Pyrasulfotole

Carcinogenicity

Description of key information

Carcinogenicity Study (OECD 451), mouse:

NOAEL (general) = 100 ppm (equivalent to 13.6 and 16.7 mg/kg bw/day in males and females, respectively)

NOAEL (carcinogenicity) = 4000 ppm (equivalent to 560 and 713 mg/kg bw/day in males and females, respectively)

Carcinogenicity Study (OECD 453), rat:

NOAEL (general) = 25 ppm (equivalent to 1.1 and 1.4 mg/kg bw/day in males and females, respectively)

NOAEL (carcinogenicity) = 2500 ppm (equivalent to 104 and 140 mg/kg bw/day in males and females, respectively)

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:

29 Jan 2003 - 21 Oct 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

other: reference to review article

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:

Secrétariat du Groupe Interministeriel des Produits Chimiques, Paris, France

Species:

rat

Strain:

Wistar

Remarks:

Rj:WI (IOPS HAN)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: R. Janvier (Le Genest St Isle, France)
- Age at study initiation: 7 weeks
- Weight at study initiation: mean group weights: 286 - 289 g (males), 193 - 196 g (females)
- Fasting period before study: no
- Housing: individually in suspended, stainless steel and wire mesh cages
- Diet: A04CP1-10 from S.A.F.E. (Scientific Animal Food and Engineering, Augy, France), ad libitum
- Water: filtered and softened tap water from the municipal water supply, ad libitum
- Acclimation period: 22 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): 10 to 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 29 Jan 2003 To: 03 Mar 2005

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DIETARY MIXTURES:

DIET PREPARATION
- Rate of preparation of diet (frequency): The test substance formulations were prepared to cover the dietary requirements over 4-weekly periods apart from the last formulation which covered the dietary needs until the end of the study. Twenty-six formulations (F1 to F26) were prepared during the study at each concentration.
Formulations F1 to F10 consisted of 4 loads of 45 kg at each concentration. In addition, an additional load (load 4 Bis) was prepared for formulation F2 to cover for accidental loss of diet. Formulations F11 to F13 consisted of 3 loads of 45 kg at each concentration. Formulations F14 to F25 consisted of 2 loads of 56 kg at each concentration. Formulation F26 consisted of 3 loads of 61 kg at each concentration.
- Mixing appropriate amounts with (Type of food): A04CP1-10 from S.A.F.E.
- Storage temperature of food: at ambient temperature

VEHICLE
- No vehicle was used.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in the diet has been demonstrated in a previous and recent 90-day rat study (M-102924-01-2). The stability of the test substance at 2 and 15000 ppm in the diet was verified for up to at least 82 days, when kept at ambient temperature which covered the period of storage and usage on this study.

Twenty-six formulations (F1 to F26) were prepared during the study at each concentration. The homogeneity of the test substance in diet was verified from the first loads at 25, 250, 1000 and 2500 ppm on the first formulation (F1) and on the first loads at 25 and 2500 ppm of formulations F6, F12, F14, F20 and F26, to demonstrate adequate formulation procedures.
The concentration was checked for all loads at all dose levels for formulations F1, F2, F3, F6, F9, F12, F14, F17, F20, F23 and F26.

Results:
- Homogeneity Analysis: 82* - 115% of nominal concentration
- Concentration Analysis: 85 - 112% of nominal concentration
* 3/266 samples were slightly outside the in-house target range of 85 - 115% of nominal concentration. This minor deviation was not considered to have affected the integrity of the study.

Duration of treatment / exposure:

24 months

Frequency of treatment:

daily, 7 days a week

Post exposure period:

not applicable

Dose / conc.:

25 ppm

Remarks:

equivalent to 1.0 and 1.4 mg/kg bw/day in males and females, respectively

Dose / conc.:

250 ppm

Remarks:

equivalent to 10 and 14 mg/kg bw/day in males and females, respectively

Dose / conc.:

1 000 ppm

Remarks:

equivalent to 41 and 57 mg/kg bw/day in males and females, respectively

Dose / conc.:

2 500 ppm

Remarks:

equivalent to 104 and 140 mg/kg bw/day in males and females, respectively

No. of animals per sex per dose:

10 (for the 6-month subchronic phase)
10 (for the 12-month chronic phase)
55 (for the carcinogenicity phase)

Control animals:

yes, plain diet

Details on study design:

- Toxicokinetic data: no data provided

- Dose selection rationale: Dose levels were selected based on the results from a previous 90-day dietary study in the rat (M-102924-01-2) where dietary administration of up to 7000 ppm in males and 12000 ppm in females resulted in increased plasma cholesterol and triglycerides, increased liver and kidney weights, yellow calculi in the urinary tract, urothelial hyperplasia, centrilobular hepatocellular hypertrophy, and diffuse thyroid follicular cell hyperplasia. The NOAEL in the rat 90-day study was 30 ppm, with a LOAEL of 1000 ppm.

- Rationale for animal assignment: On the day of randomization, animals were allocated to dose groups using a computerized randomization procedure that ensured a similar body weight distribution among groups for each sex. Selected animals were in a weight range from
243 to 325 g for the males and 169 to 221 g for the females at the start of exposure to the test substance, i.e., within ± 20% of the mean body weight on the day of randomization. Any animal deemed unsuitable for selection based on weight, ophthalmological abnormalities or health status was not used for the study.

- Section schedule rationale: At termination of the carcinogenicity phase, males were sacrificed prior to females due to a 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.

Observations and examinations performed and frequency:

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed physical examinations including palpation for masses were performed weekly from study day 1.

BODY WEIGHT: Yes
- Time schedule for examinations: At least weekly during the acclimatization period then weekly for the first 13 weeks of study, approximately every 4 weeks thereafter and prior to necropsy. An additional body weight session was scheduled for study day 673 to enable achieved test material intake figures to be calculated over this period with corresponding additional food consumption session.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations:
all animals: during acclimatization (indirect ophthalmoscopy)
all surviving animals: after approximately 3, 6, 9, 12 and 24 months (indirect ophthalmoscopy and slit lamp)
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
Blood analyses were performed on the first ten surviving rats per group in weeks 28/29, 50/51, 78/79 and 104/105.
At scheduled necropsy, blood smears were prepared for all animals not sampled for hematology. The blood smears were stained with Wright stain for possible differential white blood cell determination (on the request of the Study Pathologist).
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- Parameters examined: hematocrit, hemoglobin, leukocyte count, erythrocyte count, platelet count, prothrombin time, leukocyte differential count, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean corpuscular volume, reticulocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
Blood analyses were performed on the first ten surviving rats per group in weeks 28/29, 50/51, 78/79 and 104/105.
- Animals fasted: Yes
- Parameters examined: calcium, chloride, magnesium, inorganic phosphorus, potassium, sodium, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma glutamyltransferase, albumin, creatinine, urea, total cholesterol, glucose, total bilirubin, total protein, triglycerides

URINALYSIS: Yes
- Time schedule and for collection of urine: Prior to allocation of the animals to treatment groups, urinalysis was performed on thirty males and thirty females.
Urinalysis was performed on the first ten surviving rats per group in weeks 13/14, 26/27/28, 48/49, 80/81 and 101/102.
- Metabolism cages used for collection of urine: not specified
- Animals fasted: Yes (diet and water)
- Parameters examined: appearance, volume, specific gravity/osmolality/refractive index, pH, sediment (microscopic), protein, glucose, ketones, bilirubin, blood/red blood cells, urobilinogen, creatinine

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Necropsy included the examination of external surfaces, all orifices, all major organs, tissues and body cavities. All significant macroscopic abnormalities (including masses and their regional lymph nodes when possible) were recorded, sampled and examined microscopically.

ORGAN WEIGHTS: Yes (see Table 1 "Any other information on materials and methods incl. tables)

HISTOPATHOLOGY: Yes (see Table 1 "Any other information on materials and methods incl. tables)
Histopathological examinations were performed on all organs and tissues embedded including gross abnormalities in all animals from all groups including decedents.
For all unscheduled sacrificed or dead animals on study, the cause of death was determined when it was possible.
Initial examinations were performed by the Principal Investigator in Histopathology. Following the initial examination, a review pathologist, undertook an independent « peer-review » of representative slides and diagnoses according to standardized operating procedures. The diagnoses presented in this report represent the consensus opinion of the two pathologists. In addition, a Scientific Advisory Panel, at Experimental Pathology Laboratories, Inc. PO BOX 474, Herndon, VA 20172-0474, U.S.A., peer reviewed a selection of thyroid gland slides.

Statistics:

See "Any other information on materials and methods incl. tables"

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

During the first 6 and 12 months of treatment, treatment-related clinical signs consisted of an increased incidence of white area on the eye at the three highest dose levels in both sexes. In addition, there was an increased incidence of soiled fur/soiling around the anogenital region at the two highest dose levels in both sexes (see Tables 3 and 4 "Any other other information on results incl. tables").

During the second year, treatment-related clinical signs consisted of an increased incidence of white area on the eye in the three highest dose levels in both sexes. There was also an increased incidence of soiling around the anogenital region at the three highest dose levels in males and the two highest dose levels in females (see Table 5 "Any other other information on results incl. tables").

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

After 6 months of treatment, the mortality rate at 2500 ppm in males was slightly higher than in the control group (4/75 deceased animals versus 1/75 in the control). The mortality rate in the remaining male treated groups was very low and similar to the control group. In females there were no deaths during the first 6 months.

After 1 year of treatment the mortality rate in males at 2500 ppm was higher than in the control group (8/65 deceased animals versus 2/65 in the control). At 25 ppm in males, the mortality rate was also slightly higher than in the control group (6/65), but as the incidence at 1000 and 100 ppm was similar to the controls (2/65 in both groups), the higher incidence at 25 ppm was considered to be incidental. In females, the mortality rate was similar in the treated and the control groups.
One female from the control group (chronic phase) died due to accidental trauma.

After 2 years of treatment, the mortality rate in males at 2500 ppm was statistically significantly higher than in the control group (30/55 deceased animals versus 30/55 in the control; see Table 6). There was no statistically significant difference between the remaining male treated groups and the controls. The mortality rate in females was similar across the groups with no indication of a treatment-related effect. One male from the control group and one male at 250 ppm, together with one female at 25 ppm and 1000 ppm and two females at 2500 ppm died during anesthesia for blood sampling.
One male from the control group was sacrificed due to accidental trauma.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

(for mean body weight and body weight gain data see Table 7 "Any other other information on results incl. tables")

6-month phase
At 2500 and 1000 ppm, during the first week of treatment, mean body weight was reduced by 3% (p < 0.05) and 2%, respectively, in males, whilst mean cumulative body weight gain was reduced by 16% (p < 0.01) and 18% (p < 0.01), respectively, compared to controls. Thereafter, mean body weight gain was consistently lower than in controls by up to 56% and 60%, respectively, the effect being statistically significant on several occasions. At the end of the 6-month period (day 176), mean body weight was reduced by 6% (p < 0.01) and 5% (p < 0.01), respectively, and mean cumulative body weight gain was reduced by 10% (p < 0.01) and 9% (p < 0.01), respectively, compared to controls. In females, there was no treatment-related effect on mean body weight, body weight gain and cumulative body weight gain throughout the first 6 months of treatment. The few minor differences from controls which attained statistical significance were sporadic and were considered to be chance findings reflecting the number of comparisons made.
At 250 ppm, during the first week of treatment, mean body weight was reduced by 2% in males, whilst mean cumulative body weight gain was reduced by 14% (p < 0.01), compared to controls.Thereafter mean body weight, body weight gain and cumulative body weight gain were comparable to controls throughout the first 6 months of treatment. The few minor differences reaching statistical significance were considered to reflect inter-individual variation rather than a treatment-related effect. In females, there was no treatment-related effect on mean body weight, body weight gain and cumulative body weight gain throughout the first 6 months of treatment. The few minor differences from controls which attained statistical significance were sporadic and were considered to be chance findings reflecting the number of comparisons made.
At 25 ppm, there was no treatment-related effect on mean body weight, body weight gain and cumulative body weight gain in either sex over the first 6 months of treatment. The few minor differences from controls which attained statistical significance were sporadic and were considered to be chance findings reflecting the number of comparisons made.

