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

Repeated dose toxicity: oral

Administrative data

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

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

carcinogenicity: 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:

EPA OPPTS 870.4300 (Combined Chronic Toxicity / Carcinogenicity)

Version / remarks:

adopted in 1998

Qualifier:

according to guideline

Guideline:

other: MAFF in Japan, notification 12 Nousan n° 8147

Version / remarks:

adopted in 2000

GLP compliance:

yes

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 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:

104 weeks

Frequency of treatment:

continously (via diet)

Dose / conc.:

900 ppm

Remarks:

equivalent to approximately 35.3 and 51.2 mg/kg bw/day in males and females, respectively

Dose / conc.:

4 000 ppm

Remarks:

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

Dose / conc.:

18 000 ppm

Remarks:

equivalent to approximately 741 and 1052 mg/kg bw/day in males and females, respectively

No. of animals per sex per dose:

10 (12 month interim sacrifice)
60 (24 month sacrifice; carcinogenicity phase)

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. The monthly and overall mean achieved dosage intake for the 24 months of treatment were derived from the weekly data.

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during acclimatization phase, after approximately 12 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: first ten surviving animals
- 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: first ten surviving animals
- 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 and during the month before final sacrifice, 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 730 to 745 all surviving animals, respectively, 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 carcinogenicity 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 (4/58 animals)
It is specified in the study report that these effects may be treatment-related.

(see table 2)

Dermal irritation (if dermal study):

not examined

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 (7% during the second year), decreased mean cumulative body weight gains compared to controls (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 (15% at the end of the second year), decreased mean cumulative body weight gains compared to controls (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:

effects observed, treatment-related

Description (incidence and severity):

FEMALES
18000 ppm (carcinogenicity phase): increased incidence and severity of diffuse squamous cell hyperplasia in the cervix when compared to controls, increased incidence of focal squamous cell metaplasia in the endometrium when compared to controls, increased incidence of diffuse squamous cell hyperplasia in the vagina when compared to controls, tendency towards a higher severity of corpora lutea depletion in the ovary when compared to controls, increased incidence of focal pars distalis hyperplasia when compared to controls (within the range of the in-house historical control data and therefore considered to be incidental and not treatment-related)

(see tables 9, 10 and 11)

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

FEMALES
18000 ppm (carcinogenicity phase): slightly increased incidence of epithelial tumors in the uterus when compared to controls

(see table 12)

Details on results:

For details on results including tables see the attached document.

Relevance of carcinogenic effects / potential:

Neoplastic histopathological findings were observed at the highest dose level of 18000 ppm. The administration of the test substance at 18000 ppm in the diet for 2 years resulted in an achieved intake of 1052 mg/kg bw/day in females which exceeds the guideline limit dose of 1000 mg/kg bw/day. In addition, based on a reduction of 15% in final mean body weight and an overall 24% reduction of the mean cumulative body weight gain compared to controls, this dose level was considered to be above the Maximum Tolerated Dose (MTD), where a 10% reduction in body weight gain is considered adverse. Therefore, the effects observed in the target organs (uterus, ovary and vagina) were a late onset occurrence, observed at a dose which exceeds both the MTD and the limit dose.

Key result

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Effect level:

>= 18 000 ppm

Based on:

test mat.

Remarks:

equivalent to 741 mg/kg bw/day

Sex:

male

Basis for effect level:

other: highest dose tested

Key result

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Effect level:

4 000 ppm

Based on:

test mat.

Remarks:

equivalent to 221 mg/kg bw/day

Sex:

female

Basis for effect level:

histopathology: neoplastic

Key result

Dose descriptor:

NOAEL

Remarks:

chronic toxicity 24 month

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Version / remarks:

adopted in 2009

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 1987

Test guideline 3

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

810-161-6

Cas Number:

1229654-66-3

Molecular formula:

C22 H16 Cl F3 N10 O2

IUPAC Name:

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

Test animals

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

Administration / exposure

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)

Doses / concentrations

Doses / concentrations 1

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

Doses / concentrations 2

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

Doses / concentrations 3

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.

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

1

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

2

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

Target system / organ toxicity

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

Any other information on results incl. tables

For details on results including tables see the attached document.

Applicant's summary and conclusion

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) .