diethyl 1-(2,4-dichlorophenyl)-5-methyl-4,5-dihydro-1H-pyrazole-3,5-dicarboxylate

Endpoint summary

Description of key information

Subacute toxicity:

Oral toxicity (OECD 407), 28 days, rat: NOAEL = 102.0 mg/kg bw/day (males) and 101.4 mg/kg bw/day (females)

Oral toxicity (OECD 407), 28 days, mouse (male/female): NOAEL = 1100 mg/kg bw/day

Oral toxicity (shortened OECD 409), 30 days, dog (male/female): NOAEL = 342 mg/kg bw/day

Dermal toxicity (OECD 410), 29 days, rat (male/female): NOAEL = 300 mg/kg bw/day

 

Subchronic toxicity:

Oral toxicity (OECD 408), 90 days, rat: NOAEL 42.1 mg/kg bw/day (males) and 44.3 mg/kg bw/day (females)

Oral toxicity (OECD 408), 90 days, mouse: NOAEL 89.3 mg/kg bw/day (males) and 105.4 mg/kg bw/day (females)

Oral toxicity (OECD 409), 90 days, dog (male/female): NOAEL = 81 mg/kg bw/day

 

Chronic toxicity:

Oral toxicity (OECD 453), 104 weeks, rat: NOAEL = 48.47 mg/kg bw/d (males) and 59.98 mg/kg bw/d (females)

Oral toxicity (OECD 452), 52 weeks, dog (male/female): NOAEL = 55 mg/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

Guideline adapted to the needs of a shorter study duration (subchronic to subacute).

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

adapted to the objectives of a 30-day toxicity study

GLP compliance:

yes

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL Biological Research Laboratories Ltd., Wolferstr. 4, CH 4414 Fuellinsdorf/Switzerland
- Age at study initiation: 5 to 7 months
- Weight at study initiation: 6 - 10 kg
- Fasting period before study:
- Housing: Air-conditioned, individual kennels with a minimum of 2.0 square meters floor space.
- Diet: 350 g repelleted standard Kliba 335 dog maintenance diet ("Kliba", Klingentalmuehle AG, 4303 Kaiseraugst/Switzerland) presented at approximately 10.00 daily and withdrawn at 13.00.
- Water: Tap water, ad libitum by an automatic watering system.
- Acclimation period: 20 days under test conditions, after veterinary examination (8 days after arrival).


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3, monitored hourly; kennel floor temperature maintained at approximately 23°C
- Humidity (%): 40 - 70 %, monitored hourly
- Air changes (per hr): 20
- Photoperiod (hrs dark / hrs light): 12/12, with at least 8 hours music during the light period.

Route of administration:

oral: feed

Vehicle:

other: microgranulated feed with approx. 1:10 volume/weight ratio water added

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): every 2 weeks
- Mixing appropriate amounts with (Type of food): microgranulated feed, see details on environmental conditions
- Storage temperature of food: Food required for the first week was stored at room temperature. Food required for the second week was stored at -20°C for the first week and at room temperature during the second week (i.e. the week of use).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability, homogeneity and content of the test article in the feed was determined from the first batch of diet prepared for the study. Intercurrent sampling for analyses (homogeneity and content) was performed at 2 weeks. Analyses were performed in the RCC UMWELTCHEMIE AG, according to a method supplied by the sponsor.
The mean concentrations found were 93.0 %, 92.9 %, 93.7 % and 90.6 % for dose groups 2, 3, 4 and 5 of the nominal concentration, respectively. The homogeneity varied in the range from -3 % to +3 % of the mean concentration.

HOE 107892 SUBSTANCE TECHNICAL was found to be stable at room temperature for at least 21 days.

Duration of treatment / exposure:

males 30 days, females 31 days

Frequency of treatment:

daily during the feeding period (for 3 hours, within the feed)

Dose / conc.:

1 250 ppm

Remarks:

equivalent to 51 mg/kg bw/day

Dose / conc.:

2 500 ppm

Remarks:

equivalent to 95 mg/kg bw/day

Dose / conc.:

5 000 ppm

Remarks:

equivalent to 179 mg/kg bw/day

Dose / conc.:

10 000 ppm

Remarks:

equivalent to 342 mg/kg bw/day

No. of animals per sex per dose:

2 (3 controls)

Control animals:

yes, plain diet

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily
- Cage side observations included: viability, clinical signs (behaviour, appearance)


DETAILED CLINICAL OBSERVATIONS: No


BODY WEIGHT: Yes
- Time schedule for examinations: at least once weekly and before necropsy


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/dog/day: Yes (The food consumption was recorded daily. The daily ration was weighed before and after feeding. The daily food consumption over each week for each dog, and the group mean over each week of these values are reported.)
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No (The test article intake is the mean achieved daily test article consumption per kg of body weight, calculated from the nominal test article concentration, the average daily food consumption over one week for an individual dog and the body weight.)


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No


WATER CONSUMPTION AND COMPOUND INTAKE: No


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pretest and at 4 weeks
- Dose groups that were examined: all


HAEMATOLOGY: Yes
- Time schedule for collection of blood: pretest and at 4 weeks
- Anaesthetic used for blood collection: No
- Animals fasted: Yes, overnight
- How many animals: all
- Parameters examined: erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, reticulocyte count, nucleated erythrocytes normoblasts, Heinz bodies, methemoglobin, total leukocyte count, differential leukocyte count, red cell morphology, thromboplastin time, activated partial thromboplastin time


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: pretest and at 4 weeks
- Animals fasted: Yes, overnight
- How many animals: all
- Parameters examined: glucose, urea, creatinine, uric acid, total bilirubin, total lipids, total cholesterol, triglycerides, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, creatine kinase, alkaline phosphatase, gamma-glutamyl-transferase, calcium, phosphorus, sodium, potassium, chloride, total protein, protein electrophoresis, reduced glutathione, oxidized glutathione, total glutathione

URINALYSIS: Yes
- Time schedule for collection of urine: pretest and at 4 weeks
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, overnight
- Parameters examined: volume, specific gravity, colour, appearance, pH, protein, glucose, ketone, bilirubin, blood, urobilinogen, sediment, creatinine, creatinine clearance, lactate dehydrogenase, gamma-glutamyl-transferase, leucine aminopeptidase, sodium, potassium


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: pretest and at 4 weeks
- Dose groups that were examined: all
- Battery of functions tested: other: hearing test

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, all organs
HISTOPATHOLOGY: Yes, adrenal glands, aorta, bone - femur (including articular surface), bone marrow - femur and sternum, brain - including sections of medulla pons, cerebral and cerebellar cortex, epididymides, esophagus, eyes (with optic nerve), female mammary gland area, gall bladder, heart, kidneys, large intestine - caecum, colon and rectum, liver, lungs (infused with formalin), lymph nodes - retropharyngeal and mesenteric, ovaries, pancreas, pituitary gland, prostate gland, salivary glands - mandibular and parotid, sciatic nerve, skeletal muscle, skin, small intestine - duodenum, jejunum and ileum, spinal cord - cervical, midthoracic and lumbar, spleen, stomach, testes, thymus, thyroid gland (including parathyroid), tongue, trachea, urinary bladder, uterus, all gross lesions, tissue masses and tumours.

Statistics:

No statistical analysis was performed due to the low number of animals used in this study.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
All animals survived the 4 week treatment period. There were no treatment-related clinical signs.

BODY WEIGHT AND WEIGHT GAIN
Low weight gain was recorded during the treatment period in both males and one female at 10000 ppm. Body weight gain in the remaining groups was considered to be unaffected by treatment.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Slightly to moderately reduced mean food intake was recorded over the treatment period in males and females at 10000 ppm and in females at 5000 ppm in comparison with food intake during the pretest period. Food intake in the dogs at 1250 or 2500 ppm and the males at 5000 ppm was considered to be unaffected by treatment.
The ratio of test article intake of each group was approximately in proportion to the concentration of HOE 107892 SUBSTANCE TECHNICAL in the diet.


OPHTHALMOSCOPIC EXAMINATION
The ophthalmic parameters were unaffected by treatment.

HAEMATOLOGY
Hematology parameters were unaffected by treatment.

CLINICAL CHEMISTRY
A slight to moderate reduction in oxidized glutathione concentration in the liver was recorded in males and females at 2500, 5000 or 10000 ppm in comparison with the controls. This reduction was most pronounced in both sexes at 5000 ppm. The remaining parameters examined were considered to be unaffected by treatment.

URINALYSIS
Urinalysis parameters were unaffected by treatment.

NEUROBEHAVIOUR
Auditory perception was unaffected by treatment.

ORGAN WEIGHTS
Moderately increased liver weight was recorded in males (absolute and relative to body weight) and females (absolute and relative to brain and body weight) at 10000 ppm. There were no other changes that could be clearly associated with treatment.

GROSS PATHOLOGY
No significant findings were recorded.

HISTOPATHOLOGY: NON-NEOPLASTIC
Haemocysts were observed on the atrioventricular valve in single animals at 2500 and 5000 ppm, and in the adrenal cortex of one animal at 10000 ppm. These findings are of uncertain significance, but are most likely to be of a spontaneous nature.


HISTORICAL CONTROL DATA (if applicable)
Clinical laboratory reference values for untreated Beagle Dogs.

Dose descriptor:

NOEL

Remarks:

systemic

Effect level:

1 250 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: corresponding to 51 mg/kg bw/day average test article intake

Dose descriptor:

LOEL

Remarks:

systemic

Effect level:

2 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

Remarks on result:

other: corresponding to 95 mg/kg bw/day average test article intake

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

10 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effect observed

Remarks on result:

other: corresponding to 342 mg/kg bw/day average test article intake

Remarks:

highest dose tested

Critical effects observed:

not specified

Treatment with HOE 107892 SUBSTANCE TECHNICAL at a dietary inclusion level of 10000 ppm (approximately 342 mg/kg body weight/day in this study) was associated with slightly to moderately reduced food intake with a corresponding reduction in body weight gain, a moderate reduction in oxidized glutathione concentration in the liver, and moderately increased liver weight.

A slight reduction in food intake was also recorded in the females at 5000 ppm and a slight to moderate reduction in oxidized glutathione concentration in the liver was evident in the dogs at 2500 or 5000 ppm, respectively.

No substance-related changes could be detected at a dietary level of 1250 ppm (approximately 51 mg/kg body weight/day). Therefore, this dose level was the NOEL on this study.

Based on the above findings, a dietary inclusion level of 10000 ppm HOE 107892 SUBSTANCE TECHNICAL was considered suitable as the high dose level for a 13 week study in this species.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Principles of method if other than guideline:

Additionally, after completion of the guideline study, the activities of aminopyrine N-demethylase, pentylresorufin O-depentylase, microsomal, NADPH2-dependent cytochrome c reductase, and glutathione S-transferase as well as the contents of reduced glutathione were measured in frozen kidney samples of the mice treated with Hoe 107892 (technical substance) for 28 days in the Guideline study.

GLP compliance:

yes

Limit test:

no

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hoechst AG, Pharma Development Toxicology, Kastengrund, SPF breeding colony
- Age at study initiation: approx. 5 weeks
- Weight at study initiation: males 23.5 ± 0.6, females 20.9 ± 0.6
- Housing: in fully air-conditioned rooms in Makrolon cages (Typ 3) on soft wood granulate in groups of 5 animals
- Diet (e.g. ad libitum): Altromin 1321 mouse diet (Altromin GmbH, Lage/Lippe), in special bran feed racks, ad libitum, except for the period in which the animals were kept in diuresis cages
- Water (e.g. ad libitum): tap water in plastic bottles, ad libitum, except for the period in which the animals were kept in diuresis cages
- Acclimation period: males 6 days, females 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5 - 22
- Humidity (%): 51 - 60
- Air changes (per hr): 6
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): once prior to treatment
- Mixing appropriate amounts with (Type of food): Altromin 1321
- Storage temperature of food: room temperature

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

3 separate samples were taken from each diet mix immediately after preparation. The homogeneity and stability of the test substance preparations were guaranteed over a period of 35 days. The actually administered test substance concentrations corresponded in sufficient degree to the nominal test substance concentrations administered via the diet.

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

continuously

Dose / conc.:

100 ppm

Dose / conc.:

500 ppm

Dose / conc.:

1 000 ppm

Dose / conc.:

2 500 ppm

Dose / conc.:

5 000 ppm

Dose / conc.:

7 500 ppm

No. of animals per sex per dose:

5

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, and at weekends and on public holidays once daily
- Cage side observations included: behaviour and general health condition.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly for damage to the oral mucosa and impairment of dental growth.


BODY WEIGHT: Yes
- Time schedule for examinations: at the start of the study and then twice weekly thereafter.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Determined at the same times as the body weights. The values reported refer to the intervals between the measurements and are converted to show food consumption over 24-hour periods.
- 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: No


WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: once weekly for a period of 16 hours


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: weekly for opacity of the refracting media of the eyes
- Dose groups that were examined: all animals


HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the study period
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: all
- Parameters examined: erythrocytes, haemoglobin, haematocrit, leucocytes, thrombocytes, differential blood count, reticulocytes*, Heinz bodies*, coagulation time, MCV, MCH, MCHC (* only in control and highest dose groups)


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the study period
- Animals fasted: No
- How many animals: all, under Nembutal anaesthesia
- Parameters examined: sodium, potassium, total bilirubin, creatinine, serum glucose, urea nitrogen, chloride, ASAT, ALAT, alkaline phosphatase, gamma-GT, cholesterol, triglycerides, total protein; liver enzymes: reduced glutathione, aniline hydroxylase, pentylresorufin O-depentylase, ethylresorufin O-deethylase, NADPH cytochrome c reductase, glutathione S-transferase


URINALYSIS: Yes
- Time schedule for collection of urine: over approx. 16 hours after the night of days 19 - 20 of treatment.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, but 0.4 ml of water administered by gavage
- Parameters examined: appearance, colour, pH value, haemoglobin, protein, glucose, ketone bodies, bilirubin, urobilinogen, nitrite, ascorbic acid, sediment (sediment only tested in control and highest dose group)


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: weekly
- Dose groups that were examined: all
- Battery of functions tested: other: neurological disturbancies


OTHER:
Absolute and relative organ weights: heart, brain, lungs, testes (without epididymides)/ovaries, liver, adrenals, kidneys, spleen.
The activities of aminopyrine N-demethylase, pentylresorufin O-depentylase, microsomal, NADPH2-dependent cytochrome c reductase, and glutathione S-transferase as well as the contents of reduced glutathione were measured in frozen kidney samples collected in the Guideline study in an additional study at a later time point.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (orifices, eyes, teeth, oral mucosa and internal organs)
HISTOPATHOLOGY: Yes (heart, stomach (fore- and gland-stomach), lungs, intestine (jejunum & colon), liver, gall bladder, kidneys, urinary bladder, spleen, prostate/uterus, brain, both epididymides, both testes/ovaries, seminal vesicles, adrenals, both eyes with optic nerve, pituitary, bone marrow (femur & sternum), thyroid & parathyroid, thymus, macroscopic abnormalities)

Statistics:

The following data were evaluated statistically at the level of significance p < 0.05:
Body weights at the designated measurement times, haematology parameters (except diff. blood count and methemoglobin), clinical chemistry parameters, urinalysis (pH value), absolute organ weights, relative organ weights.
Evaluation was performed by the department Pharma Forschung Informatik with the aid of a program package for evaluating toxicological studies. Urinary pH value was evaluated by the two-sided T-test using the "Artemis" computer system. The statistical methods used in each case are given in the report. The biochemistry data of the liver were evaluated by statistic program "SAMPLE" in the Pharma Development Toxicology.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
No clinical signs of systemic toxicity could be observed and no animal died during the study.

BODY WEIGHT AND WEIGHT GAIN
Administration of the test substance had no effects on body weight gains in any of the dose groups.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food consumption was unaffected by the test substance in all treated groups up to and including 5000 ppm. In the highest dosing group, there appeared to be an increase in food consumption, particularly in the females.

Group mean of daily test substance intake (mg/kg bw/d):
Males: 0, 19.4, 97.5, 211, 502.5, 1080, 1747.5; Females: 0, 21.6, 109.0, 207, 532.5, 1135, 1987.5

WATER CONSUMPTION
No substance-related changes in any of the treated groups.

OPHTHALMOSCOPIC EXAMINATION
There were no indications of opacity of the refracting media of the eyes.

HAEMATOLOGY
Hematological examinations revealed slight decreases in erythrocyte counts, hemoglobin content and hematocrit value in the males from the 7500 ppm group. Faint signs of this effect were also recognizeable in the females from this dosing group. This finding might be related to the test substance and indicative of a marginal normochrome anaemia.
No other changes attributable to the test substance could be detected. A statistically significant increase in coagulation time among the 2500 ppm
females and the slightly higher MCHC values in all treated female groups were considered to reflect normal biological variation or due to somewhat lower control values (MCHC).

CLINICAL CHEMISTRY
The evaluation of the clinical chemistry parameters indicated no toxicologically relevant changes and gave no convincing evidence of substance-dependence in most cases. In particular, no clinico-chemical correlates of hepatotoxicity and nephrotoxicity could be established.

