1-(3-chloropyridin-2-yl)-N-[4-cyano-2-methyl-6-(methylcarbamoyl)phenyl]-3-{[5-(trifluoromethyl)-2H-tetrazol-2-yl]methyl}-1H-pyrazole-5-carboxamide

Endpoint summary

Description of key information

Subacute repeated dose toxicity, dermal (OECD 410), rat: NOAEL >= 1000 mg/kg bw/day

Combined chronic toxicity and carcinogenicity study, oral (OECD 453), rat: NOAEL (12 months) = 4000 ppm (equivalent to 184 and 246 mg/kg bw/day in males and females, respectively),

NOAEL (24 months) = 4000 ppm (equivalent to 159 and 221 mg/kg bw/day in males and females, respectively)

Subchronic repeated dose toxicity, oral (OECD 408), rat: NOAEL >= 10000 ppm (equivalent to 608 and 723 mg/kg bw/day in males and females, respectively)

Subchronic repeated dose toxicity, oral (OECD 409), dog: NOAEL = 3200 ppm (equivalent to 126 and 138 mg/kg bw/day in males and females, respectively)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 Jan 2013 - 19 Oct 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Version / remarks:

adopted in 2009

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.33 (Combined Chronic Toxicity / Carcinogenicity Test)

Version / remarks:

adopted in 1987

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.4300 (Combined Chronic Toxicity / Carcinogenicity

Version / remarks:

adopted in 1998

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Wistar Rj:WI (IOPS HAN)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: R. Janvier, Le Genest St Isle, France
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 6 weeks
- Weight at study initiation: 206 - 209 g (mean group weight males), 158 - 159 g (mean group weight females)
- Housing: 5 animals of the same sex per cage in suspended, stainless steel and wire mesh cages
- Diet: A04CP1-10 from SAFE (Scientific Animal Food and Engineering, Augy, France,) ad libitum except at designated time periods
- Water: filtered and softened tap water from the municipal water supply, ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): 10 - 15
- 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): every 4 weeks
- Mixing appropriate amounts with (Type of food): A04CP1-10
- Storage temperature of food: ambient

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The first (F1), the seventh (F7) and the thirteenth formulation (F13) prepared for the study were evaluated for homogeneity using HPLC-UV analysis. Triplicate samples were collected of the lowest and the highest dose (900 and 18000 ppm) from the top, the middle and the bottom of the formulations. The mean values obtained from the homogeneity checks were used as measured concentrations. In addition, for concentration analysis, two samples of formulations F1, F4, F7, F10, F13 and F16 were collected. One sample was analyzed and the other was stored frozen for possible verification purpose. Data indicate homogenous mixture of the test material in the diet. The measured values for the individual homogeneity samples ranged between 91 and 107% of nominal. Additionally, samples were at the targeted concentration (93 – 111% of nominal). One value at 111% was slightly out of in-house target ranges but considered as having no impact on the study results reliabilities. The stability of the test item has been demonstrated in a previous study at concentrations of 120 and 12000 ppm for up to 104 days when stored frozen and then kept for 10 days at ambient temperature (Study completion: 2011; Study number: SA 10372; Doc No: M-568723-03-1). Stability of the high dose 18000 ppm was verified during the current study over a frozen storage period of 167 days followed by 105 days of storage at room temperature. With measured concentrations of 99 and 100% of nominal following storage procedure the test substance was found to be stable.

Duration of treatment / exposure:

52 weeks (interim)
104 weeks (terminal sacrifice)

Frequency of treatment:

continously (via diet)

Dose / conc.:

900 ppm

Remarks:

equivalent to approximately 41.0 and 56.7 mg/kg bw/day in males and females over the 12-month period, respectively and equivalent to approximately 35.3 and 51.2 mg/kg bw/day in males and females over the 24-month period, respectively

Dose / conc.:

4 000 ppm

Remarks:

equivalent to approximately 184 and 246 mg/kg bw/day in males and females over the 12-month period, respectively and equivalent to approximately 159 and 221 mg/kg bw/day in males and females over the 24-month period, respectively

Dose / conc.:

18 000 ppm

Remarks:

equivalent to approximately 854 and 1147 mg/kg bw/day in males and females over the 12-month period, respectively and equivalent to approximately 741 and 1052 mg/kg bw/day in males and females over the 24-month period, respectively

No. of animals per sex per dose:

10 (12 month interim sacrifice)
60 (24 month sacrifice)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results from a previous 90-day dietary study in the rat (2012, Doc number M-443358-02-1), where no toxicity was seen at the highest dose tested of 10000 ppm equating to 608 mg/kg bw/day in males and 723 mg/kg bw/day in females. Consequently, the high dose level chosen on the current study was 18000 ppm, which over the course of the study was expected to equate to a limit dose in females and close to a limit dose in males, but was a dose that was expected to be tolerated. The low dose of 900 ppm was expected to be a dose level where no toxic findings were observed. The mid dose of 4000 ppm, provides a 4.5 fold factor between the high and low dose.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily (including weekends and public holidays) except on a limited number of occasions
- Cage side observations included: mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least daily (clinical signs), at least weekly (detailed physical examination including palpation for masses), ill health

BODY WEIGHT: Yes
- Time schedule for examinations: at least weekly during the acclimatization period, weekly for the first 13 weeks of study, approximately every 4 weeks thereafter and prior to necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE:
The weight of food supplied and of that remaining at the end of the food consumption period was recorded for each animal. Food consumption was recorded twice weekly during the first 6 weeks of treatment, then weekly up to Week 13, and once approximately every 4 weeks thereafter.The weekly mean achieved dosage intake in mg/kg bw/day for Weeks 1 to 13, then 1 week per month thereafter was calculated.

