was the rat, based on the mild liver weight increase observed at the highest dose level of 3000 ppm after 90 days of treatment. This effect was not observed in dogs after 90 days, even at the highest dose level of 50000 ppm. Since the described effects on the liver in rats were mild and not accompanied by histological changes, this effect is considered as adaptive and the highest feeding level of 10000 ppm is therefore considered the NOAEL, corresponding to 707 mg/kg bw in males and 788 mg/kg bw in females.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

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 U.K. Limited, Margate, Kent, England
- Age at study initiation: four - five weeks
- Weight at study initiation: weight range 126 - 131 g (males) and 115 - 120 g (females)
- Fasting period before study: no
- Housing: Five rats of one sex were held in each polypropylene cage (Pattern RCl from North Kent Plastics Limited) measuring 56 x 38 X 18 cm, with stainless steel mesh floors and lids
- Diet: complete low-fat powdered rodent diet (Spratt's Laboratory Animal Diet No. 2, ad libitum
- Water: public supply, water was supplied to each cage via two polythene bottles with chromium plated sipper-tubes.
- Acclimation period: seven days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2
- Humidity (%): 55 +/- 20
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 1981-01-21 To: 1981-04-27 (1981-05-07)

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
For the treated groups, the test substance was incorporated into the powdered diet to provide the nominal concentrations.
The latter were held constant throughout the treatment period. A pre-mix was prepared freshly each week and from this the different dietary concentrations were obtained by serial dilution with further quantities of diet. Homogeneity was achieved by mixing (pre-mix) for ten minutes in a Hobart Model A200, then for 15 minutes (final mix) in either a Gardner Interrupted spiral mixer for dietary concentrations of 10000 or 3000 ppm, or in a Hobart model A200 for the dietary concentration of 1000 ppm. After formulation, the diets were sealed in transparent polythene bags within light-proof polythene bans.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before commencement of treatment, the test substance content of diet mixes representative of those to be used during the study was tested for homogeneity and for stability over a four-week period. Spot checks were performed for the test substance content of diets prepared for each treatment level for Weeks 1, 4, 8 and 13.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

contiuously in feed

Dose / conc.:

1 000 ppm

Remarks:

corresponding to 71.8 mg/kg in males and 78.3 mg/kg in females

Dose / conc.:

3 000 ppm

Remarks:

corresponding to 216.4 mg/kg in males and 246.0 mg/kg in females

Dose / conc.:

10 000 ppm

Remarks:

corresponding to 707.7 mg/kg in males and 788.8 mg/kg in females

No. of animals per sex per dose:

20 male and 20 female rats in each group

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS/MORTALITY: Yes
- Time schedule: once or twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: on the day that treatment commenced and at weekly intervals thereafter.
- Group mean values were calculated at the same intervals.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- 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
- Quantity of food eaten by each cage of rats was calculated weekly by measurement of the amount of food given and that remaining in the food hoppers, together with an estimation of food scattered.

FOOD EFFICIENCY: Yes
- Weekly food conversion ratios, i.e. the weights of food consumed per unit gain in bodyweight, were calculated.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: daily visual observation of the water bottles.

OPHTHALMOSCOPIC EXAMINATION: No / No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before before commencement of treatment, 6 and 12 weeks of treatment
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: No data
- How many animals: obtained from ten male and ten female rats per group
- Parameters checked: Haemoglobin concentration (Hb), Erythrocyte count (RBC), Leucocyte count (WBC) - total, differential, Platelet count, Prothrombin time (PT), Activated partial thromboplastin time (PTTK), Mean cell volume (MCV), Mean cell haemoglobin content (MCHC)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: before commencement of treatment, 6 and 12 weeks of treatment
- Animals fasted: No data
- How many animals: obtained from ten male and ten female rats per group
- Parameters checked: Urea concentration, Glucose concentration, Total protein concentration, Electrophoretic protein fractions, Alkaline phosphatase activity, Alanine amino-transferase activity (ALT), Aspartate amino-transferase activity (AST), Total bilirubin concentration, Direct bilirubin concentration, Total cholesterol concentration, Sodium (Na) and Potassium (K) concentrations, Calcium concentration (Ca), Chloride concentration (Cl), Creatinine concentration, Phosphorus (inorganic) concentration (P)

URINALYSIS: Yes
- Time schedule for collection of urine: before commencement of treatment, 6 and 12 weeks of treatment
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters checked: Appearance, Volume, pH, Specific gravity (SG), Protein, Total reducing substances, Glucose, Ketones, Bile pigments, Urobilin, Blood, Microscopy - the sediment from centrifugation (3000 rpm)

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

- Terminal observations: All rats surviving until the end of the treatment period and the rat sacrificed prematurely were subjected to euthanasia by carbon dioxide inhalation. Necropsy followed with the minimum of delay.