Only slight increases in cholesterol from 5000 ppm onwards in the males and at 7500 ppm also in females and in triglycerides in the 7500 ppm male group may possibly reflect a substance-related effect. In view of the slight non-dose-related decrease in bilirubin in the females of the three highest dose groups a substance-dependency can also not be definitively excluded; however, the biological significance of this finding remains doubtful.
All other changes were only faintly marked, i.e. within the range of biological variation or without any dose-relationship and without toxicological correlates. The marked non-dose-related decreases in potassium and glucose in the females of all treated groups are with certainty related to non-typical high control values.

Biochemistry data indicated marked induction of drug-metabolizing enzymes from 100 ppm and 500 ppm onwards in the females and males, respectively. In addition, GSH was slightly increased in the same treatment groups; probably related to this finding, the GST was slighty increased in both sexes of the highest dose group. The latter findings may be assessed as an adaptive increase in GSH-mediated detoxication processes.

URINALYSIS
The urinary status of the animals in all treated groups remained unaffected by 4-week feeding of the test substance. There were no signs of any damage to the renal system. The urine of most of the animals was clear to cloudy and light to dark yellow, in some cases 'reddish in colour. At the end of the treatment period, the group mean pH values ranged between 7.1 and 8.3 in the males and between 5.7 and 7.0 in the females.
Examination of the urine for protein, glucose, haemoglobin, bilirubin, urobilinogen, nitrite, ascorbic acid and ketone bodies gave no indication of substance-related changes; the same applies to the examination of urinary sediments in the control and high-dose animals at the end of the treatment period.

NEUROBEHAVIOUR
There were no indications of any neurological disturbances which might have been attributed to the administration of the test substance.

ORGAN WEIGHTS
Analysis of the organ weights indicated a dose-related increase in liver weights in the males from 5000 ppm onwards and in the females from the highest dosing group (7500 ppm). This finding was considered to be treatment-related. In addition, the kidney weights appeared to be slightly lower in the females from the two highest dosing groups. No other changes in organ weights attributable to the test substance could be detected.

GROSS PATHOLOGY
A small number of gross lesions were observed in all test groups. There were no differences in the lesions found in the treated mice and in the controls.

HISTOPATHOLOGY: NON-NEOPLASTIC
The microscopic examinations indicated hepatocellular hypertrophy in mice from groups treated with 2500, 5000 and 7500 ppm. This finding was considered to be treatment-related and to reflect an induction of metabolizing enzymes.

In addition, a single cell necrosis in one mouse and a slight increase in the incidence and severity of inflammatory foci in the liver of female mice in the
7500 ppm group were diagnosed.

The other lesions observed in this study were considered to be spontaneous findings commonly diagnosed in mice of this strain and age.

OTHER FINDINGS:
In contrast to the liver, the enzyme biochemical examinations indicated no changes in renal tissue attributable to the treatment. The assessments made in report 91.1031 remained unchanged. The differences between hepatic and renal tissue may be explained by tissue-specific differences in toxicokinetics and metabolism of the test substance and/or its metabolites.

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

5 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effect observed

Remarks on result:

other: equivalent to a mean daily substance intake of approx. 1100 mg/kg bw

Dose descriptor:

LOAEL

Remarks:

systemic

Effect level:

7 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Remarks on result:

other: equivalent to a mean daily substance intake of approx. 1870 mg/kg bw

Dose descriptor:

NOEL

Remarks:

systemic

Effect level:

< 100 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no effect observed

Dose descriptor:

LOEL

Remarks:

systemic

Effect level:

100 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical biochemistry

Remarks on result:

other: equivalent to a mean daily substance intake of 22 mg/kg bw

Dose descriptor:

NOEL

Remarks:

systemic

Effect level:

100 ppm

Sex:

male

Basis for effect level:

other: equivalent to a mean daily substance intake of 19 mg/kg bw

Dose descriptor:

LOEL

Remarks:

systemic

Effect level:

500 ppm

Sex:

male

Basis for effect level:

other: clinical chemistry: marked induction of hepatic drug-metabolizing enzymes; equivalent to a mean daily substance intake of 98 mg/kg bw.

Critical effects observed:

not specified

Conclusion:

Based on the results obtained in this study, the liver proved to be the target organ after treatment with the test substance. The "No Observable Effect Level" (NOEL) for changes in hepatic physiology is considered to be slightly below 100 ppm (approx. 20 mg/kg/d). Biochemistry indicated marked induction of hepatic drug-metabolizing enzymes from 100 ppm and 500 ppm onwards in the females and males, respectively. In addition, GSH was slightly increased in the same treatment groups; probably related to this finding, the GST was slighty increased in both sexes of the highest dose group. The latter findings may be assessed as an adaptive increase in GSH-mediated detoxication processes. At dosage levels of 5000 and 7500 ppm, there was also an increase in liver weights, and hepatocellular hypertrophy was diagnosed by histopathology. These findings were considered to reflect an induction of metabolizing enzymes indicating an adaptive phenomenon rather than a toxic effect. Faint signs of this effect were already recognizable in one mouse from the 2500 ppm group. Slight adverse effects in the form of minimal hepatotoxicity (inflammatory foci and single cell necrosis), slight anaemia and changes in lipid status were noted only in the highest dose level. Thus, the "NO ADVERSE EFFECT LEVEL" is considered to be 5000 ppm, equivalent to a mean daily substance intake of approx. 1100 mg/kg body weight. No toxicologic significance could be assigned to the slight decrease in kidney weights in the females from 5000 ppm onwards in the absence of any further toxicological correlates.

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 82-1 (90-Day Oral Toxicity)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hazleton/LRE Research Products, 6321 South 6th Street, Kalamazoo, MI 49009, USA
- Age at study initiation: 5.5 to 6.5 months
- Weight at study initiation: males: 6.1 - 9.2 kg, females: 5.8 - 7.7 kg
- Fasting period before study: no
- Housing: individual kennels with minimum of 2.0 square meters floor space.
- Diet: 350 ± 1 g pelleted standard Kliba 335 dog maintenance diet ("Kliba", Klingentalmuehle AG, 4303 Kaiseraugst/Switzerland) presented at approximately 10.00 daily and withdrawn at 13.00. Results of the analyses for contaminants are included in the report.
- Water (e.g. ad libitum): tap water was supplied ad libitum by an automatic watering system.
- Acclimation period: 17 days under test conditions, after veterinary examination.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3 (floor temperature 23°C)
- Humidity (%): 40 - 70
- Air changes (per hr): 20
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

acetone

Details on oral exposure:

DIET PREPARATION
HOE 107892 SUBSTANCE TECHNICAL was dissolved in acetone and subsequently mixed with microgranulated feed. Water (approximately 1:10 volume/weight ratio) was added to aid pelleting. The pellets were dried with warm air for approximately 48 hours before storage.
- Rate of preparation of diet (frequency): every 4 -5 weeks
- Mixing appropriate amounts with (Type of food): microgranulated feed
- Storage temperature of food: dark at -20°C


VEHICLE
- Justification for use and choice of vehicle (if other than water): admixed to the diet, which is recognized as an efficacious method of absorption, anticipated route of human exposure to the test article.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability, homogeneity and content of the test article in the feed was determined in a previous 4-week feeding study (RCC Project 271754). According to the sponsor, the stability of test substance was guaranteed for 35 days at room temperature. Due to a calculation error, stability was verified for only 29 days in an additional stability analysis, which was terminated before start of treatment, but was subsequently proven over 37 days (RCC Project 291115). A sample of each diet preparation was retained for the determination of homogeneity and content. Analyses were performed in the laboratories of RCC UMWELTCHEMIE AG, according to a method provided by the sponsor.

Duration of treatment / exposure:

males: 13 weeks and 6 days, females 14 weeks

Frequency of treatment:

daily, during feeding period (10.00 to 13.00)

Dose / conc.:

400 ppm

Remarks:

equivalent to 15 mg/kg bw/day

Dose / conc.:

2 000 ppm

Remarks:

equivalent to 81 mg/kg bw/day

Dose / conc.:

10 000 ppm

Remarks:

equivalent to 339 mg/kg bw/day

No. of animals per sex per dose:

4 (additionally 2 as satellite groups at 0, 2000 and 10000 ppm)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Based on a 4-week toxicity study performed at RCC (Project no. 271754).
- Rationale for selecting satellite groups: In order to check reversibility of potentially occurring effects.
- Post-exposure recovery period in satellite groups: 4 weeks (0, 2000, 10000 ppm)

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (morning and afternoon)
- Cage side observations included: mortality, clinical signs (changes in appearance and behaviour)


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly palpation for tissue masses. A description of any lesion or mass at any examination was recorded and the subsequent progress was monitored.


BODY WEIGHT: Yes
- Time schedule for examinations: weekly, from commencement of pretest, and before necropsy.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Daily from commencement of pretest. The daily food consumption for each dog, and the weekly group means of these values are reported.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/day: Yes
- Compound intake calculated from the consumption and body weight data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at pretest, 4 weeks, 13 weeks, 4 weeks recovery.
- Dose groups that were examined: Each animal was examined for abnormalities of the eyes at least 20 minutes after the instillation of 0.5 % tropicamide solution (Mydriaticum, Dispersa AG, Hettlingen, Switzerland) using a binocular indirect Ophthalmoscope (all pupil model, Keeler Instruments Inc.) and the results were recorded. The observation area included the cornea, conjunctiva, sclera, iris, lens and fundus. Photographs were taken at the discretion of the ophthalmologist and were retained in the raw data.


HAEMATOLOGY: Yes
- Time schedule for collection of blood: at pretest, 4 weeks, 13 weeks and 4 weeks recovery. The blood samples were collected between 07.00 and 09.00 to reduce biological variation caused by circadian rhythms. Blood samples were drawn from the jugular vein into evacuated blood collection tubes.
- Anaesthetic used for blood collection: No
- Animals fasted: Yes, for approx. 18 hours before sampling, but water provided
- How many animals: all
- Parameters examined: Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Platelet count, Reticulocyte count, Nucleated erythrocytes (normoblast), Total leukocyte count, Differential leukocyte count, Red cell morphology, Coagulation (Thromboplastin time, Activated partial thromboplastin time)


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at pretest, 4 weeks, 13 weeks and 4 weeks recovery. The blood samples were collected between 07.00 and 09.00 to reduce biological variation caused by circadian rhythms. Blood samples were drawn from the jugular vein into evacuated blood collection tubes.
- Animals fasted: Yes, for approx. 18 hours before sampling, but water provided
- How many animals: all
- Parameters examined: Glucose, Urea, Creatinine, Bilirubin total, Lipids total, Cholesterol total, Triglycerides, Phospholipids, Aspartate aminotransferase, Alanine aminotransferase, Lactate dehydrogenase, Glutamate dehydrogenase, Creatine kinase, Alkaline phosphatase, Gamma-glutamyltransferase, Ornithine carbamyltransferase, Iron, Calcium, Phosphorus, Magnesium, Sodium, Potassium, Chloride, Protein total, Protein electrophoresis (Albumin, Alpha 1-globulin, Alpha 2-globulin, Beta 1-globulin, Beta 2-globulin, Sum of beta globulins, Gamma globulin, Albumin to Globulin ratio)


URINALYSIS: Yes
- Time schedule for collection of urine: at pretest, 4 weeks, 13 weeks and 4 weeks recovery. Urine was collected with the animals in metabolism cages for 24 hours. The urine samples were collected over ice. Urine samples were collected from all animals.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, 18 hours
- Parameters examined: Volume (24-hour), Specific gravity, Osmolality, Colour, Appearance, pH, Protein, Glucose, Ketone, Bilirubin, Blood, Urobilinogen, Urine Sediment, Gamma-glutamyltransferase, Lactate dehydrogenase, Alkaline phosphatase, Leucine aminopeptidase, Beta-N-Acetyl-D-glucosaminidase, Sodium, Potassium, Phosphorus, Protein total, Creatinine, Creatine clearance, Urea


NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, after 13 weeks of treatment, after 4 weeks recovery: all organs of all animals were examined and all findings recorded. Samples of the following tissues and organs were collected and fixed in neutral phosphate buffered 4 % formaldehyde solution: Adrenals, aorta, bone (femur with articular surface), bone marrow (sternum, femur), brain (medulla/pons, cerebral and cerebellar cortex), epididymides, esophagus, gall bladder, heart, kidneys, large intestine (cecum, colon, rectum), liver, lungs (infused with formalin), lymph nodes (retropharyngeal, mesenteric), mammary gland area-female, (male), ovaries, pancreas, pituitary gland, prostate gland, salivary gland (parotid, mandibular), sciatic nerve, skeletal muscle, skin, small intestine (duodenum, jejunum, ileum), spinal cord (cervical, midthoracic, lumbar), spleen, stomach, testes, thymus, thyroid gland with parathyroid, tongue, trachea, urinary bladder, uterus (with vagina) and all gross lesions. The eyes (with optic nerve) were fixed in Heidenhain's Susa solution.

HISTOPATHOLOGY: Yes: All sampled tissues (except for male mammary gland area parathyroid, optic nerve, tongue and vagina) were processed, embedded in paraffin wax and cut at a thickness of 2-4 µm. Sections were stained with haematoxylin and eosin and examined light microscopically. Bone marrow smears from one sternebrum from all animals were taken for possible future examinations.

Other examinations:

RESIDUE ANALYSIS
of blood, urine and feces: At the same intervals as the clinical laboratory investigations, 10 ml of blood was drawn from the jugular vein into heparinized tubes (143 U.S.P. Units). Following centrifugation the plasma was transferred to glass tubes and the plasma and red cells stored at -20°C in the dark. In addition, 10 ml of urine or the remainder of the urine sample from the clinical laboratory investigations and 10 g of faeces were similarly stored at RCC, Research and Consulting Company Ltd., Itingen/Switzerland for possible future investigations.

ORGAN WEIGHTS
Adrenal glands, brain (including brainstem), heart, kidneys, liver, ovaries, pituitary gland, prostate gland, spleen, testes with epididymides, thyroid gland (with parathyroid).

Statistics:

The following statistical methods were used to analyse the body weights, clinical laboratory data and organ weights. Univariate one-way analysis of variance was used to assess the significance of intergroup differences. If the variables could be assumed to follow a normal distribution, the Dunnett-Test (many to one t-test) based on a pooled variance estimate was applied for the comparison between the treated groups and the control groups.
The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables.
Individual values, means, standard deviations and statistics were rounded-off before printing. For example, test statistics were calculated on the basis of exact values for means and pooled variances and then rounded-off to two decimal places. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistic values.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
No animal died during the scheduled treatment and recovery periods. There were no clinical signs that could be related to treatment with HOE 107892 SUBSTANCE TECHNICAL.

BODY WEIGHT AND WEIGHT GAIN
Slight to moderate weight loss (from 4 to 11 % of the pretest weight) was recorded in three males and three females at 10000 ppm during the first week of treatment. Lesser weight losses, recorded in a small number of dogs from all groups, including male controls, were considered to be the result of normal biological variation.
Thereafter, with the exception of one male and one female which showed overall losses, the dogs gained weight, such that overall group mean body weight gain in the males was similar to that of the controls. However, a marked reduction in overall group mean body weight gain was recorded in the female dogs at 10000 ppm. Improved weight gain was noted in the dogs of the high dose group, particularly the females, during the recovery period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Slightly to moderately decreased group mean food intake in the males and females at 10000 ppm, respectively, in comparison with food intake pretest. Thereafter, mean food intake improved but for both sexes it remained consistently lower than that of the controls throughout the treatment period.
A slight to marked increase in food intake was noted in the dogs of the high dose group during the recovery period when compared with their mean intake during the treatment period.


OPHTHALMOSCOPIC EXAMINATION
The ophthalmic parameters were unaffected by treatment with HOE 107892 SUBSTANCE TECHNICAL. The findings were those commonly observed during canine ophthalmoscopy and were considered to be spontaneous in origin.

HAEMATOLOGY
Treatment with HOE 107892 SUBSTANCE TECHNICAL resulted in decreased red blood cell count, hemoglobin concentration, hematocrit and mean corpuscular hemoglobin concentration and increased mean corpuscular volume, platelet and reticulocyte counts and polychromasia in dogs at 10000 ppm in weeks 4 and/or 13 in comparison with the control and pretest data.
These changes were generally restricted to individual dogs although the differences in group mean corpuscular volume and mean corpuscular hemoglobin concentration from the controls were often statistically significant.
The values for these parameters in the affected dogs were generally similar to those of the controls at the end of the recovery period. Hematology parameters of dogs at 400 or 2000 ppm HOE 107892 SUBSTANCE TECHNICAL were unaffected by treatment.

CLINICAL CHEMISTRY
Treatment with HOE 107892 SUBSTANCE TECHNICAL at 10000 ppm resulted in the following changes in comparison with the control and pretest data:
- Slightly increased group mean lipids and/or cholesterol and phospholipid levels in weeks 4 and 13.
- Slightly increased group mean alkaline phosphatase activity in weeks 4 and 13.
- Slightly increased group mean iron concentration and decreased chloride concentration in the females in weeks 4 and 13 and week 13, respectively. The differences from the controls for these parameters were usually statistically significant.
In addition, markedly increased alanine aminotransferase activity was recorded in one male and one female and slightly decreased protein levels in three males and two females of the 10000 ppm group in week 13.
Slightly increased alkaline phosphatase activity and decreased total protein levels were also recorded in individual dogs at 2000 ppm.