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during acclimatization phase, after approximately 12 months and 24 months
- Dose groups that were examined:all animals (acclimatization phase), control and high dose groups (12 months), all surviving animals (24 months)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Weeks 15, 24, 25, 51, 78 or 79 and 105; puncture of the retro orbital venous plexus
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all surviving animals (12 month interim sacrifice), first ten surviving animals (24 months)
- Parameters examined: hematocrit, hemoglobin concentration, white blood cell count, red blood cell count, platelet count, prothrombin time, white blood cell differential evaluation count, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean corpuscular volume, reticulocyte count, blood smears

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Weeks 15, 24, 25, 51, 78 or 79 and 105; puncture of the retro orbital venous plexus
- Animals fasted: Yes
- How many animals: all surviving animals (12 month interim sacrifice), first ten surviving animals (24 months)
- Parameters examined: calcium, chloride, inorganic phosphorous, potassium, sodium, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma glutamyltransferase, albumin, creatinine, urea, total cholesterol, glucose (fasting), total bilirubin, total protein, triglycerides

URINALYSIS: Yes
- Time schedule for collection of urine: Weeks 13, 25 or 26, 52, 77 and 104
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
- Parameters examined: appearance, volume, specific gravity / osmolality / refractive index, pH, sediment (microscopic), protein, glucose, ketones, bilirubin, blood / red blood cells, urobilinogen

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
Bioanalytical examination: After approximately 3 months of treatment, as well as at the end of 12 months, a blood sample was collected from the sublingual vein of five suitable animals of each sex and all dose groups following anaesthesia with isoflurane. Blood was also collected from 2 control males and 2 control females to determine analytical baseline. Based on the results of a blood kinetic analysis study (2012, Doc number M-427766-02-1), blood was collected at approximately 8.00 am on the day of sampling. Plasma was prepared and determination of the test substance level and main metabolite using HPLC Tandem Mass Spectrometry (HPLC-MS/MS) analysis was conducted.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 1)
On study Days 374 to 376 for the 12-month interim kill all surviving animals dedicated to interim sacrifice/chronic group were sacrificed by exsanguination under deep anesthesia (inhalation of isoflurane). An approximately equal number of animals randomly distributed amongst all groups were sampled on each day at the interim sacrifice. Animals were diet fasted overnight prior to sacrifice. All animals, including animals either found dead or killed for humane reasons, were necropsied. The necropsy included the examination of external surfaces, all orifices, all major organs, tissues and body cavities. The organs or tissues checked in table 1 were sampled and/or weighed. All significant macroscopic abnormalities (including masses and their regional lymph nodes when possible) were recorded, sampled and examined microscopically. Two femoral bone marrow smears were prepared from sacrificed animals, one of which was stained with May-Grünwald Giemsa, but not examined as no relevant changes were observed in hematology or bone marrow histology. The second smear was stored unstained. Samples were fixed by immersion in 10% neutral buffered formalin with the exception of the eye and optic nerve, Harderian gland, epididymis and testis that were fixed in Davidson's fixative.

HISTOPATHOLOGY: Yes (see table 1)
The samples listed in table 1 (except exorbital lachrymal gland, larynx/pharynx and nasal cavities) were embedded in paraffin wax. Histological sections, stained
with hematoxylin and eosin, were prepared from all organs and tissue samples.
Histopathology examination of the chronic phase was performed as follows:
- all organs and tissue samples from animals sacrificed or found dead during the treatment period,
- all organs and tissue samples from animals of control and high dose groups,
- liver, lung, kidney and thyroid gland from animals of the intermediate dose groups,
- gross abnormalities from all animals.
For all unscheduled sacrificed or dead animals on study, the cause of death was determined when it was possible.
Histopathology examination of the carcinogemicity phase was performed on all organs and tissues embedded including gross abnormalities in all animals from all groups including decedents. For all unscheduled sacrificed or dead animals on study, the cause of death was determined when it was possible.

Statistics:

In general Bartlett test was performed. If the Bartlett test was not significant (p>0.05), means were compared using the analysis of variance (ANOVA). If the ANOVA was significant (p≤0.05), means of the exposed groups were compared to the mean of the control group using the Dunnett test (2-sided). If the Bartlett test was significant (p≤0.05), group means were compared using the Kruskal-Wallis test. If the Kruskal-Wallis test was significant (p≤0.05), means of the exposed groups were compared to the mean of the control group using the Dunn test (2-sided). If the Bartlett test was significant for body weight and average food consumption/day parameters and selected haematology parameters (red blood cell count, platelet count, white blood cell count, neutrophil count, lymphocyte count, reticulocyte count) data were transformed using the log transformation for body weight and food consumption and the square root transformation for haematology parameters before the same methods were applied as mentioned above. If the Bartlett test was significant (p≤0.05) even after log transformation, statistical analysis was proceeded as mentioned above. For survival analysis Kaplan-Meier estimation procedures were used. Dose relative trends in survival were assessed using log- rank trend test (2-sided). Pairwise comparison was assessed using Tarone tests with Scheffe adjustment for multiple comparisons (1-sided). Histopathological findings were assessed with poly-k (k=3) tests which take into account time to death and the presence of the findings. Firstly, a trend poly-k test (2-sided) was performed regarding all doses. Then, pairwise comparisons were assessed using poly-k test (1-sided) with Sidak adjustment where applicable. The Cochran-Armitage trend test (2-sided) followed by the Fisher’s exact test (1-sided) with Sidak adjustment was applied for selected clinical signs, ophthalmic and macroscopic observations.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

FEMALES
18000 ppm: increased incidence of prolapsed vagina during the second year (4/58 animals).
It is specified in the study report that these effects may be treatment-related.

(see table 2)

Mortality:

mortality observed, non-treatment-related

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

MALES
18000 ppm: decreased body weights compared to controls (3% during the first year, 7% during the second year), decreased mean cumulative body weight gains compared to controls (3 - 8% during the first year, 4 - 10% during the second year), decreased overall mean cumulative body weight gain at the end of the study compared to controls (10%)

FEMALES
18000 ppm: decreased mean body weight compared to controls (5% (not statistically significant) at the end of the first year, 15% at the end of the second year), decreased mean cumulative body weight gains compared to controls (8% during the first year, 8 - 24% during the second year), decreased overall mean cumulative body weight gain at the end of the study compared to controls (24%)

(see tables 5 and 6)

Food consumption and compound intake (if feeding study):

effects observed, non-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:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Details on results:

For details on results including tables see the attached document.

Key result

Dose descriptor:

NOAEL

Remarks:

12 months

Effect level:

4 000 ppm

Based on:

test mat.