- The external features of each animal were scrutinised and compared to any relevant comment on the clinical history report. The eyes complete with optic nerve and relevant adnexa were removed. The cranial cap was lifted and the brain dissected free of meninges. The pituitary was freed from the sella turcica and fixed separately. The ventral abdominal skin was reflected to allow observation of the subcutaneous structures, in particular, mammary glands and superficial lymph nodes. Abdominal and thoracic viscera were examined in situ and a note was made of any abnormal position, morphology or interactions. The urinary bladder was slightly inflated with fixative and the urethra ligated. After removal, the bladder was viewed by transmitted light. The mucosa was examined after fixation at the time of embedding. The entire intestinal tract was re-examined after removal. The stomach was opened along its greater curvature and rinsed with isotonic saline, prior to fixation. The caecum was similarly treated. After weighing, both kidneys and the liver were repeatedly sectioned at intervals of a few millimetres; the cut surfaces were examined. The lungs were slightly inflated with fixative via the trachea prior to immersion fixation. The spinal cord was fixed within six thoraco-lumbar vertebrae.

- Organ weight analysis (without connective tissue):
Adrenal glands, Brain, Heart, Kidneys, Liver, Lungs, Ovaries, Pituitary gland, Prostate gland, Spleen, Testes (with epididymides attached), Thymus gland, Thyroid glands, Uterus

HISTOPATHOLOGY: Yes
- Tissues preserved in fixative:
Adrenal glands, Bone, Brain, Caecum, Colon, Duodenum, Eyes and optic nerves, Extra-orbital lacrimal glands, Harderian glands, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs (including bronchi), Lymph nodes (cervical and mesenteric), Mammary gland (caudal), Oesophagus, Ovaries, Pancreas, Pituitary gland, Prostate gland, Salivary gland (left), Seminal vesicles, Skeletal muscle, Skin, Spleen, Stomach (fundus and pylorus), Testes, Thymus gland, Thyroid glands, Trachea, Urinary bladder, Uterine cervix, Uterus

- Other tissues preserved (possible future requirement for microscopic evaluation):
Aorta, Mammary gland (cranial), Sciatic nerve (left), Spinal cord, Salivary glands (right), Tongue,

- Microscopic examination: From all of the control rats, and those rats given the highest dosage of the test substance, all the tissues listed above were subjected to microscopic examination. From rats receiving the low or intermediate dosage of the test substance, the heart, liver, kidneys and macroscopic abnormalities were subjected to microscopic examination. After dehydration and embedding in paraffin wax, sections of the required tissues were cut at 5 pm thickness and stained with haematoxylin and eosin. Both glandular and non-glandular areas of the stomach, and both auricular and ventricular sections of the heart, were thus prepared.
The brain was sectioned at three levels (cerebellum, cerebral cortex and medulla). Two sections each of the liver, lungs and lymphatic tissue were prepared.

Other examinations:

Concentration measurements of test substance in dietary samples confirmed the nominal test substance concentrations.

Statistics:

Statistical evaluation, by Student's t-test using a pooled within-group error variance, has been performed on the following:
- Overall food intake for selected periods
- Bodyweight increment for selected periods
- Haematology
- Blood chemistry
- Absolute organ weights
- Bodyweight-relative organ weights.
[Unless stated, group mean values were not significantly different from controls (p > 0.05)].

Clinical signs:

no effects observed

Description (incidence and severity):

There were no signs of reaction to treatment or deaths which could be ascribed to the administration of the test substance.