The values for the affected parameters were generally similar to the control or pretest values at the end of the recovery period. However, slightly to markedly increased alanine aminotransferase, glutamate dehydrogenase and ornithine carbamyltransferase activities were noted in one male of the high dose group at the investigation during the recovery period.

URINALYSIS
Urinalysis parameters were unaffected by treatment with HOE 107892 SUBSTANCE TECHNICAL.

ORGAN WEIGHTS
Examination of the organ weight data revealed slightly to moderately increased group mean liver weight in males and females at 2000 and 10000 ppm, respectively, after 13 weeks of treatment. The differences from the controls were usually statistically significant for the absolute weight and body and brain weight ratios in the dogs at 10000 ppm and for liver to body weight ratio in the males at 2000 ppm. Liver weight in control and treated dogs was generally similar at the end of the 4-week recovery period.
The remaining organ weights were unaffected by treatment with HOE 107892 SUBSTANCE TECHNICAL.

GROSS PATHOLOGY
Retropharyngeal lymph node discolouration was seen in several animals with no clear relation to treatment. No treatment-related macroscopic findings were recorded.

HISTOPATHOLOGY: NON-NEOPLASTIC
A focal lesion consisting of haemorrhage, necrosis and inflammation was seen in the liver of one female of the high dose group. The small number of other findings recorded are within the normal range of background alterations which may be seen in untreated animals of this age and strain.

Dose descriptor:

NOEL

Remarks:

systemic

Effect level:

400 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: corresponding to 15 mg/kg bw/day

Dose descriptor:

LOEL

Remarks:

systemic

Effect level:

2 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

organ weights and organ / body weight ratios

Remarks on result:

other: corresponding to 81 mg/kg bw/day

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

2 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effect observed

Remarks on result:

other: corresponding to 81 mg/kg bw/day

Dose descriptor:

LOAEL

Remarks:

systemic

Effect level:

10 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

food consumption and compound intake

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Remarks on result:

other: corresponding to 339 mg/kg bw/day

Critical effects observed:

not specified

ASSESSMENT

In the present study, treatment with HOE 107892 SUBSTANCE TECHNICAL resulted in the following, toxicologically relevant, effects:

- A reduction in food intake with resultant body weight loss (females) at 10000 ppm.

- A slight anemia, with evidence of a compensatory hematopoiesis, in individual dogs at 10000 ppm.

- A clear increase in liver weight and clinical biochemical changes, particularly increased alkaline phosphatase activity, indicative of liver toxicity or dysfunction at 10000 ppm.

In view of these changes, the histopathological findings consisting of hemorrhage, necrosis and inflammation, in the liver of one dog of this group can, with justification, be assumed to be an expression of liver toxicity. The first indication of liver dysfunction was evident, as increased liver weight and alkaline phosphatase activity, in individual dogs at 2000 ppm. Based on these findings, 400 ppm (equivalent to 15 mg/kg body weight/day) was established as the no-effect-level (NOEL) in this study.

Reference 4

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

extended examinations

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hoechst AG, Department of Toxicology, Kastengrund, SPF breeding colony
- Age at study initiation: approx. 5 weeks (at start of preliminary study period)
- Weight at study initiation: males 126 ± 5.8 g mean, females 120 ± 5.8 g mean
- Housing: 5 per group in Makrolon cages (Type 4) on soft wood granulate (Lignocel)
- Diet (e.g. ad libitum): Altromin 1321 rat diet (Altromin GmbH, Lage/Lippe), ad libitum, except for the period in which the animals were kept in diuresis cages
- Water (e.g. ad libitum): tap water in plastic bottles, ad libitum, except for the period in which the animals were kept in diuresis cages
- Acclimation period: males 6 d, females 7d


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-22
- Humidity (%): 51-60
- Air changes (per hr): 6
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
Each 1 kg premix with 15 times the final dietary concentration was blended into 14 kg Altromin 1321 (total 15 kg) in two stages (1st stage: 1 kg premix + 4 kg diet; 2nd stage: 5 kg + 10 kg diet) with Loedige ploughshare mixers of the types M20 and FM130. The blending time for each diet mix was 30 minutes. The diet mixes were stored in closed plastic containers.
- Rate of preparation of diet (frequency): Once, premix prepared 1 day before the final diet mixes; final diet mixes were prepared 5 to 6 days before dosing
- Mixing appropriate amounts with (Type of food): Altromin 1321
- Storage temperature of food: room temperature

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The homogeneity and stability of the test substance preparations were guaranteed over a period of 35 days and the actually administered test substance concentrations corresponded in sufficient degree to the nominal test substance concentrations.

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

continuously, except for the period in which the animals were kept in diuretic cages

Dose / conc.:

100 ppm

Remarks:

equivalent to 10.1 mg/kg bw/day (males) and 10.0 mg/kg bw/day (females)

Dose / conc.:

500 ppm

Remarks:

equivalent to 52.3 mg/kg bw/day (males) and 52.3 mg/kg bw/day (females)

Dose / conc.:

1 000 ppm

Remarks:

equivalent to 102.0 mg/kg bw/day (males) and 101.4 mg/kg bw/day (females)

Dose / conc.:

2 500 ppm

Remarks:

equivalent to 258.3 mg/kg bw/day (males) and 249.0 mg/kg bw/day (females)

Dose / conc.:

5 000 ppm

Remarks:

equivalent to 518.7 mg/kg bw/day (males) and 488.2 mg/kg bw/day (females)

Dose / conc.:

7 500 ppm

Remarks:

equivalent to 773.6 mg/kg bw/day (males) and 744.3 mg/kg bw/day (females)

No. of animals per sex per dose:

5

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, once daily at weekends and on public holidays
- Cage side observations included: clinical signs, mortality, behaviour and general health condition


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly (neurological disturbances, damage of the oral mucosa, impairment of dental growth)


BODY WEIGHT: Yes
- Time schedule for examinations: at the start of the study and then twice weekly until the end of the study


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): at the start of the study and then twice weekly until the end of the study (same times as body weights). The reported values refer to the intervals between one measurement and the next and are converted to show food consumption over 24-hour periods.
- 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: No


WATER CONSUMPTION: Yes
- Time schedule for examinations: once weekly for a period of 16 hours (from 3.15 p.m. to 7.15 a.m.)


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: weekly (opacity of refracting media)
- Dose groups that were examined: all


HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the study period
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: all
- Parameters examined: erythrocytes, haemoglobin, haematocrit, leucocytes, thrombocytes, differential blood count, reticulocytes*, Heinz bodies*, coagulation time, thromboplastin time, activated partial thromboplastin time, thrombin time, methaemoglobin* (*only in control and high dose groups); MCV, MCH and MCHC were also calculated


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the study period
- Animals fasted: No
- How many animals: all
- Parameters examined: sodium, potassium, inorganic phosphorus, uric acid, total bilirubin, creatinine, serum glucose, urea nitrogen, calcium, chloride, ASAT, ALAT, alkaline phosphatase, gamma-GT, LDH, cholesterol, triglycerides, total lipids, total protein, serum electrophoresis (albumin, alpha1-globulin, beta1-globulin, gamma globulin)


URINALYSIS: Yes
- Time schedule for collection of urine: In all groups the urine accumulated over approx. 16 hours was collected from the males and females after the night of days 19 - 20 of treatment in chilled vessels.
- Metabolism cages used for collection of urine: Yes (diuresis cages)
- Animals fasted: Yes
- Parameters examined: appearance, colour, pH value, haemoglobin, protein, glucose, ketone bodies, bilirubin, urobilinogen, nitrite, ascorbic acid, sediment (only control and high dose group), volume; in addition, the following special parameters for diagnosis of nephrotoxicity, were examined in all animals: creatinine, sodium, potassium, urea, uric acid, alanine aminopeptidase, gamma-glutamyl transferase, N-acetyl-glucosaminidase, lactate dehydrogenase, creatinine clearance (endogenous clearance test, calculated value based on the concentration of creatinine in urine and serum and urine volume in mL/min)


NEUROBEHAVIOURAL EXAMINATION: No


OTHER:
After completion of the study it was decided to examine the following enzyme-biochemical parameters in liver and kidney tissue: glutathione content, cytosolic glutathione S-transferase, aminopyrine N-demethylase, pentylresorufin O-depentylase, microsomal NADPH cytochrome c reductase (results of these examinations were reported separately and amended to the report)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (orifices, eyes, teeth, oral mucosa, internal organs, organ weights)
HISTOPATHOLOGY: Yes (heart, stomach, lungs, small intestine, liver, large intestine, kidneys, urinary bladder, spleen, prostate / uterus, brain, both epididymides, both testes / ovaries, thymus, adrenals, both eyes with optic nerve, pituitary, bone marrow (femoral), thyroid (both lobes), sternum, seminal vesicles, gross lesions). All tissues were fixed in Carnoy and selected tissues (1 adrenal gland, heart, kidneys, and liver) were also fixed in formalin. The fixed tissues were embedded in paraffin. Sections were stained with haematoxylin and eosin. Microscopic examination of all organs designated in the protocol was performed on the rats of the control and the highest dose group. In the intermediate groups only gross lesions were examined microscopically.

Other examinations:

Organ weights: heart, brain, lungs, testes (without epididymides) / ovaries, liver, adrenals, kidneys, spleen, pituitary, thyroid

Statistics:

The following data were evaluated statistically at the level of significance p < 0.05:
Body weights at the designated measurement times, haematology parameters (except diff. blood count and methaemoglobin), clinical chemistry parameters (except gamma-GT), urinalysis (pH value, volume, clinical chemistry parameters), absolute organ weights, relative organ weights.
Evaluation was performed by the department Pharma Forschung Informatik with the aid of a program package for evaluating toxicological studies. The statistical methods used in each case were given on the computer printouts.

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):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

effects observed, treatment-related

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:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
Behaviour and general health condition remained normal throughout the study in all of the treated groups. There were no indications of any treatment-related neurological disturbances, impairment of dental growth or changes in the oral mucosa. No clinical signs of systemic toxicity could be observed. No treatment-related mortality was observed (one female from the 100 ppm group died on day 10 without any indications of the cause of death).

BODY WEIGHT AND WEIGHT GAIN
No effects on body weight gains up to and including 7500 ppm.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food consumption was not affected by the test substance in any of the treated groups.

WATER CONSUMPTION
Water consumption showed no substance-related changes in any of the treated groups.

OPHTHALMOSCOPIC EXAMINATION
No indications of opacity of the refracting media of the eyes were observed

HAEMATOLOGY
Slightly lower erythrocyte counts and haematocrit values were observed in the males treated at 500 ppm or higher dose levels as compared with the controls; in addition, the haemoglobin content was slightly decreased at 5000 and 7500 ppm. However, taking into account the minimal degree and the absence of a clear dose-dependency the changes noted in the groups treated up to and including 2500 ppm were considered to reflect normal biological variation. Only the decreased erythrocyte counts in connection with the decreased haemoglobin content in the males treated at 5000 and 7500 ppm were considered to be indicative of a minimal anaemia. Additional correlates indicative of haematotoxicity could not established by clinical biochemistry or pathology.

CLINICAL CHEMISTRY
Statistical analysis of the clinical chemistry parameters revealed a number of significant changes in the males. Evaluation of the data gave no convincing evidence of substance-related changes in any of the treated groups. All changes were only faintly marked, i.e. within the range of biological variation or without any dose-relationship and without toxicological correlates. In particular, no clinico-chemical correlates of haematotoxicity or nephrotoxicity could be established. Serum electrophoresis also indicated no substance-related changes.

URINALYSIS
The urinary status of the animals in all treated groups remained unaffected by the treatment. There were no signs of any damage to the renal system. The urine of most of the animals was clear to cloudy and light to dark yellow, in some cases reddish in colour. At the end of the treatment period, the group mean pH values ranged between 6.8 and 8.1 in the males and between 6.5 and 7.5 in the females. The group mean volume of urine ranged between 4.7 and 7.1 in the males and between 5.3 and 6.4 ml/16h in the females.
Examination of the urine for protein, glucose, haemoglobin, bilirubin, urobilinogen, nitrite, ascorbic acid and ketone bodies gave no indication of substance-related changes; the same applies to the examination of urinary sediments in the control and high-dose animals at the end of the treatment period.
Evaluation of the special parameters such as potassium, sodium, creatinine, urea and uric acid in the urine as well as calculation of endogenous creatinine clearance indicated no substance-related changes in any of the treated groups.
Measurements for urinary enzymes indicated a dose-dependent decrease in the excretion of gamma-glutamyl transferase in the females from 2500 ppm onwards together with a tendency for a decrease in alanine aminopeptidase - also an enzyme located in the brush-border region of the proximal renal tubule - in the same groups. These findings were considered to be substance-related indicative of functional changes in the proximal renal tubules. With a view of the reduced excretion of lactate dehydrogenase in the males of the highest dose group a substance-dependency cannot be definetively excluded. The slight decrease in N-acetyl glucosaminidase in all treated male groups was considered to be non substance-related in the absence of any dose-dependency.

ORGAN WEIGHTS
Statistical analysis of the absolute and relative organ weights indicated no significant changes in any of the treated groups as compared with the control group.

GROSS PATHOLOGY
Macroscopic examinations of the organs revealed no substance-related pathomorphological changes in any of the treated groups.

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopic examinations of the organs revealed no substance-related pathomorphological changes in any of the treated groups. In particular, no changes in organs related to haematotoxicity such as bone marrow, spleen or liver and to nephrotoxicity could be established.

OTHER FINDINGS
Test substance-treatment resulted in an induction of hepatic drug-metabolizing enzymes (aminopyrine N-demethylase, pentylresorufin O-depentylase, microsomal NADPH2-dependent cytochrome c reductase, and glutathione S-transferase. These effects were more pronounced in the males than in the females. In the kidneys, glutathione S-transferase was increased, too, and microsomal NADPH2-dependent cytochrome c reductase showed, at least, a tendency to be increased.

In the liver there were statistically significant changes in glutathione contents in some groups of the males but none in the females. These changes were of no convincing biological meaning, however. There were statistically significant increases in aminopyrine N-demethylase activities in the males at doses higher than 500 ppm, and in the females at doses higher than 1000 ppm. Pentylresorufin O-depentylase activity was raised significantly only in the males at 7500. The activities of microsomal NADPH2-dependent cytochrome c reductase were statistically significantly increased at doses higher than 100 ppm in the males and higher than 2500 ppm in the females. The activities of glutathione S-transferase were statistically significantly increased at doses higher than 500 ppm in the males and higher than 1000 ppm in the females.

In the kidneys there were some statistically significant changes in the contents of reduced glutathione without convincing evidence for a glutathione increase. Regarding the two mixed-function oxidases determined the results were not unequivocal. In the males, aminopyrine N-demethylase activities were raised at all doses but there was no clear dose dependency; in the females, enzymic activities higher than zero which did not show any trend were found only in less than five animals per group so that statistical evaluation was not possible. There was a statistically significant activity loss of pentylresorufin O-depentylase at doses higher than 2500 ppm in the males, and a statistically significant activity increase at all doses except 500 ppm in the females, but the control activities in the females were as high as the lowered activities in the males. Therefore, the reliability of these results was cosidered rather questionable. Microsomal NADPH2-dependent cytochrome c reductase showed no clear-cut changes in the males (there could be a tendency to activity increase in the highest dose) and statistically significant activity increases in the females at doses higher than 1000 ppm. The activities of glutathione S-transferase were statistically significantly increased in both males and females at doses higher than 2500 ppm.

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

1 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effect observed

Remarks on result:

other: corresponding to 102.0 mg/kg bw/day for males and 101.4 mg/kg bw/day for females

Dose descriptor:

LOAEL

Remarks:

systemic

Effect level:

2 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

urinalysis

Remarks on result:

other: corresponding to 258.3 mg/kg bw/day for males and 249.0 mg/kg bw/day for females

Critical effects observed:

not specified

Conclusion:

Based on the results obtained in this 4-week repeated-dose oral toxicity study with Hoe 107892 00 ZC99 0001 in the Wistar rat, the NO OBSERVABLE ADVERSE EFFECT LEVEL (NOAEL) is considered to be 1000 ppm (APPROX. 102 mg/kg bw/day). No signs of marked systemic toxicity could be observed at all dietary levels up to and including 7500 ppm.

