2-{2-chloro-4-mesyl-3-[(tetrahydrofuran-2-ylmethoxy)methyl]benzoyl}cyclohexane-1,3-dione

Carcinogenicity

Description of key information

Combined chronic toxicity and carcinogenicity study, oral (OECD 453), rat:

carcinogenicity: NOEL = 214 mg/kg bw/day (males) and 296 mg/kg bw/day (females)

systemic toxicity (24 month): NOAEL = 0.08 mg/kg bw/day (males) and 0.11 mg/kg bw/day (females)

Carcinogenicity study, oral (OECD 451), mouse:

carcinogenicity: NOEL = 583 mg/kg bw/day (males) and 743 mg/kg bw/day (females)

systemic toxicity (18 month): LOAEL = 21.0 mg/kg bw/day (males) and 27.1 mg/kg bw/day (females)

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 Mar 2003 - 29 Apr 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Version / remarks:

adopted 12 May 1981

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Version / remarks:

adopted 25 Jun 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Interministerial Group for Chemical Products, Paris, France

Species:

mouse

Strain:

other: C57BL/6J

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories (Saint Germain-sur-l’Arbresle, France)
- Females nulliparous and non-pregnant: not stated
- Age at study initiation: 6 weeks
- Weight at study initiation: 17.9 to 22.5 g (males), 11.5 to 19.2 g (females)
- Housing: Stainless steel, wire mesh cages. Individually housed.
- Diet: Ground and irradiated A04C-10 P1, Scientific Animal Food and Engineering, Augy, France, ad libitum.
- Water: Filtered softened tap water, ad libitum.
- Acclimation period: 13 days

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

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): 10 to 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: Approximately every 8 weeks
- Mixing appropriate amounts with: A04C-10 P1 diet
- Storage temperature of food: Ambient temperature

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test substance was extracted from the diet samples by simple homogenization with acidified acetonitrile (H2SO4 0.1%). Quantification was performed using HPLC-UV detection.

Stability in diet formulations had been demonstrated in a previous study (M-270092-01-1, 2006). The stability of the test substance at 2 ppm in the diet was verified for a period of 77 days at ambient temperature followed by 25 days of frozen storage and 5000 ppm after a period of 84 days at ambient temperature.

Homogeneity was checked for all dose levels (150, 800 and 4000 ppm) at the first diet formulation and at the low (150 ppm) and high (4000 ppm) dose levels at the fifth formulation. Samples were taken from the high, middle and low surface levels of the diet formulations. Homogeneity was also checked in a complementary mix of mid (800) and high (4000) dose levels, that was prepared during the study.

Concentrations of the diet preparations were checked at the third, fifth, seventh, ninth and tenth formulations. Control diets for all formulations were also analysed.

Results of the homogeneity and concentration checks ranged from 93 to 112%, and were consequently within the target ranges of 85 to 115%. Control diets showed no test substance contamination.

Duration of treatment / exposure:

52 weeks (interim sacrifice) and 78 weeks (terminal sacrifice)

Frequency of treatment:

continuously (via diet)

Dose / conc.:

150 ppm

Remarks:

equivalent to 21.0 mg/kg bw/day (males) and 27.1 mg/kg bw/day (females)

Dose / conc.:

800 ppm

Remarks:

equivalent to 112 mg/kg bw/day (males) and 142 mg/kg bw/day (females)

Dose / conc.:

4 000 ppm

Remarks:

equivalent to 583 mg/kg bw/day (males) and 743 mg/kg bw/day (females)

No. of animals per sex per dose:

60 (10 interim sacrifice and 50 terminal sacrifice)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Dose levels were selected after evaluation of the results from a previous 90-day dietary study in the mouse (M-233114-01-1, 2003). In this study, initial slight body weight loss, lower body weight when compared to controls, liver effects (increased weight and centrilobular hypertrophy) were observed at 7000 ppm. A dose of 2500 ppm was considered to be a No Observed Adverse Effect Level (NOAEL).

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily for moribundity and mortality (once at weekends and public holidays).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily for clinical signs. Detailed physical examinations including palpatation of masses were performed weekly. The onset, location and dimension of the masses were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly for 13 weeks and then every 4 weeks thereafter and prior to necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
- 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 weekly during the first 13 weeks of treatment, and once every 4 weeks thereafter.

FOOD EFFICIENCY: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: 3, 6, 9 and 12 months and on all surviving animals at the end of the study.
- Dose groups that were examined: Examinations were performed on 24 animals / sex / group (all animals from the interim sacrifice phase and 14 animals from the final sacrifice phase).
- Ophthalmoscopic examinations were performed using a slit lamp biomicroscope for the anterior segment of the eye and an indirect ophthalmoscope for fundus.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Weeks 53 and 54 at interim sacrifice, and Weeks 79, 80 or 81 at terminal sacrifice.
- Anaesthetic used for blood collection: Yes (Isoflurane)
- Animals fasted: Yes, overnight
- How many animals: Haematology was performed on all the surviving animals of the interim sacrifice groups and on the first ten surviving animals of the terminal sacrifice groups on Weeks 53 or 54. On Weeks 79, 80 or 81, prior to necropsy, haematology was performed on the first twenty surviving mice of the terminal sacrifice groups and blood smears were performed on all the other sacrificed animals.
- Parameters checked: Red blood cell count (RBC), haemoglobin (HGB), haematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), white blood cell count (WBC) and differential count evaluation (neutrophil (NEU), lymphocyte (LYM)) and platelet count (PLT). A blood smear was prepared and stained with Wright stain. It was examined only when the results of the haematology parameters were abnormal.
- Blood sampling: Blood was sampled by puncture of the retro-orbital venous plexus.

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
On study Days 372, 373 or 374 for the 12-month interim kill, and on study Days 553 to 564 for the carcinogenicity phase. The necropsy included the examination of all major organs, tissues and body cavities. All significant macroscopic abnormalities were recorded, sampled and examined microscopically. The animals were sacrificed by exsanguination under deep anesthesia (pentobarbital, i.p. injection of 60 mg/kg bw) after diet fasting overnight.

Organ weights: Adrenal gland, brain, epididymis, heart, kidney, liver, ovary, spleen, testis and uterus (with cervix) were weighed fresh at scheduled sacrifice only. Paired organs were weighed together.

HISTOPATHOLOGY: Yes
Tissues collected were as follows: Adrenal gland, aorta, articular surface (femoro-tibial joint), bone (sternum), bone marrow (sternum), brain, epididymis, oesophagus, exorbital (lacrymal) gland, eye and optic nerve, gallbladder, harderian gland, heart, intestine (duodenum, jejunum, ileum, caecum, colon, rectum), kidney, larynx / pharynx, liver , lung (including bronchia), lymph nodes (submaxillary, mesenteric), mammary gland, nose (nasal cavities), ovary, pancreas, pituitary gland, prostate gland, sciatic nerve, seminal vesicle (with coagulating gland), skeletal muscle skin, spinal cord (cervical, thoracic, lumbar), spleen, stomach, submaxillary (salivary) gland, testis, thymus, thyroid gland (with parathyroid gland), tongue, trachea, urinary bladder, uterus (with cervix) and vagina.

All the above mentioned samples (except exorbital lacrymal gland, larynx/pharynx and nasal cavities) were embedded in paraffin wax. Histological sections, stained with haematoxylin and eosin, were prepared from all organs and tissue samples, for histological examination.

Interim sacrifice (Week 52): Histopathological examination was performed on the liver, the gallbladder and the kidney in all groups including decedent animals.

Terminal sacrifice: Histopathological examinations were performed on all organs and tissues embedded including gross abnormalities in all animals from all groups including decedents.

Statistics:

See Attachment 1.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 4000 ppm, treatment-related clinical signs consisted of intense yellow colored urine in all males and 58 / 60 females, first observed after 5 months of treatment, and present thereafter. In males, 3 / 60 animals had soiled fur. There was a relatively high incidence of males with ocular discharge and females with prolapsed rectum at this dosage compared to the controls. The observations hunched posture and wasted appearance noted in males and / or in females were considered not to be treatment-related as there was no dose effect relationship.

In addition, observation of dental abnormality was slightly increased at the top dose when compared to controls. It was considered not to be treatment-related, since it is a common finding found in this species.

All other findings were considered to be incidental as they occurred at a similar frequency across all groups including the controls, as there was no evidence of a dose-related response or they occurred as isolated findings.

For data on clinical signs, see Table 1.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

Not applicable.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Forty five animals died prematurely up to the end of the 12-month interim sacrifice (Week 53). On Day 36, one female died due to an accidental trauma (see Table 2).

Between the interim sacrifice and final phase sacrifice, 53 animals died prematurely. The mortality rate in the low dose males of the carcinogenicity phase animals during whole study period was statistically significantly higher than the controls, but in the absence of treatment-related macroscopic or microscopic findings and of a dose-related response, this finding was considered not to be treatment-related (see Table 3).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 4000 ppm, the mean body weight of both sexes was statistically significantly decreased during the first week of treatment (p≤0.01), resulting in a mean body weight reduction of 7% in males and 8% in females. Throughout the remainder of the study, mean body weight was statistically significantly lower than the controls, at most time points for males (p≤0.01) and at several time points observed for females (p≤0.05 or p≤0.01), resulting in a final body weight reduction of 6% in males and 9% in females.

At 800 ppm, the mean body weight of males was statistically significantly reduced after eight weeks of treatment (p≤0.05), resulting in a body weight reduction of 3% compared to the controls. Thereafter, mean body weight was statistically significantly lower than the controls at several time points (p≤0.05 or p≤0.01), resulting in a final body weight reduction of 6%.

At 800 ppm in females and at 150 ppm in both sexes, mean body weight and mean body weight change were comparable with the controls.

For body weight data, see Table 4.

Terminal body weight - interim sacrifice: At 4000 ppm, mean terminal body weights were statistically significantly lower (-10%, p≤0.01) in males when compared to controls.

Terminal body weight - carcinogenicity phase: At 4 000 ppm, mean terminal body weights were statistically significantly lower in males and females when compared to controls (-4% and -8% respectively, p≤0.01). At 800 ppm, mean terminal body weights were statistically significantly lower in males when compared to controls (-3%, p≤0.05).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Dietary intake (Weeks 1-52):
150 ppm: 21.4 mg/kg bw/day (males) and 28.4 mg/kg bw/day (females)
800 ppm: 115 mg/kg bw/day (males) and 149 mg/kg bw/day (females)
4000 ppm: 599 mg/kg bw/day (males) and 772 mg/kg bw/day (females)

Dietary intake (Weeks 1-80):
150 ppm: 21.0 mg/kg bw/day (males) and 27.1 mg/kg bw/day (females)
800 ppm: 112 mg/kg bw/day (males) and 142 mg/kg bw/day (females)
4000 ppm: 583 mg/kg bw/day (males) and 743 mg/kg bw/day (females)

Food efficiency:

not examined

Description (incidence and severity):

Not applicable.