Mortality:

no mortality observed

Description (incidence):

One male rat receiving 3000 ppm was killed on humane grounds during Week 6 due to respiratory distress. Breathing appeared to be impaired by bleeding from a wound inside the upper lip and the resultant swelling of the muzzle. Necropsy did not reveal any treatment-related changes.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The bodyweight gain of rats receiving the test substance was unaffected by treatment.
Occasional inter-group differences in weight gain achieved statistical significance (p < 0.05), but these lacked a consistent pattern among the treated groups and were not dosage-related. They were, therefore, not considered to be of any biological significance.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The food consumption of treated rats was similar to that of the controls throughout the treatment period. The food consumption of females receiving 1000 ppm was marginally lower than that of the controls, but this was considered fortuitous and of no biological significance.

Food efficiency:

no effects observed

Description (incidence and severity):

The efficiency of food utilisation of treated rats, as estimated from their food conversion ratios, was broadly similar to that of the controls. The slightly higher overall food conversion ratio for females receiving 3000 ppm mainly reflected the slightly inferior weight gain of this group and was not attributed to treatment.

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

no effects observed

Description (incidence and severity):

Daily examination of water bottles did not reveal any differences in the water consumption of treated and control groups.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Haematological examination after six and 12 weeks of treatment did not reveal any effects that could be ascribed to treatment.
Such statistically significant inter-group differences as were detected lacked dosage-relationship and were not apparent on both occasions of examination.
After 12 weeks of treatment, low erythrocytic characteristics, packed cell volume, haemoglobin concentration and erythrocytic count in rat contributed to lower group mean values for these characteristics in males receiving 3000 ppm, when compared with control males. The resulting inter-group differences were not considered to be related to treatment.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Biochemical examination of plasma after six and 12 weeks of treatment did not reveal any effects that could be attributed to treatment. Such inter-group differences as were detected generally lacked a consistent trend or dosage-relationship, were not apparent on both occasions of examination and although in some cases attained statistical significance, were generally too small in magnitude to be of biological significance.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no changes in the chemical characteristics or constituents of urine examined that could be ascribed to treatment with the test substance. After 11 weeks of treatment marginally lower urine volumes recorded in treated rats were attributable to high values recorded in some animals of the control group.

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):

The absolute and body weight-relative liver weights of males and females receiving 10000 ppm were slightly higher than those of control animals.
There was no other inter-group difference in organ weights that could be ascribed to treatment. Higher body weight-relative liver weights in female rats receiving 1000 or 3000 ppm were attributed to the minimally lower body weight of these animals in comparison with control females, and were not considered to be of biological significance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no changes which associated with treatment in animals killed at termination. A range of commonplace lesions was present in the animals examined; such changes included subpleural foci and petechiae on the lungs of a few animals in control or treated groups. Slight hydronephrosis was present in a small number of male rats in the control or treated groups.
In one female animal in the lowest dosage group calculi were present in the urogenital tract. These findings are considered incidental.
In the male of Group 3 which was killed on humane grounds, a small range of commonplace lesions was present; these included pulmonary petechiae and pallor, prominent parathyroids, and enlarged and occasionally congested cervical lymph nodes.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Animals killed at termination of the study period: There were no pathological findings associated with treatment with the test substance.
(There was a range of mundane degenerative and inflammatory findings present in the tissues examined, similar in type and incidence to those considered usual in CD rats at this laboratory)
Animal killed in extremis: One male rat receiving 3000 ppm was killed on humane grounds. There were no pathological findings associated with treatment with the test substance. The cause of the terminal condition in this animal was considered to be acute periodontitis with intraepidermal oedema and haemorrhage.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

Achieved dosages, expressed as mg/kg/day, fell as the treatment period progressed, reflecting the declining ratio between food consumption and body weight. Although there were some inter-group differences in respect of growth and food consumption, the relationship between treatment levels was generally maintained in terms of the achieved dosage.

Dose descriptor:

NOAEL

Effect level:

10 000 other: ppm (mg/kg food)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Dose descriptor:

NOEL

Effect level:

3 000 other: ppm (mg/kg food)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects noted

Critical effects observed:

not specified

Achieved Doses:

(mg/kg body weight)

	Male Animals			Female Animals
Week	1000 ppm	3000 ppm	10000 ppm	1000 ppm	3000 ppm	10000 ppm
1	136.2	421.8	1329	130.6	398.9	1274
2	111.9	337.3	1119	109.5	339.4	1102
3	95.3	289.2	943	96.6	301.2	991
4	83.7	253.6	832	91.4	277.7	919
5	73.3	218	735	82.5	252.9	837
6	65.5	198.5	647	75.6	237.8	780
7	58.7	169.9	569	67.1	214.1	670
8	55.3	166.9	543	65.6	220.9	666
9	55.4	159.5	524	63.9	205	634
10	51.3	151.8	492	60.5	192.9	617
11	49.3	151.5	512	61	192.3	605
12	47.7	146.7	471	58.4	186.3	594
13	49.4	147.9	484	55.1	178.5	565
Average	71.8	216.4	707.7	78.3	246.0	788.8

