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

Carcinogenicity

Description of key information

Oral toxicity (OECD 453), 124 weeks feeding study, rat: NOAEL carcinogenic = 251.60 mg/kg bw/day (males) and 317.96 mg/kg bw/d (females) corresponding to 5000 ppm in the feed

Oral toxicity (OECD 451), 87 weeks feeding study, mouse: NOAEL carcinogenic = 350.79 mg/kg bw/day (males) and 463.35 mg/kg bw/d (females) corresponding to 2500 ppm in the feed

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

carcinogenicity: 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 451 (Carcinogenicity Studies)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 83-2 (Carcinogenicity)

Deviations:

no

GLP compliance:

yes

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: 4 weeks
- Weight at study initiation: males: 14 - 24 grams (mean: 19 grams); females: 14 - 23 grams (mean: 18 grams)
- Housing: individually in Makrolon type-1 cages with wire mesh tops and granulated softwood bedding (Lignocel, Schill AG, 4132 Muttenz/ Switzerland)
- Diet (e.g. ad libitum): pelleted standard Kliba 343 rat/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: 7 days under 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 exposure:

DIET PREPARATION
The test substance was dissolved in acetone, mixed with microgranulated feed and pelleted using a pelleting machine. 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, during which time the acetone has evaporated.
- Rate of preparation of diet (frequency): twice monthly
- Mixing appropriate amounts with (Type of food): microgranulated feed
- Storage temperature of food: at room temperature in disposable paper bags

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 before study initiation with RCC Project 284894 and was repeated with the first diet batches prepared for the study. Intercurrent sampling for analyses (homogeneity and content) were performed at the following weeks of treatment: 5, 13, 23, 29, 37, 53, 61, 69, 77, 85. Analyses were performed in the Analytical Chemistry Laboratory of RCC UMWELTCHEMIE AG according to a method supplied by the sponsor.

Duration of treatment / exposure:

females 81 weeks, males 87 weeks; interim sacrifice groups 52 weeks

Frequency of treatment:

continuously, in the feed

Post exposure period:

none

Dose / conc.:

20 ppm

Remarks:

equivalent to 2.76 mg/kg bw/day (males) and 3.78 mg/kg bw/day (females)

Dose / conc.:

100 ppm

Remarks:

equivalent to 14.08 mg/kg bw/day (males) and 18.31 mg/kg bw/day (females)

Dose / conc.:

500 ppm

Remarks:

equivalent to 70.58 mg/kg bw/day (males) and 92.01 mg/kg bw/day (females)

Dose / conc.:

2 500 ppm

Remarks:

equivalent to 350.79 mg/kg bw/day (males) and 463.35 mg/kg bw/day (females)

No. of animals per sex per dose:

70 (of which 20 were killed at interim sacrifice)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Based upon results of a subchronic toxicity study in the mouse (RCC Project 284894). In this study dietary concentrations of 500, 2500 and 7500 ppm were used. The following results were obtained: At 7500 ppm: slight anemia in both sexes; body weight reduction in males 12 % together with slight increase in food consumption; slight hepatotoxicity indicated by increases in liver weight, ASAT, ALAT, ALP, LDH, hepatocellular hyperthrophy and decrease in bilirubin. At 2500 ppm: body weight decrease in males (-9 %) and decrease in bilirubin. At 500 ppm: decrease in bilirubin.
The top dose level (2500 ppm) was expected to cause a 10 % reduction in bodyweight gains, hepatocellular hypertrophy, and slight changes in liver function in males. The mid-high dose level (500 ppm) was expected to cause minimal, if any, signs of toxicity. The lower dose levels (20 and 100 ppm) were expected to establish a "no observable effect level" (NOEL).

Observations and examinations performed and frequency:

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


DETAILED CLINICAL OBSERVATIONS: Yes, including a palpation for tissue masses
- Time schedule: weekly


BODY WEIGHT: Yes
- Time schedule for examinations: recorded weekly until week 13, twice monthly thereafter, and prior to necropsy


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): recorded weekly until week 13 and twice monthly thereafter
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes


