Methanesulfonamide, N-[2-[(4,6-dimethoxy-1,3,5-triazin-2-yl)carbonyl]-6-fluorophenyl]-1,1-difluoro-N-methyl-

Carcinogenicity

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 Jan 2010 - 10 Dec 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study (expiry data of the test item: 2012-01-18; final sacrifice (24-months): 15-28 Feb 2012)

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.4300 (Combined Chronic Toxicity / Carcinogenicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: MAFF in Japan, notification 12 Nousan n° 8147 (November, 2000) guideline

Deviations:

no

GLP compliance:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: R. Janvier, Le Genest St Isle, France
- Age at study initiation: 6 weeks old
- Weight at study initiation: 204.1-208.1 g range mean group weight males; 158.7-161.0 g mean group weight females
- Housing: by sex in groups of 5 unless reduced by mortality or isolation. The cages were suspended, stainless steel and wire mesh.
- 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, ad libitum
- Acclimation period: 13 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
The test item was incorporated into the diet (Certified Rodent Meal A04CP1-10 from SAFE) to provide the required dietary concentrations of 50, 250 or 1500 ppm. The test item formulations were prepared to cover the dietary requirements for periods of at least 5 weeks. When not in use, the diet formulations (250 and 1500 ppm) were stored at ambient temperature for a maximum of 66 days or at -20°C +/- 5°C for a maximum of 61 days followed by 10 days at ambient temperature (50 ppm).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test item in the diet has been demonstrated in previous and recent studies. The stability of the test item at 150, 500 and 15000 ppm in the diet was verified for up to 66 days when kept at ambient temperature, and also for 56 days when stored frozen and then kept for 10 days at ambient temperature. In addition, a complementary stability determination was performed during the course of the current study at the dose of 50 ppm. Formulation at 50 ppm was found to be stable after 36 days at room temperature or after 61 days of frozen storage followed by 10 days of storage at room temperature.
Sixteen formulations (F1 to F16) were prepared during the study at each concentration.
Formulations F1 consisted of 5 loads of 57 kg at each concentration.
Formulations F2 to F9 consisted of 4 loads of 56 or 57 kg at each concentration.
Formulations F10 to F15 consisted of 4 loads of 64 kg at each concentration.
Formulations F16 consisted of 2 loads of 63 kg at each concentration.
The homogeneity of the test item in diet was verified from the first loads at 50, 250 and 1500 ppm on the first formulation (F1) and on the first loads at 50 and 1500 ppm of formulations F8 and F13, to demonstrate adequate formulation procedures. The mean value obtained from the homogeneity check was taken as measured concentration. The concentration was checked for all loads at all dose levels for formulations F1, F4, F8, F10, F13 and F16.

Results:
- Homogeneity Analysis: 87-105% of nominal concentration
- Concentration Analysis: 85-106% of nominal concentration
- Stability analysis at 50 ppm: 87-94% of nominal concentration (under storage conditions of the current study).

Duration of treatment / exposure:

chronic toxicity phase: 12 months (52 weeks)
carcinogenicity phase: 24 months (104 weeks)

Frequency of treatment:

daily (7 days/week)

Remarks:

Doses / Concentrations:
50, 250 and 1500 ppm
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
1.96, 9.86 and 60.4 mg/kg bw/day (males)
Basis:
other: mean dose values as calculated from the reported body weight and food intake values

Remarks:

Doses / Concentrations:
2.81, 14.2 and 84.2 mg/kg bw/day (females)
Basis:
other: mean dose values as calculated from the reported body weight and food intake values

No. of animals per sex per dose:

chronic toxicity phase: 10
carcinogenicity phase: 60

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 (M-363159-02-1), where the NOAEL was 250 ppm of AE 1887196 in both sexes (corresponding to 16.4 and 20.0 mg/kg bw/day in males and females, respectively) and significant toxicity (essentially consisting of liver and thyroid gland changes at the histopathological examination) was observed at 5000 ppm, the highest dose level tested (corresponding to 323 mg/kg bw/day in males and 395 mg/kg bw/day in females).

Positive control:

none positive control

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were checked for moribundity and mortality twice daily (once daily on weekends or public holidays).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were observed for clinical signs at least once daily for all animals, except for some animals on their day of necropsy. The nature, onset, severity, duration and recovery of clinical signs were recorded. Cages and cage trays were inspected daily for evidence of ill health such as blood or loose feces. Detailed physical examinations including palpation for masses were performed weekly from Study day 1. The onset, location and dimension of the masses were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Each animal was weighed at least weekly during the acclimatization period then weekly for the first 13 weeks of study, approximately every 4 weeks thereafter and prior to necropsy. Body weights recorded prior to necropsy are referred to terminal body weights.