There was no relevant change in terminal body weights of treated animals when compared to controls.

Chronic phase
At 2500, 1000 and 250 ppm, there was no treatment-related effect on mean body weight gain in males during the second half of the first year of treatment. The few minor differences from controls which attained statistical significance were sporadic and were considered to be chance findings reflecting the number of comparisons made. At the end of the first year of treatment (day 344), mean body weight was reduced by 6% (p < 0.01), 5% and 3%, respectively, and mean cumulative body weight gain was reduced by 10% (p < 0.01), 8% (p < 0.05) and 5%, respectively, compared to controls. In females, mean body weight, body weight gain and cumulative body weight gain were comparable with the controls during the second half of the first year of treatment.
At 25 ppm, there was no treatment-related effect on mean body weight, body weight gain and cumulative body weight gain in either sex throughout the second half of the first year of treatment. The few minor differences from controls which attained statistical significance were sporadic and were considered to be chance findings reflecting the number of comparisons made.

There was no relevant change in terminal body weights of treated animals when compared to controls.

At 2500 ppm, throughout the second year of treatment, mean body weight was reduced by up to 14 and 8% in males and females, respectively, whilst mean cumulative body weight gain was reduced by up to 23 and 12% in males and females, respectively, compared to controls. In males, mean body weight gain was consistently lower at most time points throughout the second year of treatment, compared to the controls, the effect being statistically significant on several occasions. In females, the effect on mean body weight gain was more pronounced during the first half of the second year of treatment and was statistically significant (p < 0.01) at a few time points.
At 1000 ppm, throughout the second year of treatment, mean body weight was reduced by up to 12% and 10% in males and females, respectively, whilst mean cumulative body weight gain was reduced by up to 20 and 15% in males and females, respectively, compared to controls. In males, lower mean body weight gains were observed at most time points throughout the first 6 months of the second year of treatment, when compared to the controls, the effect being statistically significant on most occasions. In females, mean body weight gain was consistently lower at most time points throughout the second year of treatment, compared to the controls, although the effect was not statistically significant.
At 250 ppm, throughout the second year of treatment, mean body weight was reduced by up to 8% and 6% in males and females, respectively, whilst mean cumulative body weight gain was reduced by up to 13 and 9% in males and females, respectively, compared to controls. In males, lower mean body weight gains were observed at most time points throughout the first 6 months of the second year of treatment, when compared to the controls, the effect being statistically significant on a few occasions. In females, mean body weight gain was comparable to or slightly lower than the controls throughout the second year of treatment, although the effect was not statistically significant.
At 25 ppm, there was no treatment-related effect on mean body weight, body weight gain and cumulative body weight gain in either sex throughout the second year of treatment. The few minor differences from controls which attained statistical significance were sporadic and were considered to be chance findings reflecting the number of comparisons made.
Mean terminal body weights were lower and statistically different in males (-12% at 2500 ppm, -8% at 1000 ppm, -7% at 250 ppm) when compared to controls.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Mean food consumption was similar to controls throughout the chronic phase in both sexes and at all dose levels evaluated. The few minor differences from controls which attained statistical significance were sporadic and were considered to be chance findings reflecting the number of comparisons made.

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 6 month (weeks 23 to 24), ophthalmological examination treatment-related findings consisted of corneal opacity, neovascularisation and oedema of the cornea, and "snow flake-like" corneal opacity at 2500 and 1000 ppm in both sexes and 250 ppm in males, and neovascularisation of the cornea and "snow flake-like" corneal opacity in females at 250 ppm.

At the 1 year (weeks 49/50/51) and 2 year (weeks 102/103) treatment-related findings consisted of corneal opacity, neovascularisation and oedema of the cornea, and "snow flake-like" corneal opacity at 2500, 1000 and 250 ppm in both sexes.

At the two year examination the number of males at 25 ppm with corneal opacity and neovascularisation of the cornea was very slightly higher than in the control group. However, in the absence of treatment-related ocular findings at the histopathological examination, these non-specific changes were considered to be incidental.

Other ophthalmological changes were considered to be chance findings as they occurred in isolation, in a non dose-related manner or at a similar frequency in the control and treated group.

See Attachment 1 "attached background material" for tabulated ophthalmological data.

Haematological findings:

no effects observed

Description (incidence and severity):

No toxicologically relevant variation was noted in the parameters assayed at any blood sampling for any dose and either sex.
The few statistically significant differences were considered not to be relevant in view of their low magnitude, the absence of a clear dose-effect relationship and/or their isolated occurrence.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In males, higher mean total cholesterol concentrations were observed at 2500, 1000 and 250 ppm throughout the study. The increase at 25 ppm at 6 and 12 months was within historical control data (2.02 ± 0.47 at 7 months, 2.41 ± 0.57 at 12 months), and as cholesterol concentration at 25 ppm returns to normal levels for the remainder of the study, this increase was considered not to be treatment-related.

In females at 2500 ppm, at month 24, mean total cholesterol and triglyceride concentrations were higher (+63% and +135% respectively, not statistically significant) relative to the control group. In females at month 7, when compared to the controls, statistically significant differences were noted at 2500, 1000 and 250 ppm in total cholesterol and triglyceride concentrations. However in the absence of a clear dose-effect relationship and in view of the variation of the individual values, these changes were considered not to be relevant.
Other statistically significant differences were considered not to be relevant in view of their low magnitude, the absence of a clear dose-effect relationship and/or their isolated occurrence.

See Attachment 1 "attached background material" for tabulated clinical chemistry data.

Endocrine findings:

not examined

Description (incidence and severity):

Not applicable.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

When compared to the control groups, a tendency towards higher ketone levels was observed at 2500 and 1000 ppm in both sexes at all collection periods and at 250 ppm in males at month 19 and 24 only.
Throughout the study lower mean pH values were noted in males at 2500, 1000 and 250 ppm. In females lower mean pH values were noted only on month 3 - 4, at 2500 ppm (-10%, p < 0.01), 1000 ppm (-6%, p < 0.01) and 250 ppm (-6%, p < 0.01). Correlated with the lower mean pH values, lower amounts of crystals were generally observed in the urine.
Additionally, when compared to the controls, a tendency towards higher protein levels was seen in males from Month 6-7 at all dietary levels tested in this study.
Other statistically significant differences were considered not to be relevant in view of their sporadic occurrence and/or the absence of a clear dose-effect relationship.

See Attachment 1 "attached background material" for tabulated urinalysis data.

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):

Mean terminal body weights were lower and statistically different in males (-12% at 2500 ppm, -8% at 1000 ppm, -7% at 250 ppm) when compared to controls.
At 2500 ppm, 1000 ppm and at 250 ppm in males, mean liver weights were higher and statistically different for most parameters, when compared to controls (see Table 7 "Any other other information on results incl. tables"). These differences were associated with microscopic hepatocellular hypertrophy and were considered to be treatment-related.
At 2500 ppm, 1000 ppm and at 250 ppm in males, mean kidney weights were higher and statistically different for all parameters when compared to controls (see Table 8 "Any other other information on results incl. tables"). These differences were associated with relevant microscopic findings and were considered to be treatment-related.
At 2500 ppm and at 1000 ppm in males, mean spleen weights were higher and statistically different for all parameters when compared to controls. As these differences were not associated with relevant microscopic findings, they were considered not to be toxicologically significant.
All other organ weight differences were judged to be incidental in view of their individual variation.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

a/ Unscheduled deaths:
289 animals died prematurely before the end of the study (see Table 4 "Any other other information on results incl. tables").
A higher incidence of eye opacity was found in treated groups at 2500, 1000 and 250 ppm when compared to controls. This change was dose-related, was correlated with relevant histopathological findings and was considered to be treatment-related.
A higher incidence of irregular surface and paleness of the kidney was found in treated males at 2500 and 1000 ppm than in controls. These changes were correlated with relevant histopathological findings and were considered to be treatment-related.
All other gross pathology changes were considered as incidental and not treatment-related.

b/ Terminal sacrifice:
A higher incidence of eye opacity was found in treated animals at 2500, 1000 and 250 ppm than in controls. This change was dose-related, was correlated with relevant histopathological findings and was considered to be treatment related.
A higher incidence of irregular surface of the kidney was found in treated males at 2500 and 1000 ppm than in controls. This change was correlated with relevant histopathological findings and was considered to be treatment-related.
All other gross pathology changes were considered as incidental and not treatment-related.

See Attachment 2 "attached background material" for tabulated gross pathological findings.

Neuropathological findings:

not examined

Description (incidence and severity):

Not applicable.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Clear treatment-related effects were seen in the eyes, thyroid gland and pancreas in both sexes, and liver and kidney in males.
Treatment-related changes of little or no toxicological significance were seen at a higher incidence and/or severity in the sciatic nerve, skeletal muscle, pituitary gland, blood vessels, ovaries, uterus, vagina and brain.

In the eyes, the incidence of unilateral or bilateral inflammation of the cornea with regenerative hyperplasia was greater than controls in animals dosed at 2500, 1000 ppm and males at 250 ppm. Neovascularisation was greater than controls in animals dosed at 2500, 1000 or 250 ppm. In some animals there was mucous metaplasia, vacuolation or atrophy of the cornea or bilateral atrophy of the peripheral retina.

In the liver, minimal centrilobular hepatocellular hypertrophy was noted in animals dosed at 2500 and in males at 1000 or 250 ppm. This finding was considered to be an adaptive response to treatment rather than an adverse effect. The incidence of bile duct hyperplasia, fibrosis and peribiliary inflammatory cells was higher than controls in females at all dosage levels. Since these changes are a common finding in ageing rats and as there was no difference in severity and no dose-relationship, they were considered not to be treatment-related.

In the thyroid gland, colloid alteration showed a higher incidence and/or severity at 2500, 1000 or 250 ppm compared with controls. Brown pigment in follicular cells showed a higher incidence and/or severity at all dosage levels compared with controls. Alteration of colloid and brown (lipofuscin) pigment in the thyroid gland of rats were considered not to be an adverse finding, since these findings were seen in the controls and reflect a normal age-related physiological process associated with the rapid turnover of colloid which is unique to the rat. Diffuse follicular cell hypertrophy was only seen in a few females dosed at 2500, 1000 or 250 ppm but showed no effect of treatment in males. Focal follicular hyperplasia was seen in animals from all groups and the incidence was slightly greater than controls in males dosed at 1000 ppm.