WATER CONSUMPTION: No


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 52 weeks males and females, after 81 weeks females, after 87 weeks males, just prior to scheduled necropsy from retro-orbital plexus
- Anaesthetic used for blood collection: Yes (slight ether anaesthesia)
- Animals fasted: No
- How many animals: after 52 weeks ten animals per group and sex, after 81 and 87 weeks each ten animals per group. Differential blood counts were made on all surviving animals.
- Parameters examined: Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Mean corpuscular hemoglobin, Mean corpuscular
hemoglobin concentration, Platelet count, Reticulocyte count, Reticulocyte fluorescence ratios (LFR, MFR, HFR), Nucleated erythrocytes (normoblasts), Total leukocyte count, Differential leukocyte count, Red cell morphology


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 52 weeks males and females, after 81 weeks females, after 87 weeks males, just prior to scheduled necropsy, from retro-orbital plexus
- Animals fasted: No
- How many animals: after 52 weeks ten animals per group and sex, after 81 and 87 weeks each ten animals per group for clinical biochemistry parameters and another ten or remaining animals for immunoglobulin assays.
- Parameters examined: Urea, Creatinine, Bilirubin total, Aspartate aminotransferase, Alanine aminotransferase, Lactate dehydrogenase, Alkaline phosphatase, Immunoglobulin G, Immunoglobulin A, Immunoglobulin M, Immunoglobulin G1, Immunoglobulin G2a, Immunoglobulin G2b, Immunoglobulin G3


URINALYSIS: Yes
- Time schedule for collection of urine: over approximately 18 hours, at 50 weeks from those 20 animals scheduled for interim sacrifice and at 80 and 85 weeks from all surviving females and males, respectively.
- Metabolism cages used for collection of urine: Yes, each metabolism cage contained two mice of the same group to assure sufficient sample volumes when technically possible.
- Animals fasted: Yes, 20 ml tap water/kg bodyweight was administered to the animals prior to the urine collection period. The animals did not have access to food and water during the urine collection period.
- Parameters examined: Volume (18-hour), Specific gravity, Osmolality, pH, Protein, Glucose, Ketone, Bilirubin, Blood, Urobilinogen, Urine Sediment


NEUROBEHAVIOURAL EXAMINATION: No


OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (Adrenals, Aorta, Brain [including sections of medulla/pons, cerebral and cerebellar cortex], Cecum, Colon, Duodenum, Epididymides, Esophagus, Eyes with nerve and Harderian gland, Femur [including joint], Heart, Ileum, Jejunum, Kidneys, Lacrymal gland [exorbital], Larynx [if indicated by signs of toxicty], Liver with gallbladder, Lungs [infused with formalin], Lymph nodes [mandibular, mesenteric], Mammary gland area, Nasopharynx [if indicated by signs of toxicity], Ovaries, Pancreas, Pituitary gland, Prostate gland, Rectum, Salivary glands [mandibular, sublingual], Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord [cervical, midthoracic, lumbar], Spleen, Sternum with bone marrow, Stomach, Testes, Thymus, Thyroid gland [including parathyroid], Tongue, Trachea, Urinary bladder [infused with formalin], Uterus, Vagina, All gross lesions, tissue masses and tumors)

HISTOPATHOLOGY: Yes (Adrenal glands, Aorta, Bone [femur, including joint; sternum], Bone marrow [sternum], Brain, Cecum, Colon, Duodenum, Epididymides, Esophagus, Eyes, Exorbital lacrimal glands, Gallbladder, Harderian glands, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs, Lymph nodes [mandibular, mesenteric], Mammary gland area [female], Optic nerves, Ovaries, Pancreas, Parathyroid glands, Pituitary gland, Prostate, Rectum, Salivary glands [mandibular, sublingual], Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord [cervical, mid-thoracic, lumbar], Spleen, Stomach, Testes, Thymus, Thyroid gland, Tongue, Trachea, Urinary bladder, Uterus, Vagina, and all gross lesions)

Other examinations:

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

Statistics:

The following statistical methods were used to analyze the body weight, food 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 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. 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.

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:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No treatment-related mortality was observed. Neither the types nor the incidences of the clinical signs noted indicated test article related effects. The nodules and masses noted reflected the normal pattern for a chronic study with this strain of mouse and the incidence was not affected by treatment with the test article.

BODY WEIGHT AND WEIGHT GAIN
Mean body weights in the males at 2500 ppm were slightly but consistently lower as from treatment week 11 the differences being 2 grams (= approximately 4 %) on average. The difference in mean body weight from the control value attained statistical significance on week 23, only. This finding was considered to be test article related. Body weights and body weight gain in the males at 0, 20, 100 and 500 ppm were similar. Although the females of the 2500 ppm group had statistically significantly reduced mean body weights on weeks 57, 61 and 63, the body weight development in the female mice was not considered to be affected by the treatment with the test article up to and including the highest dietary concentration of 2500 ppm.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food consumption in male and female animals was not considered to be affected by treatment with the test article. The sporadic statistically significant differences noted were considered to be incidental. Test article intake was strictly proportional to dietary concentration and slightly higher in females than in males.