Conclusions:

The only changes that could confidently be ascribed to treatment with the test substance were slightly higher absolute and body weight-relative liver weights of rats receiving 10000 ppm and even these inter-group differences from controls were minimal. The liver weights of rats receiving 3000 ppm were not disturbed by treatment. There was no evidence of histopathological change in the liver or the other tissues examined. It is concluded therefore that the test substance at concentrations of 1000 and 3000 ppm is without toxicological effect in dietary administration to the rat.

Executive summary:

Groups of 20 male and 20 female CD rats were fed diets containing the test article at concentrations of 1000, 3000 or 10,000 ppm for 13 weeks. A similarly constituted group of rats received untreated diet and served as a control group. There were no signs of reaction to treatment. One male receiving 3000 ppm was killed on humane grounds; necropsy and microscopic examination of the tissues did not reveal any lesions that could be ascribed to treatment. Food and water consumption and body weight gain were unaffected by administration of test material. Food conversion ratios of the treated rats remained broadly similar to those of the controls throughout the treatment period. Haematological examination after six and 12 weeks of treatment did not reveal any changes that could be attributed to treatment. Examination of the chemical composition of the plasma after six and 12 weeks of treatment did not reveal any effects that could be attributed to treatment. Urinalysis after six and 11 weeks did not reveal any treatmentrelated effects. Macroscopic examination of the tissues of rats killed at termination did not reveal any changes that could be ascribed to treatment. The absolute and bodyweight-relative liver weights of rats receiving 10,000 ppm were slightly higher than those of controls. Microscopic examination of the tissues of rats killed at termination did not reveal any changes that could be ascribed to treatment. It is concluded that the administration of the test item at a dietary concentration of 3000 ppm elicited no manifestation of toxicity and is considered as the NOEL.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1989-10-03 to 1990-01-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)

Version / remarks:

1981

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: Toxicological principles for the safety assessment of direct food additives and color additives used in food: Guideline for Subchronic Oral Toxicity Studies, US Food and Drug Administration, Bureau of Foods, 1982.

GLP compliance:

yes

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Animal production CIBA-GEIGY Limited, 4332 Stein, Switzerland
- Age at study initiation: males 24-30 weeks, females 24-29 weeks
- Weight at study initiation: males 7.5 - 10.7 kg, females 7.1 - 11.0 kg
- Housing: Two dogs were housed together in a kennel.
- Diet: Certified pelleted standard diet NAFAG 941 Tox, 350 g/animal daily.
- Water: Tap water was given ad libitum. The drinking water quality fulfilled in the critical parameters the specifications of the "Schweizerisches Lebensmittelbuch" (Edition 1972).
- Acclimation period: 27 days between delivery and the start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): minimum room temperature of 15° C
- Humidity (%): No data.
- Air changes (per hr): No data.
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 1989-10-30 To: 1990-01-28 (1990-01-30)

Route of administration:

oral: feed

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS / DIET PREPARATION
The appropriate amounts of the test substance were added to pulverized food, mixed, added subsequently to a further portion of pulverised food and mixed homogeneously. To these premixes diet was added to attain the nominal concentrations, blended homogeneously and pelleted. Three batches of medicated feed were prepared for the 13 weeks test period. The animals in the control group were fed with similarly prepared food without the test substance. The medicated diet was stored in stainless steel containers at room temperature.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prior to the start of the study, samples of the medicated food containing the test article at concentrations of 500, 5000 and 50000 ppm, were dispatched to the Analytical Laboratories Rosental, CIBA-GEIGY Limited, Basle, Switzerland, for analysis of content and stability. Results of the stability tests
indicate that the test article stable for at least 6 weeks in dog pelleted diet at room temperature.