Reference 5

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 82-1 (90-Day Oral Toxicity)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL Biological Research Laboratories Ltd, CH 4414 Fuellinsdorf, Switzerland
- Age at study initiation: about 4 weeks
- Weight at study initiation: Males: 17-22 grams (mean: 19 grams); Females: 15-18 grams (mean: 17 grams)
- Housing: individually in Makrolon type-1 cages with wire mesh lids and standard granulated softwood bedding (Lignocel, Schill AG, 4132 Muttenz/Switzerland).
- Diet (e.g. ad libitum): pelleted standard Kliba no. 343 mouse maintenance diet (Kliba, Klingentalmuehle AG, 4303 Kaiseraugst/Switzerland), ad libitum
- Water (e.g. ad libitum): tap water ad libitum via water bottles
- Acclimation period: 6 days under test conditions, after veterinary examination.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 40-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

acetone

Details on oral exposure:

DIET PREPARATION
The test substance was dissolved in acetone, mixed with microgranulated food and pelleted using a pelleting machine. Water (approximately 1:10 volume/weight ratio) was added to aid pelleting. The pellets were dried for approximately 48 hours before storage in disposable paper bags at room temperature. The stability of the test article in the diet was guaranteed for 35 days according to the certificate of analysis AZ 04292, dated June 15, 1990.
- Rate of preparation of diet (frequency): pellets prepared every 4 weeks
- Mixing appropriate amounts with (Type of food): microgranulated food
- Storage temperature of food: room temperature


VEHICLE
- Concentration in vehicle: 100, 500, 2500, 7500 ppm

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations, the homogeneity and stability of HOE 107892 SUBSTANCE TECHNICAL in rodent feed was determined. The overall mean concentrations found were 95.7 %, 93.4 %, 96.3 % and 92.7 % of the nominal concentrations for dose groups 2, 3, 4 and 5, respectively (100, 500, 2500, 7500 ppm). The homogeneity varied in the range from -15 % to +13 % of the mean concentrations.
HOE 107892 SUBSTANCE TECHNICAL was found to be stable in rodent feed at room
temperature for at least 35 days.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

continuously

Dose / conc.:

100 ppm

Remarks:

equivalent to 17.8 mg/kg bw/day (males) and 20.8 mg/kg bw/day (females)

Dose / conc.:

500 ppm

Remarks:

equivalent to 89.3 mg/kg bw/day (males) and 105.4 mg/kg bw/day (females)

Dose / conc.:

2 500 ppm

Remarks:

equivalent to 449.0 mg/kg bw/day (males) and 523.5 mg/kg bw/day (females)

Dose / conc.:

7 500 ppm

Remarks:

equivalent to 1492.8 mg/kg bw/day (males) and 1743.1 mg/kg bw/day (females)

No. of animals per sex per dose:

20

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Based upon the results of the four week-repeated-dose oral toxicity study in mice performed by the sponsor (study 90.0424).

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: viability twice daily, clinical signs once dialy
- Cage side observations included:


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly


BODY WEIGHT: Yes
- Time schedule for examinations: recorded weekly using an on-line electronic recording system


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): recorded weekly for a 7-day period using an on-line electronic recording system. Food spillage (when detected) was also entered.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: at 13 weeks
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes, 18 hours before
- How many animals: 10/sex/group
- Parameters examined: Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Mean corpuscular hemoglobin, Mean corpuscular
hemoglobin concentration, Platelet count, Reticulocyte count, Nucleated erythrocytes (normoblasts), Total leukocyte count, Differential leukocyte
count, Red cell morphology

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at 13 weeks
- Animals fasted: Yes, 18 hours before
- How many animals: 10/sex/group
- Parameters examined: Glucose, Urea, Creatinine, Bilirubin total, Cholesterol total, Triglycerides, Aspartate aminotransferase, Alanine aminotransferase, Lactate dehydrogenase, Alkaline phosphatase, Gamma-glutamyl-transferase, Calcium, Phosphorus, Sodium, Potassium, Protein total

URINALYSIS: Yes
- Time schedule for collection of urine: at 10 weeks, urine samples were collected from all animals over approximately 18 hours
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (20 ml tap water/kg bw administered to the animals prior to the urine collection period)
- Parameters examined: Volume (18-hour), Specific gravity, Osmolality, pH, Protein, Glucose, Ketone, Bilirubin, Blood, Urobilinogen, Urine Sediment

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, after 13 weeks (Adrenal glands, Aorta, Brain - including section of medulla/pons, cerebral and cerebellar cortex, Cecum, Colon, Duodenum, Epididymides, Esophagus, Exorbital lacrymal glands, Eyes with optic nerve and Harderian gland,Femur - including articular surface, Heart, Ileum, Jejunum, Kidneys, Liver with gallbladder, Larynx, Lungs, infused with formalin, Lymph nodes - mesenteric, mandibular, Mammary gland area, Ovaries, Pancreas, Pituitary gland, Prostate gland, Rectum, Salivary gland - mandibular, sublingual, Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord - cervical, midthoracic, lumbar, Spleen, Sternum with bone marrow, Stomach, Testes, Thymus, Thyroid gland / parathyroid gland, Tongue, Trachea, Urinary bladder, infused with formalin, Uterus, Vagina, All gross lesions)

HISTOPATHOLOGY: Yes (All listed tissues, with the exception of exorbital lacrymal gland and larynx, from the control and the high dose groups, additionally adrenals, brain, heart, kidneys, liver, ovaries, pancreas, spleen and testes from the intermediate groups, and all gross lesions.)

Other examinations:

ORGAN WEIGHTS:
Adrenals, brain, heart, kidneys, liver, lungs, ovaries, pituitary, spleen, testes, thymus (if present) and thyroid

Statistics:

If the variables could be assumed to follow a normal distribution, the Dunnett test (many to one t-test) based on a pooled variance estimate was applied for the comparison between the treated groups and the control groups. The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution. For the overall spontaneous mortality data, the Fisher's exact test for 2x2 tables was applied. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Individual values, means, standard deviations and statistics were rounded off before printing. For example, test statistics were calculated on the basis of exact values for means and pooled variances and then rounded off to two decimal places. Therefore, two groups may display the same printed means for a given
parameter, yet display different test statistics values.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no treatment-related deaths during the study. Two mice died during the blood sampling procedure on day 86. There were no treatment-related clinical signs.

BODY WEIGHT AND WEIGHT GAIN
There was no statistically significant effect on body weight gain in females at any dose nor in males receiving 100 or 500 ppm. A dose-related impairment of bodyweight gain was noted for males at 2500 and 7500 ppm, the difference between group means and those of control being statistically significant.

FOOD CONSUMPTION and COMPOUND INTAKE (if feeding study)
Food intake was slightly increased for both sexes at 7500 ppm (by approximately 10 % on average). No effects were seen at lower dose levels. The proportion of animals for which significant spillage of the diet was detected, was higher at 7500 ppm than at the other dietary concentrations, at
which the proportion was similar to that of controls.

Test substance administration at dietary concentrations of 100, 500 or 2500 ppm had no effect on relative food consumption. The occasional statistically significant differences in consumption between treated and control groups are considered not to be biologically significant. In both sexes at 7500 ppm higher than control relative food consumption was recorded, which was statistically significantly different from controls for most of the study.

At dietary concentrations of 100, 500 and 2500 ppm the test article intake on a mg/kg basis was in direct proportion to the concentration in the diet whereas at 7500 ppm the amount consumed was proportionately higher.

HAEMATOLOGY
A slight anemia was indicated for both sexes at 7500 ppm by the following statistically significant differences from control means: slightly lower erythrocyte count in males, slightly lower haemoblobin in both sexes, sligthly lower haematocrit in males, slightly higher mean corpuscular volume in males, slightly higher mean corpuscular haemoglobin in males and slightly higher reticulocyte counts in both sexes.
Total leucocyte counts were lower in both sexes at all doses, with evidence of a dose-relationship in males. However, since the values were within the range of historical data for mice of this strain and age, and no evidence of selective toxicity to the myeloid series in the bone marrow was obtained, the findings were considered to be spurious. All other statistically significant differences between values for controls and other groups were considered unrelated to treatment.

CLINICAL CHEMISTRY
Test substance administration had no effect on clinical biochemistry parameters in males at 100, 500 and 2500 ppm and females at 100 ppm. The following statistically significant differences between group mean values for treated animals and control means were considered to be of toxicological significance: lower bilirubin in females at 500, 2500 and 7500 ppm, with evidence of a dose-related effect, slightly higher aspartate aminotransferase in females at 7500 ppm, slightly higher alanine aminotransferase in females at 7500 ppm, higher lactate dehydrogenase in females at 2500 and in both sexes at 7500 ppm, higher alkaline phosphatase in males at 7500 ppm, slightly higher phosphorus in females at 7500 ppm. All other statistically significant differences between values for controls and other groups were considered unrelated to treatment with test substance.

URINALYSIS
Specific gravity and osmolality were slightly increased for males at 7500 ppm.

ORGAN WEIGHTS
Differences from controls after 13 weeks of treatment, were based on examination of absolute organ weights, organ/body weight ratios and organ/brain weight ratios. There were no treatment-related effects on organ weights in females at 100, 500, or 2500 ppm and males at 100 and 500 ppm. Liver - higher weight in males and females at 7500 ppm, with evidence of a slight effect in males at 2500 ppm. Spleen - higher weight in males at 7500 ppm. Kidney - marginally lower weight for males at 2500 and 7500 ppm. All other statistically significant differences from control means were attributed to biological variation or differences in terminal bodyweights.

GROSS PATHOLOGY
Most males and a few females at 7500 ppm were found to have enlargement and dark brown discolouration of the liver.

HISTOPATHOLOGY: NON-NEOPLASTIC
Moderate hepatic centrilobular parenchymal hypertrophy was seen in both males and females at 7500 ppm. The same phenomenon was seen, at a lower incidence, in males at 2500 ppm, but females were unaffected. There was a slight increase in the severity of splenic haemosiderin deposition and splenic haematopoiesis identified at 7500 ppm. No other treatment related findings were identified.

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effect observed

Remarks on result:

other: corresponding to 89.3 mg/kg bw/day (males) and 105.4 mg/kg bw/day (females)

Dose descriptor:

LOAEL

Remarks:

systemic

Effect level:

2 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

histopathology: non-neoplastic

Remarks on result:

other: corresponding to 449.0 mg/kg bw/day (males) and 523.5 mg/kg bw/day (females)

Critical effects observed:

not specified

Mean body weight gain and terminal body weight of males:

Group of males	Mean body weight gains (g) from day 1-85	Mean terminal body weight (g)
0 ppm	16.7	43.6
100 ppm	16.2 (97)*	42.5 (97)
500 ppm	15.2 (91)	41.3 (95)
2500 ppm	13.7 (82)	39.8 (91)
7500 ppm	11.8 (71)	38.4 (88)

*in brackets: percentage of control mean

Conclusion:

The centrilobular hypertrophy and increased blood ASAT, ALAT, LDH, and ALP activities reflect an adaptation to an increased functional burden on the liver at higher dose levels (2500 ppm and 7500 ppm). The no observable adverse effect level (NOAEL) in this study was a dietary concentration of 500 ppm, corresponding to an average daily test article intake of 89.3 mg/kg bodyweight for males and 105.4 mg/kg bodyweight for females.

Reference 6

Endpoint:

chronic toxicity: oral

Remarks:

combined repeated dose and carcinogenicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPP 83-5

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd., CH-4414 Fuellinsdorf/Switzerland
- Age at study initiation: 4 weeks
- Weight at study initiation: males: 63-96 g (mean: 79 g) females: 42-74 g (mean: 58 g)
- Housing: groups of five in Makrolon type-4 cages with wire mesh tops and granulated softwood bedding (Lignocel, Schill AG, Muttenz/ Switzerland)
- Diet (e.g. ad libitum): pelleted standard Kliba 343 rat/mouse maintenance diet ("Kliba", Klingentalmuehle AG, 4303 Kaiseraugst/Switzerland), and was available ad libitum.
- Water (e.g. ad libitum): tap water ad libitum via water bottles
- Acclimation period: 7 days under test conditions, after veterinary examination.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 40 - 70 %
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

acetone

Details on oral exposure:

DIET PREPARATION
Dissolved in acetone and mixed to microgranulated food, pelleted in a Buehler pelleting machine. Water was added to each feed preparation at an approximate 1:10 volume/weight ratio to ensure pelleting, after which the pellets were dried with warm air for approximately 48 hours before storage.
- Rate of preparation of diet (frequency): monthly up to treatment week 8, and every two weeks thereafter
- Mixing appropriate amounts with (Type of food): microgranulated feed
- Storage temperature of food: room temperature


VEHICLE
- Justification for use and choice of vehicle (if other than water): admixed to the diet, which is recognized as an efficacious method of absorption.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability, homogeneity and content of the test article in feed was determined prior to study initiation with RCC Project 285028 and was repeated with the first diet batches prepared for the study. Intercurrent sampling for analyses (homogeneity and content) was performed every two months. Analyses were performed in the Analytical Chemistry Laboratory of RCC Umweltchemie AG according to a method supplied by the sponsor.

Duration of treatment / exposure:

124 weeks oncogenicity, 104 weeks chronic toxicity, 52 weeks interim sacrifice

Frequency of treatment:

continuously

Dose / conc.:

40 ppm

Remarks:

nominal in diet

Dose / conc.:

200 ppm

Remarks:

nominal in diet

Dose / conc.:

1 000 ppm

Remarks:

nominal in diet

Dose / conc.:

5 000 ppm

Remarks:

nominal in diet

No. of animals per sex per dose:

80 (50 oncogenicity 124 weeks, 20 chronic toxicity 104 weeks, 10 interim sacrifice after 52 weeks)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Based upon results of a subchronic toxicity study in the rat (RCC Project 285028), the top dose level (5000 ppm) was expected to cause a significant reduction in body weight gains, a slight anemia, urinary enzyme changes, and changes in liver function. The mid-high dose level (1000 ppm) was expected to cause minimal, if any, signs of toxicity. The lower dose levels (40 and 200 ppm) were expected to establish a "no observable effect level" (NOEL).

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: mortality twice daily, clinical signs at least once daily
- Cage side observations included: mortality, clinical signs

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly detailed clinical examination which included a palpation for tissue masses (except for acclimatization/pretest phase). A description of any lesion or mass observed at any examination was recorded and the subsequent progress monitored.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly until week 13 and every two weeks thereafter using an on-line electronic recording system consisting of a
Mettler balance connected to the RCC computer system.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
The food consumption was recorded for a 7-day period. The data were recorded weekly until week 13 and every 2 weeks thereafter using an on-line electronic recording system consisting of a Mettler balance connected to the RCC computer system.
- Food consumption for each cage determined and average daily diet consumption calculated as g food/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No, cage means were calculated as mg compound/kg bw/day.
- The relative food consumption (RFC) was calculated weekly as g food/kg bw/day.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes, mean water consumption values were calculated as g water /animal/day over the measurement intervals.
- Time schedule for examinations: once monthly in the first 3 months and every 3 months thereafter using an on-line electronic recording system, consisting of a Mettler balance connected to the RCC computer system, recorded over 24 hours.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at pretest, 26, 51, 78 and 104 weeks
- Dose groups that were examined: all dose groups of the animals scheduled for assessment of chronic toxicity (20/sex/dose, actual number examined depending on survival). After the application of a mydriatic solution (Disperse AG, Winterthur, Switzerland) the cornea, lens, anterior chamber, vitreous body and ocular fundus of both eyes were examined using a Heine Miroflex 2 Ophthalmoscope (Eisenhut Vet AG, Allschwil/Switzerland).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Animals of the interim sacrifice group at 52 weeks, animals scheduled for assessment of chronic toxicity at 26, 52, 78 and 104 weeks. Blood samples were collected between 06.00 and 09.15 to reduce biological variation caused by circadian rhythms. Blood samples were drawn from the retro-orbital plexus using a micro-hematocrit glass capillary tube. Blood smears for differential blood counts were taken for all remaining animals scheduled for assessment of carcinogenicity at 123 weeks of treatment.
- Anaesthetic used for blood collection: Yes (light ether anaesthesia)
- Animals fasted: Yes, approximately 18 hours before blood sampling but allowed access to water ad libitum, animals of the carcinogenicity group were not fasted before blood sampling.
- How many animals: All animals (10/sex/dose) scheduled for interim sacrifice and half of the animals (10/sex/dose, with the lowest identification numbers) scheduled for assessment of chronic toxicity.
- Parameters examined: erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, reticulocyte count, nucelated erythrocytes (normoblasts), total leukocyte count, differential leukocyte count, red cell morphology, coagulation (thromboplastin time, activated partial thromboplastin time)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Animals of the interim sacrifice group at 52 weeks, animals scheduled for assessment of chronic toxicity at 26, 52, 78 and 104 weeks. Blood samples were collected between 06.00 and 09.15 to reduce biological variation caused by circadian rhythms. Blood samples were drawn from the retro-orbital plexus using a micro-hematocrit glass capillary tube.
- Animals fasted: Yes, approximately 18 hours before blood sampling but allowed access to water ad libitum.
- How many animals: All animals (10/sex/dose) scheduled for interim sacrifice and half of the animals (10/sex/dose, with the lowest identification numbers) scheduled for assessment of chronic toxicity.
- Parameters examined: glucose, urea, creatinine, bilirubin total, bilirubin direct, cholesterol total, triglycerides, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, alkaline phosphatase, gamma-glutamyltransferase, calcium, phosphorus, sodium, potassium, chloride, protein total, protein electrophoresis (albumin, alpha 1-globulin, alpha 2-globulin, sum of beta-globulins, gamma-globulin).