HAEMATOLOGY
There were no changes of toxicological significance after 52, 81 and 87 weeks of treatment. All statistical differences in the results of the hematology data were considered to be incidental or unrelated to the treatment and of normal biological variation for mice of this strain and age. This conclusion was in coincidence with historical control data for this mouse strain.

CLINICAL CHEMISTRY
The assessment of clinical biochemistry data indicated no changes of toxicological significance after 52, 81 and 87 weeks of treatment nor were there any relevant treatment-related changes on immunoglobulins for females at 81 weeks and males at 87 weeks. All statistical differences in the results of the clinical biochemistry data were considered to be incidental or unrelated to the treatment and of normal biological variation for mice of this strain and age. This conclusion was in coincidence with historical control data for this mouse strain.

URINALYSIS
Urinalysis data indicated no treatment-related changes at 50, 80 and 85 weeks. All statistical differences in the results of the urinalysis data were considered to be incidental or unrelated to the treatment and of normal biological variation for mice of this strain and age. This conclusion was supported by historical control data for this mouse strain.

ORGAN WEIGHTS
In males of the 2500 ppm group, the liver weight of the animals terminated after 52 weeks as well as after 87 weeks was increased in comparison with the control group. However, the difference from the control value was statistically significant only for the liver/body weight ratio after 52 weeks. This finding was considered to be test article related and to correlate with the hepatocellular hyperthrophy diagnosed at microscopic examination. The other statistically significant differences (heart and kidneys) in organ weights and organ/brain weight ratio recorded in males at 2500 ppm after 52 weeks were not confirmed at terminal sacrifice after 87 weeks and were therefore considered to be incidental or to correlate with the slightly lower mean body weight noted in these males. No effects on the organ weights were noted in females at 2500 ppm or in the male and female animals at 20, 100 or 500 ppm.

GROSS PATHOLOGY
A number of macroscopic findings were diagnosed in mice of all groups at interim sacrifice (after 52 weeks ) as well as at termination of the study. These macroscopic findings did not distinguish treated mice from controls. No test article related effects were evident.

HISTOPATHOLOGY: NON-NEOPLASTIC
A treatment-related hepatocellular hypertrophy was diagnosed in a number of male mice of the 500 and 2500 ppm groups, a few male mice of the 100 ppm group following both the 52-week and 87-week treatment periods, and a number of female mice of the 2500 ppm group after the 81-week treatment period. No other non-neoplastic changes were diagnosed in mice after 52, 81 (females) and 87 weeks (males) of treatment, that were considered to represent a treatment-related effect of the test article. The type and incidence of all other non-neoplastic changes were considered to be incidental and are commonly diagnosed in mice of this strain and age.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
No neoplastic lesions were diagnosed in mice treated with the test article, that were considered to represent a carcinogenic effect of the test article.
Therefore, the test article did not show any oncogenic potential up to and including the highest dose level of 2500 ppm.

Dose descriptor:

NOAEL

Remarks:

neoplastic

Effect level:

>= 2 500 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: corresponding to an average daily test article intake of 350.79 mg/kg bw/day for males and 463.35 mg/kg bw/day for females

Remarks:

highest dose tested

Dose descriptor:

NOEL

Remarks:

systemic

Effect level:

20 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effect observed

Remarks on result:

other: corresponding to an average daily test article intake of 2.76 mg/kg bw/day for males and 3.88 mg/kg bw/day for females

Dose descriptor:

LOEL

Remarks:

systemic

Effect level:

100 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: corresponding to an average daily test article intake of 14.08 mg/kg bw/day for males and 18.31 mg/kg bw/day for females

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 an average daily test article intake of 70.58 mg/kg bw/day for males and 92.01 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:

body weight and weight gain

organ weights and organ / body weight ratios

Remarks on result:

other: corresponding to an average daily test article intake of 350.79 mg/kg bw/day for males and 463.35 mg/kg bw/day for females

Conclusion:

At 2500 ppm, the male animals had slightly but persistently reduced mean body weights (approximately 4%) from week 11 up to the end of the treatment period. An increase of the liver weight was evident in the males terminated after 52 weeks as well as at the end of the study. This finding was considered to be test article related and to correlate with the findings noted at microscopic examination. Centrilobular to midzonal hepatocellular hyperthrophy was diagnosed in few male mice at 100 ppm, a number of males at 500 and 2500 ppm after 52-week and 87-week treatment periods, and in a number of female animals after the 81-week treatment period. This finding was considered to be test article related but, generally, hepatocellular hyperthrophy is not considered to be a change reflective of a toxic effect of a test article, but a morphologic change consistent with an adaptive metabolic response. Based on these results, the "no observed effect level" (NOEL) was considered to be 20 ppm corresponding to an average daily test article intake of 2.76 mg/kg body weight for males and 3.88 mg/kg body weight for females. In consideration that hepatocellular hyperthrophy is not a toxic effect but an adaptive response to treatment, the dose level of 500 ppm - corresponding to an average daily test article intake of 70.58 mg/kg body weight for males and 92.01 mg/kg body weight for females - was defined to be the "no observed adverse effect level" (NOAEL).

HOE 107892 SUBSTANCE TECHNICAL did not reveal any carcinogenic effect in mice up to and including the highest dose level of 2500 ppm (corresponding to an average daily test article intake of 350.79 mg/kg body weight for males and 463.35 mg/kg body weight for females).

Reference 2

Endpoint:

carcinogenicity: 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 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 83-5 (Combined Chronic Toxicity / Carcinogenicity)

Deviations:

no

GLP compliance:

yes

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 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

Post exposure period:

none

Dose / conc.:

40 ppm (nominal)

Dose / conc.:

200 ppm (nominal)

Dose / conc.:

1 000 ppm (nominal)

Dose / conc.:

5 000 ppm (nominal)

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:

NOAEL

Remarks:

neoplastic

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

Remarks:

highest dose tested

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:

LOEL

Remarks:

systemic

Effect level:

1 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

Remarks on result:

other: corresponding to 48.47 and 59.98 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:

haematology

Remarks on result:

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

Remarks:

highest dose tested

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:

The clinical laboratory findings point at the red blood cell as target organ of the test article: anemia, more marked in the females, with an increase in hernopoietic activity as indicated by the increase in reticulocytes was noted for the animals of the highest dose group (5000 ppm). Reduction of the body weight gain was also noted in the female animals. The higher test article intake (ca. 26%) noted in the females in comparison with the concurrent males was considered to be the cause of the more pronounced effects in the females.

Another target organ may be the liver. Decrease of the total bilirubin concentration in the females at 1000 ppm and in the animals of both sexes at 5000 ppm was noted. These findings were considered to be the expression of physiological-functional changes in the absence of any other biochemical or histological correlate of liver toxicity.

No biological significance could be assigned to the changes in urinary enzyme excretion of the females at 1000 and 5000 ppm.

Histopathological examination and organ weights analysis gave no indications of test article related abnormal findings.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

251.6 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

The available information comprises adequate and reliable studies (Klimisch score 1), and is thus sufficient to fulfil the standard information requirements set out in Annex X, 8.5, of Regulation (EC) No. 1907/2006.

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The test article did not show any carcinogenic potential up to and including the highest dose level of 5000 ppm in the feed in rats and no carcinogenic effect up to and including the highest dose level of 2500 ppm in the feed in mice. Data from workers occupationally exposed to the test article confirms that there is no further evidence of a carcinogenic potential. Additionally, there was no mutagenicity observed either in vitro or in vivo. Therefore, the test item does not meet the criteria for classification as carcinogen according to Regulation (EC) No. 1272/2008. The data are conclusive but not sufficient for classification.

Additional information

There is data available from a combined chronic toxicity/carcinogenicity feeding study over 124 weeks in rats and a carcinogenicity feeding study over 87 weeks in mice. Both studies were performed according to OECD guidelines (453 and 451) and in compliance with GLP.

In rats, a number of neoplastic lesions were diagnosed 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. All other differences in the results of the hematology parameters apart from those thought to be treatment-related were conidered to be incidental or unrelated of the treatment and of normal biological variation for rats of this strain and age.

No neoplastic lesions were diagnosed in mice that were considered to represent a carcinogenic effect of the test article. Based on the results of the two conducted studies, the test item is considered to have no carcinogenic potential.