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 body weight/day for Weeks 1 to 13, then 1 week per month thereafter was calculated as follows:
Test item intake [mg/kg bw/day] = (Dosel level [ppm] x Group mean food consumption [g/day]) / (Group mean body weigth [g] at the end of the food consumption period)
The monthly and overall mean achieved dosage intake for the 24 months of treatment were derived from the weekly data.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: During the acclimatization phase, all animals were examined by indirect ophthalmoscopy. During the treatment period, funduscopic (indirect ophthalmoscopy) and biomicroscopic (slit lamp) examinations were performed on control and high dose groups at one year and on all surviving animals at two years. Each eye was examined by direct ophthalmoscopy in the first instance, and then after instillation of an atropinic agent (Mydriaticum, Merck Sharp and Dohme), each eye was re-examined by means of a slit lamp and an indirect ophthalmoscope.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was sampled from Isoflurane anesthetized animals by puncture of the retro orbital venous plexus after overnight diet fasting. Blood was collected in tubes containing EDTA.
At scheduled sacrifice (interim and final), blood smears were prepared for all animals. When possible, a blood smear was prepared for the moribund animals, just before sacrifice, except during weekends and public holidays. Blood analyses were performed on all the surviving animals of the 12-month interim sacrifice groups on Weeks 17, 26 and 50.
Blood analyses were performed on the first ten suitable surviving rats of the terminal sacrifice groups on Weeks 17, 26, 50, 78 and 104.
- Anaesthetic used for blood collection: Yes (Isoflurane (Baxter, Maurepas, France and Virbac, Carros, France))
- Animals fasted: Yes
- Parameters checked: haematocrit, haemoglobin, leukocyte count, erythrocyte count, platelet count, blood clotting measurements (activated partial thromboplastin time; clotting time; prothrombin time), leukocyte differential count, mean corpuscular HGB, mean corpuscular HGB concentration, mean corpuscular volume, reticulocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was sampled from Isoflurane anesthetized animals by puncture of the retro orbital venous plexus after overnight diet fasting. Blood was collected in tubes containing clot activator (for serum).
At scheduled sacrifice (interim and final), blood smears were prepared for all animals. When possible, a blood smear was prepared for the moribund animals, just before sacrifice, except during weekends and public holidays. Blood analyses were performed on all the surviving animals of the 12-month interim sacrifice groups on Weeks 17, 26 and 50.
Blood analyses were performed on the first ten suitable surviving rats of the terminal sacrifice groups on Weeks 17, 26, 50, 78 and 104.
- Animals fasted: Yes
- Parameters checked: electrolytes: calcium, chloride, inorganic phosphorus, potassium, sodium; enzymes: alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma glutamyltransferase; other: albumin, creatinine, urea, total cholesterol, glucose (fasting), total bilirubin, total protein, trigylcerides

URINALYSIS: Yes
- Time schedule for collection of urine: Urinalysis was performed on all the surviving animals of the 12-month interim sacrifice groups on Weeks 13 to 15, 25 and 51. Urinalysis was also performed on the first ten suitable surviving rats of the terminal sacrifice groups on Weeks 13 to 15, 25, 51, 76 or 77 and 103.
- Animals fasted: Yes (Diet and water were withdrawn during the overnight (approximately 16 hours) collection period.)
- Parameters checked: appearance, volume, specific gravity/osmolality/refractive index, pH, sediment (microscopic), protein, glucose, ketones, bilirubin, blood/red blood cells, urobilinogen

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The following organs or tissues were sampled and/or weighed at necropsy:
tongue, submaxillary (salivary) gland, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, liver, gall bladder, pancreas, trachea, lung, nasal cavities, pharynx, larynx, aorta thoracic, heart, bone marrow sternum, lymph node mesenteric, lymph node submaxillary, spleen, thymus, kidney, urinary bladder, testis, epididymis, prostate gland, seminal vesicle, ovary, uterus (with cervix), mammary gland, vagina, brain (3 sections), sciatic nerve, spinal cord (cervical, thoracic, lumbar), eyes (retina), optic nerves, pituitary gland, adrenal gland, lacrymal exorbital gland, parathyroid gland, thyroid gland, harderian gland, bone (sternum), skeletal muscle, skin, all gross lesions and masses, articular surface (femorotibial joint)

HISTOPATHOLOGY: Yes
Histopathology examinations were performed for the interim sacrifice (52 weeks of treatment) as follows:
- all organs and tissue samples from animals sacrificed or dying during the treatment period,
- all organs and tissue samples from animals of control and high dose groups,
- liver, lung, kidney and thyroid gland (identified as a target organ) from animals of the intermediate dose groups,
- gross abnormalities from all animals.
Carcinogenicity phase (104 weeks of treatment): Histopathological examinations were performed on all organs and tissues embedded including gross abnormalities in all animals from all groups including decedents.

Other examinations:

Bioanalytical examinations
At the end of the first and second year of the study, a blood sample (approximately 500 μL) was collected from the retro-orbital venous plexus of five suitable animals from the test groups for each sex at approximately 8:00 a.m. (i.e. shortly after the dark period of light/dark cycle which corresponds to the time of food intake). Similarly, a blood sample was also collected from one suitable male and female animals of the control group. Animals were not overnight dietary
fasted before blood sampling.
Prior to blood sampling, animals were anesthetized with Isoflurane. Plasmas were prepared from blood collected into heparinized vials by centrifugation and stored in the dark at approximately -20 °C until determination of the test substance level and potentially its main metabolites.