Water consumption and compound intake (if drinking water study):

not examined

Description (incidence and severity):

Not applicable.

Ophthalmological findings:

effects observed, treatment-related

Description (incidence and severity):

The only treatment-related effects noted were ocular discharge observed at 4000 ppm in males at Months 9 and 12.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

After 12 months administration at 4000 ppm, a slightly lower mean total leucocyte count (-46%, p≤0.01) was noted in females, when compared to the controls. Both absolute neutrophil (-50%, p≤0.05) and lymphocyte (-47%, p≤0.01) counts were found to be lower in this sex. In males only, mean lymphocyte count was slightly lower (absolute count: -38%, relative count: -18%, p≤0.01).
In the absence of relevant change in the absolute neutrophil count, the statistically significantly higher relative neutrophil count was considered not to be relevant.

After the 18-month treatment period, lower mean red blood cell count, haemoglobin concentration and haematocrit were noted at 4000 ppm in both sexes. The magnitude and statistical significance to the controls are included in table 5 in the field "Any other information on results incl. tables".

In addition, at 4000 ppm, higher mean corpuscular volume (+10%, p≤0.01) and mean corpuscular haemoglobin (+5%, p≤0.01) were observed in females. In the absence of a relevant change at 800 ppm, the statistically significant differences noted in erythrocyte parameters at 150 ppm in both sexes were considered not to be toxicologically relevant.

The other few statistically significant differences observed throughout the study were considered to be incidental (i.e. no dose-effect relationship) or not relevant in view of their low magnitude.

See Attachment 2 for haematology data.

Clinical biochemistry findings:

not examined

Description (incidence and severity):

Not applicable.

Endocrine findings:

not examined

Description (incidence and severity):

Not applicable.

Urinalysis findings:

not examined

Description (incidence and severity):

Not applicable.

Behaviour (functional findings):

not examined

Description (incidence and severity):

Not applicable.

Immunological findings:

not examined

Description (incidence and severity):

Not applicable.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Interim sacrifice:
At all dose levels in males and at 4000 and 800 ppm in females, mean liver to body weight ratios were statistically significantly higher when compared to controls. At 800 ppm, mean liver to brain weight ratio of females was statistically significantly higher when compared to controls. These changes were associated with relevant histopathological findings and were considered to be treatment-related.

Some other organ weight differences were statistically significant, notably for the ovaries and the uterus. As these changes were not dose-related, they were considered to be incidental and not treatment-related.

Carcinogenicity phase:
At 4000 and 800 ppm in both sexes and at 150 ppm in males, mean absolute and relative liver weights were statistically significantly higher when compared to controls. At 150 ppm in females, mean liver to body weight ratio was statistically significantly higher. These changes were considered to be treatment-related.

Other organ weight differences were judged to be incidental in view of their individual variation.

See Tables 6 and 7 for liver weight data. See Attachment 3 for mean organ weight data from the interim and carcinogenicity phases.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Interim sacrifice phase:
Unscheduled deaths - Gallbladder stones were observed in 1/5 males treated at 800 ppm and were considered to be treatment-related. The few other gross observations were considered as incidental and not treatment-related.

Interim sacrifice - Enlarged liver was observed in 1/8 and 1/10 females treated at 4000 and 150 ppm, respectively. This finding was correlated with centrilobular hepatocellular hypertrophy in the female at 4000 ppm and was considered to be treatment-related. Gallbladder stones were found in both sexes at all dose levels.

Carcinogenicity phase:
Unscheduled deaths - Enlarged liver was found in some treated animals. This finding was correlated with centrilobular hepatocellular hypertrophy at the microscopic examination in the male from the high dose group and was considered to be treatment-related. Dark liver was found in some animals and was correlated with centrilobular hepatocellular hypertrophy in animals treated at 4000 ppm and in the male treated at 800 ppm. This finding was thus considered to be treatment-related.
Gallbladder stones were observed in some treated animals. This finding was considered to be treatment-related.

Terminal sacrifice - A higher incidence of gallbladder stones (multiple, brown, grey or black colored, pinpoint or up to 0.1 cm in diameter) was observed, which were statistically significant in all treated groups when compared to controls. This change was considered to be treatment-related.
Enlarged liver was observed in treated males at 4000 and 800 ppm. A higher incidence of enlarged liver was observed in treated females at all dose levels when compared to controls and was dose-related. Dark liver was noted at all dose levels in males and at 4000 and 800 ppm in females. These changes were often correlated with centrilobular hepatocellular hypertrophy at the microscopic examination and were considered to be treatment-related.
In treated males at 4000 ppm, statistically significantly higher incidence of prominent lobulation in the liver, abnormal shape of the kidney and pale kidney were noted when compared to controls. In treated females at 4000 ppm and in both sexes at 800 ppm, statistically significantly higher incidence of enlarged spleen was noted when compared to controls. In treated females at 4000 ppm, statistically significantly higher incidence of dilatation of uterine horn(s) was noted when compared to controls. All these macroscopic changes were not correlated with relevant histopathological findings and were thus considered not to be treatment-related.

Gross pathology data is presented in Tables 8 - 10. See Attachment 4 for a summary of gross pathology findings.

Neuropathological findings:

not examined

Description (incidence and severity):

Not applicable.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Interim sacrifice:
Unscheduled deaths - No treatment-related lesion was responsible for the unscheduled deaths. Slight centrilobular hepatocellular hypertrophy was noted in one female at 4000 ppm .

Scheduled interim sacrifice - Treatment-related effects were found in the liver and gallbladder. Minimal to slight centrilobular hepatocellular hypertrophy was observed in both sexes at 4000 and 800 ppm and in males at 150 ppm. This finding was associated with higher liver weights and enlarged liver at necropsy. At 4000 ppm in females, a higher incidence of diffuse hepatocellular vacuolation concomitant with a lower incidence of mainly periportal hepatocellular vacuolation was noted. This finding was judged to be correlated with the presence of centrilobular hepatocellular hypertrophy.

Carcinogenicity phase:
Treatment-related effects were found in the liver, the gallbladder and the kidney.

Liver - Minimal to moderate centrilobular hepatocellular hypertrophy was observed in both sexes at all dose levels, with evidence of a dose-response. This finding was associated with higher liver weights and partly enlarged or dark liver at necropsy. Minimal to moderate hepatocellular single cell necrosis was noted in both sexes at 4000 ppm and in females at 800 ppm. A loss of diffuse hepatocellular vacuolation was statistically significantly observed in males at 4000 and 800 ppm, when compared to controls. At 4000 ppm in females, a higher incidence of diffuse hepatocellular vacuolation concomitant with a lower incidence of mainly periportal hepatocellular vacuolation was noted. All these changes were judged to be correlated with the presence of a gradual dose-related hepatocellular hypertrophy.

Gallbladder - Histological confirmation of macroscopic gallstones was made in both sexes at all dose levels. The higher incidence of gallstones was statistically significant at all dose levels when compared to controls. A statistically significantly higher incidence and severity of eosinophilic cytoplasmic alteration was observed in females at all dose levels when compared to controls.

Kidney - The presence of unilateral or bilateral collecting duct hyperplasia, pelvic epithelium hyperplasia and papillary necrosis was observed at all dose levels in males, with evidence of a dose-related response. Unilateral or bilateral collecting duct hyperplasia was observed in males at all dose levels. The higher incidences were found to be statistically significant (unilateral, bilateral or combined unilateral / bilateral). Unilateral or bilateral pelvic epithelium hyperplasia was observed in males at all dose levels. The higher incidences were found to be statistically significant when it was unilateral (4000 ppm and 800 ppm) or combined unilateral / bilateral (all doses). A statistical positive trend towards a higher incidence of bilateral pelvic epithelium hyperplasia was noted (p≤0.05). Unilateral or bilateral papillary necrosis was observed in males at all dose levels. The higher incidences were found to be statistically significant when it was unilateral or combined unilateral / bilateral at all dose levels. A statistical positive trend towards a higher incidence of bilateral papillary necrosis was noted (p≤0.05). These findings were considered to be treatment-related.

In the females at 4000 ppm, statistically significantly higher incidence of focal / multifocal interstitial mononuclear cell infiltrate was noted in the kidney when compared to controls. As this finding was not associated with other relevant microscopic findings in the kidney, it was considered to be without toxicological importance.

In the males at 4000 ppm, statistically significantly higher incidence of amyloid deposition in the parathyroid gland was noted when compared to controls. As amyloid deposition was significantly observed in various organs (liver, kidney, lung, thyroid gland, adrenal gland, heart, spleen, stomach, caecum, mesenteric lymph node, jejunum, ileum, colon) with a comparable incidence in control and treated animals, it was considered to be incidental and not treatment-related.

In the females at 4000 ppm, statistically significantly higher incidence of lactation / dilated ducts in the mammary gland was noted when compared to controls. As this finding was not associated with higher incidence of mammary gland hyperplasia or neoplasia and was not associated with relevant pituitary gland findings, it was considered not to be toxicologically relevant. In addition, this finding was within the range of the laboratory's historical data (see Attachment 5) and was thus considered to be without toxicological significance.

In the males, statistically significantly higher incidence of focal / multifocal fibro-osseous lesion in the spinal cord at all doses and in the articular surface at 4000 ppm was noted when compared to controls. This finding was slightly outside of the range of the laboratory's historical data (see Attachment 5). However, as this finding was observed with a low incidence and severity (minimal to slight), with a focal / multifocal distribution rather than diffuse and given that there was no higher incidence of fibro-osseous lesion in the sternum, it was considered not to be toxicologically relevant.

Histopathology data is presented in Tables 11 - 15.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

There was no effect of treatment on an earlier development or an increased incidence of tumors. The tumors in treated animals were those commonly observed in this strain and age of mouse kept under monitored environmental conditions and were considered to be incidental in origin.

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Relevance of carcinogenic effects / potential:

No treatment-related neoplastic changes were observed at any dose level tested in either sex.

Key result

Dose descriptor:

NOEL

Remarks:

carcinogenicity

Effect level:

4 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no carcinogenicity observed up to the highest dose tested

Remarks on result:

other: equivalent to 583 mg/kg bw/day (males) and 743 mg/kg bw/day (females)

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

< 150 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Adverse effects seen at all dose levels tested.