Duration of treatment / exposure:

91 to 93 days

Frequency of treatment:

once/day, 7 days a week

Dose / conc.:

500 ppm

Remarks:

corresponding to 16.8 mg/kg bw/d (m); 16.6 mg/kg bw/d (f)

Dose / conc.:

1 500 ppm

Remarks:

corresponding to 47.4 mg/kg bw/d (m); 49.0 mg/kg bw/d (f)

Dose / conc.:

5 000 ppm

Remarks:

corresponding to 154 mg/kg bw/d (m); 169 mg/kg bw/d (f)

Dose / conc.:

50 000 ppm

Remarks:

corresponding to 1571 mg/kg bw/d (m); 1682 mg/kg bw/d (f)

No. of animals per sex per dose:

4 males and 4 females

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The doses were selected based on the results of a previously conducted 3-month oral toxicity study in dogs (HRC, March 26, 1964) with the test substance. 50000 ppm was expected to result in a daily intake of about 1500 mg/kg bodyweight and was chosen as the highest dose level because higher concentrations of the test substance in the diet were expected to interfere with normal nutrition.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS/MORTALITY: Yes
- Time schedule: checked daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: examination was carried out daily

BODY WEIGHT: Yes
- Time schedule for examinations: recorded individually at weekly weighing sessions

FOOD CONSUMPTION AND COMPOUND INTAKE:
- 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

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: performed at pretest and week 13
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Pretest, Week 7, Week 13
- Anaesthetic used for blood collection: No data
- Animals fasted: yes
- How many animals: all surviving animals of each sex and group
- Parameters examined: Erythrocyte Count (RBC), Hemoglobin (Hb), Hematocrit (Hct), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Leucocyte Count (WBC), Differential Leucocyte Count, Thrombocyte Count (Plt), Prothrombin Time (PT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Pretest, Week 7, Week 13
- Animals fasted: yes
- How many animals: all surviving animals of each sex and group
- Parameters checked: Glucose, Urea, Creatinine, Total bilirubin, Total protein, Albumin, Globulins, A/G Ratio, Cholesterol, Sodium, Potassium, Calcium,
Chloride, Phosphorus inorganic, Aspartate aminotransferase (ASAT, GOT), Alanine aminotransferase (ALAT, GPT), Alkaline phosphatase (ALP), Gamma-glutamy1 transpeptidase (GGT)

URINALYSIS: Yes
- Time schedule for collection of urine: Pretest, Week 7, Week 13
- Metabolism cages used for collection of urine: No data
- Animals fasted: yes
- Parameters checked: Relative density, pH value, Protein, Glucose, Ketones, Bilirubin, Blood, Urobilinogen

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
At the end of the 3 months treatment period all scheduled control and treated dogs were bled under T-61 (Hoechst) anesthesia and subjected to detailed autopsy. Besides the weight of the exsanguinated body the following organs were weighed: brain, heart, liver, kidneys, adrenals, ovaries/testes, spleen, thymus, thyroid gland (including parathyroids) ,

The following organs and tissues were preserved in neutral buffered 4% formalin: skin, mammary area, spleen, cervical lymph node, mesenteric lymph node, popliteal lymph node, sternum with bone marrow, rib with cartilage, skeletal muscle, trachea, lung, heart, aorta, submandibular salivary gland, liver, gall bladder, pancreas, oesophagus, stomach, small intestine, large intestine, kidney, urinary bladder, prostate, testis, epididymis, vagina, uterus, ovary, pituitary gland, adrenal gland, thyroid with parathyroid gland, thymus, peripheral nerve, brain, spinal cord, eye with optic nerve, orbital gland, any tissue with gross lesions.
Because of macroscopical observation of discolored nose the muzzle together with transition to the skin was taken at the autopsy from the females of groups 1 (0 ppm) and 5 (50000 ppm).

HISTOPATHOLOGY: Yes
After the fixation, organ samples listed above were taken, embedded in paraplast, sectioned at 3-5 microns, stained with hematoxylin and eosin, and subjected to microscopical examination.

Statistics:

For each time point and parameter a trend test was conducted (Ref.) showing increasing or decreasing trends in location from control to the highest dose group. This test is sensitive to monotone dose-related treatment effects. Due to the small sample size (n = 4) no other routine statistical tests were performed.
For each group mean values and standard deviations were calculated for each parameter and time point. Reference: A. R. Jonckheere, Biometrika (1954) 41:133-145.