In the pancreas, diffuse degeneration/atrophy of acinar tissue was recorded in a small number of animals dosed at 2500 and 1000 ppm. Focal degeneration/atrophy of acinar tissue was recorded in animals from all groups but the incidence was greater than controls in females dosed at 2500 ppm. Interstitial oedema was greater than controls in a small number of animals dosed at 1000 ppm.

In the kidneys, chronic progressive nephropathy (CPN) was seen in most animals on test but the severity was slightly greater than controls in males dosed at 2500, 1000 or 250 ppm. At 25 ppm, the difference was judged not to be relevant since there was no real difference in severity when compared to controls. A higher incidence and severity of hyperplasia of the collecting ducts were noted in males dosed at 2500 or 1000 ppm.

In the sciatic nerve, diffuse atrophy and degeneration of myelin/fibres was seen in animals from all groups but the incidence and severity of atrophy were greater than controls in animals dosed at 2500, 1000 or 250 ppm, whilst the incidence of degeneration was reduced in a similar fashion. Perivascular mineralisation was seen in animals from all groups but the incidence was greater than controls in males dosed at 250 ppm. Granuloma/cholesterol clefts were seen in animals from all groups but the incidence and severity were greater than controls in females dosed at 1000 ppm. These lesions were considered to be of minor toxicological importance as they occur spontaneously in ageing laboratory rats kept under laboratory conditions and the incidence and severity of diffuse atrophy and degeneration of myelin/fibres was within the in-house historical control range.

In the skeletal muscle, atrophy of myofibres was seen in animals from all groups but the incidence and severity were greater than controls in males dosed at 2500, 1000 or 250 ppm. However, this lesion was considered to be of minor toxicological importance as it occurs spontaneously in ageing laboratory rats kept under laboratory conditions and the incidence and severity of the lesion was within the in-house historical control range.

In the pituitary gland, focal hyperplasia of the pars distalis was noted in animals from all groups but the incidence was slightly higher than controls in animals dosed at 2500 ppm. It should be noted that there was no evidence of an effect of treatment on tumours of the pars distalis and this finding was considered to have occurred by chance.

In the blood vessels, a higher incidence of arterial inflammation with degeneration or intimal proliferation was seen in a number of tissues, mainly at 2500 or 1000 ppm in males. In other tissues the incidence was very low and there was no apparent difference from controls. This
change was considered to be of little toxicological importance.

In the ovaries, depletion of corpora lutea and follicles was recorded in all groups but the incidence was slightly greater than controls in animals dosed at 2500, 1000 or 250 ppm. In the uterus, squamous metaplasia of glands was higher in incidence than controls at 2500 ppm and a decrease in vaginal mucification with an increase in focal granular cell hyperplasia at the same dose level. When compared with controls, the changes were minor in severity and low in frequency, therefore they were considered to be of minor toxicological importance.

Focal mineralisation in the brain was seen in animals from all groups but the incidence was marginally higher than controls in males dosed at 2500 ppm but due to the low incidence and severity, it was considered to be of minor toxicologically importance as this finding occurs spontaneously in ageing laboratory rats kept under laboratory conditions and the incidence and severity was within the in-house historical control range.

See Attachment 3 "attached background material" for tabulated histopathological findings (eye, liver, thyroid, kidneys and pancreas).

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the eyes, squamous cell tumours were seen in the cornea of two males dosed at 2500 ppm (one papilloma and one carcinoma). Tumours of the cornea are not reported as spontaneous lesions and the occurrence of two in males dosed at 2500 ppm were considered to be an effect of treatment. In the light of persistent corneal inflammation and regenerative hyperplasia as an effect of treatment, these tumours were considered to have occurred as a result of a nongenotoxic proliferative mechanism.
In the thyroid gland, follicular cell adenoma was seen in five males dosed at 250 ppm (p<0.05). There were, however, no thyroid follicular cell adenomas at higher doses (1000 and 2500 ppm). In the absence of a dose relationship and in the face of very low tumor incidence, these follicular cell adenomas were considered not to be treatment-related.

A variety of spontaneous tumours was noted in control and treated animals with no indication of an effect of treatment. The spectrum of these findings is generally consistent with neoplastic changes commonly encountered in ageing laboratory rats kept under laboratory conditions.

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Details on results:

Corneal opacities, occasionally accompanied by neovascularization and their histopathological correlates (keratitis, reactive epithelial hyperplasia, and vascularization) are considered a rat-specific phenomenon. Corneal changes were not seen in other species chronically treated with the test substance (i.e. mice and dogs). The test substance is an inhibitor of the HPPDase enzyme and induces increased plasma tyrosine levels. This effect is more pronounced in rats than in mice and dogs. Experimentally induced hypertyrosinemia has been shown to induce snow flake-like corneal lesions in rats but not in mice (M-210983-01-2). In mice and humans, even under conditions of strong HPPD inhibition, tyrosine concentrations will not increase to levels high enough to induce ocular toxicity and hence, this toxicity observed in the rat is inappropriate for extrapolation to humans (ECETOC TR No. 99).

Thyroid findings (increased weight, histopathological changes comprising changes in colloid, follicular cell hypertrophy and pigment deposition in the follicular epithelium) are considered a non-adverse and rat specific phenomenon. No changes of the thyroid were noted in either mice or dogs, the other two species in which repeated-dose studies with histopathological examination of the thyroid were conducted with the test substance. As the test substance through inhibition of the HPPDase enzyme increases plasma tyrosine concentration in the rat, it is quite possible that some of this increased tyrosine is taken up by the thyroid and stored in the colloid, either as free tyrosine or through either increasing the synthesis of thyroglobulin or altering its composition in terms of number of tyrosine residues per thyroglobulin molecule.
In the absence of signs of altered thyroid functions (such as effects on body weights, fertility and gestation indices or effects on offspring performance in the developmental neurotoxicity study), the observed morphological changes are considered to be non-adverse.

Relevance of carcinogenic effects / potential:

Corneal tumors seen in two males at 2500 ppm are considered to be secondary to persistent corneal inflammation and regenerative hyperplasia. Accordingly, these tumours were considered to have occurred as a result of a nongenotoxic proliferative mechanism. As the underlying corneal changes are a rat-specific phenomenon, the occurrence of corneal tumors is considered of no relevance to humans.

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

25 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects noted at 25 ppm

Remarks on result:

other: equivalent to 1.0 and 1.4 mg/kg bw/day in males and females, respectively

Key result

Dose descriptor:

LOAEL

Remarks:

general toxicity

Effect level:

250 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

gross pathology

histopathology: non-neoplastic

ophthalmological examination

organ weights and organ / body weight ratios

urinalysis

Remarks on result:

other: equivalent to 10 and 14 mg/kg bw/day in males and females, respectively

Key result

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Effect level:

2 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: neoplastic

Remarks on result:

other: equivalent to 104 and 140 mg/kg bw/day in males and females, respectively

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 ppm

System:

gastrointestinal tract

Organ:

pancreas

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Critical effects observed:

yes

Lowest effective dose / conc.:

250 ppm

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Critical effects observed:

yes

Lowest effective dose / conc.:

250 ppm

System:

eye

Organ:

cornea

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Critical effects observed:

yes

Lowest effective dose / conc.:

250 ppm

System:

endocrine system

Organ:

thyroid gland

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Critical effects observed:

no

Lowest effective dose / conc.:

250 ppm

System:

hepatobiliary

Organ:

liver

Table 2: Mean achieved test item intake per group

Mean achieved test item intake (mg/kg bw/day)
Sex	Males				Females
Dose levels (ppm)	25	250	1000	2500	25	250	1000	2500
Weeks 1 - 13	1.4	14	58	143	1.8	19	77	191
Weeks 1 - 52	1.1	11	45	114	1.5	15	63	155
Weeks 1 - 104	1.0	10	41	104	1.4	14	57	140

 Table 3: Treatment-related clinical signs after 6 months

Incidence of treatment-related clinical signs after 6 months of treatment (first day of appearance)
Sex	Male					Female
Dose level (ppm)	0	25	250	1000	2500	0	25	250	1000	2500
Group size	75	75	75	75	75	75	75	75	75	75
White area on eye	0	1 (40)	38 (13)	41 (13)	48 (6)	0	1 (14)	5 (69)	36 (41)	52 (27)
Soiled fur generalized	0	0	2 (138)	4 (138)	13 (131)	0	0	2 (159)	4 (159)	19 (159)
Soiled fur localized	0	0	0	0	6 (34)0	0	0	0	1 (167)	1 (167)
Soiled anogenital region	0	0	0	1 (68)	2 (111)	0	0	1 (104)	1 (139)	25 (35)

Table 4: Treatment-related clinical signs after 12 months

Incidence of treatment-related clinical signs after 6 months of treatment
Sex	Male					Female
Dose level (ppm)	0	25	250	1000	2500	0	25	250	1000	2500
Group size	65	65	65	65	65	65	65	65	65	65
White area on eye	1	1	48	60	59	0	2	10	41	57
Soiled fur generalized	0	1	2	4	15	0	0	2	5	20
Soiled fur localized	0	0	0	0	11	0	0	0	5	17
Soiled anogenital region	0	0	0	4	16	0	0	1	9	35

Table 5: Treatment-related clinical signs during the second year of treatment

Incidence of treatment-related clinical signs during the second year of treatment
Sex	Males					Females
Dose level (ppm)	0	25	250	1000	2500	0	25	250	1000	2500
Group size	53	49	55	53	49	54	54	55	55	55
White area on eye	6	7	46	50	48	4	6	13	40	43
Soiled anogenital region	0	2	7	6	15	3	1	0	8	20

Table 6: Mortality incidence after two years (unscheduled deaths)

Mortality incidence after 2 years of treatment
Sex	Males					Females
Dose level (ppm)	0	25	250	1000	2500	0	25	250	1000	2500
Group size	55	55	55	55	55	55	55	55	55	55
Mortality [% mortality)	30 (54.5)	38 (69.1)	25 (45.5)	29 (52.3)	40* (72.7)	28 (50.9)	19 (34.5)	25 (45.5)	22 (40.0)	26 (47.3)

* ≤ 0.0.5

Table 7: Mean body weights (BW) and cumulative body weight gains (BWG)