Remarks on result:

other: equivalent to 21.0 mg/kg bw/day (males) and 27.1 mg/kg bw/day (females)

Key result

Dose descriptor:

LOAEL

Remarks:

systemic

Effect level:

150 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Remarks on result:

other: equivalent to 21.0 mg/kg bw/day (males) and 27.1 mg/kg bw/day (females)

Critical effects observed:

yes

Lowest effective dose / conc.:

150 ppm

System:

hepatobiliary

Organ:

gall bladder

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Critical effects observed:

yes

Lowest effective dose / conc.:

150 ppm

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Clinical Signs

Table 1: Incidence of treatment-related clinical signs

Sex	Male				Female
Dose level (ppm)	0	150	800	4000	0	150	800	4000
Group size	60	60	60	60	60	60	60	60
Intense yellow colored urine	0	0	0	60**	0	0	0	58**
Prolapsed rectum	0	0	0	0	0	5*	3	8**
Soiled fur	0	0	1	3	1	1	2	0
Ocular discharge	1	4	3	8*	2	0	1	0
Hunched posture	0	5*	5*	5*	1	2	2	0
Wasted appearance	0	6*	5*	7**	0	3	5*	1
Dental abnormality	1	5	3	7*	5	2	3	1

  * p≤0.05
** p≤0.01

Mortality Incidence

Table 2: Mortality incidence up to Week 53

Dose level (ppm)	First 53 weeks (up to study Day 371)
	Male	Female
0	5/60	2/60
	(8%)	(3%)
150	9/60	6/60
	(15%)	(10%)
800	8/60	3/60
	(13%)	(5%)
4000	5/60	7/60
	(8%)	(12%)

Percentage mortality in parentheses

Table 3: Mortality incidence, whole study period (carcinogenicity phase)

Dose level (ppm)	Whole study period Carcinogenicity phase animals
	Male	Female
0	9/50	4/50
	(18%)	(8%)
150	25/50**	8/50
	(50%)	(20%)
800	14/50	9/50
	(28%)	(18%)
4000	19/50	8/50
	(38%)	(16%)

Percentage mortality in parentheses.
** p≤0.01
The statistical significance corresponding to the pairwise comparison between each treated group and the control group using survival analyses is presented in the above table.

Body Weights

Table 4: Group mean body weight (g)

Sex	Males				Females
Dose level (ppm)	0	150	800	4000	0	150	800	4000
Week 0 (% control)	20.5 -	20.5 (100)	20.6 (100)	20.6 (100)	16.8 -	16.9 (101)	16.7 (99)	16.7 (99)
Week 1 (% control)	22.3 -	22.3 (100)	22.5 (101)	20.8** (93)	18.7 -	19.1 (102)	18.6 (99)	17.2**(92)
Week 4 (% control)	23.6 -	23.5 (100)	23.5 (100)	23.0** (97)	19.0 -	19.5* (103)	19.4 (102)	18.5* (97)
Week 13 (% control)	27.8 -	27.6 (99)	27.2* (98)	26.7** (96)	22.1 -	22.6* (102)	22.7** (103)	22.3 (101)
Week 49 (% control)	31.8 -	31.0* (97)	30.9* (97)	29.9** (94)	25.8 -	26.7* (103)	27.6** (107)	25.0* (97)
Week 77 (% control)	32.4 -	31.8 (98)	30.6** (94)	30.3** (94)	28.1 -	27.4 (98)	28.0 (100)	25.6** (91)

* p≤0.05

** p≤0.01

Haematology

Table 5: Haematology parameters at study termination (Months 18 - 19)

Dose level (ppm)	4000
Sex	Males	females
Red blood cell count	-13% p≤0.01	-17% p≤0.01
Haemoglobin concentration	-13% p≤0.01	-14% p≤0.01
Haematocrit	-12% p≤0.01	-12% p≤0.01

Organ Weights

Table 6: Liver weight changes, interim sacrifice phase (% change when compared to controls)

Sex	Males			Females
Dose level (ppm)	150	800	4000	150	800	4000
Mean absolute liver weight	NC	+8% NS	NC	+11% NS	+20% NS	+15% NS
Mean liver to body weight ratio	+8% p≤0.05	+10% p≤0.05	+16% p≤0.01	NC	+11% p≤0.05	+19% p≤0.01
Mean liver to brain weight ratio	NC	+9% NS	+8% NS	+12% NS	+21% p≤0.05	+19% NS

NC = no relevant change

NS = not statistically significant

Table 7: Liver weight changes, carcinogenicity phase (% change when compared to controls)

Sex	Males			Females
Dose level (ppm)	150	800	4000	150	800	4000
Mean absolute liver weight	+8% p≤0.01	+12% p≤0.01	+16% p≤0.01	NC	+10% p≤0.01	+8% p≤0.05
Mean liver to body weight ratio	+10% p≤0.01	+16% p≤0.01	+22% p≤0.01	+8% p≤0.01	+9% p≤0.01	+17% p≤0.01
Mean liver to brain weight ratio	+8% p≤0.05	+13% p≤0.01	+19% p≤0.01	NC	+10% p≤0.01	+10% p≤0.01

NC = no relevant change

NS = not statistically significant

Gross Pathology

Interim Sacrifice:

Table 8: Gross pathology – interim sacrifice

Sex	Males				Females
Dose level of (ppm)	0	150	800	4000	0	150	800	4000
Gallbladder stone(s)	1/10	3/7	2/5	2/9	0/9	3/10	4/10	3/8

Carcinogenicity Phase:

Table 9: Gross pathology – unscheduled deaths

Sex	Males				Females
Dose level of (ppm)	0	150	800	4000	0	150	800	4000
Gallbladder stone(s)	0/9	3/25	0/14	4/19	0/4	2/10	2/9	0/8
Enlarged liver	0/9	0/25	0/14	1/19	0/4	1/10	3/9	0/8
Dark liver	0/9	1/25	1/14	3/19	0/4	1/10	0/9	1/8

Table 10: Gross pathology – terminal sacrifice

Sex	Males				Females
Dose level of (ppm)	0	150	800	4000	0	150	800	4000
Gallbladder stone(s)	3/41	13/25**	21/36**	14/31**	2/46	19/40**	31/41**	25/42**
Enlarged liver	0/41	0/25	4/36	4/31	3/46	8/40	12/41	17/42
Dark liver	0/41	1/25	2/36	4/31	0/46	0/40	2/41	2/42

**p≤0.01

Histopathology

Interim sacrifice:

Table 11: Incidence and severity of treatment-related microscopic changes in the liver, interim sacrifice

Sex	Males				Females
Dose level (ppm)	0	150	800	4000	0	150	800	4000
Number of animals examined	10	7	5	9	9	10	10	8
Centrilobular hepatocellular hypertrophy: diffuse
Minimal	0	3	3	5	0	0	4	5
Slight	0	0	0	4	0	0	1	2
Total	0	3	3*	9**	0	0	5*	7**
Hepatocellular vacuolation: diffuse
Minimal	9	7	4	8	2	1	2	5
Slight	1	0	1	0	0	0	0	0
Total	10	7	5	8	2	1	2	5
Hepatocellular vacuolation: mainly periportal: diffuse
Minimal	0	0	0	0	6	7	7	1
Slight	0	0	0	0	1	1	1	0
Total	0	0	0	0	7	8	8	1

*p≤0.05
**p≤0.01

Table 12: Incidence and severity of treatment-related microscopic changes in the gallbladder, interim sacrifice

Sex	Males				Females
Dose level (ppm)	0	150	800	4000	0	150	800	4000
Number of animals examined	8	6	5	7	8	9	9	7
Gallstones
Minimal	1	2	0	0	0	1	1	1
Slight	0	0	1	0	0	0	1	0
Total	1	2	1	0	0	1	2	1
Stones only noted at necropsy
Present	0	2	2	2	0	2	3	2
Total incidence of animals with gallstones^1
Total	1	4	3	2	0	3	5*	3

¹including stones only noted at necropsy and those found only after histological examination.
*p≤0.05

Table 13: Incidence and severity of treatment-related microscopic changes in the liver, all animals, carcinogenicity phase

Sex	Males				Females
Dose level (ppm)	0	150	800	4000	0	150	800	4000
Number of animals examined	49	50	49	50	50	49	50	50
Centrilobular hepatocellular hypertrophy: diffuse
Minimal	0	11	31	5	0	19	20	9
Slight	0	1	9	34	0	2	20	29
Moderate	0	0	0	9	0	0	0	11
Total	0	12**	40**	48**	0	21**	40**	49**
Hepatocellular single cell necrosis: focal/multifocal
Minimal	0	0	0	2	0	0	6	20
Slight	0	0	0	3	0	0	0	5
Moderate	0	0	0	1	0	0	0	0
Total	0	0	0	6*	0	0	6*	25**
Hepatocellular vacuolation: diffuse
Minimal	15	10	6	10	5	9	6	27
Slight	10	2	5	2	8	5	9	9
Moderate	0	0	1	0	0	1	0	0
Total	25	12	12**	12*	13	15	15	36**
Hepatocellular vacuolation: mainly periportal: diffuse
Minimal	0	1	1	1	2	1	4	2
Slight	1	1	0	0	23	22	13	1
Moderate	0	0	0	0	9	2	5	0
Total	1	2	1	1	34	25	22*	3**

*p≤0.05
**p≤0.01

Table 14: Incidence and severity of treatment-related microscopic changes in the gallbladder, all animals, carcinogenicity phase

Sex	Males				Females
Dose level (ppm)	0	150	800	4000	0	150	800	4000
Number of animals examined	49	48	47	49	46	49	48	50
Gallstones
Minimal	3	11	17	12	0	17	20	18
Slight	0	10	7	5	0	5	10	13
Moderate	0	0	0	1	0	4	3	4
Total	3	21**	24**	18**	0	26**	33**	35**
Eosinophilic cytoplasmic alteration: focal/multifocal
Minimal	16	13	11	11	11	11	13	20
Slight	7	14	14	13	2	18	15	11
Moderate	1	1	2	3	0	1	3	3
Total	24	28	27	27	13	30**	31**	34**

**p≤0.01

Table 15: Incidence and severity of treatment-related microscopic changes in the kidney, all animals, carcinogenicity phase