Statistics:

Mean and standard deviation were calculated for each group. All statistical analyses were carried out separately for males and females. Group means were compared at least at the 5% level of significance. Statistical analyses were carried out using Pristima System (version 6.1.0 build 19 and from April 24, 2012 version 6.3.2 build 17), except for average food consumption/day parameters, clinical signs at physical examinations, ophthalmic and macroscopic findings, survival data, neoplastic and non-neoplastic findings which were analyzed using SAS Software Release 9.2.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

at 1500 ppm: higher incidence of half-closed eyes, scabs, soild fur and reduced motor activity in both sexes; higher incidence of prostration in females; at 250 pp: higher incidence of scabs and soiled fur in females

Mortality:

mortality observed, treatment-related

Description (incidence):

at 1500 ppm: higher incidence of half-closed eyes, scabs, soild fur and reduced motor activity in both sexes; higher incidence of prostration in females; at 250 pp: higher incidence of scabs and soiled fur in females

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

at 1500 ppm: body weight parameters were reduced in males

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, treatment-related

Description (incidence and severity):

at 1500 ppm: slight increased incidence of chromodacryorrhea in males at the end of the first and second year of treatment

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

at 1500 ppm: up to Month 18, slightly lower mean triglycerides concentrations in males and slightly higher mean total cholesterol concentrations in females

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

at 1500 ppm: higher mean absolute and relative liver weights in females at the 12-month scheduled sacrifice; statistically significant higher mean liver to body weight ratio in both sexes at the 24-month scheduled sacrifice

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

at 1500 ppm: enlarged liver in females at the 12-/24-month scheduled sacrifice and higher incidence of nodule(s)/mass(es) in the liver at the 24-month scheduled sacrifice; prominent lobulation in both sexes at the 24-month unscheduled sacrifice

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

12-month sacrifice: 1500 (both sexes) and 250 (males) ppm: cellular alterations in the liver and the thyroid gland; 24-month sacrifice: 1500 ppm: cellular alterations in the liver (also at 250 ppm), the thyroid gland and the urinary bladder in both sexes

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

at 1500 ppm: a higher incidence of hepatocellular adenoma in both sexes

Details on results:

CLINICAL SIGNS
At 1500 ppm, a higher incidence of soiled fur was observed in males and females during the first (p ≤ 0.01 in males and p ≤ 0.001 in females) and second year of the study (p ≤ 0.001 in females only), when compared to controls. In addition, a higher incidence of hair loss was observed in males during the second year of the study (p ≤ 0.05) in comparison to controls.
At 250 and 50 ppm, there were no treatment-related clinical signs recorded during detailed physical examination in either sex.

Regarding clinical observations recorded at least once each day during cage side observation, there were no treatment-related clinical signs during the first year of the study in either sex. During the second year, treatment-related clinical signs are summarized as follows:
At 1500 ppm, a higher incidence of half-closed eyes, scabs, soiled fur and reduced motor activity was observed in both sexes compared to controls. In addition, a higher incidence of prostration was noted in females.
At 250 ppm, treatment-related clinical signs were recorded in females only and consisted of a slightly higher incidence of scabs and soiled fur.
At 50 ppm, there were no treatment-related clinical signs recorded at cage side observation in either sex. The increased incidence of soiled fur in males concerned different localizations (back, abdomen, anogenital region, hindlimb, head or nose) and was observed with no dose-relationship. Therefore it was considered not to be treatment-related.
Other clinical signs recorded were those commonly recorded spontaneously in rats of this age and strain (including those typically associated with the moribundity status) or were recorded in few animals only and were thus considered not to be related to AE 1887196 administration.

MORTALITY
The mortality rate in males and females allocated to the carcinogenicity phase was unaffected by treatment with AE 1887196. Mortality adjusted rates ranged from 50.1-77.9% and from 35.0-49.9% in males and females, respectively (Table 1).

BODY WEIGHT AND WEIGHT GAIN
At 1500 ppm in males, mean cumulative body weight gain was 7-8% lower than controls essentially during the second quarter of the study (Days 1-120, 1-148 and 1-175, p ≤ 0.01) and also between Days 1-400 (p ≤ 0.05). As a consequence, mean body weight was 5-6% lower than controls on the same Days (p ≤ 0.01 or p ≤ 0.05). Otherwise, mean body weight parameters in males were comparable to controls or marginally reduced (Table 2). In females, there was no treatment-related effect on body weight parameters (Table 3).
At 250 and 50 ppm in either sex, there was no treatment-related effect on body weight parameters (Table 2 and 3). The few other minor differences (reaching statistical significance) from controls were considered to reflect inter-individual variations but not treatment-related effects. In particular, the reduced body weight gains in males at the mid and low doses between Study days 92-175 compared to controls were considered not to be treatment related as they were observed with no dose-relationship and were followed by a compensatory increase in mean body weight gains from Study days 175-372.

FOOD CONSUMPTION
Mean food consumption was unaffected by treatment in either sex. The few minor differences (reaching statistical significance) from controls corresponded to incidental increases or decreases. They were considered to reflect inter-individual variations and not to be treatment-related effects.

OPHTHALMOSCOPIC EXAMINATION
At the end of the first year of treatment, an increased incidence of chromodacryorrhea was observed in high dose males (7/70 occurences compared to 0/66 in the control males; p ≤ 0.01). No treatment-related ophthalmological findings were noted in high dose females after 1 year of treatment.
At the end of the second year of treatment, a slight increased incidence of chromodacryorrhea was still observed in the high dose males (6/29 occurences compared to 2/29 in the control males; not statistically significant).
No treatment-related ophthalmological findings was noted at the mid and low dose levels in males or at any dose level in females after 2 years of treatment. Other ophthalmological changes after 1 or 2 years of treatment were considered to be chance findings as they occurred in few animals only, in a non dose-related manner or at a similar frequency in the control and treated group.

HAEMATOLOGY
No treatment-related change was noted at any dietary level in either sex. The few differences observed, even when statistically significant, were considered not to be relevant in view of their transient or incidental occurrence and their low magnitude.