Remarks:

equivalent to 184 and 246 mg/kg bw/day in males and females, respectively

Sex:

male/female

Basis for effect level:

body weight and weight gain

Key result

Dose descriptor:

NOAEL

Remarks:

24 months

Effect level:

4 000 ppm

Based on:

test mat.

Remarks:

equivalent to 159 and 221 mg/kg bw/day in males and females, respectively

Sex:

male/female

Basis for effect level:

body weight and weight gain

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

18 000 ppm

System:

female reproductive system

Organ:

ovary

uterus

vagina

Treatment related:

yes

Dose response relationship:

no

Relevant for humans:

not specified

For details on results including tables see the attached document.

Conclusions:

The No Observed Adverse Effect Level (NOAEL) over a 12- and 24-month period of dietary administration with the test substance to the Wistar rat was 4000 ppm in both sexes (equivalent to 184 and 246 mg/kg bw/day in males and females during the 12-month period, respectively and equivalent to 159 and 221 mg/kg bw/day in males and females during the 24-month period, respectively) .

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 Apr 2011 - 14 Jul 2012

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)

Version / remarks:

adopted in 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted in 2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Version / remarks:

adopted in 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: M.A.F.F. in Japan, notification 12 Nousan N°8147 guideline

Version / remarks:

adopted in 2000

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Wistar Rj:WI (IOPS HAN)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: R. Janvier, Le Genest St Isle, France
- Age at study initiation: 6 - 7 weeks
- Weight at study initiation: 251 - 253 g (males), 192 - 193 g (females)
- Housing: 5 animals of the same sex per cage in suspended, stainless steel and wire mesh cages
- Diet: A04CP1-10 (Scientific Animal Food and Engineering, Augy, France), ad libitum except at designated time periods
- Water: filtered and softened tap water from the municipal water supply, ad libitum except before urine collection when animals were water fasted overnight
- Acclimation period: 13 days

DETAILS OF FOOD AND WATER QUALITY: Routine analyses of feed and water indicated that there was no contamination which could have compromised the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): 10 - 15
- 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): single preparation
- Mixing appropriate amounts with (Type of food): A04CP1-10
- Storage temperature of food: room temperature

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dietary formulations prepared for the study were evaluated for homogeneity and concentration using HPLC-UV analysis. Duplicate samples were collected for homogeneity and concentration verification of the lowest and the highest dietary concentrations from the surface, the middle and the bottom of the formulation. In addition samples of the intermediate dose level were collected from the surface, middle and the bottom of the formulation for concentration analysis. Data indicate homogenous mixture of the test material in the diet preparation. The measured values for the individual homogeneity samples ranged between 95 and 107% of nominal. Additionally, samples were at the targeted concentration (92 – 104% of nominal). The stability of the diet preparations has been demonstrated in a preliminary study. In this study, dietary samples at 120 and 12000 ppm were taken at the beginning of the study and were kept either at room temperature or frozen for varying time periods. The test substance was found to be stable in the diet over a 104-day period at room temperature or a 94-day freezing period followed by 10 days at room temperature.

Duration of treatment / exposure:

at least 90 days (control and test groups)
at least 90 days and 4 weeks post-exposure observation period (satellite control and satellite test groups)

Frequency of treatment:

continously (via diet)

Dose / conc.:

900 ppm

Remarks:

equivalent to approximately 55 and 66 mg/kg bw/day in males and females, respectively

Dose / conc.:

3 000 ppm

Remarks:

equivalent to approximately 178 and 213 mg/kg bw/day in males and females, respectively

Dose / conc.:

10 000 ppm

Remarks:

equivalent to approximately 608 and 723 mg/kg bw/day in males and females, respectively

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results from a previous 28-day study in the rat with dietary administration of up to 8000 ppm (equivalent to 599 mg/kg/day in males and 700 mg/kg/day in females). No mortality, no clinical signs, no significant effect on body weight and food consumption, no haematological or biochemical parameter changes, no liver enzyme changes, no findings in organ weights, or at macroscopic and microscopic examinations were observed. Thus, the NOEL in the rat 28-day study was 8000 ppm.
- Post-exposure recovery period in satellite groups: 4 weeks

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (once daily on weekends or public holidays)
- Cage side observations included: moribundity and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily (clinical signs), at least weekly (detailed physical examinations)

BODY WEIGHT: Yes
- Time schedule for examinations: once during acclimatisation period, on the first day of test substance administration, then at least weekly throughout the treatment and recovery periods

FOOD CONSUMPTION AND COMPOUND INTAKE:
The weight of food supplied and of that remaining at the end of the food consumption period was recorded twice weekly during the first 6 weeks of treatment, then weekly for all animals during the treatment and recovery period. From these records the mean daily consumption was calculated. The weekly mean achieved dosage intake in mg/kg/day for each week and for weeks 1 to 13 was calculated for each sex.

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE : No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during acclimatisation period, during week 13 of the treatment period
- Dose groups that were examined: all animals (acclimatisation period ), all surviving animals of control and high dose group (treatment period)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: study days 93, 94 or 95 (main study phase), recovery day 30 or 31 (recovery animals), puncture of the retro-orbital venous plexus
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all surviving animals
- Parameters examined: hematocrit, hemoglobin, leukocyte count, erythrocyte count, platelet count, prothrombin time, leukocyte differential count, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean corpuscular volume, reticulocyte count, blood smear

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: study days 93, 94 or 95 (main study phase), recovery day 30 or 31 (recovery animals), puncture of the retro-orbital venous plexus
- Animals fasted: Yes
- How many animals: all surviving animals
- Parameters examined: general appearance of the serum, calcium, chloride, inorganic phosphorus, potassium, sodium, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma glutamyltransferase, globulin, albumin, creatinine, urea, total cholesterol, glucose, total bilirubin, total protein, triglycerides, albumin/globulin ratio (calculated)

URINALYSIS: Yes
- Time schedule for collection of urine: study days 87 or 88 (animals of the main study), recovery day 28 (animals of the recovery phase)
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes
- Parameters examined: general appearance of the urine, volume, specific gravity/ osmolality/ refractive index, pH, sediment (microscopic), protein, glucose, ketones, bilirubin, blood/ red blood cells, urobilinogen

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during study week 11 and 12
- Dose groups that were examined: all surviving animals
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 1)
On study days 93, 94 or 95 (animals of the treatment phase) or on days 30 and 31 post treatment (animals of the recovery phase), all surviving animals from all groups were sacrificed by exsanguination under deep anesthesia (isoflurane) and necropsied. The necropsy included the examination of all major organs, tissues and body cavities. The organs or tissues checked in table 1 were sampled and/or weighed.