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected from all animals of the interim sacrifice group at 52 weeks and from 10 animals/sex/dose (with the lowest identification numbers) scheduled for assessment of chronic toxicity at 26, 52, 78 and 104 weeks.
- Metabolism cages used for collection of urine: Yes, urine collected on ice during the 18-hours fasting period into a specimen vial.
- Animals fasted: Yes
- Parameters examined: volume, specific gravity, colour, appearance, pH, protein, glucose, ketone, bilirubin, blood, urobilinogen, urine sediment, gamma-glutamyltransferase, lactate dehydrogenase, alkaline phosphatase, leucine aminopeptidase, beta-N-Acetyl-D-glucosaminidase, sodium, phosphorus, potassium, protein total, creatinine, creatinine clearance, urea


NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY:
Yes, all surviving animals and all animals which died spontaneously or were killed in extremis were necropsied and descriptions of all macroscopic abnormalities were recorded. All surviving animals were weighed prior to necropsy. Necropsies were performed by experienced prosectors supervised by a veterinary pathologist. All animals surviving to the end of the observation period and all moribund animals were anesthetized by intraperitoneal injection of sodium pentobarbital and killed by exsanguination. Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4 % formaldehyde solution (the tissues between brackets need to be examined only if indicated by signs of toxicity or target organ involvement): Adrenal glands, Aorta, Brain [including sections of medulla/pons, cerebral and cerebellar cortex], Cecum, Colon, Duodenum, Epididymides, Esophagus, Eyes - with optic nerve and Harderian gland, Femur [including joint], Heart, Ileum, Jejunum, Kidneys, (Larynx), Lacrimal gland [exorbital], Liver, Lungs [infused with formalin], Lymph nodes [mandibular, mesenteric], Mammary gland area, (Nasopharynx), Ovaries, Pancreas, Pituitary gland, Prostate gland, Rectum, Salivary gland [mandibular, sublingual], Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord [cervical, midthoracic, lumbar], Spleen, Sternum with bone marrow, Stomach, Testes, Thymus - if present, Thyroid gland/parathyroid, Tongue, Trachea, Urinary bladder [infused with formalin], Uterus, Vagina, All gross lesions and tumors

Samples of liver (approximately 2 grams) and kidneys (approximately 1 gram for males and approximately 0.7 gram for females), brain (1 gram), fatty
tissue (perirenal) and skeletal muscle for possible future tests were taken from interim sacrifice-animals at scheduled necropsy. The remainder of tissues was used for histopathological evaluations. The organ samples for biochemistry were placed on ice in labelled plastic bags, rinsed in ice-cold saline (0.9% NaCl solution), blotted dry and immediately frozen in liquid nitrogen, and stored at -80°C. Only organs without macroscopical abnormalities were used for these purposes.

HISTOPATHOLOGY: Yes
The tissues were embedded in paraffin. The sections were cut at an approximate thickness of 4 micrometers and stained with hematoxylin and eosin.
Sections of the following tissues from rats scheduled for interim sacrifice as well as from all animals that died or were killed in extremis were examined microscopically (number of sections per tissue):
Adrenal glands (2), aorta (1), bone [femur with stifle joint sternum] (2), bone marrow [sternum, femur] (2), brain (1), epididymides (2), esophagus (1), exorbital lacrymal glands (2), eyes with optic nerve (2), Harderian glands (2), heart (1), kidneys (2), large intestine [cecum, colon, rectum] (3), liver (2), lungs (2), lymph nodes [mandibular, mesenteric] (2), mammary gland area [female] (1), ovaries (2), pancreas (1), parathyroid glands (2), pituitary gland (1), prostate (1), salivary glands [sublingual, mandibular] (2), sciatic nerve (1), seminal vesicles (2), skeletal muscle (1), skin (1), small intestine [duodenum, jejunum, ileum] (3), spinal cord (3), spleen (1), stomach (1), testes (2), thymus (l), thyroid gland (2), tongue (1), trachea (1), urinary bladder (1), uterus (1), vagina (1) and all gross lesions.

Other examinations:

ORGAN WEIGHTS:
Brain, Adrenals, Kidneys, Heart, Liver, Pituitary, Lungs, Ovaries, Spleen, Thyroid, Testes

Statistics:

The following statistical methods were used to analyze the body weight, food consumption, water consumption, organ weights and clinical laboratory data :
Univariate one-way analysis of variance was used to assess the significance of intergroup differences. If the variables can be assumed to follow a normal distribution, the Dunnett test (many to one t-test) based on a pooled variance estimate was applied for the comparison between the treated groups and the control groups for each sex.
The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
The exact Fisher-test was applied to ophthalmoscopic examinations and overall spontaneous mortality data.
Statistical evaluation of neoplastic lesions was performed according to Peto et al., 1980, using the test for positive trend with respect to dose rates. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables.
Individual values, means, standard deviations and statistics were rounded-off before printing. For example, test statistics were calculated on the basis of exact values for means and pooled variances and then rounded-off to two decimal places. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
The age-specific survival rate up to termination for each group and sex was calculated with Kaplan-Meier non-parametric estimates based on censored survival data, censorship being imposed on unnatural causes of death (death during blood sampling procedures, trauma, etc.).

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

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:

CLINICAL SIGNS AND MORTALITY
Survival was not affected by the test article. For distribution of unscheduled deaths within the groups see table 1. Neither the type nor the incidences of the clinical signs (mainly alopecia) or of the nodules and masses noted indicated an effect of treatment with the test article.

BODY WEIGHT AND WEIGHT GAIN
Compared with the control group, group 5 (5000 ppm,) females had statistically significantly reduced mean body weights from start of treatment throughout the recording period. These constantly reduced values were considered to be a borderline effect of test article. At this dose level, no effects on the body weight of the male animals were evident. The difference between male and female animals was considered to be caused by the higher test article intake (ca. 26%) of the females in comparison with the respective males. In the dose groups 2 (40 ppm), 3 (200 ppm) and 4 (1000 ppm), the body weight development of the male and female animals was not considered to be affected by treatment with the test article.

In the group 4 (1000 ppm) males, body weight gain was increased in comparison with that of the control group. After reaching a peak of approximately 11% at 91 weeks the difference in body weight gain nearly disappeared until the end of study. In absence of any dose relationship this finding was considered to be incidental.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Up to and including the highest dietary concentration of 5000 ppm, in both the male and female animals the differences in food consumption between the respective control group and the dose groups were not considered to be caused by treatment with the test article.
Generally, in both male and female animals of the dose groups relative food consumption compared favourably with that of the respective control animals. Although, in several cases differences from control values attained statistical significance, in particular for the group 5 (5000 ppm) animals, these findings were not considered to indicate test article related effects.

Test article intake was proportional to dietary concentrations and slightly higher in females than in males (see table 2).


WATER CONSUMPTION
In comparison with the control group, water consumption was increased in the males of group 5 (5000 ppm) during the first three recording periods (on days 17-18, 38-39 and 73-74 of treatment) and in the males of group 4 (1000 ppm) up to week 123 (last measurement). This finding was considered to be incidental and not test article related because no dose-relationship was evident between groups 4 (1000 ppm) and 5 (5000 ppm). Males of groups 1 (0 ppm), 2 (40 ppm) and 3 (200 ppm) and the female animals of all groups had similar water consumption.
As for absolute water consumption, relative water consumption was increased during the first weeks of treatment in males of groups 4 (1000 ppm) and 5 (5000 ppm). As from week 25 up to week 123 (last measurement) relative water consumption in these dose groups was similar to that of the control males. Females of group 5 (5000 ppm) had increased relative water consumption in comparison with the control group during the pretest period as well as during the treatment period. This finding was considered to be caused by the reduced mean body weights noted in the females at this dose level (see above) and was not considered to be test article related. Males of groups 2 (40 ppm) and 3 (200 ppm) and females of groups 2 (40 ppm), 3
(200 ppm) and 4 (1000 ppm) had similar relative water consumption to that of the respective controls throughout the treatment period.

OPHTHALMOSCOPIC EXAMINATION
Up to and including the highest dose level of 5000 ppm, no treatment related effects were noted in both male and female animals at ophthalmoscopic examinations.

HAEMATOLOGY
The assessment of hematological data indicated a few changes with statistical significance during the course of the treatment. The effects noted were most obvious in the rats of group 5 (5000 ppm) and were as follows:
-slightly lower erythrocyte count by 7 to 8 % on average for females at 26 and 52 weeks (a slight decrease by 8 % was also noted at 104 weeks, however, the finding did not achieve statistical significance);
-slightly lower hemoglobin concentration by 7 % on average for females at 26 weeks, by 3 to 7 % for both sexes at 52 weeks and by 10 % for females at
104 weeks (a slight decrease by 4 % was also noted for females of group 4 (1000 ppm) at 26 weeks);
-slightly lower hematocrit value by 4 to 3 % on average for females at 26 and 52 weeks;
-slightly higher mean corpuscular volume (MCV) by 5 % on average for females at 52 weeks;
-slightly lower mean corpuscular hemoglobin concentration (MCHC) by 2 to 4 % on average for both sexes at 26 and 52 weeks and by 5 % for females at 104 weeks (a slight decrease by 2 to 4 % was also noted for females of group 4 at 52 and 104 weeks);
-Higher reticulocyte count (relative and absolute) by 40 to 52 % on average for both sexes at 26 weeks, by 13 to 10 % for males and 42 to 30 % for females at 52 weeks, by 24 to 32 % for both sexes at 78 weeks, and 72 to 44 % for females at 104 weeks. In addition, a slight increase by 14 to 16 % on average was noted for males of group 4 at 26 weeks and by 92 to 69 % at 104 weeks.

The findings noted were considered treatment-related and suggest slight anemia with an increase in hemopoietic activity as indicated by the increase in reticulocytes. Furthermore, there was no effect on differential blood counts after 123 weeks of treatment. All other differences in the results of the hematology parameters were considered to be incidental or unrelated to the treatment and of normal biological variation for rats of this strain and age.

CLINICAL CHEMISTRY
For clinical biochemistry data there were no changes of toxicological significance during the course of the treatment. The only change of note was a
slight decrease in the total bilirubin concentration by 11 % on average for males of group 5 (5000 ppm) at 26 weeks, by 21 to 24 % for both sexes at 52 weeks and by 30 to 34 % for both sexes at 78 weeks. A slight decrease by 26 to 28 % was also noted for females of group 4 (1000 ppm) at 52 and 78 weeks. For the direct bilirubin concentration, a slight decrease by 24 to 20 % on average was noted for both sexes of groups 4 and 5 at 52 weeks and by 28 to 33 % for females of groups 4 and 5 at 78 weeks. The lower bilirubin concentrations were not considered of toxicological significance but more likely an expression of the functional activity of reticuloendothelial cells (bilirubin production) and hepatic cell function (bilirubin removal).
All other differences in the results of the clinical biochemical parameters were considered to be incidental or unrelated to the treatment and of normal biological variation for rats of this strain and age.

URINALYSIS
For urinalysis data the following effects were recorded in the rats of groups 4 (1000 ppm) and/or 5 (5000 ppm) during the course of the treatment:
-Decreased gamma-glutamyltransferase, alkaline phosphatase, leucine aminopeptidase and B-N-acetyl-D-glucosaminodase activity (females).
However, in contrast to the increase in urinary excretion of enzymes (enzymuria) which reflect lesional changes of renal tubuli, the biological significance of the above changes - slightly reduced urinary enzyme excretion - of membrane bound and lysosomal enzymes remains doubtfully, particularly in the absence of any other measurable biochemical abnormality and any histological correlate.

ORGAN WEIGHTS
No treatment related effects on the organ weights of the animals were noted in all dose groups. The statistically significant differences noted were considered to be incidental or for the group 5 (5000 ppm) females to correlate with the slightly reduced terminal body weight.

GROSS PATHOLOGY
A number of gross lesions were observed in the rats that died during the course of the study and in those that were sacrificed on schedule. The type, incidence, and severity of these gross lesions did not distinguish treated rats from controls.

HISTOPATHOLOGY: NON-NEOPLASTIC
A number of non-neoplastic lesions were diagnosed. The type, incidence, severity and organ distribution of these non-neoplastic changes did not distinguish treated rats from controls.
The non-neoplastic lesions were primarily inflammatory, degenerative, and/or hyperplastic changes. These lesions mainly affected the endocrine, reproductive, and large parenchymatous organs.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
A number of neoplastic lesions were diagnosed in the rats at scheduled and unscheduled necropsy. The type, incidence, severity and organ distribution were considered to be similar in both treated and control rats. The same was true for the number of primary neoplasms, the number of rats with more than one primary neoplasm, the number of rats with metastases, and the number of benign and malignant neoplasms per dose group and sex.

HISTORICAL CONTROL DATA (if applicable)
All other differences in the results of the hematology parameters apart from those thought to be treatment-related were considered to be incidental or unrelated to the treatment and of normal biological variation for rats of this strain and age.

Key result

Dose descriptor:

NOEL

Remarks:

systemic

Effect level:

200 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effect observed

Remarks on result:

other: corresponding to 9.80 and 12.07 mg/kg bw/day for males and females, respectively.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

1 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no observed adverse effect

Remarks on result:

other: corresponding to 48.47 and 59.98 mg/kg bw/day for males and females, respectively.

Key result

Dose descriptor:

LOAEL

Remarks:

systemic

Effect level:

5 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

Remarks on result:

other: corresponding to 251.60 and 317.96 mg/kg bw/day for males and females, respectively.

Key result

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Effect level:

5 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no observed adverse effect

Remarks on result:

other: corresponding to 251.60 and 317.96 mg/kg bw/day for males and females, respectively.

Remarks:

highest dose tested

Key result

Critical effects observed:

not specified

Table1: Distribution of unscheduled deaths:

	Number of unscheduled deaths		Survival (Kaplan-Meier Estimate)
Concentration (ppm)	Males	Females	Males	Females
0	25	30	60 %	49 %
40	29	33 (A)	53 %	49 %
200	29	33	53 %	43 %
1000	32	29	48 %	55 %
5000	22	32	65 %	47 %

(A) = One female died during anaesthesia

Table 2: Test article intake:

	Average test article intake up to 52 weeks (mg/kg/day)
Concentration (ppm)	Males	Females
40	1.92	2.38
200	9.80	12.07
1000	48.47	59.98
5000	251.60	317.96

Conclusion:

Based on the results obtained in this study, the "No observed effect level" (NOEL) of HOE 107892 SUBSTANCE TECHNICAL in the rat was considered to be 200 ppm in the diet, corresponding to an average daily intake of 9.80 mg/kg for males and 12.07 mg/kg for females. In consideration that the findings in the clinical biochemistry investigations expressed physiological-functional changes the "No observed adverse effect level" (NOAEL) was considered to be 1000 ppm in the diet, corresponding to an average daily intake of 48.47 mg/kg for males and 59.98 mg/kg for females. Up to and including the highest dietary concentration of 5000 ppm, there was no indication of any oncogenic potential of the test article.

Reference 7

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 82-1 (90-Day Oral Toxicity)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL Biological Research Laboratories Ltd, CH 4414 Fuellinsdorf, Switzerland
- Age at study initiation: about 5 weeks
- Weight at study initiation: males: 74-95 g (mean: 83 g), females: 67-87 g (mean: 76 g)
- Fasting period before study: no
- Housing: behind a barrier system, groups of five in Makrolon type-4 cages with wire mesh lids and standard granulated softwood bedding (Lignocel, Schill AG, 4132 Muttenz/Switzerland).
- Diet (e.g. ad libitum): pelleted standard Kliba no. 343 rat maintenance diet (Kliba, Klingentalmuehle AG, 4303 Kaiseraugst/Switzerland), ad libitum
- Water (e.g. ad libitum): tap water ad libitum via water bottles
- Acclimation period: 7 days under test conditions, after veterinary examination.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 40 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

acetone

Details on oral exposure:

DIET PREPARATION
HOE 107892 SUBSTANCE TECHNICAL was dissolved in acetone, mixed with microgranulated food and pelleted using a pelleting machine. Water (approximately 1:10 volume/weight ratio) was added to aid pelleting. The pellets were dried for approximately 48 hours before storage in disposable paper bags at room temperature. The pellets were prepared every four weeks.
- Rate of preparation of diet (frequency): every 4 weeks
- Mixing appropriate amounts with (Type of food): microgranulated food
- Storage temperature of food: room temperature


VEHICLE
- Justification for use and choice of vehicle (if other than water): admixed to the diet, which is recognized as an efficacious method of absorption.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test article in the diet was established prior to initiation of treatment (RCC Project 271754). In the current study, the stability of
the test article in the diet was ascertained over a period of 5 weeks with the first food preparations. Intercurrent sampling for analyses (homogeneity and content) was performed on each food preparation. Analyses were performed in the RCC UMWELTCHEMIE Analytical Chemistry Laboratory, according to a method supplied by the sponsor.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

continuously

Dose / conc.:

100 ppm

Remarks:

equivalent to 8.1 mg/kg bw/day (males) and 8.6 mg/kg bw/day (females)

Dose / conc.:

500 ppm

Remarks:

equivalent to 42.1 mg/kg bw/day (males) and 44.3 mg/kg bw/day (females)

Dose / conc.:

2 500 ppm

Remarks:

equivalent to 206.7 mg/kg bw/day (males) and 223.0 mg/kg bw/day (females)

Dose / conc.:

7 500 ppm

Remarks:

equivalent to 660.6 mg/kg bw/day (males) and 708.9 mg/kg bw/day (females)

No. of animals per sex per dose:

10 (additionally 10 per satellite group)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Based upon the results of a repeated-dose (4 week) oral toxicity study in rats performed by the sponsor (study 90.0423).
- Rationale for selecting satellite groups: In order to check reversibility of potentially occurring effects.
- Post-exposure recovery period in satellite groups: 4 weeks (0, 2500, 7500 ppm)

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: mortality twice daily, clinical signs daily
- Cage side observations included: mortality, clinical signs


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly palpation for tissue masses. A description of any lesion or mass at any examination was recorded and the subsequent progress was monitored.