CLINICAL CHEMISTRY
At 1500 ppm, up to Month 18, slightly lower mean triglycerides concentrations were noted in males and slightly higher mean total cholesterol concentrations were noted in females. The other differences, even if statistically significant, were considered not to be treatment-related in the absence of a dose-effect relationship or as they were transient.

URINALYSIS
No treatment-related change was observed at any dietary level in either sex. The few differences observed, even when statistically significant, were considered not to be relevant in view of their transient or incidental occurrence.

ORGAN WEIGHTS AND TERMINAL BODY WEIGHT - 12-month chronic phase
There was no change in mean terminal body weight in treated animals when compared to controls. At 1500 ppm, mean absolute and relative liver weights were statistically significantly higher in females when compared to controls. These changes were considered to be treatment-related.
At 1500 ppm and at 50 ppm, mean absolute kidney weight and mean kidney to brain weight ratio were statistically significantly higher in females when compared to controls (+15 to +16% and +14 to +15%, at 1500 ppm and 50 ppm, respectively). Since these changes were not dose-related and not associated with microscopic findings, they were considered to be incidental and not treatment-related. The few other organ weight changes, even if statistically significant, were also considered as incidental and not treatment-related.

ORGAN WEIGHTS AND TERMINAL BODY WEIGHT - 24-month carcinogenicity phase
There was no change in mean terminal body weight in treated animals when compared to controls.
At 1500 ppm, mean liver to body weight ratio was statistically significantly higher in both sexes when compared to controls. These changes were associated with relevant histopathological findings and were considered to be treatment-related. All other organ weight changes, even if statistically significant, were considered as incidental and not treatment-related since they were not associated with relevant histopathological findings and/or were not dose-related.

GROSS PATHOLOGY - 12-month chronic phase
UNSCHEDULED DEATHS
7 animals died before the end of the study. Two males were found dead on Study days 331 and 364. One male was killed for humane reasons on Study day 347. Two males had an accidental trauma on Study days 221 and 344. Two females died during anesthesia on Study days 114 and 344. All the macroscopic findings observed were considered as incidental and not treatment-related.
TERMINAL SACRIFICE
At 1500 ppm, enlarged liver was noted in both sexes (Table 5). This finding was correlated with hepatocellular centrilobular hypertrophy in females only and was considered to be treatment-related in this sex only. All other macroscopic changes were considered as incidental and not treatment-related.

GROSS PATHOLOGY - 24-month carcinogenicity phase
UNSCHEDULED DEATHS
241 animals died before the end of the study. At 1500 ppm in both sexes, prominent lobulation was noted in the liver (Table 6). This finding was correlated with relevant microscopic findings in 3/4 males and 4/6 females and was considered to be treatment-related. A higher incidence of enlarged liver was noted in both sexes. However, this finding was correlated with relevant microscopic findings in males only and was considered to be treatment-related in this sex. One male had a nodule/mass in the liver (Table 6). It was correlated with hepatocellular adenoma at the microscopic examination. All other macroscopic changes were considered as incidental and not treatment-related.
TERMINAL SACRIFICE
At 1500 ppm, a higher incidence of nodule(s)/mass(es) in the liver was noted in treated females when compared to controls (Table 7). This finding was correlated with relevant microscopic findings (hepatocellular adenoma in three females and eosinophilic foci in one female) and was considered to be treatment-related. Enlarged liver was noted in one female and it was correlated with microscopic hepatocellular hypertrophy.

HISTOPATHOLOGY: NON-NEOPLASTIC - 12-month chronic phase
Treatment-related non neoplastic findings were observed in the liver and the thyroid gland. In the liver at 1500 ppm, a higher incidence of eosinophilic focus(i) of hepatocellular alteration was observed in both sexes. Minimal to slight hepatocellular hypertrophy; minimal to moderate hepatocellular macrovacuolation and minimal hepatocellular brown pigment were noted in females only. Minimal to slight hepatocellular single cell necrosis was observed in both sexes. At 250 ppm, a slightly higher incidence of eosinophilic focus(i) of hepatocellular alteration was observed in males only. At 50 ppm, no treatment-related findings were observed in the liver.
All other microscopic changes on 12-month chronic phase animals were observed at low incidence and/or with no dose-relationionship or were thus considered not to be treatment-related.

HISTOPATHOLOGY: NON-NEOPLASTIC - 24-month carcinogenicity phase
Treatment-related non neoplastic findings were observed in the liver, thyroid gland and urinary bladder (Tables 9-11). In the liver at 1500 ppm, a higher incidence and severity of eosinophilic focus(i) of hepatocellular alteration was observed in both sexes (p≤0.001), compared to controls. Minimal to slight hepatocellular hypertrophy was noted in both sexes (p ≤ 0.001). Minimal to moderate hepatocellular macrovacuolation was noted mainly in females (p ≤ 0.001 in females). A higher incidence and/or severity of hepatocellular brown pigment was observed in both sexes (p ≤ 0.001 or p ≤ 0.05) and a higher incidence of hepatocellular single cell necrosis was observed in females only (p ≤ 0.05). At 250 ppm, a higher incidence and severity of eosinophilic focus(i) of hepatocellular alteration (p≤0.05 in females), minimal hepatocellular hypertrophy (p ≤ 0.01) and minimal to slight hepatocellular macrovacuolation (p ≤ 0.01 in females) was noted in both sexes in comparison to controls. A slightly higher incidence of hepatocellular brown pigment was also observed in females (not statistically significant). At 50 ppm, there were no treatment-related non-neoplastic findings in the liver.
In the thyroid gland at 1500 ppm, a higher incidence and severity of colloid alteration (p ≤ 0.01) and a slightly higher incidence of follicular cell hypertrophy (not statistically significant) were observed in both sexes. At 250 ppm and 50 ppm, there were no treatment-related non-neoplastic findings in the thyroid gland.
In the urinary bladder at 1500 ppm, a slightly higher incidence and severity of diffuse transitional cell hyperplasia was observed in females (not statistically significant). A slightly higher incidence and/or severity of suburothelial mononuclear cell infiltrate was observed in both sexes (not statistically significant). At 250 ppm and 50 ppm, there were no treatment-related non-neoplastic findings in the urinary bladder.
All other microscopic changes on 24-month carcinogenicity phase animals, even if statistically significant, were observed at low incidence and/or with no dose-relationionship or were thus considered not to be treatment-related.