HISTOPATHOLOGY: Yes
1. Treatment phase: The samples listed in table 1 (except exorbital lachrymal gland, larynx/pharynx and nasal cavities) were embedded in paraffin wax. Histological sections, stained with hematoxylin and eosin, were prepared from all organs and tissue samples from all the animals in the control and high dose groups. Additionally, sections from liver, kidney, lung, thyroid gland and from significant gross findings observed at necropsy were prepared for all the animals in all intermediate dose groups. An additional slide (of one animal) treated with special stain (Periodic acid Schiff) was prepared for the thyroid. All the tissues processed were examined.
2. Recovery phase: Only liver, kidney, lung, thyroid gland, macroscopic observations and all organs collected from one animal found dead were processed and embedded in paraffin wax for all the animals. The histological slides were prepared and stained with hematoxylin and eosin. No tissues were examined.

Other examinations:

Test substance plasma analysis
During week 12 of the main study, a blood sample was collected from the sublingual vein of 5/10 animals of each group (not fasted). Based on the results of a blood kinetic study, the relevant time point for blood collection was determined to be 4:00 p.m. A blood sample was also collected from 2/10 male animals of the control group for validation of the analytical method. Prior to blood sampling, animals were anesthetized with isoflurane. Plasma was prepared from blood for possible determination of the test item and its major metabolites using HPLC-UV analysis.

Statistics:

Mean and standard deviation were calculated for each group.
1. Main study: The treated groups were compared with the control group using the Bartlett test. If the Bartlett test was not significant (p>0.05), means were compared using the analysis of variance (ANOVA). If the ANOVA was not significant (p>0.05), no further analysis was performed. If the ANOVA was significant (p≤0.05), means were compared using the Dunnett test (2-sided). If the Bartlett test was significant (p≤0.05), group means were compared using the non- parametric Kruskal-Wallis test. If the Kruskal-Wallis test was not significant (p>0.05), no further analysis was performed. If the Kruskal-Wallis test was significant (p≤0.05), means were compared using the Dunn test (2-sided). If the Bartlett test was significant for body weight parameters and selected hematology parameters data were transformed using the log transformation (body weight) or the square root transformation (hematology). The Bartlett test was again used for the transformed data and statistical analysis was proceeded as mentioned above. For pH as urinalysis parameter the Kruskal-Wallis test was used followed by a Dunn test (2-sided) in case of significance (p≤0.05).
2. Recovery: The high dose group was compared with the control group using the F test. If the F test was not significant (p>0.05), means were compared using the T-test (2-sided). If the F test was significant (p≤0.05), means were compared using the modified t- test (2-sided). If the F test was significant (p≤0.05) for body weight parameters and selected hematology parameters data were transformed using the log transformation (body weight) or the square root transformation (hematology). The F test was again used for the transformed data and statistical analysis was proceeded as mentioned above. For pH as urinalysis parameter group means were compared using the non-parametric Mann-Whitney test (2-sided).

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Body weight and weight changes:

effects observed, non-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:

effects observed, non-treatment-related

Haematological findings:

effects observed, non-treatment-related

Clinical biochemistry findings:

effects observed, non-treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

For details on results including tables see the attached document.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 10 000 ppm

Based on:

test mat.

Remarks:

equivalent to 608 and 723 mg/kg bw/day in males and females, respectively

Sex:

male/female

Basis for effect level:

other: highest dose tested

Key result

Critical effects observed:

no

For details on results including tables see the attached document.

 

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 Jul 2013 - 28 Jan 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.27 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)

Version / remarks:

adopted in 2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3150 (90-Day Oral Toxicity in Non-rodents)

Version / remarks:

adopted in 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: M.A.F.F. in Japan, notification 12 Nousan N°8147 guideline

Version / remarks:

adopted in 2000

GLP compliance:

yes (incl. QA statement)

Remarks:

Groupe Interministeriel Des Produits Chimiques, Ivry-sur-Seine, France

Limit test:

no

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Marshall BioResources, North Rose, New-York, USA
- Age at study initiation: 8 - 9 months
- Weight at study initiation: 6.9 - 9.0 kg (males), 5.4 - 7.2 kg (females)
- Housing: individual in stainless steel kennels with a floor surface area of 2 m², pair housed overnight (when possible, same sex and dose group), weekly supervised exercise (separated by sex and dose group)
- Diet: certified canine meal 125C3-P1 (Scientific Animal Food and Engineering, Augy, France), 330 g moistened with 470 g water, daily for approximately 1.5 h
- Water: filtered and softened tap water from the municipal water supply, ad libitum
- Acclimation period: 27 days

DETAILS OF FOOD AND WATER QUALITY: Routine analyses of feed and water indicated that there was no contamination which could have compromised the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 21
- Humidity (%): 40 - 70
- Air changes (per hr): 15
- 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): two preparations per concentration during the study
- Mixing appropriate amounts with (Type of food): canine meal 125C3-P1
- Storage temperature of food: room temperature

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dietary formulations prepared for the study were evaluated for homogeneity and concentration using HPLC-UV analysis. Duplicate samples were collected for homogeneity and concentration verification of the lowest and the highest dietary concentrations from the surface, the middle and the bottom of the formulation. In addition single samples of the intermediate dose level were collected from the surface, middle and the bottom of the formulation for concentration analysis. Data indicate homogenous mixture of the test material in the diet preparation. The measured values for the individual homogeneity samples ranged between 93 and 98% of nominal. Additionally, samples were at the targeted concentration (92 – 104% of nominal).
The stability of test item in the diet at concentrations of 200 and 25000 ppm has been demonstrated in a previous study over a 74-day period under storage and usage conditions similar to those of the current study. In addition, the stability of the test item at concentrations of 200 and 25000 ppm in the moistened diet distributed to the dog has been determined for a period which corresponded approximately to the time of food preparation and distribution.

