1,3,5-Triazine-2,4-diamine, N2-[(1R,2S)-2,3-dihydro-2,6-dimethyl-1H-inden-1-yl]-6-(1-fluoroethyl)-

Carcinogenicity

Description of key information

OECD 453, rat, 104 wks, diet: NOAELtoxicity (m/f) = 12/17 mg/kg bw/day; LOAELtoxicity = 167 mg/kg bw/day; NOAELcarcinogenicity (m/f) ≥ 414/452 mg/kg bw/day 
OECD 451, mouse, ≥78 wks, diet: NO(A)ELtoxicity (m/f) = 34/42 mg/kg bw/day; LOAELtoxicity (m/f) = 142/168 mg/kg bw/day; NOAELcarcinogenicity(m/f) ≥ 142/168 mg/kg bw/day

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Reason / purpose for cross-reference:

reference to same study

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: M.A.F.F. Notification 12 Nousan No 8147 guideline (November, 2000)

Deviations:

no

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.
- Age at study initiation: approx. 6 weeks old
- Weight at study initiation: 224-226 g mean group weight (males), 167-169 g mean group weight (females)
- Fasting period before study: not applicable, feeding study
- Housing: By sex in groups of 5 unless reduced by mortality or isolation. The cages were suspended, stainless steel and wire mesh. Each cage was identified by cards bearing the animals' unique identification number.
- Diet: A04CP1-10 from S.A.F.E. (Scientific Animal Food and Engineering, Augy, France), ad libitum except at designated time periods. Feed was stored in an identified room controlled for temperature and humidity. Diet was used only until the date of expiry.
- Water: Filtered and softened tap water from the municipal water supply, ad libitum.
- Acclimation period: 15 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): Seventeen formulations (F1 to F16 + F7bis) were prepared during the study. The test substance formulations were prepared to cover the dietary requirements over 6 weekly periods apart from the formulation 7bis and the last formulation which covered the dietary needs for two weeks and until the end of the study, respectively.
- Mixing appropriate amounts with (Type of food): A04CP1-10 from S.A.F.E. (Scientific Animal Food and Engineering, Augy, France)
- Storage temperature of food: When not in use, the diet formulations were stored at room temperature.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance (batch number KM03-8286.1-B/M36) in the diet was demonstrated in a previous study (SA 04029). The stability of the test substance at 20 and 10000 ppm in the diet was verified for up to 92 days, when kept at ambient temperature which covered the period of storage and usage for this study.

Seventeen formulations (F1 to F16 + F7bis) were prepared during the study which consisted of 1 to 6 loads at each concentration.
The homogeneity of the test substance in diet was verified on the first load at each concentration on the first formulation (F1), on the first load at 300 and 10000 ppm of formulations F7 and F13 and on the first load at 6000 ppm of formulation F7bis, to demonstrate adequate formulation procedures. The mean value obtained from the homogeneity check was taken as measured concentration. The concentration was checked on formulations F1, F4, F7 and F7bis, F10, F13 and F16 for all loads at all concentrations not checked for homogeneity.

Homogeneity and concentration of BCS-AA10717 in the diet were within the in-house target range of 85 to 115% of nominal concentration (homogeneity 85-109%; concentration 88-113%). Therefore all study mixes were considered acceptable for use on study.

Duration of treatment / exposure:

104 weeks

Frequency of treatment:

continuously

Post exposure period:

none

Dose / conc.:

300 ppm

Remarks:

corresponding to 12 mg/kg bw/day for males and 17 mg/kg bw/day for females over 24-month period

Dose / conc.:

3 000 ppm

Remarks:

corresponding to 118 mg/kg bw/day for males and 167 mg/kg bw/day for females over 24-month period

Remarks:

corresponding to 414 mg/kg bw/day for males and 661/452 mg/kg bw/day for females over 24-month period; dietary level lowered to 6000 ppm in females from week 41 onwards due to marked clinical signs)

No. of animals per sex per dose:

70 (60 + 10 for interim 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 (SA 04093), where dietary administration of up to 10000 ppm in males and females resulted in reduced mean body weight in both sexes, reduced food consumption in females, increased TSH levels in males on Week 3, higher liver weights in both sexes and follicular cell hypertrophy of the thyroid gland in males at the end of treatment. In addition, one female was sacrificed prematurely on Day 20. A dose level of 200 ppm represented the No Observed Effect Level in males. For females, a dose level of 5000 ppm represented the No Observed Adverse Effect Level.