Sex	Males				Females
Dose level (ppm)	0	150	800	4000	0	150	800	4000
Number of animals examined	50	50	49	50	50	49	50	50
Collecting ducts hyperplasia: unilateral
Minimal	0	3	0	2	0	1	0	0
Slight	0	1	3	5	0	0	0	0
Total	0	4*	3	7**	0	1	0	0
Collecting ducts hyperplasia: bilateral
Minimal	0	1	1	1	0	0	0	0
Slight	0	1	3	7	0	0	0	0
Moderate	0	3	4	4	0	0	0	0
Total	0	5*	8**	12**	0	0	0	0
Collecting duct hyperplasia: unilateral/bilateral
Minimal	0	4	1	3	0	1	0	0
Slight	0	2	6	12	0	0	0	0
Moderate	0	3	4	4	0	0	0	0
Total	0	9**	11**	19**	0	1	0	0
Pelvic epithelium hyperplasia: unilateral: focal/multifocal
Minimal	0	4	7	6	0	0	0	0
Total	0	4	7**	6*	0	0	0	0
Pelvic epithelium hyperplasia: bilateral: focal/multifocal
Minimal	0	1	1	2	0	0	0	0
Slight	0	0	2	1	0	0	0	0
Total	0	1	3	3	0	0	0	0
Pelvic epithelium hyperplasia: unilateral/bilateral: focal/multifocal
Minimal	0	5	8	8	0	0	0	0
Slight	0	0	2	1	0	0	0	0
Total	0	5*	10**	9**	0	0	0	0
Papillary necrosis: unilateral: focal/multifocal
Minimal	0	1	1	4	0	0	0	0
Slight	0	0	3	4	0	0	0	0
Moderate	0	2	4	2	0	0	0	0
Marked	0	1	0	0	0	0	0	0
Total	0	4*	8**	10**	0	0	0	0
Papillary necrosis: bilateral: focal/multifocal
Minimal	0	0	0	1	0	0	0	0
Slight	0	2	1	0	0	0	0	0
Moderate	0	0	2	2	0	0	0	0
Marked	0	0	0	1	0	0	0	0
Total	0	2	3	4	0	0	0	0
Papillary necrosis: unilateral/bilateral: focal/multifocal
Minimal	0	1	1	5	0	0	0	0
Slight	0	2	4	4	0	0	0	0
Moderate	0	2	6	4	0	0	0	0
Marked	0	1	0	1	0	0	0	0
Total	0	6**	11**	14**	0	0	0	0

*p≤0.05
**p≤0.01

Conclusions:

The study was performed under GLP conditions and according to OECD TG 451. No treatment-related neoplastic changes were observed at any dose level tested in either sex. Thus, under the conditions of this study the NOEL in terms of carcinogenic potential was 4000 ppm for male and female mice (equivalent to 583 and 743 mg/kg bw/day for males and females, respectively). A NOAEL for general toxicity could not be established due to findings noted in liver and gallbladder of both sexes and kidney in males at all dose levels. The LOAEL was therefore determined to be 150 ppm (equivalent to 21.0 and 27.1 mg/kg bw/day in males and females, respectively).

Reference 2

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 Oct 2002 - 22 Dec 2005

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 12 May 1981

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Version / remarks:

adopted 25 Jun 2018

Deviations:

yes

Remarks:

no details on whether feed and water were analysed for contaminants

GLP compliance:

yes (incl. QA statement)

Remarks:

INTERMINISTERIAL GROUP FOR CHEMICAL PRODUCTS, Paris, France

Species:

rat

Strain:

other: Wistar Rj:WI (IOPS HAN)

Sex:

male/female

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): A04C-10P1
- Storage temperature of food: 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 R146) in the diet was demonstrated in a previous study (2002) at concentrations of 1.25 and 15000 ppm for at least 59 days under frozen storage followed by 11 days storage at room temperature. The stability of the test substance (batch number PFI 0226) was also checked during the present study at concentrations of 2 and 5000 ppm for at least 77 days storage at room temperature.
The homogeneity of the test substance in the diet was verified for all first loads of the formulation F1, for the extreme concentrations on the first loads (2 and 5000 ppm) of the formulations F6, F13, F18 and F24, on the first load at 50 ppm of the formulation F6ter and on the first load at 2 ppm of the formulations F16bis and F17 to demonstrate adequate formulation procedures. 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 results obtained from the homogeneity checks on the first loads were taken as measured concentration. Additionally, during the study, the concentration of all loads at each dietary level was verified (if not submitted for homogeneity check) prior to administration to the animals for the formulations F1, F2, F3, F6, F9, F13, F15, F16, F18, F21 and F24. The concentration of the preparations was also checked on the second loads of all supplemental preparations made for one week administration, on loads 2 to 4 at 50 ppm of the formulation F6ter, and on loads 2 to 4 at 2 ppm of the formulation F17.

Duration of treatment / exposure:

104 weeks

Frequency of treatment:

continuously (via diet)

Dose / conc.:

2 ppm

Remarks:

equivalent to approximately 0.08 and 0.11 mg/kg bw/day in males and females, respectively

Dose / conc.:

50 ppm

Remarks:

equivalent to approximately 2.03 and 2.83 mg/kg bw/day in males and females, respectively

Dose / conc.:

1 500 ppm

Remarks:

equivalent to approximately 62.4 and 88.6 mg/kg bw/day in males and females, respectively

Dose / conc.:

5 000 ppm

Remarks:

equivalent to approximately 214 and 296 mg/kg bw/day in males and females, respectively

No. of animals per sex per dose:

50 (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 (2002).

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily (including weekends and public holidays)
- 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), except on Week 21 for females where physical examination was done twice for males in error.

BODY WEIGHT: Yes
- Time schedule for examinations: on Days 3, 7 and 10 during the acclimatisation 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 and at Month 4, 5, 12, 24 and at the end of reversibility phase
- Dose groups that were examined: all animals (acclimatization phase), all surviving animals (4, 5, 12, 24 months and after reversibility phase)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: all animals: Weeks 13 - 14, 22 - 24, 51 - 53; recovery group: on Week 69 (Day 91 of recovery phase); terminal sacrifice: Weeks 78 and 105 - 107; 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 (recovery group and 24 month terminal sacrifice)
- Parameters examined: Red blood cell count, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, reticulocyte count, white blood cell total and differential counts and platelet count, blood smears, prothrombin time and activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: all groups: Weeks 13 - 14, 22 - 24, 51 - 53; recovery group: on Week 69 (Day 91 of recovery phase); terminal scrifice group: Weeks 78 and 105 - 107; puncture of the retro orbital venous plexus
- Animals fasted: Yes
- How many animals: all surviving animals (12 month interim sacrifice), first ten surviving animals (recovery group and 24 month terminal sacrifice)
- Parameters examined: Total bilirubin, glucose, urea, creatinine, total cholesterol, triglycerides, chloride, sodium, potassium, calcium and inorganic phosphorus concentrations, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and gamma-glutamyltransferase activities, total protein and albumin
URINALYSIS: Yes
- Time schedule for collection of urine: all groups: Weeks 11 - 12, 25 - 26 and 49 - 50; recovery groups: Week 68 (day 87 of recovery phase); terminal sacrifice group: Week 77 - 79 and 104
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
- Parameters examined: appearance, volume, pH, urinary refractive index, glucose, bilirubin, ketone bodies, occult blood, protein, urobilinogen, urinary sediment (microscopic exmination for grading of: red blood cells, white blood cells, epithelial cells, bacteria, casts and crystals)

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 1)
On Days 729 to 752 of the dosing phase for the terminal sacrifice, all surviving animals were sacrificed by exsanguination under deep anesthesia (pentobarbital, intraperitoneal injection of 60 mg/kg bw). At termination, males were sacrificed prior to females due to the higher mortality rate in this sex. Consequently, an approximately equal number of males or females randomly distributed amongst the groups were sampled on each day taking into account the mortality. Animals were diet fasted overnight prior to sacrifices. All animals, including animals either found dead or killed for humane reasons, were necropsied. The necropsy included the examination of all major organs, tissues and body cavities. The organs or tissues checked in table 1 were sampled and/or weighed. All significant macroscopic abnormalities (including masses and their regional lymph nodes when possible) were recorded, sampled and examined microscopically. Samples were fixed by immersion in neutral buffered 10% formalin with the exception of the eye, optic nerve, Harderian gland, epididymis and testis that were fixed in Davidson's fixative. 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.


HISTOPATHOLOGY: Yes (see table 1)
Samples (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 (whether or not a tumor was 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 not significant (p>0.05), the group means were considered homogeneous and no further analysis was performed. If the ANOVA was significant (p≤0.05), means of the exposed groups were compared to the mean of the control group using the Dunnett test (2-sided). If the Bartlett test was significant (p≤0.05), group means were compared using the Kruskal-Wallis test. If the Kruskal-Wallis test was not significant (p>0.05), group means were considered homogeneous and no further analysis was performed. If the Kruskal-Wallis test was significant (p≤0.05), means of the exposed groups were compared to the mean of the control group using the Dunn test (2-sided). If the Bartlett test was significant for body weight and average food consumption/day parameters 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.
Individual statistic methods used for specific parameters are descibed in `Any other information on materials and methods´.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Carcinogenicity phase:
MALES
50 ppm: focal swelling (10/48; principally of hindlimb), reduced motor activity (12/48) and white area on eyes (41/48)
1500 ppm: focal swelling (21/45; principally of hindlimb), white area on eyes (45/45) and reduced motor activity (9/45; not statistically significant)
5000 ppm: focal swelling (16/42; principally of hindlimb), reduced motor activity (11/42), skin lesions (13/42) and white area on eyes (41/42)

FEMALES
50 ppm: white area on eyes (42/50)
1500 ppm: white area on eyes (45/49)
5000 ppm: white area on eyes (38/49), chromodacryorrhea (10/49) and soiled anogenital region (6/49)

For more details see table 2 in section "any other information on results incl. tables".

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

Not applicable.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Cummulative mortality incidence after 2 years:
MALES
0 ppm: 29/50 animals after 2 years
2 ppm: 33/50 animals after 2 years
50 ppm: 40/50 animals after 2 years
1500 ppm: 34/50 animals after 2 years
5000 ppm: 33/50 animals after 2 years

FEMALES
0 ppm: 23/50 animals after 2 years
2 ppm: 27/50 animals after 2 years
50 ppm: 14/50 animals after 2 years
1500 ppm: 17/50 animals after 2 years
5000 ppm: 13/50 animals after 2 years

For more details see table 3 in section "any other information on results incl. tables".

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

MALES
2 ppm: Mean body weight and body weight gain were comparable to the controls.
50 ppm: Compared to control mean body weight and mean body weight gain were slightly lower by up to 11 and 17% (p<0.01 or p<0.05), respectively.
1500 ppm: Compared to control mean body weight and mean body weight gain were slightly lower by up to 12 and 17% (p<0.01 or p<0.05), respectively.
5000 ppm: Compared to the control mean body weight and mean body weight gain were lower by 12 and 17% (p<0.01 or p<0.05), respectively.