Duration of treatment / exposure:

at least 90 days

Frequency of treatment:

once daily (for about 1.5 h), 7 days/week

Dose / conc.:

800 ppm

Remarks:

equivalent to 25.6 and 29.9 mg/kg bw/day in males and females, respectively

Dose / conc.:

3 200 ppm

Remarks:

equivalent to 126 and 138 mg/kg bw/day in males and females, respectively

Dose / conc.:

12 800 ppm

Remarks:

equivalent to 440 and 485 mg/kg bw/day in males and females, respectively

No. of animals per sex per dose:

4

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dose levels were selected from results obtained in a subacute toxicity study previously performed in dogs (2012, Doc Number M-440096-01-1), where the test substance was administered for 28 days in the diet at 0, 2000, 6000 and 17000 ppm. At 17000 ppm, clinical chemistry determinations revealed increased total cholesterol concentrations in 2/2 animals from both sexes, when compared to their own pre-test results. At necropsy, slight red foci were observed in the stomach in 1/2 females. This change was correlated with slight glandular erosion/necrosis in the stomach observed at the microscopic examination. At 6000 ppm, the same clinical chemistry and histopathology findings were observed. At 2000 ppm, no treatment-related change was noted in either sex. Thus in the current study a high dose level of 12800 ppm was expected to result in clear signs of toxicity, but not exceed a Maximal Tolerated Dose. A mid dose of 3200 ppm provides a four-fold factor between the high and low dose levels and a low dose of 800 ppm was expected to be a No Observed Adverse Effect Level.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (once daily on weekends and public holidays)
- Cage side observations included: mortality, moribundity, ill-health, behavioural changes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily (clinical signs), weekly (detailed examination in an open area), daily (examination of the kennels for vomitus, diarrhea or blood)

BODY WEIGHT: Yes
- Time schedule for examinations: at least weekly prior to feeding and before final necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
The weight of food supplied to each animal and that remaining were recorded daily throughout the treatment period. From these records, the mean weekly consumption was calculated for each dog. The group mean achieved dosage for each sex, expressed as mg/kg body weight/day, was calculated for each week and the overall mean subsequently derived for weeks 1 to 13.

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during acclimatization phase and at the end of the treatment
- Dose groups that were examined: all surviving animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Once before dosing and on Days 43 or 44 and 87 or 88, blood samples were taken from all animals in all groups by puncture of the jugular vein.
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined: hematocrit, hemoglobin concentration, white blood cell count, red blood cell count, platelet count, activated partial thromboplastin time, prothrombin time, leukocyte differential count, mean corpuscular hemoglobin, mean corpuscular volume, reticulocyte count and % reticulocytes, mean corpuscular hemoglobin concentration, blood smear

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Once before dosing and on Days 43 or 44 and 87 or 88, blood samples were taken from all animals in all groups by puncture of the jugular vein.
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined: general appearance of the serum, calcium, chloride, inorganic phosphorus, potassium, sodium, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase, albumin, creatinine, urea, total cholesterol, glucose, total bilirubin, total protein, triglycerides, globulin concentrations and albumin/globulin ratio (calculated)

URINALYSIS: Yes
- Time schedule for collection of urine: once before dosing and on days 45 or 46 and 85, 86 or 87
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes
- Parameters examined: appearance, volume, specific gravity/ osmolality/ refractive index, pH, microscopic examination of sediment, protein, glucose, ketones, bilirubin, blood/ red blood cells, urobilinogen

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 1)
On Days 92 to 95, all animals from all groups were tranquilized (intramuscular injection of a combination of zolazepam with the dissociative anesthetic tiletamine) followed by intravenous injection of pentobarbital. Animals were then exsanguinated and necropsied. All animals were diet fasted prior to sacrifice.The necropsy included the examination of all major organs, tissues and body cavities. Macroscopic abnormalities were recorded, sampled and examined microscopically. Organs and tissues checked in table 1 were sampled and/or weighed at necropsy. Samples were fixed by immersion in 10% neutral buffered formalin with the exception of the eye, optic nerve, epididymis and testis that were fixed in Davidson’s fixative.

HISTOPATHOLOGY: Yes
All samples listed in table 1 (except larynx/pharynx which was not processed), were embedded in paraffin wax. Histological sections, stained with hematoxylin and eosin (HE), were prepared from all the animals in all groups. Histopathological examinations were performed in all animals from all groups.

Other examinations:

Bioanalytical examination
At the end of the study, a blood sample was collected from the jugular or any suitable vein of two surviving dogs per group and per sex approximately 4 hours after food distribution (i.e. time of peak plasma level determined in a previous study, (2012, Doc number M-440096-01-1)). Plasma was prepared from blood for determination of the test item and its potential major metabolites using HPLC/Tandem Mass Spectrometry. Analysis in plasma samples was performed as recommended in Commission Regulation (EU) N° 283/2013 of 1 March 2013 setting out the data requirements for active substances, in accordance with Regulation (EC) N° 1107/2009.

Statistics:

Mean and standard deviation (SD) were calculated for each group. Group means were compared at least at the 5% level of significance. For body weight change parameters mean and SD were calculated for each group and per time period. In general Bartlett test was performed to compare the homogeneity of group variances. If the Bartlett test was not significant (p>0.05), means were compared using the analysis of variance (ANOVA). If the ANOVA was not significant (p>0.05), the group means were considered to be homogeneous and no further analysis was performed. If the ANOVA was significant (p≤0.05), means of the exposed groups were compared to the mean of the control group using the Dunnett test (2-sided). If the Bartlett test was significant (p≤0.05), group means were compared using the non- parametric Kruskal-Wallis test. If the Kruskal-Wallis test was not significant (p>0.05), the group means were considered to be homogeneous and no further analysis was performed. If the Kruskal-Wallis test was significant (p≤0.05), means of the exposed groups were compared to the mean of the control group using the Dunn test (2-sided). If the Bartlett test was significant for body weight and average food consumption/day parameters, selected haematology parameters (red blood cell count, platelet count, white blood cell count, neutrophil count, lymphocyte count, reticulocyte count) data were transformed using the log transformation for body weight and food consumption and the square root transformation for haematology parameters before the same methods were applied as mentioned above. If the Bartlett test was significant (p≤0.05) even after log transformation, statistical analysis was proceeded as mentioned above. For the pH as quantitative urinalysis parameter the Kruskal-Wallis test was applied mentioned above.
If one or more group variance(s) equaled 0, means were compared using non-parametric procedures.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