Group mean body weights (BW) and cumulative body weight gains (BWG) (g)
Dose levels (ppm)	0	25	250	1 000	2 500
Males
Initial BW (Day 1) (%C)	287	287 (100)	288 (100)	289 (101)	286 (100)
BW Week l (Day 8) (%C)	336	336 (100)	330 (98)	329 (98)	327 * (97)
BW Week 13 (Day 92) (%C)	548	565 (103)	539 (98)	526 * (96)	529 (97)
BW Week 25 (Day 176) (%C)	632	645 (102)	624 (99)	602 ** (95)	597 ** (94)
BW Week 53 (Day 372) (%C)	704	714 (101)	688 (98)	664 * (94)	661 ** (94)
BW Week 77 (Day 540) (%C)	737	733 (99)	688 * (93)	664 ** (90)	647 ** (88)
Final BW (Day 708) (%C)	651	636 (98)	624 (96)	619 (95)	576 (88)
BWG Week 1 (Days 1 to 8) (%C)	49	50 (102)	42 ** (86)	40 ** (82)	41 ** (84)
BWG Weeks 1-13 (Days 1 to 92) (%C)	261	279 * (107)	252 (97)	237 ** (91)	243 * (93)
BWG Weeks 14-25 (Days 92 to 176) (%C)	83	76 (92)	86 (104)	77 (93)	70 ** (84)
BWG Weeks 26-53 (Days 176 to 372) (%C)	70	70 (100)	64 (91)	67 (96)	65 (93)
BWG Weeks 54-77 (Days 372 to 540) (%C)	41	29 (71)	1 ** (2)	-2 ** (nc)	-14 ** (nc)
BWG Weeks 78-101 (Days 540 to 708) (%C)	-73	-82 (112)	-47 (64)	-38 (52)	-60 (82)
Overall BWG (Days 1 to 708) (%C)	365	353 (97)	339 (93)	331 (91)	298 (82)
Females
Initial BW (Day 1) (%C)	196	193 (98)	193 (98)	194 (99)	193 (100)
BW Week l (Day 8) (%C)	214	216 (101)	212 (99)	210 (98)	209 (98)
BW Week 13 (Day 92) (%C)	297	302 (102)	300 (101)	296 (100)	299 (101)
BW Week 25 (Day 176) (%C)	320	328 (103)	330 (103)	327 (102)	331 (103)
BW Week 53 (Day 372) (%C)	367	367 (100)	369 (101)	357 (97)	366 (100)
BW Week 77 (Day 540) (%C)	437	432 (99)	423 (97)	410 (94)	423 (97)
Final BW (Day 708) (%C)	452	443 (98)	423 (94)	409 (90)	415 (92)
BWG Week 1 (Days 1 to 8) (%C)	17	22** (129)	20 (118)	17 (100)	16 (94)
BWG Weeks 1-13 (Days 1 to 92) (%C)	102	109** (107)	107 (105)	102 (100)	105 (103)
BWG Weeks 14-25 (Days 92 to 176) (%C)	22	26 (118)	30** (136)	31** (141)	33** (150)
BWG Weeks 26-53 (Days 176 to 372) (%C)	47	36 (77)	40 (85)	29** (62)	33* (70)
BWG Weeks 54-77 (Days 372 to 540) (%C)	70	65 (93)	57 (81)	53 (76)	57 (81)
BWG Weeks 78-101 (Days 540 to 708) (%C)	23	21 (91)	12 (52)	4 (17)	-2 (nc)
Overall BWG (Days 1 to 708) (%C)	254	249 (98)	231 (91)	217 (85)	223 (88)

 C            control

nc          not calculated

*  Statistically different (p ≤ 0.05) from the control

** Statistically different (p ≤ 0.05) from the control

Table 8: Mean liver weights

Liver weight changes at terminal sacrifice (% change when compared to controls) carcinogenicity phase
Sex	Males					Females
Dose level (ppm)	0	25	250	1000	2500	0	25	250	1000	2500
Mean absolute liver weight	12.93 -	13.28 (+3%)	15.53 (+20%) **	15.83 (+22%) **	15.09 (+17%)	10.18 -	10.31 (+1%)	10.29 (+1%)	10.45 (+3%)	10.57 (+4%)
Mean liver to body weight ratio	2.035 -	2.154 (+6%)	2.626 (+29%) **	2.694 (+22%) **	2.676 (+31%) **	2.520 -	2.542 (+1%)	2.747 (+9%)	2.794 (+11%)	2.781 (+10%)
Mean liver to brain weight ratio	544.287 -	560.064 (+3%)	673.789 (+24%) **	699.504 (+29%) **	670.738 (+23%) **	483.664 -	477.191 (-1%)	486.820 (+1%)	492.835 (+2%)	507.566 (+5%)

** Statistically different (p ≤ 0.05) from the control

Table 9: Mean kidney weights

Kidney weight changes at terminal sacrifice (% change when compared to controls) carcinogenicity phase
Sex	Males					Females
Dose level (ppm)	0	25	250	1000	2500	0	25	250	1000	2500
Mean absolute kidney weight	3.44 -	3.67 (+7%)	3.99 (+16%)	4.17 (+21%) **	4.21 (+22%)**	2.24 -	2.31 (+3%)	2.50 (+12%)	2.48 (+11%)	2.36 (+5%)
Mean kidney to body weight ratio	0.524 -	0.550 (+5%)	0.607 (+16%) **	0.634 (+21%) **	0.656 (+25%) **	0.691 -	0.697 (+1%)	0.718 (+4%)	0.709 (+3%)	0.701 (+1%)
Mean kidney to brain weight ratio	148.544 -	159.365 (+7%)	180.785 (+22%) **	184.084 (+24%) **	185.672 (+25%) **	109.349 -	113.074 (+3%)	119.435 (+9%)	119.840 (+10%)	114.760 (+5%)

*  Statistically different (p ≤ 0.05) from the control

** Statistically different (p ≤ 0.05) from the control

Conclusions:

The study was performed under GLP conditions and according to OECD TG 453 (adopted 1981). Treatment-related neoplastic findings of the cornea were observed at 2500 ppm but were considered to be secondary to continuous corneal inflammation and ulceration. These cornea effects are a rat specific phenomeon and to be due to a non-genotoxic proliferative mechanism. They were considered to have no relevance to humans. Accordingly, up to and including the highest administered dose, there was no indication of neoplastic findings which are relevant to man and the NOAEL for carcinogenicity is considered to be 2500 ppm (104 mg/kg bw/day in males, 140 mg/kg bw/day in females).The NOAEL for general toxicity in the 24-month phase of the study was considered to be 25 ppm (corresponding to 1.0 mg/kg bw/day for males and 1.4 mg/kg bw/day for females), based on test substance related effects at 250 ppm, including decreased body weights, reduced urinary pH, increased liver and kidney weights, histopathological effects in cornea, kidney and thyroid.

Reference 2

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 Sep 2003 to 07 Oct 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 June 2018

Deviations:

yes

Remarks:

no details on whether feed and water were analysed for contaminants

GLP compliance:

yes (incl. QA statement)

Remarks:

Groupe Interministeriel des Produits Chimiques, 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 (l'Arbresle, France)
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 6 weeks
- Weight at study initiation: 17.3 - 24.3 g (males), 15.2 - 20.0 g (females)
- Fasting period before study: no
- Housing: By sex in groups of 3 from arrival until day 8 of the acclimatization phase, then individually. Animals were housed in suspended, stainless steel and wire mesh cages.
- Diet: A04C-10 PI from S.A.F.E. (Scientific Animal Food and Engineering, Augy, France), ad libitum
- Water: filtered and softened tap water from the municipal water supply, ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): 10 to 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 03 Sep 2003 To: 04 Apr 2005

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DIETARY MIXTURES:
The test substance was incorporated into the diet to provide the required dietary concentrations of 100 and 1000 ppm for both sexes or 4000 ppm for males and 6000 ppm for females from Day 1 to Day 70 reduced to 4000 ppm from Day 71 onwards due to a high mortality observed in females of high dose. For formulation sequences F1 and F2, one load of 60 kg was prepared at 0, 100 and 1000 ppm and one load of 30 kg was prepared at 4000 and 6000 ppm.
For formulation sequences F3 to F10, one load of 60 kg was prepared at the concentration levels of 0, 100, 1000 and 4000 ppm. The 6000 ppm preparation was no longer prepared since it was the highest dose for female mice, which showed high mortality at that dose and it was reduced to a 4000 ppm dose level from Day 71 onwards.
For that reason, in order to cover extra needs for the preparation at 4000 ppm before the third formulation and also in order to offset accidental spillage of the 4000 and 1000 ppm preparations, a complementary formulation (F2 bis) was made, consisting of one load of 10 kg at the 1000 ppm dose level and two loads of 10 kg at the 4000 ppm dose level. Another extra formulation (F3 bis) was made to accommodate the change of dose level from 6000 ppm to 4000 ppm for female animals and consisted of one load of 20 kg at 4000 ppm.

DIET PREPARATION
- Rate of preparation of diet (frequency): 10 formulations were prepared during the study at each concentration
- Mixing appropriate amounts with: Certified Rodent Meal A04C-10 PI from S.A.F.E.
- Storage temperature of food: at ambient temperature

VEHICLE
- No vehicle was used.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in the diet has been demonstrated in a previous 90 day rat study (M-102924-01-2). The stability of the test substance at 2 and 15000 ppm in the diet was verified for up to 82 and 95 days, respectively, when kept at ambient temperature.
The homogeneity of the test substance in the diet was verified at all dose levels (100, 1000, 4000 and 6000 ppm) on the first formulation, at 4000 ppm on the third formulation and at 100 and 4000 ppm on the sixth formulation. In addition, the homogeneity of the supplementary preparations F2 bis was also checked on the first load of 1000 and 4000 ppm to demonstrate adequate formulation procedures.

During the study, the concentration of each dietary level was verified prior to administration to the animals for the following formulations: F1, F2 bis (first load preparation at 1000 and 4000 ppm and second load preparation at 4000 ppm), F3, F6 and F9. In addition, particulate matter (stones) found in the gall bladder, kidney, urinary bladder of test animals were analyzed for the presence of test substance.

Results:
- Homogeneity and concentration analysis: Homogeneity and concentration of the test substance in the diet ranged between 89 to 115% of nominal concentration and were therefore within the in-house target range of 85 to 115% of nominal concentration.
- Stability analysis: Stability was demonstrated in a previous study (M-102924-01-2)

Duration of treatment / exposure:

18 months for carcinogenicity phase ("terminal sacrifice")
12 months for interim sacrifice

Frequency of treatment:

daily, 7 days a week

Post exposure period:

not applicable

Dose / conc.:

100 ppm

Remarks:

equivalent to 13.6 and 16.7 mg/kg bw/day in males and females, respectively

Dose / conc.:

1 000 ppm

Remarks:

equivalent to 137 and 168 mg/kg bw/day in males and females, respectively

Dose / conc.:

4 000 ppm

Remarks:

equivalent to 560 and 713 mg/kg bw/day in males and females, respectively

Dose / conc.:

6 000 ppm

Remarks:

initial high dose for females; reduced to 4000 ppm after 10 treatment weeks due to high mortality level

No. of animals per sex per dose:

50 for carcinogenicity phase
10 for interim sacrifice

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Dose levels were selected based on the results from previous 28-day dietary study in the mouse and 90-day dietary study in the mouse (M-103284-01-4).
In the 28-day study, treatment-related changes were observed in the urinary bladder of a proportion of the male exposed to 5000 ppm where a diffuse urothelial hyperplasia was found in 3/10 animals. This finding was associated with a diffuse submucosal granulation tissue and with a diffuse suburothelial mixed-cell infiltrate. These observations were correlated to a gritty content found macroscopically in the urinary bladder of two of the three affected male animals. These findings, observed in males, were regarded as being toxicologically relevant, while in females, no adverse effects were observed at 5000 ppm.
In the 90-day study, the only treatment-related finding consisted of an increase in urinary pH value observed in females treated at 3000 ppm. This effect was regarded as being non-toxicologically relevant.
Therefore, it was anticipated that the low and high doses chosen of 100 (both sexes) and 4000 ppm for the males or 6000 ppm for the females would constitute a No-Observed-Effect-Level (NOEL) and a maximum tolerated dose (MTD), respectively, with the intermediate dose of 1000 ppm serving to confirm any dose response relationships that may have emerged.