FEMALES:
2 ppm: Mean body weight and body weight gain were comparable to the controls.
50 ppm: Compared to control mean body weight and mean body weight gain were slightly lower by up to 8 and 12% (p<0.01 or p<0.05), respectively.
1500 ppm: Compared to control mean body weight and mean body weight gain were slightly lower by up to 16 and 23% (p<0.01 or p<0.05), respectively.
5000 ppm: Compared to the control mean body weight and mean body weight gain were lower by 16 and 23% (p<0.01 or p<0.05), respectively.

For more details see tables 4 and 5 in section "any other information on results incl. tables".

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Mean food consumption was similar to controls during the second year of the study in both sexes and at all dose levels. The few minor increases (reaching statistical significance) in males at 5000 ppm were considered to reflect inter-individual variation rather than a treatment-related effect.

Food efficiency:

not examined

Description (incidence and severity):

Not applicable.

Water consumption and compound intake (if drinking water study):

not examined

Description (incidence and severity):

Not applicable.

Ophthalmological findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of 2 years of exposure, in the 50, 1500 and 5000 ppm dose groups the incidence of corneal opacity, neovascularization and oedema of the cornea and snow flake-like corneal opacity (not observed in males at 50 and 15000 ppm) was statistically elevated in both sexes.
The type of corneal lesions observed in the rat (together with the eye keratitis noted at the microscopic examination), are based on characteristics of the compound that inhibits 4-hydroxyphenylpyruvic acid dioxygenase (4-HPPDase), an enzyme of the tyrosine catabolic pathway. These lesions are related to an increase in plasma tyrosine level caused by a blockade of the 4-HPPDase enzyme in the rat. However, the rat is a species particularly sensitive to inhibition of the 4-HPPDase enzyme and is atypical in its susceptibility to develop tyrosine-related eye lesions. Therefore, although these lesions (corneal lesions and eye keratitis) were treatment-related, they were considered not to be toxicologically relevant to man.

For more details see table 6 in section "any other information on results incl. tables".

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Second year:
MALES: Increased mean total cholesterol concentrations (+36%; p<0.05) at 5000 ppm after 18 month but reversible within 24 month

FEMALES: Increased mean total cholesterol concentrations (+27%; p<0.05) at 5000 ppm after 18 month but reversible within 24 month

Endocrine findings:

not examined

Description (incidence and severity):

Not applicable.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

MALES: A tendency towards higher ketone level at all sampling periods at 50, 1500 and 5000 ppm; lower mean pH values at Months 18 and 24 (50, 1500 and 5000 ppm); higher protein levels and lower amounts of crystals at Month 18 only (50, 1500 and 5000 ppm)

FEMALES: Higher ketone level at all sampling periods at 50, 1500 and 5000 ppm

Behaviour (functional findings):

not examined

Description (incidence and severity):

Not applicable.

Immunological findings:

not examined

Description (incidence and severity):

Not applicable.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

MALES: higher mean abosolute and/or relative liver and kidney weights (statistically significant for most parameters) at 50,1500 and 5000 ppm compared to control; decreased mean absolute brain weight at 50, 1500 and 5000 ppm (not toxicological relevant since they were observed for one parameter only, were opposite in both sexes and were not associated with any change at the macroscopic and microscopic examinations)

FEMALES: higher mean abosolute and relative liver weights at 1500 and 5000 ppm compared to control; lower absolute and mean kidney to brain weights at 1500 ppm (non-treatment related since no dose-relationship); decresed ovary weights at 5000 ppm (this increase was attributable to a bilateral mass noted on ovary of this female, which contributed by itself to the increase in ovary weights in this group) and 50 and 1500 ppm (not toxicological relevant since it was observed for this parameter only and was not associated with any change at the macroscopic and microscopic examinations); increased brain to body weight at 1500 and 5000 ppm (not toxicological relevant since they were observed for one parameter only, were opposite in both sexes and were not associated with any change at the macroscopic and microscopic examinations)

For more details see table 7 in section "any other information on results incl. tables".

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- Unscheduled death:
MALES:
50 ppm: eye opacity
1500 ppm: eye opacity
5000 ppm: eye opacity, pale appearance of animals

FEMALES
1500 ppm: eye opacity
5000 ppm: irregular surface of kidney, skin allopecia (not toxicological relevant since no relevant microscopic changes occured)

- Terminal sacrifice:
MALES
50 ppm: eye opacity, irregular surface on kidney
1500 ppm: eye opacity, irregular surface on kidney (not statistically significant)
5000 ppm: eye opacity, irregular surface on kidney

FEMALES
50 ppm: eye opacity, enlarged pituitary gland (not toxicologically relevant since no dose-relationship)
1500 ppm: eye opacity
5000 ppm: eye opacity, enlarged pituitary gland (not toxicologically relevant since no dose-relationship)

For more details see tables 8 and 9 in section "any other information on results incl. tables".

Neuropathological findings:

not examined

Description (incidence and severity):

Not applicable.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

MALES:
Kidney: minimal to severe chronic progressive nephropathy at 50, 1500 and 5000 ppm (no relevance for human since age related, spontaneously occuring in rats and a high incidence in control gorup)

Pancreas: minimal to moderate interstitial inflammation (focal/multifocal) in the pancreas at 1500 and 5000 ppm (not toxicologically relevant since no real difference in severity to controls); minimal to severe acinar atrophy/fibrosis (focal/multifocal) at 1500 and 5000 ppm (statistically significant)

Thyroid gland: colloid alteration and brown pigment in the cytoplasm of the thyroid follicular cells (not toxicologically relevant since similar alterations are present in control rats, indicating that these changes likely reflect a normal physiologic process associated with the unique rapid turnover of colloid) and follicular cell hypertrophy at 50, 1500 and 5000 ppm

Eye: marked keratitis at 50, 1500 and 5000 ppm (not toxicological relevant to human, see comment at chronic phase); atrophy/degeneration (multifocal/diffuse) in sciatic nerve at 50, 1500 and 5000 ppm (non-treatment related since high incidence in control groups and no dose-dependency)

Skeletal muscle: minimal to marked myofiber atrophy/degeneration at 50, 1500 and 5000 ppm (non-treatment related since secondary effect from sciatic nerve atrophy),

Hindleg/hindpawn: ulcerative necrotising inflammation at 1500 and 5000 ppm (indirect effect with minor toxicological relevance)

Liver: angiectasis (focal/multifocal) and extramedullary hematopoiesis (multifocal) at 1500 and 5000 ppm (not toxicologically relevant since age related and no dose-dependency); minimal to slight centrilobular hepatocellular hypertrophy (diffuse) at 1500 ppm (adaptive response)

Brain: minimal to slight mineralization (focal/multifocal) at 5000 ppm (non-treatment-related since also observed in control groups and within range of historical control data)

Pituitary gland: cyst(s)/pseudocyst(s) in pars distalis at 5000 ppm (not toxicologically relevant since it was a spontaneous finding)

Urinary bladder: proteinaceous material at 5000 ppm of animals found dead (not toxicologically relevant since it is a common finding and was obtained in isolation for this parameter)

FEMALES:
Pancreas: minimal to moderate interstitial inflammation (focal/multifocal) at 50, 1500 (not significant) and 5000 ppm (not toxicologically relevant since no real difference in severity to controls); minimal to severe acinar atrophy/fibrosis (focal/multifocal) at 2 (within range of historical data), 50, 1500 and 5000 ppm

Thyroid gland: colloid alteration, brown pigment in the cytoplasm of the thyroid follicular cells (not toxicologically relevant since similar alterations are present in control rats, indicating that these changes likely reflect a normal physiologic process associated with the unique rapid turnover of colloid) and follicular cell hypertrophy at 50, 1500 and 5000 ppm

Eye: marked keratitis at 50, 1500 and 5000 ppm (not toxicological relevant to human, see comment at chronic pahse); atrophy/degeneration (multifocal/diffuse) in sciatic nerve at 1500 ppm (non-treatment related since high incidence in control groups and no dose-dependency)

Skeletal muscle: minimal to marked myofiber atrophy/degeneration at 1500 and 5000 ppm (non-treatment related since secondary effect from sciatic nerve atrophy)

Liver: bile duct hyperplasia (focal/multifocal) at 50, 1500 and 5000 ppm (not toxicologically relevant since age related and no dose-dependency)

Ovaries: minimal to marked diffuse atrophy at 5000 ppm (non-treatment-related since within the range of historical control data)

For more details see table 10 in section "any other information on results incl. tables".

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Relevance of carcinogenic effects / potential:

No treatment-related neoplastic changes were observed at any dose level tested in either sex.

Key result

Dose descriptor:

NOEL

Remarks:

carcinogenicity

Effect level:

5 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no carcinogenicity observed up to the highest dose tested

Remarks on result:

other: equivalent to approximately 214 and 296 mg/kg bw/day over the 24-month period in males and females, respectively

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity 24 month

Effect level:

2 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at this dose level

Remarks on result:

other: equivalent to 0.08 and 0.11 mg/kg bw/day for males and females, respectively

Key result

Dose descriptor:

LOAEL

Remarks:

systemic toxicity 24 month

Effect level:

50 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: equivalent to 2.03 and 2.83 mg/kg bw/day for males and females, respectively

Table 2. Treatment-related clinical signs during carcinogenicity phase

	Males					Females
Dose level (ppm)	0	2	50	1500	5000	0	2	50	1500	5000
Second year (carcinogenicity phase)
Number of animals examined	21	17	10	16	17	27	22	36	33	36
Focal swelling	3	3	10*	21**	16**	nc	nc	nc	nc	nc
Reduced motor activity	5	6	12*	9	11*	nc	nc	nc	nc	nc
Skin lesions	7	10	6	9	13*	nc	nc	nc	nc	nc
White area on eye	3	3	41**	45**	41**	2	6	42**	45**	38**
Chromodacryorrhea	nc	nc	nc	nc	nc	1	1	3	4	10**
Soiled anogenital region	nc	nc	nc	nc	nc	0	0	0	3	6*

*•: p<0.05; **: p<0.01; nc: not concerned or no treatment-related change.