HISTOPATHOLOGY: NEOPLASTIC - 12-month chronic phase
No treatment-related neoplastic findings were observed at the end of the 12-month chronic phase.

HISTOPATHOLOGY: NEOPLASTIC - 24-month carcinogenicity phase
Treatment-related neoplastic findings were observed in the liver in the high dose groups only (Table 8). At 1500 ppm, a higher incidence of hepatocellular adenoma was observed in both sexes when compared to controls (p ≤ 0.01 in females only). Since the incidences were out of the range of inhouse historical control data, it was considered to be treatment-related. All other neoplastic findings were considered to be incidental and not treatment-related.

BIOANALYTICAL EXAMINATIONS
Plasma samples were analyzed for AE 1887196 and its main metabolite concentrations at the end of the first and second year of the study. The purpose was to confirm the absorption of the parent compound and/or its metabolites by checking their presence in the blood. In the control and treated groups, all concentrations for the parent compound were below the method validation limit. The values in the treated groups showed a dose-related increase in AE 1887196 metabolite concentrations (BCS-BP19252, BCS-BQ87904 and BCS-AA10030), as detectable levels were observed in the high and mid dose groups, with slightly higher levels in females than in males.

Dose descriptor:

NOEL

Remarks:

over a 24-month period

Effect level:

250 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: equivalent to 9.86 mg/kg bw/day weight/day in males and 14.2 mg/kg bw/day in females at 1500 ppm: neoplastic changes in the liver consisting of a higher incidence of hepatocellular adenoma in both sexes

Remarks on result:

other: Effect type: carcinogenicity (migrated information)

Dose descriptor:

NOEL

Remarks:

over a 24-month period

Effect level:

50 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: equivalent to 1.96 mg/kg bw/day in males and 2.81 mg/kg bw/day in females

Remarks on result:

other: Effect type: toxicity (migrated information)

Dose descriptor:

NOEL

Remarks:

over a 12-month period

Effect level:

50 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: equivalent to 1.96 mg/kg bw/day

Remarks on result:

other: Effect type: toxicity (migrated information)

Dose descriptor:

NOAEL

Remarks:

over a 12-month period

Effect level:

250 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: equivalent to 14.2 mg/kg bw/day based on the limited clinical signs noted at cage side observation only

Remarks on result:

other: Effect type: toxicity (migrated information)

Table 1: Mortality

CARCINOGENICITY PHASE : Whole study period MORTALITY INCIDENCE (UNSCHEDULED DEATH : ADJUSTED RATES #)
AE 1887196 dosage level (ppm)	0	50	250	1500
Males
Group size	60	60	60	60
Mortality incidence (adjusted rates)	30 (50.1%)	31 (52.7%)	40 (77.9%)	32 (53.3%)
Killed for humane reasons	7	10	11	20
Found dead	23	21	29	12
Females
Group size	60	60	60	60
Mortality incidence (adjusted rates)	21 (35.0%)	26 (43.9%)	26 (44.3%)	29 (49.9%)
Killed for humane reasons	12	15	22	24
Found dead	9	11	4	5

#Kaplan-Meier estimated rates after adjustment for censored animals (accidental trauma, dead during anesthesia). Statistical analysis was conducted on mortality incidence only.

Table 2: Males: group mean body weights (BW) and cumulative body weight gains (BWG) (g)

Group mean body weights (BW) and cumulative body weight gains (BWG) (g)
Dosage level (ppm)	0	50	250	1500
Males
Initial BW (Day 1) (%C)	207	208 (100)	204 (98)	206 (99)
BW Week 2 (Day 8) (%C)	264	266 (101)	266 (101)	266 (101)
BW Week 14 (Day 92) (%C)	538	537 (100)	537 (100)	518 (96)
BW Week 25 (Day 175) (%C)	621	609 (98)	604 (97)	589** (95)
BW Week 54 (Day 372) (%C)	713	719 (101)	723 (101)	690 (97)
BW Week 78 (Day 540) (%C)	763	757 (99)	771 (101)	732 (96)
BW Week 105 (Day 729) (%C)	679	676 (100)	700 (103)	668 (98)
BWG Week 1-2 (Days 1 to 8) (%C)	56.6	57.8 (102)	61.4** (108)	60.1* (106)
BWG Weeks 1-14 (Days 1 to 92) (%C)	331	329 (99)	333 (101)	312 (94)
BWG Weeks 14-25 (Days 92 to 175) (%C)	83	72** (87)	67** (81)	71** (86)
BWG Weeks 25-54 (Days 175 to 372) (%C)	93	109** (117)	118*** (127)	101 (108)
BWG Weeks 54-78 (Days 372 to 540) (%C)	37	109** (117)	47 (128)	36 (97)
BWG Weeks 78-105 (Days 540 to 729) (%C)	-65	-68 (nc)	-59 (nc)	-54 (nc)
Overall BWG Weeks 1-105 (Days 1 to 729) (%C)	474	470 (99)	496 (105)	465 (98)