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals checked for moribundity and mortality twice daily (once daily on weekends or public holidays). All animals were observed for clinical signs at least once daily.
- Cage side observations included: Nature, onset, severity, duration and recovery of clinical signs. Cages and cage trays were inspected daily for evidence of ill health such as blood or loose feces.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least weekly, including palpitation for masses. 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 acclimatisation period, then weekly for the first 13 weeks, approximately every 4 weeks thereafter and prior to necropsy (terminal body weight). Additionally, a supplementary body weight session was done on day 582.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, twice weekly for the first 6 weeks of the study, then approximately weekly up to week 13, then every 4 weeks thereafter.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Compound intake calculated from mean group food consumption and group mean body weight at the end of the food consumption period as mg/kg bw /day

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: During the acclimatisation phase by indirect ophthalmoscopy; during the treatment period (after approximately 12 months) by funduscopic (indirect ophthalmoscopy) and biomicroscopic (slit lamp) examinations. During re-examination 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 and an indirect ophthalmoscope.
- Dose groups that were examined: all animals during acclimatisation phase and all surviving males from the control and the high dose groups and all surviving females from all groups after approximately 12 months of treatment. In addition, all surviving animals were re-examined after approximately 24 months of treatment.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On weeks 12 or 13, 23 or 24 and 51or 52 from the interim sacrifice groups, and on weeks 12 or 13, 23 or 24, 51 or 52, 78 and 105 from the terminal sacrifice groups. Blood (0.5 mL) was sampled by puncture of the retro-orbital plexus into tubes containing EDTA. When possible, a blood smear was prepared for the moribund animals, just before sacrifice. At terminal sacrifice blood smears were prepared for all animals.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight dietary fasting.
- How many animals: On all surviving animals of the 12-month interim sacrifice groups and on the first ten suitable surviving rats of the terminal sacrifice groups.
- Parameters examined: Hematocrit, hemoglobin concentration, leukocyte count, erythrocyte count, platelet count, prothrombin time, leukocyte differential count, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean corpuscular volume, reticulocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On weeks 12 or 13, 23 or 24 and 51or 52 from the interim sacrifice groups, and on weeks 12 or 13, 23 or 24, 51 or 52, 78 and 105 from the terminal sacrifice groups. Blood was sampled by puncture of the retro-orbital plexus into tubes containing lithium heparin (for plasma), clot activator (for serum; 1.3 mL and 0.5 mL, respectively) and sodium citrate for coagulation (0.9 ml). In addition, blood samples were taken at Weeks 3 and 14 for T3, T4 and TSH determinations in animals allocated to the chronic phase.
- Animals fasted: Yes, overnight.
- How many animals: On all surviving animals of the 12-month interim sacrifice groups and on the first ten suitable surviving rats of the terminal sacrifice groups.
- Parameters examined: Calcium, chloride, inorganic phosphorus, potassium, sodium, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma glutamyltransferase, albumin, creatinine, urea, total cholesterol, glucose (fasting), total bilirubin, total protein, triglycerides. Globulin concentrations and albumin/globulin ratio values were calculated.

URINALYSIS: Yes
- Time schedule for collection of urine: On all the surviving animals of the 12-month interim sacrifice groups on weeks 14 or 15, 22 or 23 and 50 or 51 and on the first ten suitable surviving rats of the terminal sacrifice groups on weeks 14 or 15, 22 or 23, 50 or 51, 77 and 104.
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes, diet and water were withdrawn during the overnight (approximately 16 hours) collection period.
- Parameters examined: Appearance, volume, specific gravity/ osmolality / refractive index, pH, sediment (microscopic), protein, glucose, ketones, bilirubin, blood / red blood cells, urobilinogen

NEUROBEHAVIOURAL EXAMINATION: No, neurotoxicological potential of the test substance was assessed in other studies.