BODY WEIGHT: Yes
- Time schedule for examinations: weekly using an on-line electronic recording system consisting of a Mettler balance connected to the RCC computer.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Recorded weekly using an on-line electronic recording system consisting of a Mettler balance connected to the RCC computer. Food spillage (when detected) was also entered into the computer system. Such values were disregarded for the calculation of means. The relative food consumption (RFC) was recorded weekly, as well. Nominal test article intake (TAI) was calculated for each cage.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/day: Yes
- Compound intake calculated from the consumption and body weight data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at pretest, at 11 weeks and at 16 weeks. After the application of a mydriatic solution (Disperse AG, Winterthur/Switzerland) the cornea, lens, anterior chamber, vitreous body and ocular fundus of both eyes were examined under dimmed light using a Heine Miroflex 2 Ophthalmoscope (Eisenhut Vet AG, Allschwil/Switzerland).
- Dose groups that were examined: all animals


HAEMATOLOGY: Yes
- Time schedule for collection of blood: from all main study animals at 13 weeks and from all satellite animals at 17 weeks. Blood samples were collected between the hours of 07.15 and 08.40 a.m. to reduce biological variation caused by circadian rhythms. Blood samples were drawn from the retro-orbital plexus.
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes, for approx. 18 hours before sampling, but water provided
- How many animals: all
- Parameters examined: Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Platelet count, Reticulocyte count, Nucleated erythrocytes (normoblast), Total leukocyte count, Differential leukocyte count, Red cell morphology, Coagulation (Thromboplastin time, Activated partial thromboplastin time)


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: from all main study animals at 13 weeks and from all satellite animals at 17 weeks. Blood samples were collected between the hours of 07.15 and 08.40 a.m. to reduce biological variation caused by circadian rhythms. Blood samples were drawn from the retro-orbital plexus.
- Animals fasted: Yes, for approx. 18 hours before sampling, but water provided
- How many animals: all
- Parameters examined: Glucose, Urea, Creatinine, Uric acid, Bilirubin total, Bilirubin direct, Lipids total, Cholesterol total, Triglycerides, Aspartate aminotransferase, Alanine aminotransferase, Lactate dehydrogenase, Creatine kinase, Alkaline phosphatase, Gamma-glutamyltransferase, Calcium, Phosphorus, Sodium, Potassium, Chloride, Protein total, Protein electrophoresis (Albumin, Alpha 1-globulin, Alpha 2-globulin, Sum of beta globulins, Gamma globulin, Albumin to Globulin ratio)


URINALYSIS: Yes
- Time schedule for collection of urine: on ice from all main study animals at 13 weeks and from all satellite group animals at 17 weeks during the 18-hours fasting period into a specimen vial using a metabolism cage.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, 18 hours
- Parameters examined: Volume (18-hour), Specific gravity, Osmolality, pH, Protein, Glucose, Ketone, Bilirubin, Blood, Urobilinogen, Urine Sediment, Gamma-glutamyltransferase, Lactate dehydrogenase, Alkaline phosphatase, Leucine aminopeptidase, Beta-N-Acetyl-Dglucosaminidase, Sodium, Potassium, Phosphorus, Protein total, Creatinine, Creatine clearance, Urea,


NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes: All animals, all macroscopic abnormalities were recorded. Samples of the following tissues and organs were collected and fixed in neutral phosphate buffered 4 % formaldehyde solution: Adrenal glands, Aorta, Brain - including section of medulla/pons, cerebral and cerebellar cortex, Cecum, Colon, Cervix, Duodenum, Epididymides, Esophagus, Exorbital lacrymal glands, Eyes with optic nerve and Harderian gland, Female mammary gland area, Femur - including articular surface, Heart, Ileum, Jejunum, Kidneys, Liver, Larynx, Lungs (infused with formalin), Lymph nodes - mesenteric, mandibular, Ovaries, Pancreas, Pituitary gland, Prostate gland, Rectum, Salivary gland - mandibuler, sublingual, Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord - cervical, midthoracic, lumbar, Spleen, Sternum with bone marrow, Stomach, Testes, Thymus, Thyroid gland / parathyroid gland, Tongue, Trachea, Urinary bladder (infused with formalin), Uterus, Vagina, All gross lesions

HISTOPATHOLOGY: Yes: The following tissues were trimmed, processed and embedded in paraffin wax, and sectioned at a thickness of 2-4 µm. All aforementioned tissues, with the exception of exorbital lacrymal gland and larynx, from groups 1 and 5 (Main test and Recovery). Adrenals, brain, heart, kidneys, liver, ovaries, pancreas and testes from group 4 (Main test and Recovery) and groups 2 and 3. All gross lesions. Sections were stained with haematoxylin and eosin and examined light microscopically.

Other examinations:

ORGAN WEIGHTS
The following organ weights were recorded on the scheduled dates of necropsy: adrenals, brain, heart, kidneys, liver, lungs, ovaries, pituitary, spleen, testes, thymus and thyroid.

Statistics:

The following statistical methods were used to analyze the body weights, food consumption, organ weights and clinical laboratory data:
If the variables could be assumed to follow a normal distribution, the Dunnett test (many to one t-test) based on a pooled variance estimate was applied for the comparison between the treated groups and the control groups.
The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
For ophthalmoscopy data, the Fisher's exact test for 2 x 2 tables was applied. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Individual values, means, standard deviations and statistics were rounded off before printing. For example, test statistics were calculated on the basis of exact values for means and pooled variances and then rounded off to two decimal places. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

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:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

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:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no unscheduled deaths during the study. There were no clinical signs which could be attributed to the administration of HOE 107892.

BODY WEIGHT AND WEIGHT GAIN
The administration of 100, 500 or 2500 ppm HOE 107892 had no effect on body weight gain in either sex. Bodyweight gains were slightly impaired at 7500 ppm. Increased weight gains were recorded for high-dose animals in the 4 week treatment-free period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
The administration of HOE 107892 had no effect on food consumption. The administration of 100, 500 or 2500 ppm HOE 107892 had no effect on relative food consumption in either sex. Because of the slight impairment of body weight gain at 7500 ppm, without any effect on absolute food consumption, relative food consumption was slightly higher than in controls at this dietary concentration of HOE 107892 for most of the study duration.

As expected in an experiment in young rats, relative food consumption and, therefore, test article intake was initially high during the growing phase of the animals and fell gradually as growth slowed. At dietary concentrations of 100, 500 and 2500 ppm the amount of HOE 107892 consumed on a mg/kg body weight basis was in direct proportion to the concentration in the diet whereas at 7500 ppm the amount of HOE 107892 consumed
was proportionately higher. At each dietary concentration of HOE 107892 females consumed 5-8 % more than males, on a mg/kg basis.


OPHTHALMOSCOPIC EXAMINATION
The administration of HOE 107892 had no effect on any ophthalmoscopy parameter.

HAEMATOLOGY
The administration of 100 or 500 ppm HOE 107892 had no effect on hematology parameters in either sex. A slight anemia was indicated for both sexes at 2500 and 7500 ppm by the following statistically differences from control means: lower erythrocyte count and hemoglobin in males and females at 2500 and 7500 ppm, lower hematocrit in males and females at 7500 ppm, higher mean corpucular volume in males and females at 7500 ppm, higher mean corpuscular hemoglobin in males at 7500 ppm, and higher reticulocyte counts in males and females at 7500 ppm.

Following the 4 week treatment-free period, there were no toxicologically significant differences between group means except that mean corpuscular volume in males which had received 7500 ppm was slightly higher than in controls. All other statistically significant intergroup differences were considered to be unrelated to treatment with HOE 107892.

CLINICAL CHEMISTRY
The administration of HOE 107892 at dietary concentrations of 100 or 500 ppm in males or 100 ppm in females had no effect on clinical biochemistry parameters. At the end of the 13 week treatment period the following statistically significant differences between group mean values for animals treated with HOE 107892 and control means were considered to be of toxicological significance: Glucose - slightly lower in females at 7500 ppm, Urea - slightly lower in females at 7500 ppm, Gamma-glutamyl-transferase - slightly higher in females at 7500 ppm, Creatinine - slightly higher in males at 7500 ppm, Calcium - slightly lower in males at 7500 ppm, Sodium - slightly higher in males and females at 7500 ppm and females at 2500 ppm, Chloride - slightly lower in males at 7500 ppm. Protein - slightly lower in both sexes at 7500 ppm.

Following the 4 week treatment-free period, there were no significant differences between group means. All other statistically significant intergroup differences were considered to be unrelated to treatment with HOE 107892. In particular, the slightly increased phosphorus levels observed for
females at 500, 2500 and 7500 ppm were clearly within the range of normal biological variation and not associated with corresponding changes in calcium levels.

URINALYSIS
No effects on urinalysis parameters were seen in males. After 13 weeks of treatment, urinary Gamma-glutamyl-transferase and Leucine aminopeptidase were lower, and urinary Lactate dehydrogenase higher in females at 7500 ppm than in control females, these differences being statistically significant. Other intergroup differences occurred in recovery group animals, but were considered to be of no toxicological significance because similar differences were not present at week 13. The higher Gamma-glutamyl-transferase in males which had received 2500 or 7500 ppm was considered to be statistically significantly different from controls only because of a fall in control value between weeks 13 and 17.

ORGAN WEIGHTS
There were no treatment-related effects on organ weights. Because of the impairment of body weight gain in males at 7500 ppm the relative weights of brain, pituitary, lung, kidney, spleen and testes were statistically significantly higher than in male controls, but this is of no toxicological significance. The statistically significantly higher thyroid weights for females at 100 ppm were also considered to be of no significance in the absence of similar changes at higher dose levels.

GROSS PATHOLOGY
The administration of HOE 107892 had no effect on the appearance of any organ or tissue at necropsy in either sex. In particular, no findings indicative of hematoxicity or nephrotoxicity could be established.

HISTOPATHOLOGY: NON-NEOPLASTIC
The administration of HOE 107892 had no effect on the microscopic appearance of any tissue in either sex.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effect observed

Key result

Dose descriptor:

LOAEL

Remarks:

systemic

Effect level:

2 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

haematology

Key result

Critical effects observed:

not specified

ASSESSMENT

Systemic toxicity of HOE 107892 substance technical was indicated by impaired body weight gains at 7500 ppm. The following potential targets of HOE 107892 Substance Technical were identified:

Erythrocytes:

The hematological changes seen at 7500 ppm indicate a slight anemia, the loss of erythrocytes and consequent fall in hemoglobin and hematocrit being compensated for by the increase in hemopoietic activity, shown by the increase in circulating reticulocytes for both sexes at 7500 ppm. Slightly reduced erythrocyte counts and hemoglobin values were also recorded for both sexes at 2500 ppm. Practically all signs of the anemia resolved during the treatment-free recovery period.

Kidneys:

The slight changes in plasma concentrations of sodium and chloride, point to changes in renal function. The lower urinary gamma-glutamyl-transferase and leucine aminopeptidase values and the increase in lactate dehydrogenase in high dose females may reflect an early impact on the proximal tubular cells, although the absence of any histological changes in the kidneys indicates that the changes in that organ were probably functional rather than structural. It is significant to note that the intergroup differences were absent from animals allowed a 4 week treatment-free recovery period.

Conclusion:

A dietary concentration of 500 ppm (corresponding to an average daily test article intake of 42.1 mg/kg for males and 44.3 for females) is a no observable adverse effect level for the test article in this study.

Reference 8

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 83-1 (Chronic Toxicity)

Deviations:

no

GLP compliance:

yes

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hazleton-LRE, 6321 South 6th Street, Kalamazoo, MI 49009 / USA
- Age at study initiation: 5 to 7 months
- Weight at study initiation: males: 5.9 - 8.6 kg; females: 5.6 - 7.9 kg
- Housing: Air-conditioned, individual kennels with a minimum of 2.0 square meters floor space.
- Diet: 350 g repelleted standard Kliba 335 dog maintenance diet ("Kliba", Klingentalmuehle AG, 4303 Kaiseraugst/Switzerland) presented at approximately 10.00 daily and withdrawn at 13.00.
- Water: Tap water, ad libitum by an automatic watering system.
- Acclimation period: 6 (16 dogs) or 8 (44 dogs) weeks under test conditions, after veterinary examination


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3, monitored hourly; kennel floor temperature maintained at approximately 23°C
- Humidity (%): 40 - 70 %, monitored hourly
- Air changes (per hr): 20
- Photoperiod (hrs dark / hrs light): 12/12, with at least 8 hours music during the light period.

Route of administration:

oral: feed

Vehicle:

acetone

Details on oral exposure:

DIET PREPARATION
The test substance was dissolved in acetone and subsequently mixed with microgranulated feed. Water (approximately 1:10 volume/weight ratio) was added to aid pelleting. The pellets were dried with warm air for approximately 48 hours before storage.
- Rate of preparation of diet (frequency): once in the first monthand every 2 weeks thereafter
- Mixing appropriate amounts with (Type of food): microgranulated feed
- Storage temperature of food: at -20°C in the dark in disposable paper bags until the week of use.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability, homogeneity and content of the test article in the feed was determined in a previous 13-week feeding study (RCC Project 285120) and 4-week feeding study (RCC Project 271754). The Sponsor guaranteed the stability of test substance in the feed for 35 days at room temperature. This was verified in an additional stability analysis, where it was proven stable for over 37 days, performed on the first batch of diet prepared for this study. Intercurrent samples were retained at two-monthly intervals for the determination of homogeneity and content. Analyses were performed in the laboratories of RCC UMWELTCHEMIE AG. The overall mean concentrations were found to be 94.5 %, 96.1 %, 95.1 % and 94.9 % of the nominal concentrations for the 60, 300, 1500 and 7500 ppm dose groups, respectively.
The homogeneity varied in the range from -7 % to +6 % of the mean concentrations.

Duration of treatment / exposure:

males: 52 weeks and 4 or 6 days; females: 52 weeks and 5 days or 53 weeks

Frequency of treatment:

daily during the feeding period (for 3 hours, within the feed)

Dose / conc.:

60 ppm

Remarks:

equivalent to 2.2 mg/kg bw/day

Dose / conc.:

300 ppm

Remarks:

equivalent to 11 mg/kg bw/day

Dose / conc.:

1 500 ppm

Remarks:

equivalent to 55 mg/kg bw/day

Dose / conc.:

7 500 ppm

Remarks:

equivalent to 271 mg/kg bw/day

No. of animals per sex per dose:

6

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Selected by the sponsor based on results obtained in a 13-week toxicity study (RCC Project 285120) conducted at RCC.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily
- Cage side observations included: viability, change in behaviour or appearance


DETAILED CLINICAL OBSERVATIONS: No


BODY WEIGHT: Yes
- Time schedule for examinations: weekly from commencement of pretest and before necropsy


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): daily from commencement of pretest. The daily ration was weighed before and after feeding. The daily food consumption over each week for each dog, and the weekly group means of these values are reported.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No


WATER CONSUMPTION: No


OPHTHALMOSCOPIC EXAMINATION: Yes, the observation area included the cornea, conjunctiva, sclera, iris, lens and fundus. Photographs were taken.
- Time schedule for examinations: pretest and at 13, 27, 51 weeks
- Dose groups that were examined: all


HAEMATOLOGY: Yes
- Time schedule for collection of blood: pretest and at 13/14, 26/27, 52/53 weeks
- Anaesthetic used for blood collection: No
- Animals fasted: Yes, overnight, but allowed access to water ad libitum
- How many animals: all
- Parameters examined: Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Platelet count, Reticulocyte count, Nucleated erythrocytes (normoblasts), Total leukocyte count, Differential leukocyte count, Red cell morphology, Coagulation (Thromboplastin time, Activated partial thromboplastin time)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: pretest and at 13/14, 26/27, 52/53 weeks
- Animals fasted: Yes, overnight, but allowed access to water ad libitum
- How many animals: all
- Parameters examined: Glucose, Urea, Creatinine, Bilirubin total, Lipids total, Cholesterol total, Triglycerides, Phospholipids, Aspartate aminotransferase, Alanine aminotransferase, Lactate dehydrogenase, Glutamate dehydrogenase, Creatine kinase, Alkaline phosphatase, Gamma-glutamyl-transferase, Ornithine carbamyltransferase, Iron, Calcium, Phosphorus, Magnesium, Sodium, Potassium, Chloride, Protein total, Protein electrophoresis (Albumin, Alpha 1-globulin, Alpha 2-globulin, Beta 1-globulin, Beta 2-globulin, Sum of beta globulins, Gamma globulin, Albumin to Globulin ratio)


URINALYSIS: Yes
- Time schedule for collection of urine: pretest and at 13/14, 26/27, 52/53 weeks
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, overnight, but allowed access to water ad libitum
- Parameters examined: Volume/24 h, Specific gravity, Osmolality, Color, Appearance, Crystals, pH, Protein, Glucose, Ketone, Bilirubin, Blood, Urobilinogen, Urine Sediment, Creatinine, Creatine clearance, Urea, Alkaline phosphatase, Lactate dehydrogenase, Gamma-glutamyltransferase, Leucine aminopeptidase, beta-N-Acetyl-D-glucosaminidase, Sodium, Potassium, Phosphorus, Protein total


NEUROBEHAVIOURAL EXAMINATION: No


OTHER:
Additionally, 10 g of faeces, a plasma sample, a red blood cell sample and 10 ml of urine were stored at -20°C in the dark at RCC for possible future investigations.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (Adrenal glands, aorta, bone - femur (including articular surface), bone marrow - sternum and femur, brain - including medulla/pons, cerebral and cerebellar cortex, epididymides, esophagus, eyes (with optic nerve), female mammary gland area, (male mammary gland area), gall bladder, heart, kidneys, large intestine (cecum, colon, rectum), liver, lungs (infused with formalin), lymph nodes (retropharyngeal, mesenteric), ovaries, pancreas, pituitary gland, prostate gland, salivary glands - parotid and mandibular, sciatic nerve, skeletal muscle, skin, small intestine (duodenum, jejunum, ileum), spinal cord - cervical, midtnoracic and lumbar, spleen, stomach, testes, thymus, thyroid gland (including parathyroid gland), tongue, trachea, urinary bladder, uterus (with vagina) and all gross lesions.)