Description (incidence and severity):

Greyish discoloration of the feces was observed for animals of both sexes of the high dose group (50000 ppm) during the whole treatment period and for animals of group 4 (5000 ppm) during days 2 and 3. This observation was considered to reflect the excretion of the test article in the feces. Blue discoloration of the tip of the nose was observed for animals of both sexes of the high dose group (50000 ppm) from week 7 onwards. However, this discoloration was removable when washed with water and microscopical examination of the skin, made only in females as a representative sample, did not reveal any abnormalities which could account for the discoloration. Therefore, this finding is presumably due to a local deposition of dust particles from the medicated pellets and is of no toxicological relevance. A variable degree of diarrhea occurred intermittently for control and treated animals but showed no relationship to treatment. Except for the above, the appearance and behaviour of treated dogs was similar to that of the controls.

Mortality:

no mortality observed

Description (incidence):

None of the animals died in the course of this study or had to be sacrificed in moribund condition.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Bodyweight development of treated groups was comparable to controls.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No influence on food consumption could be seen for treated groups.

Food efficiency:

no effects observed

Description (incidence and severity):

Food consumption ratios of treated groups were comparable to controls.

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

The examination of conjunctiva, sclera, cornea, lens and fundus did not reveal any substance related findings. No alterations of the pupillary reflex could be seen in control or treated dogs. Detailed examination of the third eyelid under local anesthesia revealed conjunctivitis follicularis in all male and female dogs of the control and treated groups at pretest and at week 13. This finding is, therefore, not considered to be of experimental relevance.

Haematological findings:

no effects observed

Description (incidence and severity):

The treatment had no effects on the hematological parameters investigated.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Markedly increased activities of alanine aminotransferase and aspartate aminotransferase occurred in two female dogs of group 5 (50000 ppm) at week 7. These findings were not confirmed at termination of the study. In the absence of corroborative changes in histopathology and in organ weights no toxicological relevance was attributed to these transient changes.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No changes attributable to the treatment were observed in urinalysis.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The treatment had no effect on investigated organ weights and ratios.

Gross pathological findings:

no effects observed

Description (incidence and severity):

At the end of treatment period all animals from group 5 (50000 ppm) had staining of the nose. Histopathological examination performed on females of this group (see Symptoms), did not reveal any abnormalities which could account for this observation. The occurrence of all the other macroscopical findings was similar to those commonly found in our colony of the Beagle dogs and was regarded to be of no experimental significance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

All reported microscopical changes were incidental in nature and not related to application of the test article. Microscopical examination of the nasal mucosa and its transition to the normal skin in females of group 5 (50000 ppm) did not reveal any abnormalities which could account for staining of the nose reported at the autopsy.

Dose descriptor:

NOEL

Effect level:

1 571 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: The dietary level of the test substance producing no findings of toxicological relevance can be defined as 50000 ppm (equivalent to an average daily intake of 1571 mg/kg body weight for males).

Dose descriptor:

NOEL

Effect level:

1 682 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: The dietary level of the test substance producing no findings of toxicological relevance can be defined as 50000 ppm (equivalent to an average daily intake of 1682 mg/kg body weight for females).

Critical effects observed:

not specified

ANALYTICAL INVESTIGATIONS

Based on the results of analytical determinations of the test substance in the food, the content of active ingredient in all samples was found to be in agreement with the nominal concentrations. The overall mean concentrations, expressed as a percentage of the nominal concentrations were found to be 99.3%, 97.1%, 100.5% and 100.8% for dose groups 2, 3, 4 and 5, respectively. The batches were considered to be homogeneous.

A stability test indicate that the test substance is stable for at least 6 weeks in dog pelleted diet at room temperature.

Corrected for the analytically found test substance concentrations in the diet and rounded to 3 or 4 meaningful digits, the mean daily intake of the test substance was 16.8, 47.4, 154 and 1571 mg/kg bodyweight for males, and 16.6, 49.0, 169 and 1682 mg/kg bodyweight for females.

Conclusions:

Based on the above findings, the dietary level producing no findings of toxicological relevance can be defined as 50000 ppm, equivalent to an average daily intake of 1571 mg/kg bodyweight for males and 1682 mg/kg bodyweight for females.