Table 3. Mortality incidence after 1 and 2 years of treatment (unscheduled deaths of animals)

	Second year of treatment (carcinogenicity phase)
Males
Number of animals	50	50	50	50	50
Mortality	29	33	40	34	33
Females
Number of animals	50	50	50	50	50
Mortality	23	27	14	17	13

Table 4. Body weights and body weight gains in males

Dose level [ppm]	0	2		50			1500		5000
	g	g	% of control		g	% of control	g	% of control	g	% of control
BODY WEIGHTS
Initial (Day 1)	218	218	100		217	100	216	99	215	99
Week 1 (Day 8)	276	278	101		278	101	270	98	261**	95
Week 13 (Day 92)	535	547	102		538	101	504**	94	500**	93
Week 25 (Day 176)	630	639	101		631	100	602*	96	585**	93
Week 49 (Day 344)	708	709	100		706	100	682	96	677*	96
Week 77(Day 372)	737	742	101		685*	93	664**	90	671**	91
Final (Day 708)	660	679	103		606	92	600*	91	630	95
BODY WEIGHT GAINS
Week 1 (Days 1 – 8)	58	61	105		61	105	54**	93	46**	79
Weeks 1 – 13 (Days 1 – 92)	318	329	103		320	101	288**	91	284**	89
Weeks 13 – 25 (Days 92 – 176)	95	91	96		94	99	96	101	86	91
Weeks 25 - 49 (Days 176 – 344)	79	73	92		75	95	81	103	92	116
Weeks 49 – 77 (Days 344 – 540)	38	36	95		-17**	nc	-11	nc	-11**	nc
Weeks 77 – 101 (Days 540 – 708)	-47	-54	115		-64	136	-67	143	-44	94
Overall (Days 1 – 708)	446	463	104		392	88	383	86	418	94

*: p≤0.05; **: p≤0.01; nc: not calculated

 

Table 5. Body weights and body weight gains in females

Dose level [ppm]	0	2		50			1500		5000
	g	g	% of control		g	% of control	g	% of control	g	% of control
BODY WEIGHTS
Initial (Day 1)	162	162	100		162	100	162	100	163	101
Week 1 (Day 8)	190	190	100		189	99	185*	97	182**	96
Week 13 (Day 92)	291	291	100		289	99	280**	96	282*	97
Week 25 (Day 176)	327	324	99		325	99	317*	97	318*	97
Week 49 (Day 344)	363	356	98		359	99	345*	95	341**	94
Week 77(Day 372)	433	428	99		412	99	385**	89	393**	91
Final (Day 708)	474	483	102		438	92	405**	85	401**	85
BODY WEIGHT GAINS
Week 1 (Days 1 – 8)	28	28	100		26	93	23**	82	20**	71
Weeks 1 – 13 (Days 1 – 92)	129	128	99		127	98	118**	91	119**	92
Weeks 13 – 25 (Days 92 – 176)	37	33	89		36	97	37	100	36	97
Weeks 25 - 49 (Days 176 – 344)	36	33	92		34	94	27	75	23*	64
Weeks 49 – 77 (Days 344 – 540)	71	69	97		52	73	43**	61	45**	63
Weeks 77 – 101 (Days 540 – 708)	42	52	124		32	76	28	67	13**	31
Overall (Days 1 – 708)	309	321	104		276	89	245**	79	239**	77

*: p≤0.05; **: p≤0.01; nc: not calculated

Table 6. Ophthalmological findings

	Males					Females
Dose level (ppm)	0	2	50	1500	5000	0	2	50	1500	5000
Month 4 (Weeks 14 - 15)
Number of animals examined	75	60	60	59	63	75	60	60	59	75
Corneal opacity	0	0	12**	14**	8**	0	0	5*	7**	4
Neovascularization of the cornea	0	0	30**	38**	34**	1	1	30**	47**	23**
Oedema of the cornea	0	0	12**	12**	6**	1	2	1	1	2
Corneal opacity « snow flake »	0	0	22**	27**	29**	0	0	29**	41**	22**
Month 5 (Weeks 21 – 22)
Number of animals examined	75	60	59	59	63	75	60	60	59	75
Corneal opacity	0	0	23**	21**	22**	0	0	7**	25**	9**
Neovascularization of the cornea	0	0	33**	40**	39**	1	0	40**	50**	37**
Oedema of the cornea	0	0	23**	22**	22**	1	1	4	24**	9**
Corneal opacity « snow flake »	0	0	13**	22**	18**	0	0	35**	28**	32**
Month 12 (Weeks 50 – 51)
Number of animals examined	75	60	59	59	63	75	59	60	58	74
Corneal opacity	0	0	36**	37**	36**	1	1	21**	41**	28**
Neovascularization of the cornea	0	0	44**	45**	49**	1	1	46**	53**	50**
Oedema of the cornea	0	0	36**	39**	45**	1	2	26**	45**	37**
Corneal opacity « snow flake »	0	0	8**	8**	17**	0	0	27**	16**	24**
Week 13 of recovery phase
Number of animals examined	15	-	-	-	11	14	-	-	-	14
Neovascularization of the cornea	0	-	-	-	2	0	-	-	-	6**
Month 24 – 25 (Weeks 104 – 106)
Number of animals examined	21	17	11	17	17	29	25	37	33	39
Corneal opacity	0	0	10**	16**	11**	0	3	15**	26**	19**
Neovascularization of the cornea	0	0	10**	17**	17**	0	3	30**	32**	30**
Oedema of the cornea	0	0	10**	17**	17**	0	3	20**	31**	25**
Corneal opacity « snow flake »	0	0	0	1	7**	0	0	16**	12**	12**

*: p≤0.05; **: p≤0.01

Table 7. Changes in Organ weights

	Males					Females
Dose level (ppm)	0	2	50	1500	5000	0	2	50	1500	5000
Second year (carcinogenicity phase)
Number of animals examined	21	17	10	16	17	27	22	36	33	36
Mean absolute liver weight (g)	12.6	13.5	14.0	15.7**	15.7**	10.5	11.0	10.6	10.1	10.7
Mean liver to bw ratio (%)	2.09	2.09	2.51**	2.84**	2.74**	2.40	2.52	2.63	2.69*	2.81**
Mean absolute kidney weight (g)	0.665	0.673	0.801	0.921**	0.812*	0.709	0.706	0.725	0.736	0.766
Mean kidney to bw ratio (%)	168.4	182.4	196.7*	225.7**	208.3**	143.1	139.2	137.1	128.8**	137.9

*: p≤0.05; **: p≤0.01

Table 8. Macroscopic changes at terminal sacrifice

	Males					Females
Dose level (ppm)	0	2	50	1500	5000	0	2	50	1500	5000
Second year (carcinogenicity phase)
Eye opacity	1/21	1/17	7/10**	9/16**	14/17**	1/27	3/23	22/36**	30/33**	23/37**
Irregular surface on kidney	2/21	2/17	7/10**	6/16	7/17*	nc	nc	nc	nc	nc

*: p≤0.05; **: p≤0.01

Table 9. Macroscopic changes at unschedules sacrifice (incidence) in second year (carcinogenicity phase)

	Males					Females
Dose level (ppm)	0	2	50	1500	5000	0	2	50	1500	5000
Eye opacity	1/29	3/33	10/40*	13/34**	11/33**	1/23	1/27	1/14	6/17	3/13
Irregular surface on kidney	nc	nc	nc	nc	nc	0/23	0/27	1/14	1/17	3/13*

*: p≤0.05; **: p≤0.01

 Table 10. Microscopic changes at terminal sacrifice

	Males					Females
Dose level (ppm)	0	2	50	1500	5000	0	2	50	1500	5000
Second year (carcinogenicity phase)
Follicular cell hypertrophy in thyroid gland	3/50	6/47	16/48**	19/50**	27/48**	1/50	3/50	22/50**	20/49**	19/50**
Chronic progressive nephropathy	30/50	35/50	43/50**	42/50**	38/50**	16/50	17/50	25/50	20/50	24/50
Acinar atrophy/fibrosis in the pancreas	18/50	24/48	23/49	33/49**	36/48**	13/50	22/50*	28/50**	32/48**	31/50**
Unilateral or bilateral keratitis in the eye	1/49	1/50	41/49**	47/49**	42/50**	1/50	2/50	36/50**	44/49**	38/50**
Ulcerative necrotising inflammation in hindleg and/or hindpawn of samples	3/7	3/7	7/13	13/13	18/21	1/1	0/0	3/3	3/5	2/4

*: p≤0.05; **: p≤0.01

 

Conclusions:

The study was performed under GLP conditions and according to OECD TG 453. No treatment-related neoplastic changes were observed at any dose level tested in either sex. Thus, under the conditions of this study the NOAEL in terms of carcinogenic potential was 5000 ppm for males and female rats (equivalent to 214 and 296 mg/kg bw/day for males and females, respectively). Due to adverse effects on the exocrine tissue of the pancreas and the thyroid gland, the NOAEL for males and females in terms for systemic toxicity over a 24-month period of dietary administration with the test substance to the Wistar rat was 2 ppm (equivalent to 0.08 and 0.11 mg/kg bw/day in males and females, respectively).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

214 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

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

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available data on carcinogenicity do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.

Additional information

 Combined Chronic and Carcinogenicity Study (OECD 453), Rat

The oncogenic potential and the chronic toxicity of the test substance were assessed in a study performed according to OECD TG 453 and in compliance with GLP (M-270092-01-1). Groups of 50 male and 50 female Wistar Rj:WI (IOPS HAN) rats were fed a diet containing 0, 2, 50, 1500 and 5000 ppm of the test substance for at least 104 weeks. Seventy-five rats per sex were used in the control and high dose groups (10 animals/sex designated for the interim sacrifice after 52 weeks, 15 animals/sex for the recovery group sacrifice after 68 weeks and 50 animals/sex for the final sacrifice after 104 weeks). Sixty rats per sex were used in the intermediate groups (10 animals/sex for the interim sacrifice and 50 animals/sex for the final sacrifice). Observations and examinations included mortality and clinical signs, detailed physical examinations including palpation for masses, body weight, food consumption, ophthalmological examinations, test substance analysis in the blood, haematology, clinical chemistry determinations and urinalysis. At the scheduled chronic, recovery and carcinogenicity phase sacrifice, selected organs were weighed and designated tissues sampled and examined microscopically. The mean achieved dose levels of the test substance received by the animals over the 12-month period were approximately 0.09, 2.33, 72.0 and 245 mg/kg bw/day in males and 0.13, 3.21, 99.6 and 337 mg/kg bw/day in females. The mean achieved dose levels of test substance received by the animals over the 24-month period were approximately 0.08, 2.03, 62.4 and 214 mg/kg bw/day in males and 0.11, 2.83, 88.6 and 296 mg/kg bw/day.