Sacrifice and pathology:

GROSS PATHOLOGY:
Yes, on study days 376 to 378 for the 12-month interim sacrifice and on study days 734 to 747 for the 24-month terminal sacrifice; all surviving animals dedicated to the interim or terminal sacrifice were sacrificed by exsanguination while under deep anesthesia (inhalation of Isoflurane). An approximately equal number of animals randomly distributed amongst all groups were sampled on each day of the interim or terminal sacrifice. Animals were diet fasted overnight prior to sacrifice. All animals, including unscheduled decedent animals, were necropsied. Necropsy included: examination of external surfaces, all orifices, all major organs, tissues and body cavities. All significant macroscopic abnormalities (including masses and their regional lymph nodes when possible) were recorded, sampled and examined microscopically. Organs examined: Tongue, submaxillary gland, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, liver, pancreas, trachea, lung, nasal cavities, pharynx, larynx, thoracic aorta, heart, bone marrow (sternum), mesenteric lymph node, submaxillary lymph node, spleen, thymus, kidney, urinary blader, testis, epididymis, prostate gland, seminal vesicle, ovary, uterus (with cervix), mammary gland, vagina, brain, sciatic nerve, spinal cord (cervical, thoracic, lumbar), eyes (retina), optic nerves, pituitary gland, adrenal, gland, lachrimal exorbital gland, parathyroid gland, Harderian gland, bone (sternum), skeletal muscle, skin, all gross lesions and masses (incl. their lymph nodes if possible), articular surface (femorotibial joint).

HISTOPATHOLOGY:
Yes, the following organs were examined: Tongue, submaxillary gland, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, liver, pancreas, trachea, lung, thoracic aorta, heart, bone marrow (sternum), mesenteric lymph node, submaxillary lymph node, spleen, thymus, kidney, urinary blader, 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, parathyroid gland, Harderian gland, bone (sternum), skeletal muscle, skin, all gross lesions and masses (incl. their lymph nodes if possible), articular surface (femorotibial joint), and all significant macroscopic abnormalities (including masses and their regional lymph nodes when possible). Additionally, gross abnormalities were analysed.
Samples were fixed by immersion in neutral buffered 10% formalin with the exception of the eye and optic nerve, Harderian gland, epididymis and testis that were fixed in Davidson's fixative.

Other examinations:

ORGAN WEIGHTS: Liver, heart, spleen, thymus, kidney, testis, epididymis, prostate gland, ovary, uterus, brain, pituitary gland, adrenal gland, thyroid gland

Statistics:

Statistical analyses were carried out separately for males and females by the Bartlett Test. If results were not significant (p>0.05) the ANOVA Test was performed. If this test was not significant, statistical analyses were stopped and group means were considered homogeneous. If the ANOVA was significant, the Dunnet Test (2-sided) was performed. If the Bartlett Test turned out to be significant (p≤0.05), the Kruskal-Wallis Test was performed, followed by the Dunn Test (2-sided), if results were significant; if not, testing was stopped.

Group means of body weight and average food consumption/day parameters, hematology (red blood cell count, platelet count, white blood cell count, neutrophil count, lymphocyte count, reticulocyte count) and hormonal parameters were compared using the Bartlett Test. If results were not significant then the procedure described above was followed. If the result of the Bartlett Test was significant, data was transformed either by log-transformation (body weight and food consumption and hormonal parameters) or square root transformation (hematology parameters). Then the Bartlett Test was performed on transformed data. If results were not significant an ANOVA Test was performed on log-transformed data. If the test was not significant group means were considered homogeneous and testing was stopped; if the ANOVA was significant the Dunnett Test (2-sided) was performed on transformed data. If the result of the Bartlett Test on transformed data was significant (p≤0.05), a Kruskal-Wallis Test was performed. If the result was not significant, group means were considered homogeneous and testing was stopped; if the result was significant (p≤0.05), the Dunn Test (2-sided) was performed.