HISTOPATHOLOGY: Yes (All sampled tissues (except for male mammary gland area and tongue) from all dose groups were trimmed, processed, embedded in paraffin wax and sectioned at a thickness of 2-4 micrometers. Sections were stained with haematoxylin and eosin and examined using a light
microscope.)

Other examinations:

ORGAN WEIGHTS (paired organs were weighed separately):
Adrenal glands, brain (including brainstem), heart, kidneys, liver, ovaries, pituitary gland, prostate gland, spleen, testes with epididymides, thymus, thyroid gland (with parathyroid).

Statistics:

Body weights, clinical laboratory data and organ weights were analyzed by the following procedures: Univariate one-way analysis of variance was used to assess the significance of intergroup differences. If the variables could be assumed to follow a normal distribution, the Dunnett-Test (many to one t-test) based on a pooled variance estimate was applied for the comparison between the treated groups and the control groups. The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Individual values, means, standard deviations and statistics were rounded-off before printing. For example, test statistics were calculated on the basis of exact values for means and pooled variances and then rounded-off to two decimal places. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistic values.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no treatment-related clinical signs observed. One female at 300 ppm was found dead at the first mortality control on treatment day 314 (week 45). Vomitus was present in the pen. Histopathological examination revealed evidence of circulatory failure.

BODY WEIGHT AND WEIGHT GAIN
There was no effect on body weight that could be attributed to treatment with the test substance.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Slightly decreased group mean food intake was recorded for the males at 7500 ppm in the first two weeks of treatment in comparison with food intake
pretest. Two dogs were most markedly affected. Thereafter, mean food intake improved although continued reductions in food intake were recorded for those two dogs in the first 24 weeks and throughout the treatment period, respectively. There was no effect on food intake, when compared with the control and pretest consumption, in the remaining dogs that could be definitely related to treatment. Slight reductions in overall food intake were recorded in individual male dogs of the remaining treated groups. However, an association with treatment is considered unlikely in the absence of any dose-relationship.


OPHTHALMOSCOPIC EXAMINATION
The ophthalmic parameters were unaffected by treatment with the test substance. The findings were those commonly observed during canine ophthalmoscopy and are considered to be spontaneous in origin.

HAEMATOLOGY
Assessment of the hematology data revealed the following changes in comparison with the control and pretest data: Slightly decreased mean red blood cell count, hemoglobin concentration and hematocrit in females at 7500 ppm at each investigation. The differences from the controls for red blood cell count and hemoglobin concentration were statistically significant in weeks 13/14 and 52/53. One female was most markedly affected, such that a slight anemia was evident. Slightly to moderately increased platelet count in the males and females at 7500 ppm at each investigation during the treatment period. The differences from the controls were significant at each investigation for females and in week 52/53 in the males. There were no other changes that could be related to treatment with the test substance.

CLINICAL CHEMISTRY
A slight, but consistent, decrease in mean plasma urea and bilirubin concentrations in the females at 7500 ppm. The differences from the controls were statistically significant for urea in week 52/53 and for bilirubin in weeks 26/27 and 52/53. Slightly to moderately increased mean alkaline phosphatase activity in the males and, more particularly, in the females at 7500 ppm at each investigation during the treatment period. The differences from the controls were statistically significant with the exception of the males in weeks 26/27. The comparison with the concurrent control data revealed an average 3.7-fold increase in the females, whereas, a 1.9-fold increase was evident in the males. Indication of marginally to slightly increased alkaline phosphatase activity was also seen in the females of 1500 ppm, when compared with the concurrent control and lower dose groups. A slight disturbance of the protein-electrophoretic pattern in the females at 7500 ppm, particularly in week 52/53. This disturbance was characterized by changes in the relative proportions and absolute levels of the following: decreased albumin and increased Beta-2 globulins with a low albumin/globulin ratio.

There were no other changes that could be related to test substance treatment. Slightly increased alanine aminotransferase, glutamate dehydrogenase and ornithine carbamyltransferase activities were recorded in one female at 300 ppm, and slightly to markedly increased aspartate aminotransferase, alanine aminotransferase and creatine kinase activities in one female at 7500 ppm in week 13/14. A relationship to treatment of these findings in single dogs is considered unlikely in the absence of any dose-relationship and as similar increases occasionally occur in untreated animals.

URINALYSIS
No changes in comparison with the control and pretest data considered to be attributable to treatment with the test substance.

ORGAN WEIGHTS
Moderately to markedly increased group mean liver weight in the males and females at 7500 ppm respectively. The organ-to-body weight ratios revealed a more pronounced increase in the females (+65 %) than in the males (+22 %). The differences from the controls were statistically significant for the absolute weights and body and brain weight ratios with the exception of liver to brain weight ratio in the males. Slightly increased thyroid gland weight and decreased prostate gland weight in the males at 7500 ppm. There were no other changes considered to be attributable to treatment with the test substance.

GROSS PATHOLOGY
No treatment-related macroscopic findings were recorded in any group.

HISTOPATHOLOGY: NON-NEOPLASTIC
A minimal intrahepatic cholestasis was recorded in two males and two females at 7500 ppm. All other histopathologic lesions recorded in this study were considered to be incidental and in accordance with the normal range of background pathology in beagle dogs.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

1 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effect observed

Remarks on result:

other: corresponding to 55 mg/kg bw/day

Key result

Dose descriptor:

LOAEL

Remarks:

systemic

Effect level:

7 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

food consumption and compound intake

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Remarks on result:

other: corresponding to 271 mg/kg bw/day

Key result

Critical effects observed:

not specified

Conclusion:

In the present study, treatment with HOE 107892 SUBSTANCE TECHNICAL at 7500 ppm resulted in the following toxicologically relevant effects:

- A transient reduction in food intake on commencement of treatment in the males with more persistent decrease in two dogs.

- Slightly decreased red cell parameters (females) and increased platelet count.

- A clear increase in liver weight, clinical biochemical changes, particularly increased alkaline phosphatase activity, and minimal intrahepatic cholestasis indicative of liver dysfunction or toxicity.

The increase in thyroid gland weight and decrease in prostate gland weight in the males at 7500 ppm are of uncertain significance in the absence of correlating histopathological findings.

A similar trend to increased alkaline phosphatase activity was also apparent in the females at 1500 ppm, but was not associated with increased liver weight or histopathological changes.

There is no association between the death of one female at 300 ppm and the administration of HOE 107892 SUBSTANCE TECHNICAL.

Based on these findings, 1500 ppm (equivalent to 55 mg/kg bw/d) was established as the no-observed-adverse-effect-level (NOAEL) in this study.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

48.47 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

The available information comprises adequate, reliable (Klimisch score 1) and consistent studies, and is thus sufficient to fulfil the standard information requirements set out in Annex IX-X, 8.6, of Regulation (EC) No. 1907/2006.

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

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Deviations:

yes

Remarks:

occlusive dressing, extended program of histopathology

Qualifier:

according to guideline

Guideline:

EPA OPP 82-2 (Repeated Dose Dermal Toxicity -21/28 Days)

Deviations:

yes

Remarks:

occlusive dressing, extended program of histopathology

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hoechst AG, Kastengrund, SPF breeding colony
- Age at study initiation: approx. 8 weeks
- Weight at study initiation: males 190 ± 2.4 g; females 181 ± 2.6 g
- Housing: in Makrolon cages (Type 3) on soft wood granulate, one animal per cage
- Diet (e.g. ad libitum): Altromin 1324 pelleted rat diet (Altromin GmbH, Lage/ Lippe), ad libitum, except for the period in which the animals were kept in diuresis cages
- Water (e.g. ad libitum): tap water in plastic bottles, ad libitum, except for the period in which the animals were kept in diuresis cages
- Acclimation period: 8 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 23
- Humidity (%): 25 - 75
- Air changes (per hr): approx. 12 per hour
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

occlusive

Vehicle:

polyethylene glycol

Details on exposure:

TEST SITE
- Area of exposure: dorsal
- % coverage: 10
- Type of wrap if used: occlusive bandage (3-layer cellulose bandage, aluminium foil, Fixomull-Stretch and Elastoplast from Beiersdorf/Hamburg)
- Time intervals for shavings or clipplings: At the start of the study and subsequently at least once weekly with an electric clipper


REMOVAL OF TEST SUBSTANCE
- Washing (if done): with warm water
- Time after start of exposure: 6 hours


TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 100, 300, 1000 mg/kg
- Concentration (if solution): 50, 150, 500 mg/ml
- Constant volume or concentration used: yes


VEHICLE
- Justification for use and choice of vehicle (if other than water): handling at dosing
- Amount(s) applied (volume or weight with unit): 2 ml/kg


USE OF RESTRAINERS FOR PREVENTING INGESTION: yes, occlusive dressing

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Immediatly after preparation and once more at last day of use for application, the active ingredient contents and homogeneity of all formulations
were examined by an analytical laboratory. According to report of 23 October 1991, the homogeneity and stability of the test substance preparations were guaranteed for a period of 18 days and the actually administered test substance concentrations corresponded in high degree to the nominal concentrations.

Duration of treatment / exposure:

29 days

Frequency of treatment:

6 h/day, 5 days/week

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 (additionally 5 males and females in control and high dose recovery groups)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The selection of dosages was based on the results of an acute dermal toxicity study in rats, which yielded a LD50-value of > 4000 mg/kg body weight (Report No. 90.0451).
- Post-exposure recovery period in satellite groups: 15 days

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations included: behaviour, general health condition (clinical signs), mortality


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly for neurological disturbances, damage to the oral mucosa, and impairment of dental growth.


DERMAL IRRITATION (if dermal study): Yes, based on scores of OECD 404
- Time schedule for examinations: prior to each application


BODY WEIGHT: Yes
- Time schedule for examinations: at start of the study and then twice weekly


FOOD CONSUMPTION: Yes, at the start of the study and then twice weekly (same days as body weight)
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data


WATER CONSUMPTION: Yes
- Time schedule for examinations: once weekly for a period of 16 hours


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: weekly for opacity of the refracting media of the eyes
- Dose groups that were examined: all


HAEMATOLOGY: Yes
- Time schedule for collection of blood: one day after the last application (recovery groups at the end of the 2-week recovery period)
- Anaesthetic used for blood collection: Yes (Nembutal), at retrobulbar venous plexus
- Animals fasted: No data
- How many animals: all (a few parameters only for control and high dose group)
- Parameters examined: erythrocytes, haemoglobin, haematocrit, MCV, MCH, MCHC, leucocytes, thrombocytes, differential blood count, reticulocytes*, Heinz bodies*, coagulation time, thromboplastin time, activated partial thromboplastin time, thrombin time, methaemoglobin* (*only performed for control and high dose group)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: one day after the last application (recovery groups at the end of the 2-week recovery period): after blood sampling from the retrobulbar venous plexus for haematological examination, the animals were killed by opening of the vena cava cranial is under
Nembutal anaesthesia (50 mg/kg body weight, i.p.) and exsanguinated.
- Animals fasted: No data
- How many animals: all
- Parameters examined: sodium, potassium, inorganic phosphorus, uric acid, total bilirubin, creatinine, serum glucose, urea nitrogen, calcium, chloride, ASAT, ALAT, alkaline phosphatase, gamma-GT, LDH, cholesterol, triglycerides, total lipids, total protein, electrophoresis


URINALYSIS: Yes
- Time schedule for collection of urine: in all main groups during the night (about 16 hours) before day 25 of the study. The urine of the recovery animals was not collected, since no effects were observed in the animals of the main groups.
- Metabolism cages used for collection of urine: Yes, individual diuresis cages
- Animals fasted: Yes
- Parameters examined: appearance, colour, pH value, haemoglobin, protein, glucose, ketone bodies, bilirubin, urobilinogen, nitrite, ascorbic acid, sediment (performed only for control and high dose group), volume of urine


NEUROBEHAVIOURAL EXAMINATION: No


OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (skin, orifices, eyes, teeth, oral mucosa and internal organs)
HISTOPATHOLOGY: Yes, of control and high dose animals (heart, uterus/ovaries, lungs, adrenals, liver, pituitary, kidneys, thyroid (both lobes), spleen, bone marrow (femur), brain, sternum, both testes, skin (treated & untreated, approx. 2 x 2 cm), seminal vesicle, prostate gland, special macroscopic abnormalities); slides of treated skin, liver, kidneys, testes and macroscopic lesions were read of animals from intermediate groups

Other examinations:

ORGAN WEIGHTS
The absolute weights of the following organs were determined and the relative weights per 100 g body weight calculated: heart, brain, lungs, testes/ovaries, liver, adrenals, kidneys, pituitary, spleen, thyroid

Statistics:

The following data were evaluated statistically at the level of significance p < 0.05:
body weights at the designated measurement times, haematology parameters (except diff. blood count and methaemoglobin), clinical chemistry parameters (except gamma-GT), urinalysis (pH value, volume), absolute organ weights, relative organ weights.
Evaluation was performed by PHARMA DEVELOPMENT INFORMATIC with the aid of a program package for evaluating toxicological studies. The statistical methods used in each case were given on the computer printouts.

Clinical signs:

no effects observed

Dermal irritation:

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):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

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:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
Behaviour and general health condition remained normal throughout the study in all of the treated groups. There were no indications of any neurological disturbances, impairment of dental growth or changes in the oral mucosa which might have been attributed to administration of the test substance. There were no deaths at any time during the study.

BODY WEIGHT AND WEIGHT GAIN
The test substance had no effect on body weight gains in animals of either sex in any of the treatment groups.

FOOD CONSUMPTION
The test substance had no effect on the food consumption of animals of either sex in any of the treatment groups.

WATER CONSUMPTION
Water consumption was variable. No effects could be observed as a result of treatment with the test substance.

OPHTHALMOSCOPIC EXAMINATION
There were no indications of opacity of the refracting media of the eyes.

HAEMATOLOGY
At the end of the treatment period slight decreases in erythrocyte counts, haemoglobin content and haematocrit value in the females from the highest dose group. This finding might be related to the test substance and indicative of a marginal anaemia. This effect could not be observed at the end of the recovery period. Additional correlates indicative of haematoxicity could not established by clinical biochemistry or pathology. No other changes attributable to the test substance could be detected.

CLINICAL CHEMISTRY
Statistical analysis revealed only a decrease in total bilirubin in the females of the highest dose group at the end of treatment. This effect showed a dose-dependency and was therefore considered to be related to the treatment with the test compound. No other changes were noted at the end of treatment.
Evaluation of the serum electrophoresis data gave no convincing evidence of substance-related changes at the end of treatment. Only a marginal increase in globulines was noted in the females treated at 300 and 1000 mg/kg body weight. However, no biological significance could be assigned to this change.

URINALYSIS
The urinary status of all animals remained unaffected by dermal treatment with the test substance. There was no evidence of any damage to the renal system. The urine of the large majority of animals in all groups was clear and yellow in colour. At the end of the treatment period, the group mean pH values varied between 6.7 and 6.9 in the males and between 6.2 and 6.8 in the females. The group mean urinary volume varied between 6.1 and 7.4 ml in the males and between 2.8 and 4.3 ml/16h in the females. Examination of the urine for protein, glucose, haemoglobin, bilirubin, urobilinogen, ascorbic acid, nitrite and ketone bodies gave no indication of substance-related changes; the same applies to the examination of urinary sediments in the
control and high-dose animals at the end of the treatment period.