Over the first 12 months of the study, the mortality rate was low (1-5 per dose group) at most dose levels for both sexes with no evidence of a treatment or dose-related increase. In the male high dose group 16/75 animals were found dead or were sacrificed prematurely for humane reasons, principally within the first 6 weeks of the study. The main clinical signs in the early decedent males consisted of ocular or nasal discharge, soiled nose, soiled fur or anogenital region, wasted appearance, together with the usual signs associated with morbidity. Macroscopic examination indicated a haemorrhagic lesion as the cause of premature death in at least 8 of these males. During the second year of the study, no treatment-related effect was noted on the mortality incidence. After 2 years, 14-40 animals per dose group of 50 animals were found dead or sacrificed prematurely.

High dose group - 5000 ppm

Treatment-related clinical signs observed in a significant number of animals consisted of an increased incidence of white area on eyes (neovascularization as found in ophthalmologic examination, see below), localized soiled fur, soiled anogenital region, hair loss, reduced motor activity, cold appearance, general pallor, wasted appearance, and dental abnormality during the first year of the study in males and/or females. During the second year, the treatment-related clinical signs observed in a significant number of animals were: white area on eyes, skin lesions, reduced motor activity and focal swelling (principally of hindlimb) in males, and of white area on eyes, chromodacryorrhea and soiled anogenital region in females. At the end of the recovery phase, all clinical signs were found to be reversible, with the exception of white area on eyes that was observed in a single male only, and were therefore not considered to be adverse effects.

During the first week of treatment, mean body weight was lower by 5 and 4% and mean body weight gain by 21 and 29%, in males and females, respectively, compared to controls. This trend was observed throughout the course of the study, leading to a final body weight reduction on Day 708 of 5 and 15% in males and females, respectively, and to a body weight gain reduced by 6 and 23% in males and females, respectively, when compared to controls. No relevant changes in body weight were noted during the recovery phase; indicating that these effects were reversible. The food consumption was slightly reduced by up to 6% during the first week of treatment in both sexes, but was similar to controls thereafter. No significant changes in food consumption were noted during the recovery phase. The ophthalmological examinations revealed a high incidence of corneal opacity, neovascularization and oedema of the cornea and snow flake-like corneal opacity in both sexes throughout the study. After 13 weeks of recovery, the corneal opacity, oedema of the cornea and snow flake-like corneal opacity were completely reversible, whereas neovascularization of the cornea was still observed in some high dose  animals from both sexes. The type of corneal lesions observed at 5000, 1500 and 50 ppm in the rat (together with the eye keratitis noted at the microscopic examination), are characteristics of a compound such as the test substance that inhibits 4-hydroxyphenylpyruvic acid dioxygenase (4-HPPDase), an enzyme of the tyrosine catabolic pathway. These lesions are related to an increase in plasma tyrosine level caused by a blockade of the 4-HPPDase enzyme in the rat. However, the rat is a species particularly sensitive to inhibition of the 4-HPPDase enzyme and is atypical in its susceptibility to develop tyrosine-related eye lesions. Therefore, although these lesions (corneal lesions and eye keratitis) were treatment-related, they were considered not to be toxicologically relevant to humans (as discussed further in the repeated dose toxicity endpoint summary).

The clinical chemistry assessments showed a higher mean total cholesterol concentration up to Month 18 of the study in both sexes, together with a higher mean triglycerides concentration up to Month 12 in males and up to Month 3 in females, in comparison to controls. All changes noted were reversible after the 3-month recovery period. In the absence of clear changes in the histopathological examination, they were considered to be treatment-related, but not adverse, effects.

Urinalysis showed higher ketone level throughout the study in both sexes, and lower pH values during the first year in both sexes and during the second year in males, when compared to controls. In addition, higher protein level and a lower amount of crystals were observed in males up to Month 18, compared with controls. After 13 weeks of recovery, only the tendency towards a lower pH value was observed in females, while the rest of the above-mentioned changes were reversible. The observed effects on urinary parameters were considered not to be adverse, due to the reversibility and lack of corresponding histopathological effects.

At the end of the chronic phase (12 months) the mean absolute and/or relative kidney weights were higher in males at 5000 ppm (statistically significant) in comparison to controls. These changes were considered to be treatment-related.  Mean absolute and relative liver weights were higher in males at 5000 ppm (statistically significant), in comparison to controls. These changes were considered to be treatment-related. However, in view of the limited changes noted at the microscopic observation, they were considered to have no toxicological relevance. Mean absolute brain weight was decreased by 6% (p<0.05) in males at 5000 ppm, in comparison to controls. This change was considered not to be toxicologically relevant, since it was observed for this parameter only and was not associated with any change at the macroscopic and microscopic examinations.  All organ weight changes detected at the end of the chronic phase were found to be reversible. The few changes noted after the 3-month recovery period were considered to be incidental in view of their individual variation.

At the end of the carcinogenicity phase (24 months), the mean absolute and relative liver weights were increased by 25 and to 31%, respectively, in males, while the relative liver weight was increased by 17% in females. These changes were considered to be a treatment-related, adaptive response to the test substance exposure. Mean absolute and relative kidney weights were increased by 16 and 22%, respectively, in males, in comparison to controls. During the macroscopic examination, toxicologically relevant changes were noted in the kidneys. The incidence of irregular surface on kidney was increased in females (3), in comparison to controls (0). In the kidney, the incidence of minimal to severe chronic progressive nephropathy was statistically significantly higher in males (38), compared with controls (30). However, this change is a common age-related and spontaneously occurring disease in the rat and was observed with a high incidence in control males (30/50). This change is not considered to be toxicologically relevant. The histopathological examination showed toxicologically relevant changes in the pancreas and thyroid gland, in comparison to controls. In the pancreas, effects were observed in the exocrine tissue, with the incidence of acinar atrophy/fibrosis higher in both sexes (36 for males, 31 for females vs. 18 for males, 13 for females in control). In the thyroid gland, the incidence of follicular cell hypertrophy was elevated in both sexes (27 for males, 19 for females vs. 3 for males, 1 for females in control). Other changes noted during the histopathological examination were considered not to be toxicologically relevant. The overall incidence of neoplastic findings observed after the chronic phase was very low and did not indicate any treatment related relationship. No neoplastic finding was observed after the recovery phase on the organs examined. The overall incidence of neoplastic findings observed in animals from the carcinogenicity phase was within expectations for a two-year study and did not indicate any treatment-relationship.

1500 ppm

The treatment-related clinical signs consisted of a significantly increased incidence of white area on eyes in both sexes, together with soiled anogenital region in 4/60 females during the first year. During the 2nd year, a significantly increased number of males had white area on eyes, reduced motor activity and focal swelling (principally of hindlimb), compared with controls; while an increased number of females had a white area on eyes, compared with control.

The mean body weight was reduced by up to 6% during the first year of treatment in both sexes, in comparison to controls, while the mean body weight gain was reduced by up to 11 and 18% throughout the first year in males and females, respectively. The effect persisted in both sexes, as the final body weight on Day 708 was reduced by 9 and 15% in males and females, respectively, when compared to controls. The body weight gain was reduced by 14 and 21% in males and females, respectively, when compared to controls. The food consumption was unaffected by treatment.

The ophthalmological examinations revealed a high incidence of corneal opacity, neovascularization and oedema of the cornea and snow flake-like corneal opacity in both sexes throughout the study. These observations are considered not to be relevant to humans, as described above.

At the end of the chronic phase (12 months) the clinical chemistry evaluation showed higher mean total cholesterol and/or triglycerides concentrations were noted in both sexes (statistically significant on most occasions), in comparison to controls. Lower mean alkaline phosphatase activity was noted in males at Months 3 and 6 (statistically significant). These changes were considered to be treatment-related. However, since the decrease was observed only transiently and in the absence of any changes at the histopathological examination, they were considered not to be toxicologically relevant. Slight variations were also observed in males in total protein and albumin concentrations and consequently in globulin concentrations and albumin/globulin ratio (calculated parameters), principally at Month 3. However, the magnitude of the variations relative to the controls was less pronounced throughout the sampling periods. Therefore, these differences were considered to be meaningless and not to be toxicologically relevant. After the carcinogenicity phase no changes were observed.

The urinalysis showed higher ketone level on most occasions during the study in both sexes, compared with control. Lower mean pH values compared to controls were observed in both sexes throughout the first year and only in males throughout the second year, though no clear dose related effect was noted in males, a lower amount of crystals was observed up to Month 18, together with a higher protein level at Months 6, 12 and 18.

At the end of the chronic phase (12 months), the mean absolute and relative liver weights were higher in males (statistically significant only for the relative liver weight), in comparison to controls. These changes were considered to be treatment-related. However, in view of the limited changes noted at the microscopic observation, they were considered to have no toxicological relevance. Mean absolute and/or relative kidney weights were higher in males (statistically significant for the relative weight parameter), in comparison to controls. These changes were considered to be treatment-related. The mean absolute brain weight was decreased by 7% (p<0.01) in males at 1500 ppm, in comparison to controls. This change was considered not to be toxicologically relevant, since it was observed for this parameter only and was not associated with any change at the macroscopic and microscopic examinations.

At the end of the carcinogenicity phase (24 months), the mean absolute and relative liver weights were higher in males (25% and 36%, respectively, p<0.01), when compared to controls. Mean relative weight was also higher in females (12%, p<0.05), in comparison to controls. There were no toxicologically relevant findings during the macroscopic examination. The histopathological examination showed toxicologically relevant changes in the pancreas and thyroid gland, in comparison to controls. In the pancreas, effects were observed in the exocrine tissue, with the incidence of acinar atrophy/fibrosis higher in both sexes (33 in males and 32 in females vs. 18 in males and 13 in females in controls). In the thyroid gland, the incidence of follicular cell hypertrophy was elevated in both sexes (19 in males and 20 in females vs. 3 in males and 1 in females in controls). Other changes noted during the histopathological examination were considered not to be toxicologically relevant.

The overall incidence of neoplastic findings observed after the chronic phase was very low and did not indicate any treatment related relationship. No neoplastic finding was observed after the recovery phase on the organs examined. The overall incidence of neoplastic findings observed in animals from the carcinogenicity phase was within expectations for a two-year study and did not indicate any treatment-relationship.

50 ppm

Treatment-related clinical signs were limited to a significantly increased incidence of white area on eyes in both sexes throughout the study, together with a significant increase in the incidence of reduced motor activity and focal swelling (principally of hindlimb) in males during the second year. The Body weight parameters were comparable to control values in both sexes during the first year of treatment.

Throughout the second year, the mean body weight was reduced by up to 11 and 8% and the mean body weight gain by up to 17 and 12% in males and females, respectively, compared to controls. The food consumption was unaffected by treatment.