MALES
12800 ppm: increased salivation in 2/4 animals on 4 and 6 occasions, respectively
3200 ppm: increased salivation in 3/4 animals on one ore more occasions

FEMALES
3200 ppm: increased salivation in 3/4 animals on one ore more occasions
(see table 2)

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

FEMALES
12800 ppm: decreased mean body weight gain between days 1 - 8, 1 - 29, 1 - 57 and decreased overall mean body weight gain (days 1 - 92) compared to controls

(see table 3)

Food consumption and compound intake (if feeding study):

effects observed, non-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, non-treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

With the exception of the increased alkaline phosphatase activity at 12800 ppm in both sexes all further differences in clinical biochemistry were considered to be incidental, not test substance-related since the parameters are within the range of expected variations for Beagle dogs of this age kept under laboratory conditions.

(see table 7)

Urinalysis findings:

effects observed, non-treatment-related

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

For details on results including tables see the attached document.

Key result

Dose descriptor:

NOAEL

Effect level:

3 200 ppm

Based on:

test mat.

Remarks:

equivalent to 126 and 138 mg/kg bw/day in males and females, respectively

Sex:

male

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

Key result

Critical effects observed:

no

For details on results including tables see the attached document.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

159 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 VIII-IX, 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

Study period:

03 Feb - 04 Mar 2015

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)

Version / remarks:

adopted in 1981

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3200 (Repeated Dose Dermal Toxicity -21/28 Days)

Version / remarks:

adopted in 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

National Institute for Quality- and Organizational Development in Healthcare and Medicines, National Institute of Pharmacy, Budapest, Hungary

Limit test:

no

Species:

rat

Strain:

other: Crl:(WI) Wistar

Details on species / strain selection:

The Wistar rat is a standard species and strain used on dermal toxicity studies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approximately 8 weeks
- Weight at study initiation: 291 - 336 g (males), 193 - 249 g (females)
- Housing: up to 5 animals of the same sex per cage during acclimatisation period and individual during treatment in type II polycarbonate cages, deep wood sawdust bedding (Grade 5, Johannes Brandenburg GmbH und Co. KG, Goldenstedt, Germany)
- Diet: ssniff® SM R/M-Z+H "Autoclavable complete diet for rats and mice – breeding and maintenance" (ssniff Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water from municipal supply, ad libitum
- Acclimation period: 12 days (males), 13 days (females)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

semiocclusive

Vehicle:

water

Details on exposure:

TEST SITE
- Area of exposure: scapulae (shoulders) to the wing of the ileum (hipbone) and half way down the flank on each side of the animal
- % coverage: not less than 10%
- Type of wrap if used: porous gauze dressing (6 - 8 plies), additional non-irritating plastic wrap and restrainers (Lomir jackets with inserts)
- Time intervals for shavings or clippings: 20 and 18 h before the test for males and females, respectively

REMOVAL OF TEST SUBSTANCE
- Washing: lukewarm water
- Time after start of exposure: 6 h

TEST MATERIAL
- Amount applied: not specified
- Constant volume or concentration used: not specified
- For solids, paste formed: yes

VEHICLE
- Lot/batch no.: 2171213 (TEVA Co.)

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily, 6 h/day

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

300 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected by the Sponsor in consultation with the Study Director based on available data and information from previous experimental work, including the results of a 14-day repeated-dose dermal range-finding study in the rat, where no clear signs of toxicity were observed at any dose level tested, including the highest dose level of 1000 mg/kg bw/day. In addition, in an acute dermal toxicity study in the rat, the LD50 value was > 2000 mg/kg bw for both sexes, with no treatment-related findings (2013, Doc number M-513347-01-1). In the 90-day dietary study in the rat, no treatment-related findings were observed up to the highest dose level tested of 1000 ppm (equates approximately to 608 mg/kg bw/day in males and 723 mg/kg bw/day in females; 2012). Therefore, the dose levels selected on the current study were 100, 300 and 1000 mg/kg bw/day, where the limit dose of 1000 mg/kg bw/day was expected to be tolerated without causing death or overt suffering, the low dose was expected to be a clear NOAEL and the mid dose provides an approximate 3-fold factor between the dose levels.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations included: morbidity, mortality, ill health

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily following patch removal (general clinical observations), once before the first exposure and than at least weekly (detailed examinations)

DERMAL IRRITATION: Yes
- Time schedule for examinations: at least daily following the patch removal

BODY WEIGHT: Yes
- Time schedule for examinations: the day before the first exposure and then at least weekly

FOOD CONSUMPTION:
Food consumption was determined by re-weighing the non-consumed diet at weekly intervals. The weekly food consumption was calculated.

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: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once pre-treatment and on day 27
- Dose groups that were examined: all dose groups (pre-treatment), control and high dose (day 27)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to necropsy on day 28
- Anaesthetic used for blood collection: Yes (pentobarbital)
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined: red blood cell (erythrocyte) count, haemoglobin concentration, haematocrit, mean corpuscular (erythrocyte) volume, mean corpuscular (erythrocyte) haemoglobin, mean corpuscular (erythrocyte) haemoglobin concentration, red cell (erythrocyte) volume, platelet (thrombocyte) count, mean platelet (thrombocyte) volume, reticulocyte count, white blood (leukocyte) count, neutrophils, lymphocyte, monocyte, basophil, eosinophil, large unstained cells, blood smears, activated partial thromboplastin time, prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to necropsy on day 28
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined: glucose, total bilirubin, urea, cholesterol, triglycerides, creatinine, phosphorus, sodium, potassium, calcium, chloride, total protein, albumin, albumin/globulin ratio, aspartate aminotransferase activity, alanine aminotransferase activity, alkaline phosphatase activity, gamma glutamyltransferase activity, bile acids