- Rationale for animal assignment (if not random): On the day of randomization, animals were allocated to dose groups using a computerized randomization procedure that ensured a similar body weight distribution for each dose group. The acceptable body weight range for each dose group was ± 20% of the mean body weight on the day of randomization. Any animal deemed unsuitable for selection based on weight, ophthalmological abnormalities or health status was not used for the study.

Observations and examinations performed and frequency:

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed physical examinations including palpation for masses were performed weekly from study day 1.

BODY WEIGHT: Yes
- Time schedule for examinations: on days 3, 8 and 14 during the acclimatization period then weekly for the first 13 weeks of study, approximately every 4 weeks thereafter and prior to necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations:
all animals: during acclimatization (indirect ophthalmoscopy)
all animals from interim sacrifice phase and the first surviving 14 animals from the final sacrifice phase: after approximately 3, 6, 9, 12 months all surviving animals: at approximately 18 months
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
week 53 or 54: all surviving animals of the interim sacrifice groups and of the first ten surviving animals of the terminal sacrifice groups
week 79 or 80: the first twenty surviving mice of the terminal sacrifice groups
At scheduled necropsy, blood smears were prepared for all animals not sampled for hematology. The blood smears were stained with Wright stain for possible differential white blood cell determination (on the request of the Study Pathologist).
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- Parameters examined: haematocrit, haemoglobin, leukocyte count, erythrocyte count, platelet count, leukocyte differential count, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, mean corpuscular volume

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Necropsy included the examination of external surfaces, all orifices, all major organs, tissues and body cavities. All significant macroscopic abnormalities (including masses and their regional lymph nodes when possible) were recorded, sampled and examined microscopically.

ORGAN WEIGHTS: Yes (see Table 1)

HISTOPATHOLOGY: Yes (see Table 1)
No histopathological examination was performed for animals at the 12-month interim sacrifice.

Other examinations:

Particulate matter (stones) found in the gall bladder, kidney, urinary bladder of test animals were analyzed for the presence of test substance.

Statistics:

See "Any other information on materials and methods incl. tables"

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At the high dose, treatment-related effects consisted of hardness urinary bladder area observed in the majority of animals of both sexes, soiled fur, reduced motor activity, distended abdomen, prostration, laboured respiration and cold to touch observed in both sexes, and tremors, red urine in males, and rapid respiration observed in females (see Table 3 in "Any other information on results incl. tables").

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

Not applicable.

Mortality:

mortality observed, treatment-related

Description (incidence):

At 6000 ppm in females, the mortality was high at the beginning of the study with 7/60 females dying during the first 10 weeks. Therefore, it was decided to treat females of the high dose at a lower dose-level of 4000 ppm from Week 11 onwards. When compared to controls, the overall mortality rate throughout the study was increased in both sexes treated at 4000 ppm before the interim sacrifice and final sacrifice. Therefore, there was a treatment-related effect at 4000 ppm in either sex. At 1000 or 100 ppm, the mortality rate was comparable to controls (see Table 4 in "Any other information on results incl. tables").

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 4000 ppm, mean body weight of both sexes was statistically significantly decreased, in males from Week 21 onwards (p < 0.01), resulting in a final body weight reduction of 8% in males, and in females in Week 77 (p < 0.01) only, resulting in a final body weight reduction of 5%.
At 1000 ppm, the mean cumulative body weight gain of males was frequently statistically significantly decreased (p < 0.01 or p < 0.05).
At 100 ppm in both sexes and at 1000 ppm in females, mean body weight and mean body weight change were similar to the controls.

At the 12-months interim sacrifice, there was no change in mean terminal body weights in treated animals, when compared to controls.
At the 18-month sacrifice, at 4000 ppm, mean terminal body weights were statistically significantly decreased for males (-8%, p < 0.01) and for females (-6%, p < 0.01) when compared to controls.

Food consumption and compound intake (if feeding study):

no effects observed

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:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Lower mean red blood cell count, haemoglobin concentration and haematocrit were noted in females at 4000 ppm throughout the study (see Table 5 in "Any other information on results incl. tables").
Additionally, slight variations were noted in mean corpuscular volume (+4%, p < 0.01) and mean corpuscular haemoglobin concentration (-5%, p < 0.01) after 18-months. In males at 4000 ppm, only slight tendencies towards lower red blood cell counts (-7%, p < 0.05) and haemoglobin concentrations (-6%, not statistically significant) were noted after 18 months .
After 12 months at 4000 ppm, slightly lower mean total leucocyte count (-36%, p < 0.05) was noted in males only. This variation was not observed after 18 months and therefore was considered less likely to be adverse.
The other few statistically significant differences observed throughout the study were considered not to be toxicologically relevant in view of their low magnitude.
At month 19, two females from the 4000 ppm group had very high total leucocyte count (with large and atypical lymphocytes). At histopathology examination, these findings were correlated with an incidental malignant lymphoma of the haematopoietic system, for both animals.
No relevant change was observed at 1000 and 100 ppm in either sex.

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):

12-months interim sacrifice
At 4000 ppm for males, mean absolute and relative kidney weights were statistically significantly increased, when compared to controls.

All other organ weight differences were considered to be incidental in view of their individual variations.

18-month carcinogenicity phase
Mean absolute and relative liver weights were statistically significantly increased in males and females at all dose levels for almost all parameters, when compared to controls. These changes were considered to be treatment-related only at 4000 and 1000 ppm, since at 100 ppm the liver weight change was very low and not correlated with concomitant microscopic hepatocellular hypertrophy.

At 4000 ppm in males, mean absolute and relative kidney weights were statistically significantly increased when compared to controls. The kidney weight changes were considered to be toxicologically relevant.

Some higher spleen weights were observed in both sexes, mostly at 4000 ppm but it was considered not to be treatment-related since there were no associated histological findings. All other organ weight differences were considered to be incidental in view of their individual variation.

See Table 6 in "Any other information on results incl. tables" for selected absolute and relative organ weights.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

12-months interim sacrifice
a) Unscheduled deaths
At 4000 ppm, one male was found dead on Day 240 with multiple stones (yellow) into the urinary bladder which were considered as the cause of death of the animal. Three females were found dead, respectively on Days 60, 61 and 75. All of them had some urinary bladder stones (yellow, hard for 2/3 animals) which were considered as the cause of death of the animals.

All other deaths in intermediate groups in both sexes were judged to be incidental. Nodules inside the right kidney, bilateral pelvic dilatation, abnormal shaped right kidney, distended urinary bladder and multiple stones in the urinary bladder were observed for a low dose female, but these stones were morphologically different (white) compared to the 4000 ppm group (hard and yellow). They were considered to be fortuitous and not treatment-related.

b) Terminal sacrifice
At 4000 ppm, stone(s) in renal pelvis were observed in one male and 4/7 females. In addition, enlarged or small kidneys, renal pelvic dilatation, pale kidneys or renal cyst(s) were observed in both sexes. Stone(s) in urinary bladder were observed in 8/9 males and 6/7 females and distended urinary bladders were observed in 8/9 males and 4/7 females.
Some gallbladder stone(s) were found in males and/or females at 4000, 1000 and 100 ppm.
All other gross pathology changes were considered as incidental and not treatment-related.

18-month carcinogenicity phase
a) Unscheduled deaths
One hundred and nine animals prematurely died before the end of the study. The higher mortality rate at 4000 ppm in both sexes was largely due to the presence of stones within the urinary tract, causing acute renal failure (by urinary blockage) or chronic renal failure (by concomitant chronic kidney/urinary bladder inflammation). At 4000 ppm, stones in the kidneys were found in some animals at 4000 ppm in both sexes and enlarged or small kidneys, renal pelvic dilatation, pale kidneys or renal cyst(s) were also observed in both sexes. Stones in the urinary bladder and distended urinary bladders were found in some animals at 4000 ppm in both sexes. Stones in the gallbladder were found in 3/8 males at 1000 ppm.

b) Terminal sacrifice
At 4000 ppm, stones in the kidneys, small kidneys, renal pelvic dilatation, pale kidneys or renal cyst(s) were observed in both sexes. Stones in the urinary bladder were found in 24/25 males and 11/20 females at 4000 ppm. In addition, distended urinary bladders were observed in some males at 4000 ppm.
Stones in the gallbladder were found with a higher incidence in treated animals of both sexes.

See Attachment 1 "attached background material" for tabulated gross pathological findings.

Neuropathological findings:

not examined

Description (incidence and severity):

Not applicable.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

12-months interim sacrifice:
No microscopic examination was performed.

18-month carcinogenicity phase:
a) Unscheduled deaths
At 4000 ppm, 24/25 deaths in males and 23/30 deaths in females were attributed to the presence of urinary stones. All other deaths in intermediate doses were considered to be incidental.

b) Terminal sacrifice
Higher incidences and/or severity of amyloid deposition were observed in various organs in females. Some minor changes in incidence were seen in the heart of males and the stomach of females at 4000 ppm, and in the spleen of males at 4000 and 1000 ppm.

c) Scheduled and unscheduled deaths
Direct treatment-related effects were found in the liver, gallbladder, kidney, urinary bladder, prostate (prostatic urethra) and ureters.
Indirect treatment-related effects were also found in various organs (thymus, lymph nodes) in the lymphoid system (apoptotic bodies and/or lymphoid atrophy) following stone-induced stress.

Liver: Minimal to moderate centrilobular hepatocellular hypertrophy was observed in both sexes at 4000 and 1000 ppm, with a dose response relationship. A loss of diffuse hepatocellular vacuolation was observed in males at 4000 and 1000 ppm and at 4000 ppm in females. At 4000 ppm in both sexes a higher incidence of periportal hepatocellular vacuolation was noted. These changes were judged to be correlated with the presence of a gradual dose-related hepatocellular hypertrophy. A higher incidence of interstitial mixed cell infiltrate was noted in males dosed at 4000 ppm.
A higher incidence of lobar torsion was noted in males dosed at 4000 ppm (4/50) when compared to controls (1/50) but this finding was judged to be due to chance and not treatment-related.

Gallbladder: Higher incidences of epithelial hyperplasia and gallstones were noted in both sexes in all doses. Crystalline gallstone corresponds to birefringent stone under polarized light.