Executive summary:

In the present study a total of 40 pedigree Beagle dogs was used (4 males and 4 females per dose group). The test article was

administered mixed in the diet at doses of 0, 500, 1500, 5000 and 50000 ppm (= mg/kg food). Based on the concentration of the test compound in the diet samples (confirmed by chemical analysis) the calculated mean daily intake was 16.8, 47.4, 154 and 1571 mg/kg bodyweight for males and 16.6, 4 9.0, 169 and 1682 mg/kg bodyweight for females. None of the animals died in the course of this study or had to be sacrificed in moribund condition. No signs and symptoms of toxicological relevance occurred in treated animals. No influence on bodyweight development was recorded for treated groups. No influence on food consumption could be seen for treated groups. Food consumption ratios of treated groups were comparable to controls. Ophthalmoscopic examination revealed no reaction to the treatment. The treatment had no effects on the hematological parameters investigated and no findings of toxicological relevance were noted in blood chemistry. No changes attributable to the treatment were observed in urinalysis. The treatment had no effect on investigated organ weights and ratios. Pathological examination of control and treated animals did not reveal any abnormalities which could be attributed to the effect of the test article. Based on the above findings, the dietary level producing no findings of toxicological relevance can be defined as 50000 ppm, equivalent to an average daily intake of 1571 mg/kg bodyweight for males and 1682 mg/kg bodyweight for females.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

707 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral – sub-chronic dietary studies:

In a subchronic toxicity study in rats, groups of 20 male and 20 female CD rats were fed diets containing the test substance at concentrations of 1000, 3000 or 10,000 ppm for 13 weeks. A similarly constituted group of rats received untreated diet and served as a control group. There were no signs of reaction to treatment. One male receiving 3000 ppm was killed on humane grounds; no toxicological cause could be determined by post-mortem examination. Food and water consumption were unaffected by administration as well as body weight gain. Food conversion ratios of the treated rats remained broadly similar to those of the controls throughout the treatment period. Hematological examination and examination of the chemical composition of the plasma after six and 12 weeks of treatment did not reveal any effects that could be attributed to treatment. Urinalysis after six and 11 weeks did not reveal any treatment-related effects. Macroscopic examination of the tissues of rats killed at termination did not reveal any changes that could be ascribed to treatment. The absolute and relative liver weights of rats receiving 10,000 ppm were slightly higher than those of controls. Microscopic examination of the tissues of rats killed at termination did not reveal any changes that could be ascribed to treatment. Based on this results, it is concluded that the administration of the test substance at a dietary concentration of 3000 ppm elicited no manifestation of toxicity and represented the NOEL. Based on the mild effects on liver weights, with no correlation in histology, the highest dose level of 10000 ppm can be regarded as NOAEL. The calculated average intake at this dose level is 707 mg/kg body weight for males and 788 mg/kg body weight for females.

A second subchronic dietary study performed with rats demonstrated that the test substance is tolerated by the young, rapidly growing rat without detectable signs of toxicity. 10 male and 10 female rats received the test article in feed at dose levels of 50, 5000 and 50000 ppm, the latter was increased to 100,000 ppm after 7 weeks of treatment due to lack of toxicity. No toxic symptoms were observed and there were no mortalities. This indicates an unusually low level of toxicity in this species. Regarding haematology, there were no essential differences between control and investigated test animals. Although changes in organ weight/body weight ratio occurred at all levels of administration, it was not considered that these are necessarily associated with treatment with the test substance based on the lack of a dose response relationship. The microscopic examination did not reveal evidence of cellular damage in the organs exhibiting this change. A NOAEL of 100000 ppm (estimated at 5000 mg/kg body weight) was postulated.

In a GLP-compliant sub-chronic toxicity study following OECD guideline 408, a total of 40 pedigree Beagle dogs were used (4 males and 4 females per dose group). The test substance was administered admixed in the diet at dose levels of 0, 500, 1500, 5000 and 50000 ppm (= mg/kg food) for 90 days. Based on the concentration of the test substance in the diet samples (confirmed by chemical analysis) the calculated mean daily intake of the test substance was 16.8, 47.4, 154 and 1571 mg/kg bodyweight for males and 16.6, 4 9.0, 169 and 1682 mg/kg bodyweight for females. None of the animals died in the course of this study or had to be sacrificed in moribund condition. No signs and symptoms of toxicological relevance occurred in treated animals. No influence on bodyweight development was recorded for treated groups. No influence on food consumption could be seen for treated groups and the food consumption ratios of treated groups were comparable to controls. Ophthalmoscopic examination revealed no reaction to the treatment. The treatment had no effects on the hematological parameters investigated. Concerning blood chemistry and urinalysis, no findings of toxicological relevance were noted. The treatment had no effect on investigated organ weights and ratios. Furthermore, pathological examination of control and treated animals did not reveal any abnormalities which could be attributed to the effect of the test substance. Conclusively, a five percent dietary inclusion level (50000 ppm) of the test substance exceeded the required dose level of a limit test (1000 mg/kg bodyweight) and was well tolerated and without effects on behaviour, food consumption and body weight development. Based on the above findings, the dietary level of the test substance producing no findings of toxicological relevance can be defined as 50000 ppm, equivalent to an average daily intake of 1571 mg/kg body weight for males and 1682 mg/kg body weight for females.