URINALYSIS: Yes
- Time schedule for collection of urine: prior to necropsy on day 28
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes
- Parameters examined: leukocyte, nitrite, pH, protein, glucose, urobilinogen, bilirubin, ketones, blood/erythrocytes, specific gravity, sediment, volume

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: day 23 (last week of treatment)
- Dose groups that were examined: all dose groups
- Battery of functions tested: sensory activity, grip strength, motor activity

OTHER
Prior to necropsy, the oestrous cycle of all females was determined by taking vaginal smears, which were prepared and stained with 1% aqueous methylene blue solution. The smears were examined with a light microscope, in order to provide information regarding the stage of oestrous cycle at the time of sacrifice and assist in histological evaluation of oestrogen sensitive tissues.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Macroscopic examination of the tissues and organs and collection for histopathological examination where abnormalities were recorded. The following organs were collected and weighed: adrenals, brain, epididymides, heart, kidneys, liver, ovaries, pituitary, prostate including seminal vesicles with coagulating glands, spleen, testes, thymus, thyroids with parathyroids, uterus including cervix

HISTOPATHOLOGY: Yes
The following organs and tissues were collected: aorta, adrenals, brain, epididymides, external lachrymal glands, eyes with the optic nerves, femur with marrow, harderian glands, heart, kidneys, large intestine, larynx, liver, lungs with bronchi, lymph nodes, mammary gland (inguinal), nose/nasal cavity, oesophagus, ovaries with oviduct, pancreas, pharynx, pituitary, prostate, salivary glands, sciatic nerve, seminal vesicles with coagulating glands, skeletal muscle (quadriceps), skin and subcutis (inguinal), small intestine, spinal cord, spleen, sternum with marrow, stomach, testes, thymus, thyroid with parathyroids, tongue, trachea, treated skin area, urinary bladder, uterus, vagina

The eyes with the optic nerves and testes with epididymides were retained in modified Davidson’s fixative, all other organs in 10% buffered formalin solution.
Bone marrow smears were stained with Giemsa but not examined as there were no haematology findings. The retained tissues and organs were embedded in paraffin wax, sections were cut at 4-6 μm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope. Full histopathology was performed on the selected list of organs in control and four high dose animals, and in all macroscopic abnormalities in all groups.

Statistics:

Mean and standard deviations values were calculated. The statistical analysis was performed using SPSS PC+4.0 software. The heterogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan’s Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. If the data was not normal distributed, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there was a positive result, the inter-group comparisons were performed using Mann-Whitney U-Test.

Clinical signs:

no effects observed

Dermal irritation:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-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:

no effects observed

Clinical biochemistry findings:

effects observed, non-treatment-related

Urinalysis findings:

effects observed, non-treatment-related

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Gross pathological findings:

effects observed, non-treatment-related

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

For details on results including tables see the attached document.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no treatment-related effects at highest dose tested

Key result

Critical effects observed:

no

For details on results including tables see the attached document.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

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 VIII-IX, 8.6, of Regulation (EC) No 1907/2006.

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Reliable short-term, sub-chronic and chronic studies regarding repeated dose toxicity are available for the test substance.

Oral:

52-week/2 -year chronic toxicity feeding study in the rat

The chronic toxicity of the test substance was assessed in a study performed according to OECD Guideline 453 and in compliance with GLP (M-568723-03-1). Groups of 70 male and 70 female Wistar Rj:WI (IOPS HAN) rats were fed diet containing 0, 900, 4000, and 18000 ppm of the test substance. Ten males and 10 females from each group were allocated to the chronic (12 month) phase and were necropsied after 52 weeks of treatment. The remaining 60 animals/sex/group were allocated to the carcinogenicity (24 month) phase of the study, continued treatment until final sacrifice of the study after at least 105 weeks of treatment. The mean achieved dose levels of thetest substance received by the animals over the 12-month period were approximately 41.0, 184 and 854 mg/kg/day in males and 56.7, 246 and 1147 mg/kg/day in females, corresponding to the dietary levels of 900, 4000 and 18000 ppm in both sexes. The mean achieved dose levels of the test substance received by the animals over the 24-month period were approximately 35.3, 159 and 741 mg/kg/day in males and 51.2, 221 and 1052 mg/kg/day in females, corresponding to the dietary levels of 900, 4000 and 18000 ppm in both sexes. During the 12-months of treatment, no treatment-related clinical signswereobservedandthemortalityratewaslowacrossallgroups.Therewerenotreatment-related effects on the following parameters, at any dose-level tested: food consumption, ophthalmic changes at the 12 and 24 -month examination, hematology, clinical chemistry or urinalysis parameters after approximately 3, 6 and 12, 18 or 24 months of treatment. In addition, no treatment-related organ weight changes were observed at either the12-month interim or 24-month terminal sacrifice.No macroscopic or microscopic changes were observed in animals allocated to the12-month interim sacrifice phase and no macroscopic changes were observed at the 24-month terminal sacrifice,which could be attributable to treatment.

In conclusion, adverse treatment-related effects were limited to the high dose of 18000 ppm in both sexes.In males, these effects were limited to an effect on body weight parameters whereas in females, these effects also included non-neoplastic changes in the uterus, ovary and vagina. However, in females, the administration of the test item at this dose level in the diet for 2 years resulted in an achieved intake of 1052 mg/kg bw/day which exceeds the guideline limit dose of 1000 mg/kg bw/day infemales. Therefore, the effects observed in the target organs (uterus, ovary and vagina) were a late onsetoccurrence, observed at a dose which exceeds both the MTD and the limit dose.The No Observed Adverse Effect Level (NOAEL) over a12-month period o fdietary administration with the test substance to the Wistar rat was 4000 ppm in both sexes (equivalent to 184 mg/kg body weight/day in males and 246 mg/kg body weight/day in females). The No Observed Adverse Effect Level (NOAEL) over a 24- month period of dietary administration with the test substance to the Wistar rat was 4000 ppm in both sexes (equivalent to 159 mg/kg body weight/day in males and 221 mg/kg body weight/day infemales).