Kidney: Presence of pelvic stones was noted in both sexes in animals dosed at 4000 ppm. Some urinary stones (kidney or prostate) appeared to be birefringent stones under polarized light. In males and/or females at 4000 ppm, there were numerous renal findings related to the local irritation induced by these stones. Those secondary changes were classified as hyperplastic such as:
- collecting duct hyperplasia,
- pelvic epithelium hyperplasia.
Others secondary changes associated or urinary blockage were defined as:
- papillary fibrosis/atrophy and cortex/medulla atrophy/fibrosis/scar,
- suburothelial mixed cell infiltrate and interstitial hemorrhages,
- glomerular chamber and cortical tubular dilatation, pelvic dilatation and papillary necrosis.
Higher incidence and severity of unilateral cortical basophilic tubules were noted in females dosed at 4000 ppm. Higher incidence in males dosed at 100 ppm was judged not to be treatment-related but incidental, as there was no dose relationship and the severity was graded minimal. Higher incidences of parenchymal cysts and medullary tubular mineralization were noted in females at 4000 ppm. Collecting duct concretions and arteritis/periarteritis were also noted in both sexes dosed at 4000 ppm.
In addition, unilateral atrophy/fibrosis/scar was noted in females at 1000 ppm: this was judged not to be treatment-related since it was unilateral and only observed in females.

Urinary bladder: At 4000 ppm, presence of vesical stones (intraluminal in both sexes or intraglandular in males) was noted in both sexes.
Presence of numerous stone-induced findings was noted in males and/or females at 4000 ppm: Secondary hyperplastic related local irritation induced by the stones was defined as:
- simple, nodular/glandular, squamous and atypical hyperplasias.
Secondary changes due to stone-induced irritation or urinary blockage were defined as:
- vesical distension,
- muscular hemorrhage(s)/necrosis, vascular congestion, interstitial edema and adenomyosis,
- intramuscular inflammatory cell infiltrate; suburothelial, serosal or interstitial mixed cell infiltrate.

Prostate: At 4000 ppm, presence of intra-urethral stones was noted. Associated to the presence of these stones, there were numerous prostatic findings which were related to the chronic irritation: simple hyperplasia and adenomyosis.

Ureters (only sampled when macroscopically abnormal): At 4000 ppm, presence of stones within the ureters was noted in males dosed at 4000 ppm. The fact that no stones were found in the ureters of females was considered to be fortuitous, since some stone-induced findings due to chronic irritation or urinary blockage were noted at 4000 ppm in males and/or in females and were defined as: simple or nodular/glandular urothelial hyperplasia as well as dilatation and adenomyosis.

Some indirect treatment-related effects were noted in various organs and were judged to be caused by stone-induced stress (caused by urinary blockage or inflammation), causing lymphoid depletion (atrophy and/or lymphocytic apoptosis):
- in the thymus, higher incidence of apoptotic bodies was noted in both sexes at 4000 ppm (29/46 for males and 29/48 for females; p<0.01 for both sexes), notably in unscheduled animals (14/22 in males and 14/28 in females), when compared to controls (8/48 for males and 4/46 in females),
- in the spleen, higher incidence of lymphoid apoptotic bodies was noted in both sexes at 4000 ppm (16/49 for males; p<0.01 and 16/50 for females; p<0.05), all in unscheduled animals (16/24 in males and 16/30 in females), when compared to controls (0/50 for males and 2/50 in females). Higher incidence of lymphoid atrophy was also noted in both sexes at 4000 ppm (4/49 for males and 5/50 in females for all animals), all in unscheduled animals (4/24 in males and 5/30 in females), when compared to controls (0/50 for males and 1/50 in females),
- in the mesenteric lymph nodes, higher incidence of lymphoid apoptotic bodies was noted in both sexes at 4000 ppm (12/49 in males and 14/48 in females; p<0.01 for both sexes), almost in unscheduled animals (10/24 in males and 14/28 in females), when compared to controls (0/50 for males and 0/49 in females),
- in the submaxillary lymph nodes higher incidence of apoptotic bodies was noted in both sexes at 4000 ppm (11/50 in males and 14/49 in females; p<0.01 for both sexes), almost in unscheduled animals (11/25 in males and 13/29 in females), when compared to controls (0/50 for males and 1/50 in females).

Amyloid deposition: An increase in the incidence and/or severity of amyloid deposition was seen in a number of tissues, at doses of 4000, 1000 or 100 ppm in females. This change was judged not to be treatment-related and was considered not to be toxicologically relevant considering that:
- the high sensitivity of the C57BL/6J mice for amyloid deposits in males and females for which the pathology is known to occur spontaneously,
- the change was not systematically dose-related,
- there was no effect in common sites for amyloid deposition in the C57/BL6 mouse like the ileum or the spleen,
- this deposit was found only in one sex in this study and no inflammatory pathology specific to female mice has been found which could be related to this amyloid deposition.

Heart: Higher incidences of epicardial mixed cell infiltrate and myocardial fibrosis were noted in males dosed at 4000 ppm. Considering the low incidence and the low severity of theses changes observed only in one sex and the fact that they are found spontaneously in control, they were considered not to be toxicologically-relevant and not treatment-related.

Stomach: Higher incidence of arteritis/periarteritis was noted in females dosed at 4000 ppm. Considering the low incidence of this change observed only in one sex and the fact that it is found spontaneously in controls at the same severity, it was considered not to be toxicologically-relevant and not treatment-related.

Spleen: Higher incidence of plasmacytosis was noted in males dosed at 4000 and 1000 ppm. Considering the low incidence of this change observed only in one sex and the fact that it is found spontaneously in controls at the same severity, it was considered not to be toxicologically-relevant and not treatment-related.

Epididymis:
In the epididymis, higher incidence of bilateral oligospermia was noted in animals dosed at 4000 ppm (6/50, p < 0.05), when compared to controls (1/50). As there was neither higher incidence of bilateral testicular degeneration in animals dosed at 4000 ppm (when compared to controls) nor concomitant epididymal epithelium degeneration, this finding was judged to be incidental and not treatment-related.

All other findings were considered as incidental and not treatment-related.

See Attachment 2 "attached background material" for tabulated histopathological findings (liver, gallbladder and kidney).
See Attachment 3 "attached background material" for tabulated histopathological findings (urinary bladder, prostate and ureters).

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Transitional cell carcinomas or papillomas in the urinary bladder of both sexes and the prostatic urethra were noted at 4000 ppm (see table 7). In the light of the secondary inflammatory and hyperplastic changes induced by the urinary stones, these tumours were considered as the result of a non-genotoxic proliferative mechanism (see Table 7 in "Any other information on results incl. tables" for treatment related findings).

All other tumors in treated animals were those commonly observed in this strain of mouse at this age and were considered to be incidental in origin.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

The test substance was mainly detected in stones of the kidney and in the urinary bladder, whilst only traces of the test substance were detected in the gall bladder.

Relevance of carcinogenic effects / potential:

The urinary bladder and prostatic urethra tumors noted at 4000 ppm are considered to be the result of a non-genotoxic proliferative mechanism caused by continued irritation and inflammation due to urinary tract stones.

Key result

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Effect level:

4 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: neoplastic

Remarks on result:

other: equivalent to 560 and 713 mg/kg bw/day in males and females, respectively

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects seen at 100 ppm.

Remarks on result:

other: equivalent to 13.6 and 16.7 mg/kg bw/day in males and females, respectively

Key result

Dose descriptor:

LOAEL

Remarks:

general toxicity

Effect level:

1 000 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 137 and 168 mg/kg bw/day in males and females, respectively

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 ppm

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 ppm

System:

hepatobiliary

Organ:

gall bladder

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Critical effects observed:

yes

Lowest effective dose / conc.:

4 000 ppm

System:

urinary

Organ:

bladder

kidney

ureter

other: prostatic urethra

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Table 2: Mean achieved test item intake per group

Mean achieved test item intake (mg/kg bw/day)
Sex	Males			Females
Dose levels (ppm)	100	1000	4000	100	1000	6000*/4000
Weeks 1 - 13	15.9	161	643	19.3	199	1114
Weeks 1 - 52	14.0	141	575	17.4	175	788
Weeks 1 - 80	13.6	137	560	16.7	168	713

* Females were treated at 6000 ppm from Day 1 to Day 70 and 4000 ppm from Day 71 onwards

Table 3: Treatment-related clinical signs

Incidence of treatment-related clinical signs (first day of appearance)
Sex	Male				Female
Dose level (ppm)	0	100	1000	4000	0	100	1000	6000*/4000
Group size	60	60	60	60	60	60	60	60
Soiled fur (generalized and localized)	1 (540)	0	2 (125)	43 (216)	2 (496)	0	3 (245)	9 (245)
Reduced motor activity	1 (461)	0	2 (132)	8 (268)	3 (474)	1 (238)	3 (169)	12 (35)
Prostration	1 (468)	0	0	5 (60)	0	0	1 (362)	3 (160)
Tremors	0	1 (97)	1 (117)	4 (91)	0	1 (252)	0	1 (274)
Red urine	0	0	0	4 (64)	0	0	0	1 (314)
Laboured respiration	0	0	0	4 (60)	0	0	0	4 (90)
Rapid respiration	1 (41)	0	1 (139)	0	0	0	0	4 (90)
Cold to touch	1 (468)	0	0	6 (60)	3 (371)	0	1 (532)	5 (35)
Abdomen distended	0	1 (538)	1 (280)	9 (357)	0	1 (545)	1 (231)	3 (469)
Hardness urinary bladder area	0	0	0	52 (210)	0	1 (238)	0	33 (217)

* Females were treated at 6000 ppm from Day 1 to Day 70 and 4000 ppm from Day 71 onwards

Table 4: Mortality incidence (Unscheduled deaths)

Dose level (ppm)	Mortality incidence
	First 53 weeks (up to study Day 367) All animals		Whole study period Carcinogenicity phase animals
	Male	Female	Male	Female
0	3/60 (5%)	5/60 (8%)	7/50 (14%)	15/50 (30%)
100	5/60 (8%)	4/60 (7%)	8/50 (16%)	8/50 (16%)
1000	6/60 (10%)	6/60 (10%)	8/50 (16%)	8/50 (16%)
6000*/4000	14/60 (23%)	21/60 (35%)	25/50 (50%)	30/50 (60%)

* Females were treated at 6000 ppm from Day 1 to Day 70 and 4000 ppm from Day 71 onwards

Table 5: Selected erythrocyte parameters in female mice

		Erythrocyte values (mean ± standard deviation) in female mice (% variation when compared to controls)
Dose levels (ppm)		0	100	1000	6000#/4000
Parameter	Time
Red blood cell count (1012/L)	Month 12/13	9.50 ± 0.81 -	9.44 ± 0.64 NC	9.40 ± 0. NC	9.00 ± 0.93 (-5%)
	Month 19	9.17 ± 0.54 -	8.97 ± 0.44 NC	8.82 ± 0.47 NC	7.70 ± 1.06** (-16%)
Haemoglobin concentration (g/dL)	Month 12/13	14.5 ± 1.2 -	14.4 ± 0.9 NC	14.5 ±0.5 NC	13.6 ± 1.4** (-6%)
	Month 19	13.8 ± 0.7 -	13.6 ± 0.7 NC	13.2 ± 0.6 NC	11.6 ± 1.6** (-16%)
Haematocrit (L/L)	Month 12/13	0.481 ± 0.028 -	0.478 ± 0.026 NC	0.478 ± 0.019 NC	0.461 ± 0.043** (-4%)
	Month 19	0.435 ± 0.021 -	0.435 ± 0.022 NC	0.429 ± 0.017 NC	0.385 ± 0.043** (-13%)