Quantitative urinalysis parameters (pH) were analysed by the Kruskal-Wallis Test. If results were not significant (p>0.05) group means were considered homogeneous and testing was stopped. If results were significant (p≤0.05) the Dunn Test (2-sided) was performed.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

adverse

Mortality:

mortality observed, treatment-related

Description (incidence):

adverse

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

adverse

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

not adverse

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):

adverse

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

not adverse

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

not adverse

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

not adverse

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

not adverse

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

not adverse

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

adverse

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
10000/6000 ppm:
After 2 years of treatment mortality incidence was increased in females (35/60) when compared to controls (22/60). During the first three weeks of treatment and over the second year of study, a treatment-related increased incidence of animals killed for animal welfare reasons was observed in females. Factors contributing to the death of these animals were clinical conditions, i.e. severe neurological clinical signs and reduced body weight gain or loss.

During the first year, a range of treatment-related clinical signs was observed in up to 62/70 females, starting from the second week of treatment, and consisted of dilated pupil, limited use of limbs, uncoordinated movements, tremors, hindlimbs splayed, low alertness, soiled fur (localized at the nose), soiled nose, soiled anogenital region, wasted appearance and laboured, rapid and/or noisy respiration. Additionally, clinical signs usually associated with morbidity (reduced motor activity, general pallor, hunched posture) were noted in a few females. The incidence of treatment-related clinical signs in females was clearly lowered after the change of dose level from 10000 to 6000 ppm (Week 41), with up to 36/67 females remaining affected at the end of the first year of study. During the second year of treatment, dilated pupil, limited use of limbs, tremors, hindlimbs splayed, low alertness, soiled fur, nose or anogenital region, or wasted appearance were observed in up to 22/56 males and 39/56 females. In addition, clinical signs usually associated with morbidity were noted in a few males (piloerection) and females (noisy or laboured respiration and general pallor).

3000 ppm:
The mortality rate in females was considered unaffected by treatment; there were no effects on mortality in males.

There were no treatment-related clinical signs in males. Throughout the study, a few females showed treatment-related clinical signs, starting from the sixth week of treatment, which consisted of dilated pupil, limited use of limbs, tremors, hindlimbs splayed, low alertness and/or noisy respiration.

300 ppm:
No effects.

BODY WEIGHT AND WEIGHT GAIN
10000/6000 ppm:
The mean body weight was lower than controls at each time point from Week 2 to Week 102 by 5 to 15% (p≤0.05 or p≤0.01) in males and 4 to 20% (p≤0.01 or p≤0.05 on most occasions) in females, compared to controls. In males, the mean cumulative body weight gain was lower than in the control group between Weeks 1 to 3 (-30%, p≤0.01), 26 to 54 (-15%, not statistically significant) and 54 to 78 (-83%, p≤0.01). In females, the mean cumulative body weight gain was lower than in the control group between Weeks 1 to 3 (-59%, p≤0.01), 54 to 78 (-55%, p≤0.01) and 78 to 102 (-81%, p<0.01). Overall, the mean cumulative body weight gain between Weeks 1 to 102 was reduced by 9% (not statistically significant) in males and 31% (p<0.01) in females, compared to controls. At the end of the chronic phase (12 months), mean terminal body weight was lower (-13%) in males, when compared to controls. At the end of the carcinogenicity phase (24 months), mean terminal body weight was lower (-18%) in females, when compared to controls.

3000 ppm:
There were no treatment-related effects on body weight parameters in males. Mean body weight in females was lower than controls from week 78 to week 102 by 5 to 10% (p≤0.05 on weeks 98 and 102). Mean body weight gain was reduced during the first week of treatment by 21%, compared to controls. Thereafter, mean cumulative body weight gain was lower than in the control group between weeks 54 to 78 (-32%, p≤0.01) and 78 to 102 (-47%, not statistically significant). Overall, mean cumulative body weight gain between weeks 1 to 102 was reduced by 14% (p≤0.05).

300 ppm:
No effects.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
10000/6000 ppm:
Mean food consumption was reduced during the first two weeks of treatment by up to 34% in males and 37% in females, compared to controls. Thereafter, mean food consumption was unaffected by treatment in males and slightly reduced in females at most intervals during the next 38 weeks of treatment by up to 12%. After the change in dose level, from Week 41 (Day 281) onwards, mean food consumption was unaffected by treatment.