%C: % versus control calculated on raw data; *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001; nc: not calculated

 

Table 3: Females: group mean body weights (BW) and cumulative body weight gains (BWG) (g)

Group mean body weights (BW) and cumulative body weight gains (BWG) (g)
Dosage level (ppm)	0	50	250	1500
Females
Initial BW (Day 1) (%C)	474	159 (99)	159 (99)	159 (99)
BW Week 2 (Day 8) (%C)	161	187 (99)	185 (99)	184 (98)
BW Week 14 (Day 92) (%C)	290	293 (101)	292 (101)	291 (100)
BW Week 25 (Day 175) (%C)	316	323 (102)	321 (102)	322 (102)
BW Week 54 (Day 372) (%C)	354	367 (104)	362 (102)	367 (104)
BW Week 78 (Day 540) (%C)	408	418 (102)	428 (105)	431 (106)
BW Week 105 (Day 729) (%C)	438	445 (102)	450 (103)	441 (101)
BWG Week 1-2 (Days 1 to 8) (%C)	26.9	27.6 (103)	26.6 (99)	25.7 (96)
BWG Weeks 1-14 (Days 1 to 92) (%C)	129	134 (104)	133 (103)	132 (102)
BWG Weeks 14-25 (Days 92 to 175) (%C)	26	30 (114)	30 (112)	31 (119)
BWG Weeks 25-54 (Days 175 to 372) (%C)	38	43 (115)	41 (109)	45 (119)
BWG Weeks 54-78 (Days 372 to 540) (%C)	55	54 (98)	63 (114)	66 (119)
BWG Weeks 78-105 (Days 540 to 729) (%C)	38	38 (101)	22 (59)	21 (56)
Overall BWG Weeks 1-105 (Days 1 to 729) (%C)	276	287 (104)	290 (105)	283 (103)

%C: % versus control calculated on raw data; *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001; nc: not calculated

 

Table 4: The mean achieved dosage intake per group

Sex	Males			Females
Dosage level [ppm]	50	250	1500	50	250	1500
Week period 1-13	3.24	16.1	97.2	4.00	20.0	119
Week period 1-52	2.31	11.5	70.5	3.14	15.8	94.7
Week period 1-80	1.96	9.86	60.4	2.81	14.2	84.2

 

Table 5: Gross pathology: chronic phase

Incidence of macroscopic changes in the liver, scheduled sacrifice, chronic phase
Sex	Males				Females
Dosage level [ppm]	0	50	250	1500	0	50	250	1500
Enlarged	0/8	0/9	0/9	2/9	0/9	0/10	0/9	2/10

 

Table 6: Gross pathology: carcinogenicity phase, unscheduled sacrifice

Incidence of macroscopic changes in the liver, unscheduled sacrifice, carcinogenicity phase
Sex	Males				Females
Dosage level [ppm]	0	50	250	1500	0	50	250	1500
Enlarged	1/31	0/34	1/41	3/32	1/21	0/26	1/27	3/29
Prominent lobulation	0/31	0/34	1/41	4/32	0/21	0/26	2/27	6/29
Nodule(s)/mass(es)	0/31	0/34	0/41	1/32	0/21	0/26	0/27	0/29

 

Table 7: Gross pathology: carcinogenicity phase, scheduled sacrifice

Incidence of macroscopic changes in the liver, scheduled sacrifice, carcinogenicity phase
Sex	Males				Females
Dosage level [ppm]	0	50	250	1500	0	50	250	1500
Enlarged	0/29	0/26	0/19	1/28	0/39	0/34	0/33	1/31
Nodule(s)/mass(es)	0/29	2/26	0/19	0/28	1/39	0/34	1/33	4/31

 

Table 8: Microscopic pathology: carcinogenicity phase, neoplastic findings

Incidence of neoplastic findings in the liver, 24-month carcinogenicity phase
Sex	Males				Females
Dosage level [ppm]	0	50	250	1500	0	50	250	1500
Number of animals examined	60	60	60	60	60	60	60	60
Hepatocellular adenoma, total	0	2	2	4	0	2	2	8**
Hepatocellular carcinoma, total	0	0	0	0	1	0	0	0
Hepatocellular adenoma + carcinoma, total	0	2	2	4	1	2	2	8

**: p ≤ 0.01

Table 9: Microscopic pathology: carcinogenicity phase, non-neoplastic findings in the liver

Incidence and severity of microscopic changes in the liver, 24-month carcinogenicity phase
Sex	Males				Females
Dosage level [ppm]	0	50	250	1500	0	50	250	1500
Number of animals examined	60	60	60	60	60	60	60	60
Eosinophilic focus(i) of hepatocellular alteration
Minimal	18	19	27	31	18	12	24	36
Slight	3	1	1	10	0	2	5	10
Moderate	0	0	2	2	0	0	0	0
Total	21	20	30	43***	18	14	29*	46***
Hepatocellular hypertrophy: centrilobular
Minimal	0	1	9	20	0	0	8	27
Slight	0	0	0	4	0	0	0	22
Total	0	1	9**	24***	0	0	8**	49***
Hepatocellular macrovacuolation: mainly centrilobular: diffuse
Minimal	0	0	3	3	0	0	6	16
Slight	0	0	0	0	0	0	2	12
Moderate	0	0	0	0	0	0	0	2
Total	0	0	3	3	0	0	8**	30***
Hepatocellular brown pigment: focal
Minimal	0	0	0	5	6	4	13	28
Slight	0	0	0	0	0	0	0	7
Moderate	0	0	0	0	1	0	0	0
Total	0	0	0	5*	7	4	13	35***
hepatocellular single cell necrosis: focal
Minimal	2	3	1	4	1	2	1	6
Slight	0	0	0	0	0	0	0	1
Total	2	3	1	4	1	2	1	7*