# Females were treated at 6000 ppm from Day 1 to Day 70 and 4000 ppm from Day 71 onwards

NC: no relevant change

**: p ≤ 0.01, *: p ≤ 0.05

Table 6: Selected absolute and relative organ weights

	Dose level (ppm)
	Males				Females
Finding	0	100	1000	4000	0	100	1000	6000#/4000
	12-month sacrifice
Terminal body weight (g)	27.6	28.5	26.5	26.1	22.3	23.4	23.3	22.9
Abs. kidney weight (g)	0.419	0.432	0.402	0.762*	0.342	0.347	0.358	0.376
Rel. kidney weight (% body weight)	1.52	1.52	1.52	2.91**	1.54	1.49	1.54	1.64
	Terminal sacrifice
Terminal body weight (g)	28.1	28.6	28.0	25.9**	24.7	24.4	24.1	23.2**
Abs. liver weight (g)	1.19	1.25*	1.29*	1.22	1.34	1.36	1.39	1.39
Rel. liver weight  (% body weight)	4.23	4.38*	4.61**	4.73**	5.43	5.59	5.77**	6.01**
Abs. kidney weight (g)	0.474	0.472	0.465	0.901**	0.405	0.405	0.420	0.432
Rel. kidney weight (% body weight)	1.69	1.66	1.66	3.46**	1.64	1.66	1.74	1.87

# Females were treated at 6000 ppm from Day 1 to Day 70 and 4000 ppm from Day 71 onwards

**: p ≤ 0.01, *: p ≤ 0.05

Table 7: Treatment-related neoplastic microscopic findings

	Dose level (ppm)
	Males				Females
Finding	0	100	1000	4000	0	100	1000	6000#/4000
	Urinary bladder
No. of animals examined	50	49	50	50	49	47	50	49
M - transitional cell carcinoma	0	0	0	8**	0	0	0	2
B - transitional cell papilloma	0	0	0	3	0	0	0	2
	Urethra (prostatic)
M - urethral transitional cell carcinoma	0	0	0	1

# Females were treated at 6000 ppm from Day 1 to Day 70 and 4000 ppm from Day 71 onwards

**: p < 0.01

Conclusions:

The study was performed under GLP conditions and according to OECD TG 451 (adopted 1981). Based on the increased mortality at 4000 ppm in both males and females, the top dose of 4000 ppm was considered to have exceeded the Maximum Tolerated Dose. The NOAEL was established at 100 ppm (13.6 mg/kg bw/day in males, 16.7 mg/kg bw/day in females), as despite gallbladder concretions at all doses there was no increase at 100 ppm in epithelial hyperplasia in the gallbladder. The LOAEL was 1000 ppm. The neoplastic findings in the urinary tract were further evaluated in an additional regulatory toxicology position paper (M-284616-01-1, 2007). As above, the treatment-related neoplastic findings of the urinary tract were observed at 4000 ppm but were considered to be secondary to the administration of high doses of the test substance which precipitated in the urinary tract, causing continuous local irritation and to be due to a non-genotoxic proliferative mechanism. This is strongly supported by the fact that these tumours were only seen at the highest dose and in animals with urinary tract stones.. However, they are not considered to be relevant to human conditions, where much lower doses of the test substance are expected. Accordingly, up to and including the highest administered dose, there was no indication of neoplastic findings which are relevant to man. Accordingly, the NOAEL for carcinogenicity is considered to be 4000 ppm (560 mg/kg bw/day in males, 713 mg/kg bw/day in females).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

104 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

The available information comprises adequate and reliable studies (Klimisch score 1), and is thus sufficient to fulfil the standard information requirements set out in Annex X, 8.9.1, of Regulation (EC) No 1907/2006.

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

Data on the mode of action and human relevance for HPPDase inhibitors, specifically in relation to effects seen in the eye and thyroid gland are presented in the section Additional Toxicological Information, as well as being discussed in detail within the endpoint summary for Repeated Dose Toxicity (Additional Information field).

Justification for classification or non-classification

The available data on the carcinogenicy potential of 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl)methanone do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.

Additional information

 Reliable studies to assess the carcinogenetic potential of 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl)methanone are available in mice and rats.

 Carcinogenicity study in the C57BL/6J mouse by dietary administration:

In this GLP-conform study conducted according to OECD 451, 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl)methanone was incorporated into rodent diet and administered at concentrations of 0, 100, 1000, and 4000 ppm to groups of 60 male C57BL/6J mice (M-267521-01-2). Groups of 60 females received 0, 100, 1000, and 6000 ppm for the first 10 weeks of the study. However, mortality at 6000 ppm was considered to be excessive and this dose was decreased to 4000 ppm from week 11 onwards. These concentrations resulted in doses of 0, 13.6, 137, and 560 mg/kg bw/day for males and 0, 16.7, 168, and 713 mg/kg bw/day for females. Clinical signs and mortality were monitored twice daily on weekdays and once daily on weekends and holidays. Body weights were measured on the first day of treatment, weekly for the first 13 weeks of the study, and then at 4-week intervals through the end of the study. Ophthalmological examinations were conducted on all animals during the acclimatization phase and on all surviving animals at the end of the study. At 3, 6, 9, and 12 months, ophthalmoscopy was conducted on 24 animals per sex and treatment group. At the end of the one-year treatment period, blood was collected for haematology from the surviving animals in the one-year sacrifice group and from the first 10 surviving animals of the terminal sacrifice group. At the end of the study, blood was collected from the first 20 surviving animals of the terminal sacrifice group. At both time points, blood was collected after overnight fasting. At the 12-month and terminal sacrifices, a gross necropsy was conducted, selected organs were weighed, and organ and tissue samples were taken for histopathological examination (terminal sacrifice only).

In all sections, the female high dose group will be referred to as having received the test substance at a dietary concentration of 4000 ppm, although these animals received a dietary concentration of 6000 ppm for the first 10 weeks of the study.

There was a treatment-related increase in mortality in both males and females at 4000 ppm, but mortality at 100 and 1000 ppm were similar to observations in control animals. There were a number of treatment-related clinical signs, most related to the excretion of parent compound in the urine. These included hardness in the area of the urinary bladder, soiled fur, reduced motor activity, laboured or rapid respiration, and red urine. Body weight and body weight gain were reduced at 4000 ppm in both males and females. Body weight was unaffected at 1000 ppm in males, however body weight gain was statistically significantly decreased compared to controls at a number of time points. As the males in the 1000 ppm treatment group were slightly heavier at the beginning of the study than their concurrent controls, decreased body weight gain over the course of the study resulted in similar terminal body weights. This effect was considered to be test substance-related. There was no biologically significant effect on body weight or body weight gain in females at 1000 ppm or on either males or females at 100 ppm. There was no effect of treatment on food consumption at any dose level.

Neither males nor females showed any treatment-related ophthalmological findings at any dose level. Red cell count, haemoglobin, haematocrit, and mean corpuscular haemoglobin concentration were decreased in females at 4000 ppm at 18 months, with decreases in most parameters also at 12 months. Mean corpuscular volume was slightly increased in females at 4000 ppm at 18 months. In males at 4000 ppm, similar haematological effects were generally only observed at 18 months. These findings are considered to be treatment-related.

There was no effect of treatment on terminal body weight at the 12-month sacrifice. After 18 months, however, there was a treatment-related decrease in terminal body weight at 4000 ppm in both males and females. At 12 months, males at 4000 ppm showed statistically significantly increased absolute and relative kidney weight compared to controls. At 18 months, absolute and relative liver weights at 1000 and 4000 ppm were increased in males with a slight effect in females, and absolute and relative kidney weights were increased at 4000 ppm in males only. There were no other treatment-related effects on organ weights.

Prior to the 12-month sacrifice, one male and three females out of the animals dedicated to the 12-month satellite group at 4000 ppm were found dead (male on day 240, females on days 60, 61, and 75). All of these animals had yellow urinary bladder stones. In the 12-month satellite animals sacrificed as scheduled, one male and four out of 7 females at 4000 ppm were found to have stone(s) in the renal pelvis. Both males and females at 4000 ppm showed enlarged or small kidneys, dilation of the renal pelvis, pale kidneys, or renal cyst(s). Stones or gritty content were also observed in the urinary bladders of nearly all animals sacrificed at 4000 ppm at 12 months, along with bladder distension in the majority of these animals. Gallbladder concretions were observed at 100 and 1000 ppm in males and 1000 and 4000 ppm in females, among animals sacrificed as scheduled at 12 months. Among the animals in the 18-month study group, the majority of those which died unscheduled at 4000 ppm were found to have died due to acute or chronic renal failure, due to urinary tract blockage or chronic kidney and/or urinary bladder inflammation, respectively. Stones were found in the kidney and/or urinary bladder of these animals; other findings at necropsy of unscheduled deaths were enlarged or small kidneys, renal pelvic dilation, pale kidneys, renal cyst(s), distension of the urinary bladder, and gallbladder concretions. Similar findings were observed in animals sacrificed on schedule at 18 months.

Microscopic examination was not conducted at 12 months. At 18 months, histopathologic findings included increased incidence in males and females at 1000 and 4000 ppm of centrilobular hepatocellular hypertrophy. In both males and females at all doses but in the absence of a dose relationship, there was a slight increase in the incidence of gall bladder epithelial hyperplasia. Treatment-related histopathological findings in the urinary tract were limited to 4000 ppm in both males and females and included epithelial hyperplasia, fibrosis and scarring, and inflammatory cell infiltration. These findings were related to the urinary tract calculi observed at this dose level in the majority of animals. Separate analysis of the gallstones revealed them to be composed primarily of cholesterol.

The significance of the formation of gallstones was discussed in an additional regulatory toxicology paper position paper (M-301557-01-1, 2007). The mechanism leading to gallstone formation is unclear. However, the mouse strain used (C57BL/6J) is close to one used for research into gallstone mechanisms (C57L), and it may be that even a slight additional burden on the animal’s physiology leads to the increase in gallstone formation. Their relationship to direct toxic effects of the test substance is questionable given the lack of a dose-response relationship over a dose range from 14 to 500 mg/kg bw/day. Additionally, in the absence of increased histopathological findings in either the gallbladder or the liver which would indicate either localized tissue damage or generalized derangement of normal handling of cholesterol and bile acids, their biological significance is questionable at best. It is therefore concluded that the gallstones observed in the study are not an adverse effect.

Treatment-related neoplastic findings at 4000 ppm were limited to the urinary tract of males and females, and were comprised of transitional cell carcinomas and papillomas of the urinary bladder and urethra. In all cases, these tumours were related to the presence of urinary tract stones. These tumours were considered to be the result of a non-genotoxic proliferative mechanism due to the concurrent presence of secondary inflammation and hyperplastic findings in the same tissues, induced by the urinary stones.