3000 ppm:
There were no treatment-related effects on food consumption in males. Mean food consumption was reduced in females during the first two weeks of treatment by up to 12%, compared to controls.

300 ppm:
No effects.

OPHTHALMOSCOPIC EXAMINATION
10000/6000 ppm:
After 12 months treatment-related findings were recorded in females only and consisted of mydriasis and absence of pupillary reflex in 14/66 animals. After 24 months 4 out of these 14 females surviving to terminal sacrifice showed ophthalmological abnormalities consisting of mydriasis, pupillary reflex absent, corneal opacity, edema of the cornea, nuclear opacity of lens, hyperreflectivity in retina, and/or hyaloid remnant in vitreus. In males, the findings mydriasis and pupillary reflex absent were noted in 3/26 animals.

3000 ppm:
Mydriasis was observed in 1/69 females after 12 months which was considered to be incidental and not treatment-related.

300 ppm:
No effects.

HAEMATOLOGY
10000/6000 pm:
Slightly higher total leukocyte and lymphocyte counts were noted in females at month 3, 6 and 12. These changes were low in magnitude and were not observed during the second year of treatment, they were therefore considered not to be toxicologically relevant.

3000 ppm:
Slightly higher total leukocyte and lymphocyte counts were noted in females at month 3, 6 and 12. These changes were low in magnitude and were not observed during the second year of treatment, they were therefore considered not to be toxicologically relevant.

300 ppm:
No effects.

CLINICAL CHEMISTRY
10000/6000 ppm:
There was a range of slight treatment-related changes. Mean inorganic phosphorus concentrations were higher in both sexes at months 3, 6 and 12. Lower mean total bilirubin concentrations were observed in females throughout the first year of treatment (months 3, 6 and 12) and in males at months 6, 12, 18 and 24. Higher potassium concentration was noted in males only at month 24. Lower mean glucose concentrations (at months 3, 6 and 12) and higher mean total cholesterol and triglycerides concentrations (at months 3 and 6) were noted in females only. At the hormonal assessment, there was a slightly higher TSH level in males (+49%) on Week 3 only.

3000 ppm:
Higher mean inorganic phosphorus concentrations and higher mean total cholesterol concentrations were observed in females only, the effect being less pronounced than in the high dose group. These changes either occurred sporadically or the magnitude of effects declined with time, and, in the absence of associated histopathological findings, they were considered to be of minor relevance. In addition, slightly lower mean total bilirubin concentrations were noted at month 12, 18 and 24 in males and at month 3, 6 and 12 in females but were considered not to be adverse as they do not represent any functional impairment in the test organism.

300 ppm:
The only treatment-related effect was a lower mean total bilirubin concentrations noted in males at month 24. Total bilirubin concentration decrease was considered not to be adverse in the absence of microscopic changes and as it does not represent any functional impairment in the test organism.

URINALYSIS
10000/6000 ppm:
Urinalysis assessment revealed slightly higher mean urinary volumes in females throughout the first year of treatment, when compared to the controls.

3000 + 300 ppm:
No treatment-related changes in either sex.

ORGAN WEIGHTS
10000/6000 ppm:
At the end of the chronic phase (12 months), mean terminal body weight was lower (-13%) and mean liver to body weight ratio was higher (+23%) in males, when compared to controls. At the end of the carcinogenicity phase (24 months), mean liver to body weight ratio was higher by 18 and 14%
in males and females, respectively, when compared to controls. This change was considered to be associated with lower terminal body weight in females but also with macroscopically enlarged liver and with microscopic hepatocellular hypertrophy in both sexes.

3000 ppm:
At the end of the chronic phase (12 months), mean liver to body weight ratio was higher (+12%) in males, when compared to controls.

300 ppm: No effects

GROSS PATHOLOGY
10000/6000 ppm:
At the end of the chronic phase (12 months) treatment-related macroscopic findings consisted of enlarged liver in 2/9 males and 3/10 females. For animals allocated to the carcinogenicity phase of the study which died or were prematurely sacrificed, a higher incidence of dark liver and kidney was noted in both sexes. At the end of the carcinogenicity phase (24 months) macroscopically enlarged liver was observed in both sexes. Additionally, a higher incidence of dark liver or kidney was noted in both sexes or in females only, respectively.