***: p ≤ 0.001;**: p ≤ 0.01; *: p ≤ 0.05

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

9.86 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 X, 8.9.1, of Regulation (EC) No 1907/2006.

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available data on carcinogenicity of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.

Additional information

The oncogenic potential of Triafamone was assessed in both the mouse and rat. The studies were performed between 2010 and 2013, following the current OECD guidelines and in compliance with the GLP requirements. 

In a combined chronic toxicity and carcinogenicity study, Triafamone was administered to Wistar rats (70 animals/sex/dose group) via the diet at 50, 250 and 1500 ppm, corresponding to 1.96, 9.86 and 60.4 mg/kg bw/day in males and 2.81, 14.2 and 84.2 mg/kg bw/day in females, over a 24-month period.

After 52 weeks, 10 males and 10 females from each group allocated to the chronic (12 month) phase were necropsied at the scheduled interim sacrifice. The remaining 60 animals/sex/group, allocated to the carcinogenicity (24-month) phase of the study, continued treatment until final sacrifice of the study after at least 104 weeks of treatment, when surviving animals were necropsied.

Liver was confirmed to be the main target organ in both sexes, females appearing to be more susceptible than males.

Dietary administration of Triafamone over a 24-month period to the Wistar rat induced neoplastic changes in the liver consisting of a higher incidence of hepatocellular adenoma in both sexes compared to controls at the highest dose level tested of 1500 ppm (equivalent to 60.4 mg/kg bw/day in males and 84.2 mg/kg bw/day in females).

Treatment-related non neoplastic findings were observed in the liver, thyroid gland and urinary bladder. In the liver, a higher incidence and severity of eosinophilic focus(i) of hepatocellular alteration was noted in both sexes (p≤0.001), in association with minimal to slight hepatocellular hypertrophy in both sexes (p≤0.001), minimal to moderate hepatocellular macrovacuolation mainly in females (p≤0.001 in females), a higher incidence and/or severity of hepatocellular brown pigment in both sexes (p≤0.001 or p≤0.05) and a higher incidence of hepatocellular single cell necrosis in females only (p≤0.05). In the thyroid gland, a higher incidence and severity of colloid alteration (p≤0.01) and a slightly higher incidence of follicular cell hypertrophy were observed in both sexes. In the urinary bladder, a slightly higher incidence and severity of diffuse transitional cell hyperplasia was observed in females and a slightly higher incidence and/or severity of suburothelial mononuclear cell infiltrate was observed in both sexes.

There were no neoplastic changes at the lower doses. At 250 ppm (equivalent to 9.86  mg/kg bw/day in males and 14.2 mg/kg bw/day in females treatment-related non neoplastic findings were observed in the liver only and consisted of a higher incidence and severity of eosinophilic focus(i) of hepatocellular alteration (p≤0.05 in females), minimal hepatocellular hypertrophy (p≤0.01) and minimal to slight hepatocellular macrovacuolation (p≤0.01 in females) in both sexes together with a slightly higher incidence of hepatocellular brown pigment in females.

The No Observed Effect Level (NOEL) for neoplastic changes was 250 ppm in both sexes (equivalent to 9.86 mg/kg bw/day in males and 14.2 mg/kg bw/day in females.

Over a 24-month period of dietary administration with Triafamone to the Wistar rat, the dose level of 50 ppm (equivalent to 1.96 mg/kg bw/day in males and 2.81 mg/kg bw/day in females) was a NOEL in both sexes.

 

In a combined chronic and carcinogenicity study, groups of 60 male and 60 female C57BL/6J mice were fed diet containing 0, 50, 500 or 5000 ppm of Triafamone for 52 weeks. After 52 weeks, 10 males and 10 females from each group allocated to the chronic phase of the study were necropsied at the scheduled interim sacrifice. The remaining 50 animals/sex/group, allocated to the carcinogenicity phase of the study, continued treatment until the scheduled final sacrifice of the study after at least 78 weeks of treatment. The mean intake of Triafamone over 18 months was 0, 6.9, 70 and 710 mg/kg bw/day in males and 0, 8.6, 89 and 871 mg/kg bw/day in females, at 0, 50, 500 and 5000 ppm, respectively).

There were no neoplastic findings up to the highest dose level of 5000 ppm.

A higher incidence of mortality was observed in males at 5000 ppm only (p≤0.05). Dark urine was observed throughout the treatment period in both sexes (p≤0.001). Mean body weight and mean cumulative body weight were significantly lower than controls throughout most study intervals in males and in females.

At the 18-month terminal sacrifice of the carcinogenicity phase of the study, absolute and relative kidney were 11 to 15% lower than controls in males (p≤0.01).