 

90-day sub-chronic toxicity feeding study in the dog

The potential toxicity of the test substance was assessed in a 90-day sub-chronic toxicity feeding study in the beagle dog performed according to OECD Guideline 409 and in compliance with GLP (M-495692-02-1). Three groups of 4 male and 4 female dogs received the test substance at doses of 800, 3200 or 12800 ppm (equating to 25.6, 126 and 440 mg/kgbw/day in males and 29.9, 138 and 485 mg/kg bw/day in females) for at least 13 weeks via the diet. The selection o fdose levels was based on a short-term toxicity study combining oral administration via gavage for 12 days and dietary administration for 14 days as well as a 28-day short-term toxicity feeding study in the same species. A control group of 4 males and 4 females received untreated diet. Up to 12800 ppm, no mortalities, no treatment-related changes at the ophthalmological examination, hematology determinations, urinalysis, gross observation and microscopic examination were noted. At 12800 ppm, the only treatment-related clinical sign consisted of increased salivation noted for 2 dogs on 4 and 6 occasions, respectively. Reduced body weight and body weight gain occurred during the first week and on several occasions throughout the study reaching statistical significance in females only. The weekly mean food consumption was marginally affected in females only. Clinical chemistry determination revealed higher mean alkaline phosphatase activity in males and females of the high dose group compared to controls. At 3200 ppm, the only treatment-related clinical sign consisted of increased salivation noted on one or more occasions for 3 male dogs at the weekly clinical examinations and on 2 occasions for one male dog at the monthly physica lexaminations.This finding in isolation was considered not to be adverse. No treatment-related changes occurred in the low dose groups (800ppm). Thus, under the conditions of this study, a dietary level of 3200 ppm (equivalent to 126 and 138 mg/kg bw/day in males and females, respectively) administered to dogs for at least 13 weeks is considered to be a NOAEL in males and a NOEL infemales.

 

90-day sub-chronic toxicity feeding study in the rat

The potential toxicity of the test substance was assessed in a 90 -day sub-chronic toxicity feeding study in Wistar Rj:WI (IOPS HAN) rats performed according to OECD Guideline 408 and in compliance with GLP (M-443358-02-1). Three groups of 10 male and 10 female rats received the test substance at doses of 900, 3000 or 10000 ppm (equating approximately to 55, 178 and 608 mg/kg body weight/day in males and 66, 213 and 723 mg/kg body weight/day in females) for at least 90 days via the diet. Theselection of dose levels was based on a 28-day short-term toxicity feeding study in the same species. A control group of 10 males and 10 females received untreated diet. In addition, 10 animals/sex were added to the control and high dose group to assess the reversibility of any effects observed at the high dose level. Up to the highest dose of 10000 ppm, no treatment-related effects were observed for any of the parameters assessed. The mean concentration values of the test substance in plasma were in the range of 0.266 to 0.406 mg/L in males and 0.875 to 0.915 mg/L in females. Thus, under the conditions of this study, the NOAEL of the test substance following continuous dietary administration to rats for 90 days is 10000 ppm (equivalent to 608 and 723 mg/kg bw/day in males and females, respectively).

 

90-day sub-chronic toxicity feeding study in the mouse

The potential toxicity of the test substance was assessed in a 90-day sub-chronic toxicity feeding study in C57BL/6J mice performed according to OECD Guideline 408 and in complianc ewith GLP

(M-448164-02-1). Three groups of 10 male and 10 female mice received the test substance at doses of 900, 2700 or 6000 ppm (equating approximately to 145, 426 and 973 mg/kg bw/day in males and 179, 544 and 1224 mg/kg bw/day in females) for at least 90 days via the diet. The selection of dose levels was based on a 28-day short-term toxicity feeding study in the same species. A control group of 10 males and 10 females received untreated diet. Up to the highest dose of 6000 ppm, no treatment-related effects were observed for any of the parameters assessed. Thus, under the conditions of this study, the NOEL of the test substance following continuous dietary administration to mice for at least 90 days is 6000 ppm (equivalent to 973 and 1224.2 mg/kg bw/day in males and females, respectively).

Final conclusion: 90-day feeding studies with rats and mice did not show adverse effects up to the highest dose tested resulting in NOAEL(male/female) of 608/723 mg/kg bw/day for rats and 973/1224 mg/kg bw/day for mice, respectively. A 90-day feeding study with dogs showed no organ specific toxicity but only reduced body weight gain and some changes in clinical-chemistry parameter which were not associated with histopathological findings. A NOAEL (male/female) of 126/138mg/kgbw/day was derived for dogs. A chronic toxicity feeding study in rats is available showing adverse effects on uterus, vagina and ovary in the highest dose. Both male and female rats showed also reduced body weights in the highest dose tested. Thus, leading to a NOAEL (male/female) of 159/221 mg/kgbw/day. Taking together, the effects found in the chronic study are considered as the most relevant because the reproductive organs are affected. In all 90-day study no severe organ toxicity was observed. Even though the NOAEL from the 90-day dog study is the lowest it can be considered that the NOAEL of the chronic study in rats is the most sensitive and is therefore used for risk assessment. Furthermore, a DNEL derived from the NOAEL observed in the 90-day study with dogs would not be lower than the DNEL derived from the chronic ratstudy.

 

Dermal:

28-day short-term toxicity study in the rat

The potential toxicity of the test substance was assessed in a 28-day short-term dermal toxicity study in Crl:(WI) rats performed according to OECD Guideline 410 and in compliance with GLP (M-534165-01-1). The test substance was moistened with water and applied to the skin of groups of 10 male and 10 female rats at doses of 100, 300 or 1000 ppm for approximately 6 h/day, daily for 28 consecutive days under semi-occlusiv econditions. The selection of dose levels was based on a 14-day short-term dermal toxicity study in the same species. A control group of 5 males and 5 females were treated similarly with water. Up to the highest dose of 1000 ppm, no treatment-related effects were observed for any of the parameters assessed. Thus, under the conditions of this study, the NOAEL of the test substance following daily dermal administration to rats for a period of 28 days is 1000 mg/kg bw/day in males and females.

 

Justification for classification or non-classification

The available data on oral and dermal repeated dose toxicity do not meet the criteria for classification according to Regulation (EC) No 1272/2008, and is therefore conclusive but not sufficient for classification.