3000 ppm:
For animals allocated to the carcinogenicity phase of the study which died or were prematurely sacrificed, a higher incidence of dark kidney was noted at the macroscopic examination in females.
At the end of the carcinogenicity phase (24 months), higher incidences of enlarged liver were noted in both sexes.

300 ppm:
No effects.

HISTOPATHOLOGY: NON-NEOPLASTIC
10000/6000 ppm:
At the end of the chronic phase (12 months), centrilobular to panlobular hepatocellular hypertrophy was noted in 6/9 males and in 5/10 females. In the thyroid gland, follicular cell hypertrophy was noted in 3/9 males and 1/10 females, associated in males with a higher incidence and severity of colloid alteration, when compared to controls.
At the microscopic examination of all animals allocated to carcinogenicity phase, treatment-related findings were noted in the liver, kidney, thyroid gland, eye, pituitary gland and brain. In the liver, hepatocellular centrilobular to panlobular hypertrophy and higher incidence of centrilobular macrovacuolation were observed in both sexes. In addition, a higher incidence of hepatocellular single cell necrosis, multinucleated hepatocytes, hepatocytic brown pigments as well as decreased incidence of periportal macrovacuolation was noted in females only. In the kidney, a higher incidence and severity of microscopic intracellular brown pigments was noted in females. In the thyroid gland, higher incidences of follicular cell hypertrophy were observed in males and colloid alteration in males and females. In the eye, higher incidences of bilateral retinal atrophy and peripheral retinal atrophy were noted in females. Retinal atrophy was mainly characterized by degeneration of outer plexiform layer, outer nuclear layer and rod/cones lamina, affecting more the peripheral than the central retina. In the pituitary gland and the brain, degenerative change concerning the neurohypophysis was observed; higher incidences of pars nervosa and median eminence vacuolation were noted in the pituitary gland and the brain, respectively, in both sexes.

3000 ppm:
At the end of the chronic phase (12 months), centrilobular to panlobular hepatocellular hypertrophy was noted in 1/10 males and in 1/10 females.

At the end of the carcinogenicity phase (24 months), treatment-related findings were observed in the liver, thyroid gland and pituitary gland. In the liver, higher incidences of microscopic hepatocellular centrilobular to panlobular hypertrophy were observed in both sexes. Additionally, in females, higher incidences of multinucleated hepatocytes, centrilobular macrovacuolation and decreased incidence of periportal macrovacuolation were noted. In the thyroid gland, higher incidences of follicular cell hypertrophy were observed in males and colloid alteration in males and females at the microscopic examination. In the pituitary gland, higher incidences of pars nervosa vacuolation were noted in both sexes.

300 ppm:
No effects.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
No treatment-related increase in neoplastic lesions of any type in any organ was observed.

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

12 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: calculated from 300 ppm in feed (corresponding to 17 mg/kg bw/day for females)

Dose descriptor:

LOAEL

Remarks:

systemic toxicity

Effect level:

167 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

histopathology: neoplastic

Remarks on result:

other: calculated from 3000 ppm in feed (corresponding to 118 mg/kg bw/day for males)

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Effect level:

>= 414 mg/kg bw/day

Based on:

test mat.

Sex:

male

Remarks on result:

other: calculated from 10000 ppm in feed

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Effect level:

>= 452 mg/kg bw/day

Based on:

test mat.

Sex:

female

Remarks on result:

other: calculated from 6000 ppm in feed; reduced from 10000 ppm in week 41 due to marked clinical signs

Critical effects observed:

yes

Lowest effective dose / conc.:

167 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Critical effects observed:

yes

Lowest effective dose / conc.:

167 mg/kg bw/day (nominal)

System:

central nervous system

Organ:

brain

pituitary gland

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Critical effects observed:

yes

Lowest effective dose / conc.:

167 mg/kg bw/day (nominal)

System:

endocrine system

Organ:

thyroid gland

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

CONCLUSION:

No treatment-related effects were noted on the mortality rate in males at any dose level. No treatment-related cause of death or macroscopic or microscopic changes were established for the few animals allocated to the chronic phase (12 months) of the study which died or were humanely sacrificed before the end of the first year of treatment. No treatment-related neoplastic changes were observed in either sex at any dose level.