At the microscopic examination, there were only non-neoplastic findings noted in the liver, kidney and urinary bladder. In the liver, a lower incidence and severity of hepatocellular macrovacuolation (mainly periportal) was noted in females. In the kidney, decreased incidence and severity of corticoepithelial vacuolation was noted in males. In the urinary bladder, a higher incidence and severity of minimal vacuolation within the surface layer of the urothelium were noted in males (p≤0.01).

The dose level of 500 ppm (equivalent to 70 mg/kg bw/day in males and 89 mg/kg bw/day in females) was considered to be a No Observed Adverse Effect Level (NOAEL) in both sexes over an 18-month period of dietary administration based on mortality, clinical signs, reduced body weight gain and histopathological findings in the liver and kidney observed at 5000 ppm.

In the rat carcinogenicity study the incidence of hepatocellular adenoma was increased in both sexes at 1500, as summarized in the below table:

Incidence of neoplastic findings in the liver
Sex	Males				Females
Triafamone (ppm)	0	50	250	1500	0	50	250	1500
Number of animals examined	60	60	60	60	60	60	60	60
Hepatocellular adenoma
Total	0	2	2	4	0	2	2	8**
Hepatocellular carcinoma
Total	0	0	0	0	1	0	0	0
Hepatocellular adenoma + carcinoma
Total	0	2	2	4	1	2	2	8

**: p≤0.01.

The results of the rat carcinogenicity study with Triafamone indicate that the liver tumours are triggered by a Phenobarbital-like mode of action. Phenobarbital is the prototype of several rodent hepatocarcinogens that induce tumours by a non-genotoxic and non-cytotoxic mechanism. The sequence of key events includes chemical binding to the CAR/PXR receptors, induction of CYP2B and CYP3A isoenzymes, hepatocellular hypertrophy, increase in hepatocellular proliferation and ultimately the induction of proliferative lesions in the liver, including foci, adenomas, and finally carcinomas. The available data for Triafamone provide indicate with high degree of confidence that liver tumours in the rat are developed through a Phenobarbital-like mode of action. The activation of CAR/PXR by Triafamone has not been investigated directly, but by measuring the induction of hepatic xenobiotic metabolising enzymes, particularly of CYP2B and CYP3A forms. Results of the 28-day hepatotoxicity study (M-344191-02-1) showed the induction of CYP2B and CYP3A at doses equivalent to approximately 38 mg/kg bw/day in the female rats. In the carcinogenicity study performed with rats, tumours were observed at doses equivalent of 80 mg/kg bw/day (i. e. above induction) in females but not at doses equivalent to 14 mg/kg bw/day (i. e. well below the induction). Increased liver weight and liver hypertrophy were observed at doses equivalent to 80 mg/kg bw/day in female rats after dietary exposure for 90-days and 1-year. In addition to the liver findings observed in the short-term and subchronic study, a significant increase in altered hepatic foci (i. e. proliferative pre-neoplastic lesions) was recorded at the top dose (equivalent to 80 mg/kg bw/day) of Triafamone at the end of both the chronic and carcinogenicity phase of the rat cancer bioassay. All of the key events described for CAR/PXR-induced liver tumours were identified in Triafamone exposed rats in a temporal- and dose-responsive manner with the final event of hepatocellular tumours observed in the carcinogenicity study in the top dose rats. There was a higher incidence in the female rats of the 1500 ppm groups because Triafamone was shown to induce in females CAR/PXR-dependent isoenzymes at lower doses than in males, and the typical phenobarbital-like pleiotropic response was consistently more pronounced in the females in all the dietary toxicity studies.

Other plausible modes of action for Triafamone liver tumour formation that are likely to be relevant to humans can be effectively excluded (Cohen, 2010; Holsapple et al., 2006; Meek et al., 2003). In fact Triafamone is not genotoxic, hepatotoxicity studies in rodents showed no induction of other types of enzymes by other receptors (PPARα, AhR) or evidence of cytotoxicity.

The exact mechanism by which CAR/PXR activation leads to increased cell proliferation remains unclear. In humans, binding to the CAR/PXR receptors and induction of CYP isozymes following Phenobarbital administration occurs (Lambert et al., 2009). However, unlike in rodents, there is no evidence that this results in an increase in cell proliferation in the human liver. Thus, CAR/PXR activation leads to an increase in cell proliferation in rodents, which is a critical key event in the carcinogenic process in rodents, but it does not occur in humans. Furthermore, extensive epidemiologic investigations at exposure levels in humans that are comparable to those used in rodent bioassays are negative with respect to increased cancer risk (Whysner et al., 1996).

In conclusion, based on the results of the toxicity studies with Triafamone just reviewed, there is much evidence to support a mode of action for Triafamone-induced rat liver tumour formation involving CAR/PXR activation leading to the induction of CYP2B and CYPA3 forms, liver hypertrophy, increased cell proliferation, altered hepatocellular foci and ultimately in tumour formation. Already in the short-term studies the hepatotoxic effects due to CYP2B and CYP3A induction were more marked in the females and this explain the higher incidence of hepatocellular adenoma observed in the female rats at the end of the carcinogenicity study.

This mode of action is similar to that established for phenobarbital and related compounds. There is significant evidence from both epidemiological data in human and from specific mechanistic studies that liver tumours induced by phenobarbital do not occur in human liver. Therefore, the liver tumours observed in the rat carcinogenicity study with Triafamone are considered to be not relevant to humans.

Justification for selection of carcinogenicity via oral route endpoint:
The selected study is the most adequate and reliable study with the lowest dose descriptor.

Carcinogenicity: via oral route (target organ): digestive: liver