 Conclusions

Administration of 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl)methanone to male and female mice via dietary incorporation, at 4000 ppm, led to urinary tract stone formation, increased mortality generally due to either acute or chronic renal failure secondary to urinary tract stone development, decreased body weight and body weight gain, increased kidney and liver weights in males, renal pelvis dilation, and urothelial hyperplasia throughout the urinary tract. Liver weight and centrilobular hepatocellular hypertrophy were increased compared to controls in males at 1000 and 4000 ppm, and centrilobular hepatocellular hypertrophy was increased in females at 4000 ppm only. Gallbladder stones demonstrated to be cholesterol were observed in both males and females at all doses with no relationship to dose. Treatment-related neoplastic findings of the urinary tract were related to the presence of urinary tract stones and were only observed at 4000 ppm. These neoplasias were considered to be secondary to the administration of high doses of the test substance, as the urinary tract stones were shown to be composed of unmetabolized test substance, and were due to a non-genotoxic proliferative mechanism.

Based on the increased mortality at 4000 ppm in both males and females, the top dose of 4000 ppm was considered to have exceeded the Maximum Tolerated Dose. The NOAEL for general toxicity was established at 100 ppm (13.6 mg/kg bw/day in males, 16.7 mg/kg bw/day in females), as despite gallbladder concretions at all doses there was no increase at 100 ppm in epithelial hyperplasia in the gallbladder. The LOAEL was 1000 ppm. The neoplastic findings in the urinary tract were further evaluated in additional regulatory toxicology position papers (M-284616-01-1 and M-301557-01-1, 2007). As above, the treatment-related neoplastic findings of the urinary tract were observed at 4000 ppm but were considered to be secondary to the administration of high doses of the test substance which precipitated in the urinary tract, causing continuous local irritation and to be due to a non-genotoxic proliferative mechanism. This is strongly supported by the fact that these tumours were only seen at the highest dose and in animals with urinary tract stones. However, they are not considered to be relevant to human conditions, where much lower doses of the test substance are expected. Accordingly, up to and including the highest administered dose, there was no indication of neoplastic findings which are relevant to man. Accordingly, the NOAEL for carcinogenicity is considered to be 4000 ppm (560 mg/kg bw/day in males, 713 mg/kg bw/day in females).

 Carcinogenicity study in the Wistar rat by dietary administration:

In this GLP-conform study conducted according to OECD 453, 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl)methanone was incorporated into rodent diet and administered at concentrations of 0, 25, 250, 1000, and 2500 ppm to groups of 75 male and female Wistar rats (M-267037-01-2). In addition to the carcinogenicity phase, the study also included a 6-month and 1-year repeated-dose toxicity phase. Only the results of the carcinogenicity phase are summarized here. The repeated-dose results are summarized in the respective endpoint summary.

The administered concentrations provided final doses of 0, 1.0, 10, 41, and 105 mg/kg bw/day for males and 0, 1.4, 14, 57, and 141 mg/kg bw/day for females. Body weight and food consumption were measured weekly for the first 13 weeks of the study, then every 4 weeks through the remainder of the study until necropsy. Ophthalmological examinations were conducted on all animals during acclimatization and at months 3, 6, 12, 18, and 24 of treatment. Blood for haematological and clinical chemical examination was collected after 6, 12, and 24 months from overnight-fasted rats. Urine was collected overnight at 3, 6, 12, 18, and 24 months from animals fasted overnight of food and water.

A treatment-related increase in mortality was observed in males at 2500 ppm. There was no treatment-related effect on mortality in other male treatment groups or in females at any dose level. Treatment-related clinical signs of toxicity included white area on the eye and soiled fur in one or more areas. An increased incidence of these findings was observed at 250, 1000, and 2500 ppm in both males and females. Body weight and body weight gain were reduced in males and females at the higher doses, with a greater effect observed in males than in females. There was no effect on food consumption at any dose in either males or females.

Treatment-related findings (corneal opacity, neovascularization of the cornea, oedema of the cornea, and "snow flake" corneal opacities) were seen during ophthalmoscopy in both males and females. At 6 and 12 months, increased incidence of these findings was seen only at 250, 1000, and 2500 ppm. At 24 months, these findings were increased compared to controls at all doses in males. However, at 25 ppm there was only a slight increase in corneal opacity, neovascularization of the cornea, and oedema of the cornea in males in neovascularization of the cornea in females (the significance of these findings at 25 ppm are discussed in the repeated dose toxicity endpoint summary). The ocular findings are related to tyrosinemia resulting from inhibition of 4 -hydroxyphenylpyruvate dioxygenase (HPPDase), a key enzyme in the tyrosine catabolic pathway. As humans are capable of metabolizing and excreting excess tyrosine while rats are much less capable of this metabolism, the corneal effects of HPPDase inhibitors are considered to be relevant for rats but not for man (for a detailed justification see endpoint summary for repeated dose toxicity).

There was no treatment-related effect on haematology at any dose or time point in either male or female rats. Plasma cholesterol was biologically and statistically significantly increased at 250 ppm and above at months 7 and 12, and was biologically and/or statistically significantly increased at 1000 and 2500 ppm at 18 and 24 months. This effect on plasma cholesterol was considered to be the only treatment-related clinical chemistry finding. Although plasma cholesterol was statistically significantly increased in males at 25 ppm at the 7-month time point, this value was within historical control data and cholesterol concentrations in this dose group returned to control levels by the 12- month time point; therefore, this increase was considered not to be treatment-related.

During urinalysis, higher ketone concentrations were noted in both males and females at 1000 ppm and 2500 ppm at all collection periods, and in males at 250 ppm at months 19 and 24 only. These were due to the triketone structure of the molecule, which is largely excreted unchanged in the urine. Urine pH was decreased at 250 ppm and above in males at all time points, while in females at 250 ppm and above urine pH was decreased only at the 3-month time point. Urinary protein was increased in males at all doses from Month 7 onwards.

Liver and kidney weight was statistically significantly increased in males only at the higher doses in this study. There were no biologically significant effects on organ weights in females at any group. At necropsy, treatment-related findings were observed in the eyes, liver, and kidney. Eye opacities seen in both males and females were related to increased tyrosinemia and are considered not to be relevant for human risk assessment. An increased incidence of pale kidneys or irregular surface of the kidney was noted in males at 1000 and 2500 ppm.

 Non-neoplastic microscopic findings: Treatment-related findings were observed in the eyes, liver, pancreas, thyroid gland, and kidneys. Findings observed in other organs were considered to be of little to no toxicological relevance or not to be treatment-related.

In the eyes, the incidence of corneal inflammation was increased in males at 250 ppm and above, and in females at 1000 and 2500 ppm. At 24 months, there was a very slight increase in corneal inflammation in males at 25 ppm and in females at 250 ppm. Regenerative hyperplasia of the cornea was increased in males at 250 ppm and above, and in females at 1000 and 2500 ppm, only. Neovascularization of the cornea was increased in males 250 ppm and above, and in females at 250 ppm and above. There was an increase in males in the incidence of mucous metaplasia of the cornea at 250 ppm and above. Corneal atrophy was increased in males at 250 ppm and above and in females at 1000 and 2500 ppm, and peripheral retinal atrophy was increased in both males and females at 250 ppm and above (the significance of the ocular findings are discussed in the repeated dose toxicity endpoint summary).

Centrilobular hepatocellular hypertrophy was increased in males from 250 ppm. In females centrilobular hepatocellular hypertrophy was only reported in one animal at 2500 ppm. In the pancreas, diffuse acinar degeneration / atrophy was reported in both males and females; the incidence of diffuse acinar degeneration / atrophy was increased in males and females at 1000 and 2500 ppm. The incidence of focal acinar degeneration / atrophy was increased in females at 2500 ppm.

In the thyroid, the incidence of altered colloid was increased in males and females from 250 ppm and above. Pigment deposition in the follicular cells was increased in both males and females in all dietary groups. The incidence of focal follicular cell hyperplasia was slightly increased in males from 250 ppm. In females, this finding was only at a very low incidence. No clear relationship of follicular cell hyperplasia to dose was evident. Diffuse follicular cell hypertrophy was noted in a few males and females from 250 ppm, again in the absence of a clear dose-relationship. The observed thyroid findings are considered a rat-specific phenomenon of HPPDase inhibitors without relevance for humans (for a detailed justification see endpoint summary for repeated dose toxicity).

There was no substantive dose-related increase in the incidence of chronic progressive nephropathy in males at 24 months, and the incidence in control animals at 24 months of 80% indicates that this is a very common finding in the aging male rat. Chronic progressive nephropathy was observed in females, with no dose relationship. Hyperplasia of the collecting ducts was increased in males at 1000 and 2500 ppm.

 The only treatment-related neoplastic finding was that of squamous cell tumours (one papilloma, one carcinoma) observed in two males in the 2500-ppm dose group. These tumours were considered to have resulted from the corneal inflammation and regenerative hyperplasia resulting from tyrosinemia, and are not relevant to human risk assessment. The incidence in high dose males is attributed to the fact that male rats generally have a higher plasma tyrosine concentration than females after an equivalent dose of an HPPDase inhibitor, and males are known to be more sensitive than females to the development of specific tyrosine-related lesions including corneal opacities and neovascularization (M-284620-01-1 and M-301557-01-1, 2007). Other tumours which were observed showed no relationship to dose or were considered to be consistent with those tumours found in ageing rats, and were not evaluated as related to treatment.

Conclusions

Based on increased mortality at 2500 ppm in males, this dose is considered to have exceeded the Maximum Tolerated Dose. Treatment-related findings were seen in the eyes, liver, kidney, thyroid, and pancreas. Findings in the eye were related to the biochemical mechanism of the test substance as an HPPDase inhibitor leading to increased tyrosinemia in rats, and were considered not to be relevant for humans. The observations in the liver, including centrilobular hepatocellular hypertrophy, were evaluated as being adaptive responses to treatment rather than adverse effects. The finding of chronic progressive nephropathy is considered to be a rodent-specific finding not relevant to man. Similarly, the findings of colloid alteration and pigment deposition were considered to be specific to the rat and not indicative of an adverse finding, but to be normal findings in ageing rats. The increased incidence of acinar degeneration / atrophy in the pancreas was only observed at 1000 and 2500 ppm, and may indicate a tyrosine-linked effect as well, as these findings were not observed in the mouse or dog, which are not as sensitive as the rat to the effects of tyrosinemia. The only treatment-related neoplastic findings, squamous cell tumours of the cornea, were evaluated as being due to a nongenotoxic proliferative mechanism and to be secondary to rat-specific increased plasma tyrosine concentrations not relevant to man.

The NOAEL for general toxicity in this study was therefore considered to be 25 ppm (1.0 mg/kg bw/day in males and 1.4 mg/kg bw/day in females), while the LOAEL was 250 ppm (10 mg/kg bw/day in males and 14 mg/kg bw/day in females). Treatment-related neoplastic findings of the cornea were observed at 2500 ppm but were considered to be secondary to continuous corneal inflammation and ulceration. These cornea effects are a rat specific phenomenon and to be due to a non-genotoxic proliferative mechanism. They were considered to have no relevance to humans. Accordingly, up to and including the highest administered dose, there was no indication of neoplastic findings which are relevant to man. Accordingly, the NOAEL for carcinogenicity is considered to be 2500 ppm (104 mg/kg bw/day in males, 140 mg/kg bw/day in females).