Over a 24-month period of dietary administration with the test substance, a dose level of 300 ppm was considered to be a No Observed Adverse Effect Level (NOAEL) in males and females (equivalent to 12 and 17 mg/kg body weight/day, respectively). The NOAEL for carcinigenicity was determined to be higher than a dietary dose level of 6000 ppm in females and of 10000 ppm in males (corresponding to ≥414 and ≥452 mg/kg bw/day in males and females, respectively).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

452 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.

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The test substance did not show any carcinogenic potential up to and including the highest dose level of 10000 ppm in the feed in rats. Additionally, there was no mutagenicity observed either in vitro or in vivo.

Therefore, the test article does not have to be classified as having a carcinogenic potential for man according to the criteria of the Regulation (EC) No 1272/2008.

Additional information

There is data available from a Combined Chronic Toxicity and Carcinogenicity Study according to OECD guideline 453 in rats and a Carcinogenicity Study according to OECD guideline 451 in mice.

In the combined study, which was chosen as key study, the rats received doses of 300, 3000 and 10000/6000 ppm, corresponding to 12, 118 and 414 mg/kg bw/day for males and 17, 167, and 661/452 mg/kg bw/day for females, for 104 weeks in their feed. Both sexes of the high-dose group started with a dietary level of 10000 ppm, but due to marked clinical signs in the high dose females their dose had to be reduced to 6000 ppm from week 41 onwards. There was an increased incidence of mortality in high dose females, and many animals had to be killed for animal welfare reasons due to clinical conditions like severe neurological clinical signs and reduced body weight gain or loss. The incidence of treatment-related clinical signs was clearly reduced after reduction of the dose. Typical clinical signs in both males and females were dilated pupils, limited use of limbs, tremors, splayed hindlimbs, low alertness, soiled fur, nose or anogenital region or wasted appearance, and in lower incidences piloerection, noisy or laboured respiration and general pallor, and these clinical signs were also observed in the mid-dose group. Furthermore, reduced body weights compared to controls were observed, and histopathology at the end of the carcinogenicity phase demonstrated hepatocellular hypertrophy, multinucleated hepatocytes and centrilobular macrovacuolation; in the thyroid gland follicular hypertrophy and colloid alteration were observed; in the pituitary gland vacuolation of the pars nervosa was observed in both sexes. Therefore, the LOAEL for toxicity was determined to be 167 mg/kg bw/day. No treatment-related increases in neoplastic lesions were observed in any organ and either sex at any dose level. Therefore, the NOAEL for carcinogenicity was determined to be greater than 452 mg/kg bw/day, calculated from 10000 ppm in feed for female rats.

In the second study mice received doses of 50, 250 and 1000 ppm, corresponding to 6.8, 34 and 142 mg/kg bw/day for males and 8.4, 42 and 168 mg/kg bw/day for females, for at least 78 weeks in their feed. No signs of toxicity were observed up to and including a dose level of 250 ppm. This dose level was determined to be the NO(A)EL for toxicity, corresponding to 34 mg/kg bw/day. At a dose level of 1000 ppm clinical signs of toxicity were observed in females. Body weight and body weight gain were decreased throughout the study, at terminal sacrifice after 18 months of treatment prominent lobulation in the liver and red or black foci in the stomach were found. Histopathology demonstrated hyperplasia and necrosis in the kidney, a higher incidence of hepatocellular vacuolation in the liver and of glandular erosion/necrosis in the stomach. Therefore, the LOAEL for toxicity was determined to be 142 mg/kg bw/day. In females a reduction of lymphoma and adenoma was observed in the hematopoietic system and the pituitary gland, respectively, at the high-dose only. No change in the incidence of tumours was noted in high-dose males. In conclusion, the NOAEL for carcinogenicity was considered to be higher than 142 mg/kg bw/day for mice in this study.

Considering the results of both studies the test substance did not exhibit any carcinogenic potential in rats and mice up to and including the highest doses tested.