The ophthalmological examinations showed a high incidence of corneal opacity, neovascularization and oedema of the cornea and snow flake-like corneal opacity in both sexes throughout the study. These observations are considered not to be relevant to humans, as described above.

The clinical chemistry evaluation showed a significantly higher mean total cholesterol level up to Month 12 of the study in males and a significantly higher mean triglycerides concentration on one or two sampling periods in both sexes, in comparison to controls.

The urinalysis showed higher ketone level on most occasions during the study in both sexes and lower pH values during the first year in both sexes and also during the second year in males, when compared to controls. In addition in males, a lower amount of crystals was observed up to Month 18, together with a higher protein level at Months 6, 12 and 18.

At the end of the chronic phase (12 months), the mean kidney weights were increased by between 17 to 22% in males, in comparison to controls. There were no toxicologically relevant findings during the macroscopic examination. The few changes noted during the histopathological examination were considered to be incidental.

At the end of the carcinogenicity phase (24 months), the mean relative liver and kidney weights were significantly increased by 20% each in males, when compared to controls. There were no toxicologically relevant findings during the macroscopic examination. The histopathological examination revealed toxicologically relevant changes in the pancreas and thyroid gland, in comparison to controls. In the pancreas, effects were observed in the exocrine tissue, with the incidence of acinar atrophy/fibrosis higher in females (28 vs. 13 in controls). In the thyroid gland, the incidence of follicular cell hypertrophy was elevated in both sexes (16 in males and 22 in females vs. 3 in males and 1 in females in controls). Other changes noted during the histopathological examination were considered to be incidental.

The overall incidence of neoplastic findings observed after the chronic phase was very low and did not indicate any treatment related relationship. No neoplastic finding was observed after the recovery phase on the organs examined. The overall incidence of neoplastic findings observed in animals from the carcinogenicity phase was within expectations for a two-year study and did not indicate any treatment-relationship.

2 ppm

No toxicologically relevant changes were noted throughout the course of the study in either sex for any of the parameters evaluated.

In conclusion, no treatment-related neoplastic changes were observed at any dose level tested in either sex. Thus, under the conditions of this study the NOEL in terms of carcinogenic potential was 5000 ppm for males and female rats (equivalent to 214 and 296 mg/kg bw/day, respectively). Due to adverse effects on the exocrine tissue of the pancreas and the thyroid gland, the NOAEL for males and females in terms for systemic toxicity over a 24-month period of dietary administration with the test substance to the Wistar rat was 2 ppm (equivalent to 0.08 and 0.11 mg/kg bw/day in males and females, respectively). The histopathological changes observed in thyroid, such as follicular cell hypertrophy resulted from excessive metabolism of thyroid hormones by phase II conjugation enzymes induced by the treatment with the test substance, as demonstrated in study M-400191-01-2 (details of which are provided within the repeated dose toxicity endpoint summary).

Carcinogenicity Study (OECD 451), Mouse

The carcinogenic potential of the test substance was assessed in a study performed according to OECD TG 451 and in compliance with GLP (M-268436-01-1). Groups of 60 male and 60 female C57BL/6J mice were fed a diet containing 0, 150, 800 or 4000 ppm of the test substance. After 53 weeks, 10 males and 10 females from each group allocated to the interim sacrifice phase of the study were necropsied. 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 (equating to a mean intake over 18 months of 0, 21.0, 112 and 583 mg/kg bw/day in males and 0, 27.1, 142 and 743 mg/kg bw/day in females, at 0, 150, 800 and 4000 ppm, respectively). Mortality and clinical signs were checked daily. Additionally, detailed physical examinations including palpation for masses were performed weekly throughout treatment. Body weight and food consumption were measured for the first 13 weeks of the study, then monthly thereafter. Ophthalmological examinations were performed after 3, 6, 9 and 12 months of treatment and at study termination. Haematology determinations were performed at approximately 12 and 18 months from designated animals. Where possible, blood smears were taken from moribund animals. All animals were subjected to necropsy, with selected organs weighed at scheduled interim and final sacrifice. Designated tissues were fixed and examined microscopically.

There was no treatment-related effect on mortality, or on an earlier development or increased incidence of tumours, in any dose group, throughout the study.

4000 ppm:

Treatment-related clinical signs consisted of intense yellow coloured urine observed in all males and 58/60 females, first observed after 5 months of treatment, and present thereafter. In addition, prolapsed rectum in 8/60 females, ocular discharge in 8/60 males and soiling fur in 3/60 males were also noted. The mean body weight of both sexes was decreased during the first week of treatment, resulting in a mean body weight reduction of 7% in males and 8% in females. Throughout the remainder of the study mean body weight was lower than the controls, at most time points for males and on a number of occasions in females, resulting in a final body weight reduction of 6% in males and 9% in females. After 12 months of treatment, haematology evaluation revealed a 46% decrease of mean total leucocyte count and 47 to 50% decrease in absolute lymphocyte and neutrophil counts in females, whilst in males, only absolute lymphocyte count was decreased by 38%. After 18 months of treatment, mean red blood cell count, haemoglobin concentration and haematocrit were decreased in both sexes and mean corpuscular volume and mean corpuscular haemoglobin were increased in females only.

At the 12-month interim sacrifice, mean terminal body weight was reduced by 10% in males. Mean liver to body weight ratio was increased by 16% in males and 19% in females. Macroscopic observations consisted of gallstones observed in 2/9 males and 3/8 females (often multiple, pinpoint, brown or grey coloured). In addition, enlarged liver was observed in 1/8 females. Microscopically, minimal to slight centrilobular hepatocellular hypertrophy was observed in both sexes. In females, a higher incidence of diffuse hepatocellular vacuolation concomitant with a lower incidence of mainly periportal hepatocellular vacuolation was noted. This finding was judged to be correlated with the presence of centrilobular hepatocellular hypertrophy. In addition, higher incidence of gallbladder stones was noted in both sexes.  

At the 18-month terminal sacrifice, mean terminal body weights were reduced by 4% in males and 8% in females. Mean absolute and relative liver weights were increased by 16 to 22% in males and 8 to 17% in females. Macroscopic observations consisted of gallstones observed in 18/50 males and 25/50 females (often multiple, pinpoint, brown, grey or black coloured). In addition, enlarged liver was observed in 5/50 males and 17/50 females. Dark liver was also observed in 7/50 males and 3/50 females. Microscopically, non-neoplastic findings consisted of changes observed in the liver, gallbladder and kidney. In the liver, centrilobular hepatocellular hypertrophy was noted in 48/50 males and 49/50 females. In addition, hepatocellular single cell necrosis was noted in both sexes, together with a decreased incidence of diffuse hepatocellular vacuolation in males and an increased incidence of diffuse hepatocellular vacuolation in females concomitant with a decreased incidence of mainly periportal hepatocellular vacuolation. Gallstones were observed in 18/49 males and 35/50 females. In addition, in the gallbladder, an increased incidence and severity of eosinophilic cytoplasmic alteration was noted in females. In the kidney, males were noted to have unilateral or bilateral collecting duct hyperplasia associated with pelvic epithelium hyperplasia and papillary necrosis.

800 ppm

The mean body weight of males was reduced after eight weeks of treatment, resulting in a body weight reduction of 3% compared to the controls. Thereafter, mean body weight was lower than the controls at several time points, resulting in a final body weight reduction of 6%.

At the 12-month interim sacrifice, mean liver to body weight ratio was increased by 10% in males and 11% in females. In addition, mean liver to brain weight ratio was increased by 21% in females. Macroscopic observations consisted of gallstones observed in 3/10 males (including unscheduled death animals) and 4/10 females. Microscopically, minimal to slight centrilobular hepatocellular hypertrophy was observed in both sexes. In addition, higher incidence of gallbladder stones was noted in both sexes.

At the 18-month terminal sacrifice, mean terminal body weights were reduced by 3% in males. Mean absolute and relative liver weights were increased by 12 to 16% in males and 9 to 10% in females. Macroscopic observations consisted of gallstones observed in 21/50 males and 33/50 females. In addition, enlarged liver was observed in 4/50 males and 15/50 females and dark liver was observed in 3/50 males and 2/50 females.

Microscopically, non-neoplastic findings consisted of changes observed in the liver, gallbladder and kidney. In the liver, centrilobular hepatocellular hypertrophy was noted in 40/49 males and 40/50 females and hepatocellular single cell necrosis was noted in females only. In addition, a decreased incidence of diffuse hepatocellular vacuolation was noted in males. In the gallbladder, a higher incidence of gallstones was observed for both sexes compared to the controls. In addition, the incidence and severity of eosinophilic cytoplasmic alteration was increased in females. In the kidney, males were noted to have unilateral or bilateral collecting duct hyperplasia associated with pelvic epithelium hyperplasia and papillary necrosis.

150 ppm

At the 12-month interim sacrifice, mean liver to body weight ratio was increased by 8% in males. Macroscopic observations consisted of gallstones observed in 3/7 males and 3/10 females. Microscopically, minimal centrilobular hepatocellular hypertrophy was observed in males. In addition, higher incidence of gallbladder stones was noted in both sexes.

At the 18-month terminal sacrifice, mean absolute and relative liver weights were increased by 8 to 10% in males. In females, only the mean liver to body weight ratio was increased by 8%. Macroscopic observations consisted of gallstones observed in 16/50 males and 21/50 females. In addition, enlarged liver was observed in 9/50 females. Dark liver was also observed in 2/50 males and 1/50 females.

Microscopically, non-neoplastic findings consisted of changes observed in the liver, gallbladder and kidney. In the liver, centrilobular hepatocellular hypertrophy was noted in both sexes. A decreased incidence of diffuse hepatocellular vacuolation was also noted in males only. In the gallbladder, a higher incidence of gallstones was observed for both sexes compared to the controls. In addition, a higher incidence and severity of eosinophilic cytoplasmic alteration was noted in females when compared to controls. In the kidney, males were noted to have unilateral or bilateral collecting duct hyperplasia associated with pelvic epithelium hyperplasia and papillary necrosis.

In conclusion, dietary administration of over an 18-month period to the C57BL/6J mouse, at dose levels up to 4000 ppm (equivalent to 583 mg/kg bw/day in males and 743 mg/kg bw/day in females) did not induce any treatment related neoplastic changes. Thus, under the conditions of the study the NOEL in regard to carcinogenic potential was 583 and 743 mg/kg bw/day in males and females, respectively.

A NOAEL for general toxicity could not be established due to findings noted in liver and gallbladder of both sexes and kidney in males at all dose levels. The LOAEL was therefore determined to be 150 ppm (equivalent to 21.0 and 27.1 mg/kg bw/day in males and females, respectively).