ORGAN WEIGHTS
Statistical analysis of the absolute and relative organ weights indicated no changes in any of the treated groups as compared with the control group.

GROSS PATHOLOGY
Neither topically nor systemically any adverse reaction attributable to the test compound administered was to be found at morphological investigation of skin and internal organs.

HISTOPATHOLOGY: NON-NEOPLASTIC
Neither topically nor systemically any adverse reaction attributable to the test compound administered was to be found at microscopical investigation of skin and internal organs.

OTHER FINDINGS
The test substance proved to be non-irrating to the skin and histopathology indicated also no changes attributable to the test compound.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effect observed

Key result

Dose descriptor:

LOAEL

Remarks:

sytemic toxicity

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

haematology

Key result

Critical effects observed:

not specified

Haematology parameters:

	Dose level (mg/kg bw/d)
	males				females
Parameter	0	100	300	1000	0	100	300	1000
Erythrocytes (10e12/L)	6.65	6.79	7.08	6.76	6.85	6.32	6.64	6.10*
Hb (g/L)	131	137	139	137	131	130	128	125
Haematocrit	0.40	0.41	0.42	0.41	0.42	0.40	0.40	0.38
MCH (10e-12g)	20	20	20	20	19	21*	19	21*
MCHC (g/L)	327	333	331	336	314	327*	321	330*

* p<= 0.05

Conclusion:

Based on the results of this repeated-dose dermal toxicity study (21 treatments in 29 days) with Hoe 107892 00 ZC94 0001 in the Wistar rat, the NO OBSERVABLE ADVERSE EFFECT LEVEL (NOAEL) for systemic toxicity is considered to be 300 mg/kg bw/day.

Repeated dermal treatment with 1000 mg/kg body weight caused a marginal anaemia and a decrease in total bilirubin in the female rat.

Under the conditions of this study, the test substance in the form of a suspension in PEG 400 proved to be non-irritating to the treated skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises an adequate, reliable (Klimisch score 1) and consistent study, and is thus sufficient to fulfil the standard information requirements set out in Annex IX-X, 8.6, of Regulation (EC) No. 1907/2006.

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Deviations:

yes

Remarks:

occlusive dressing, extended program of histopathology

Qualifier:

according to guideline

Guideline:

EPA OPP 82-2 (Repeated Dose Dermal Toxicity -21/28 Days)

Deviations:

yes

Remarks:

occlusive dressing, extended program of histopathology

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hoechst AG, Kastengrund, SPF breeding colony
- Age at study initiation: approx. 8 weeks
- Weight at study initiation: males 190 ± 2.4 g; females 181 ± 2.6 g
- Housing: in Makrolon cages (Type 3) on soft wood granulate, one animal per cage
- Diet (e.g. ad libitum): Altromin 1324 pelleted rat diet (Altromin GmbH, Lage/ Lippe), ad libitum, except for the period in which the animals were kept in diuresis cages
- Water (e.g. ad libitum): tap water in plastic bottles, ad libitum, except for the period in which the animals were kept in diuresis cages
- Acclimation period: 8 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 23
- Humidity (%): 25 - 75
- Air changes (per hr): approx. 12 per hour
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

occlusive

Vehicle:

polyethylene glycol

Details on exposure:

TEST SITE
- Area of exposure: dorsal
- % coverage: 10
- Type of wrap if used: occlusive bandage (3-layer cellulose bandage, aluminium foil, Fixomull-Stretch and Elastoplast from Beiersdorf/Hamburg)
- Time intervals for shavings or clipplings: At the start of the study and subsequently at least once weekly with an electric clipper


REMOVAL OF TEST SUBSTANCE
- Washing (if done): with warm water
- Time after start of exposure: 6 hours


TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 100, 300, 1000 mg/kg
- Concentration (if solution): 50, 150, 500 mg/ml
- Constant volume or concentration used: yes


VEHICLE
- Justification for use and choice of vehicle (if other than water): handling at dosing
- Amount(s) applied (volume or weight with unit): 2 ml/kg


USE OF RESTRAINERS FOR PREVENTING INGESTION: yes, occlusive dressing

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Immediatly after preparation and once more at last day of use for application, the active ingredient contents and homogeneity of all formulations
were examined by an analytical laboratory. According to report of 23 October 1991, the homogeneity and stability of the test substance preparations were guaranteed for a period of 18 days and the actually administered test substance concentrations corresponded in high degree to the nominal concentrations.

Duration of treatment / exposure:

29 days

Frequency of treatment:

6 h/day, 5 days/week

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 (additionally 5 males and females in control and high dose recovery groups)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The selection of dosages was based on the results of an acute dermal toxicity study in rats, which yielded a LD50-value of > 4000 mg/kg body weight (Report No. 90.0451).
- Post-exposure recovery period in satellite groups: 15 days

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations included: behaviour, general health condition (clinical signs), mortality


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly for neurological disturbances, damage to the oral mucosa, and impairment of dental growth.


DERMAL IRRITATION (if dermal study): Yes, based on scores of OECD 404
- Time schedule for examinations: prior to each application


BODY WEIGHT: Yes
- Time schedule for examinations: at start of the study and then twice weekly


FOOD CONSUMPTION: Yes, at the start of the study and then twice weekly (same days as body weight)
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data


WATER CONSUMPTION: Yes
- Time schedule for examinations: once weekly for a period of 16 hours


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: weekly for opacity of the refracting media of the eyes
- Dose groups that were examined: all


HAEMATOLOGY: Yes
- Time schedule for collection of blood: one day after the last application (recovery groups at the end of the 2-week recovery period)
- Anaesthetic used for blood collection: Yes (Nembutal), at retrobulbar venous plexus
- Animals fasted: No data
- How many animals: all (a few parameters only for control and high dose group)
- Parameters examined: erythrocytes, haemoglobin, haematocrit, MCV, MCH, MCHC, leucocytes, thrombocytes, differential blood count, reticulocytes*, Heinz bodies*, coagulation time, thromboplastin time, activated partial thromboplastin time, thrombin time, methaemoglobin* (*only performed for control and high dose group)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: one day after the last application (recovery groups at the end of the 2-week recovery period): after blood sampling from the retrobulbar venous plexus for haematological examination, the animals were killed by opening of the vena cava cranial is under
Nembutal anaesthesia (50 mg/kg body weight, i.p.) and exsanguinated.
- Animals fasted: No data
- How many animals: all
- Parameters examined: sodium, potassium, inorganic phosphorus, uric acid, total bilirubin, creatinine, serum glucose, urea nitrogen, calcium, chloride, ASAT, ALAT, alkaline phosphatase, gamma-GT, LDH, cholesterol, triglycerides, total lipids, total protein, electrophoresis


URINALYSIS: Yes
- Time schedule for collection of urine: in all main groups during the night (about 16 hours) before day 25 of the study. The urine of the recovery animals was not collected, since no effects were observed in the animals of the main groups.
- Metabolism cages used for collection of urine: Yes, individual diuresis cages
- Animals fasted: Yes
- Parameters examined: appearance, colour, pH value, haemoglobin, protein, glucose, ketone bodies, bilirubin, urobilinogen, nitrite, ascorbic acid, sediment (performed only for control and high dose group), volume of urine


NEUROBEHAVIOURAL EXAMINATION: No


OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (skin, orifices, eyes, teeth, oral mucosa and internal organs)
HISTOPATHOLOGY: Yes, of control and high dose animals (heart, uterus/ovaries, lungs, adrenals, liver, pituitary, kidneys, thyroid (both lobes), spleen, bone marrow (femur), brain, sternum, both testes, skin (treated & untreated, approx. 2 x 2 cm), seminal vesicle, prostate gland, special macroscopic abnormalities); slides of treated skin, liver, kidneys, testes and macroscopic lesions were read of animals from intermediate groups

Other examinations:

ORGAN WEIGHTS
The absolute weights of the following organs were determined and the relative weights per 100 g body weight calculated: heart, brain, lungs, testes/ovaries, liver, adrenals, kidneys, pituitary, spleen, thyroid

Statistics:

The following data were evaluated statistically at the level of significance p < 0.05:
body weights at the designated measurement times, haematology parameters (except diff. blood count and methaemoglobin), clinical chemistry parameters (except gamma-GT), urinalysis (pH value, volume), absolute organ weights, relative organ weights.
Evaluation was performed by PHARMA DEVELOPMENT INFORMATIC with the aid of a program package for evaluating toxicological studies. The statistical methods used in each case were given on the computer printouts.

Clinical signs:

no effects observed

Dermal irritation:

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):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

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:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
Behaviour and general health condition remained normal throughout the study in all of the treated groups. There were no indications of any neurological disturbances, impairment of dental growth or changes in the oral mucosa which might have been attributed to administration of the test substance. There were no deaths at any time during the study.

BODY WEIGHT AND WEIGHT GAIN
The test substance had no effect on body weight gains in animals of either sex in any of the treatment groups.

FOOD CONSUMPTION
The test substance had no effect on the food consumption of animals of either sex in any of the treatment groups.

WATER CONSUMPTION
Water consumption was variable. No effects could be observed as a result of treatment with the test substance.

OPHTHALMOSCOPIC EXAMINATION
There were no indications of opacity of the refracting media of the eyes.

HAEMATOLOGY
At the end of the treatment period slight decreases in erythrocyte counts, haemoglobin content and haematocrit value in the females from the highest dose group. This finding might be related to the test substance and indicative of a marginal anaemia. This effect could not be observed at the end of the recovery period. Additional correlates indicative of haematoxicity could not established by clinical biochemistry or pathology. No other changes attributable to the test substance could be detected.

CLINICAL CHEMISTRY
Statistical analysis revealed only a decrease in total bilirubin in the females of the highest dose group at the end of treatment. This effect showed a dose-dependency and was therefore considered to be related to the treatment with the test compound. No other changes were noted at the end of treatment.
Evaluation of the serum electrophoresis data gave no convincing evidence of substance-related changes at the end of treatment. Only a marginal increase in globulines was noted in the females treated at 300 and 1000 mg/kg body weight. However, no biological significance could be assigned to this change.

URINALYSIS
The urinary status of all animals remained unaffected by dermal treatment with the test substance. There was no evidence of any damage to the renal system. The urine of the large majority of animals in all groups was clear and yellow in colour. At the end of the treatment period, the group mean pH values varied between 6.7 and 6.9 in the males and between 6.2 and 6.8 in the females. The group mean urinary volume varied between 6.1 and 7.4 ml in the males and between 2.8 and 4.3 ml/16h in the females. Examination of the urine for protein, glucose, haemoglobin, bilirubin, urobilinogen, ascorbic acid, nitrite and ketone bodies gave no indication of substance-related changes; the same applies to the examination of urinary sediments in the
control and high-dose animals at the end of the treatment period.

ORGAN WEIGHTS
Statistical analysis of the absolute and relative organ weights indicated no changes in any of the treated groups as compared with the control group.

GROSS PATHOLOGY
Neither topically nor systemically any adverse reaction attributable to the test compound administered was to be found at morphological investigation of skin and internal organs.

HISTOPATHOLOGY: NON-NEOPLASTIC
Neither topically nor systemically any adverse reaction attributable to the test compound administered was to be found at microscopical investigation of skin and internal organs.

OTHER FINDINGS
The test substance proved to be non-irrating to the skin and histopathology indicated also no changes attributable to the test compound.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effect observed

Key result

Dose descriptor:

LOAEL

Remarks:

sytemic toxicity

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

haematology

Key result

Critical effects observed:

not specified

Haematology parameters:

	Dose level (mg/kg bw/d)
	males				females
Parameter	0	100	300	1000	0	100	300	1000
Erythrocytes (10e12/L)	6.65	6.79	7.08	6.76	6.85	6.32	6.64	6.10*
Hb (g/L)	131	137	139	137	131	130	128	125
Haematocrit	0.40	0.41	0.42	0.41	0.42	0.40	0.40	0.38
MCH (10e-12g)	20	20	20	20	19	21*	19	21*
MCHC (g/L)	327	333	331	336	314	327*	321	330*

* p<= 0.05

Conclusion:

Based on the results of this repeated-dose dermal toxicity study (21 treatments in 29 days) with Hoe 107892 00 ZC94 0001 in the Wistar rat, the NO OBSERVABLE ADVERSE EFFECT LEVEL (NOAEL) for systemic toxicity is considered to be 300 mg/kg bw/day.

Repeated dermal treatment with 1000 mg/kg body weight caused a marginal anaemia and a decrease in total bilirubin in the female rat.

Under the conditions of this study, the test substance in the form of a suspension in PEG 400 proved to be non-irritating to the treated skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises one adequate, reliable (Klimisch score 1) and consistent study, and is thus sufficient to fulfil the standard information requirements set out in Annex IX-X, 8.6, of Regulation (EC) No 1907/2006.

Additional information

There are several studies available on repeated dose toxicity in rats, dogs and mice via the oral route, ranging from 28 days up to 1 and 2 year feeding studies in dogs and rats, respectively. The NOAEL for systemic toxiciy was comparable in the chronic studies for both rats and dogs with 48.47 mg/kg bw/d for males and 59.98 mg/kg bw/d for females in the rat (corresponding to 1000 ppm in the feed), and 55 mg/kg bw/d (males/females, corresponding to 1500 ppm in the feed) in the dog. In mice, there was a slightly higher NOAEL of 89.3 mg/kg bw/d for males and 105.4 mg/kg bw/d for females observed after an exposure period of 13 weeks.

Several observations were consistently made across all species and all exposure periods, those being effects on hematological parameters like slightly reduced erythrocyte counts, hemoglobin and hematocrit, which occurred already after 4 weeks of treatment. These findings reflect a tendency towards slight anemia, with an increase of reticulocytes, indicative of an increase in hemopoietic activity. These symptoms of slight anemia were shown to resolve during the 4-week recovery period subsequently to the subchronic treatment in dogs and rats.

Furthermore, slight increases in alkaline phosphatase, aspartate aminotransferase and alanine aminotransferase in parallel with a decrease in bilirubin concentrations were observed especially in subchronic studies in dogs and mice, and by trend in the chronic study in dogs, indicative of liver toxicity or dysfunction especially in the high dose animals of the respective studies. Additionally, mice demonstrated hepatocellular hypertrophy after subchronic treatment in all treatment groups in all studies. These findings were accompanied by the observation of increased liver weight and reduced bilirubin levels in the blood in more or less all studies, indicative of increased hepatic cell function. Interestingly, rats seem to be less sensitive for effects on liver parameters, as they only demonstrated increases in liver weight and a decrease in bilirubin, but no change of liver enzymes indicative of possible liver damage.

In contrast, rats showed a decreased excretion of urinary enzymes like gamma-glutamyl transferase and leucine aminopeptidase in the chronic and subchronic studies, and a decrease in excretion of gamma-glutamyl transferase and alanine aminopeptidase in the subacute study, all enzymes located in the brush-border region of the proximal renal tubule. These findings may reflect an early impact on the proximal renal tubular cells, although the absence of any histological changes in the kidneys indicates that the changes in that organ were probably functional rather than structural. However, as the excretion of these enzymes is reduced, the toxicological relevance has to be questioned, the actual effect is unclear. It is significant to note that these differences were absent from animals allowed a 4 week treatment-free recovery period in the subchronic study.

In addition to the aforementioned observations, impairment of body weight gain (or even weight loss in dogs upon subchronic treatment) was observed in chronic and subchronic studies in rats and in the subchronic study in mice, furthermore in the subacute study in dogs. In dogs only, the reduction of body weight gain was accompanied by reduced food consumption, whereas the latter was not impaired in rats and mice.

 

Furthermore, data from a subactue dermal study in rats is available, in which a NOAEL of 300 mg/kg bw/d for males and females was determined. The only findings were slight decreases in erythrocyte counts, hemoglobin content and hematocrit value in the high dose females, which were indicative of a marginal anemia. These effects could not be observed after the 15 day treatment-free recovery period. A decrease in total bilirubin was also observed in those animals. These findings seem to reflect the same trend as those observed via the oral route, just less pronounced. Therefore, the dermal route seems to be of minor relevance for the test substance than the oral route. The test substance did not produce irritating effects to the skin, and there were no histopathological changes attributable to the test compound.

 

Repeated application via inhalation was not performed, as there was no indication of toxicity in the acute inhalation study up to the highest technically administrable dose. Furthermore, the low vapour pressure and the lack of inhalable particles imply that inhalation is not the most relevant route of exposure, and there is extensive data available from oral studies. Therefore, repeated dose toxicity testing via inhalation has been waived.

Justification for classification or non-classification

The available data on oral and dermal repeated dose toxicity showed that the test item induces slight and reversible alterations on haematological and clinicochemical parameters. The effects were rather of adaptive than adverse nature, no distinct target organ was unequivocally identified. Therefore, the available data on repeated dose toxicity with the test substance do not meet the criteria according to Regulation (EC) No. 1272/2008. The data are conclusive but not sufficient for classification.