 

A further oral toxicity study in dogs (2 male and 2 female per dose group) demonstrated that up to a dosage of 2500 mg/kg/day administered in capsules for 12 weeks (on 6 days per week), the symptomatology was minimal. Most of the dogs receiving 2500 mg/kg bw/d passed loose, yellow colored stools. At 250 mg/kg bw/d, stools were often loose. No other symptoms were seen. The majority of these dogs had an eczematous condition ('mange') which was variable in extent but usually mild and localised to abdominal wall. There were considerable differences between the amounts of weight gained over the 13 week dosing period by individual animals, but there were no consistent differences that could be related to dosage with the test substance. The mean weight gains were remarkably similar for each group of animals. Regarding food consumption, there were no striking or consistent differences between the various subgroups. For hematology and biochemistry, no abnormalities were recorded which suggest toxicity. Gross pathology and histopathology demonstrated no findings which could be attributed to dosing with the test substance (no abnormalities observed). Thus, a NOAEL of 2500 mg/kg bw/day can be postulated.

 

Oral – chronic dietary studies:

In a oral carcinogenicity study, male and female (35/sex) rats were fed continuously with the test substance at a dietary concentration of 1000 ppm over a period of 104 weeks. A group of rats fed the diet without test material served as control. Treated animals remained indistinguishable from untreated controls in respect of growth performance, mortality rate and general condition; terminal hematology, biochemistry, urinalysis and histology revealed no evidence of any reaction to treatment. In particular, the total and differential tumor incidence was similar in the treated and untreated animals. By exposure to ultra-violet light, fluorescent material was demonstrable in body fat, and (to a much lesser extent) in the eyes of treated animals. Optic lens opacities of trefoil or Y-shaped configuration were evident in 10/36 treated rats surviving to termination. The incidence of these opacities was higher in the treated group than in the concurrent controls of this experiment, and indeed higher than that previously recorded among animals of the same age and strain. Whilst this might be suggestive of an effect of the test substance, the number of surviving animals in each group is small and this restriction possibly precludes the drawing of any hard and fast conclusion. Most of these eyes were examined histologically, but only senility changes were found. The incidence of other types of opacities in this experiment was similar for both treated and control groups. In male rats only, a minor degree of liver enlargement was apparently associated with treatment. There was no evidence of altered liver histology, serum alkaline phosphatase activity or serum glutamate pyruvate transaminase activity in these animals. Based on the results, a NO(A)EL for toxicity of 1000 ppm was derived.

In a second chronic study a dietary concentration of 1000 ppm was fed to groups of 52 mice per sex over a period of 52 weeks, followed by observation for 26 weeks. 52 rats per sex fed diets without the test article served as control group. Throughout the investigation, mortality rates and prime causes remained grossly comparable for the treated and untreated groups. No treatment-related trends were seen in growth or food consumption. The examination of the treated mice under UV illumination revealed fluorescent deposits in adipose tissue. After 26 weeks withdrawal from treatment, fluorescence was somewhat reduced, although still evident. Furthermore, there was no evidence to suggest that the test substance administered at 1000 ppm in the diet, might be carcinogenic for mice; tumor incidence was unaffected by treatment with the test substance. It was concluded that there was no evidence of adverse reaction to treatment, and that deposition of fluorescent material constituted the sole treatment-related finding. Based on the presented results, a NO(A)EL for toxicity of 1000 ppm was derived.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. Based on the available data the test substance is not considered to be classified for repeated dose toxicity (oral) under Regulation (EC) No 1272/2008.