Benzamide, N-[[4-[(cyclopropylamino)carbonyl]phenyl]sulfonyl]-2-methoxy-

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Remarks:

combined repeated dose and carcinogenicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 Nov 2003 to 11 Dec 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 Nov 2003 to 11 Dec 2006

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Version / remarks:

adopted 12 May 1981

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Version / remarks:

adopted 25 Jun 2018

Deviations:

yes

Remarks:

no details on whether feed and water were analysed for contaminants

GLP compliance:

yes (incl. QA statement)

Remarks:

INTERMINISTERIAL GROUP FOR CHEMICAL PRODUCTS, Paris, France

Species:

rat

Strain:

other: Wistar Rj:WI (IOPS HAN)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: R. Janvier, Le Genest St Isle, France
- Age at study initiation: 6 weeks
- Weight at study initiation: 221 - 222 g mean group weight males; 163 - 165 g mean group weight females
- Housing: By sex in groups of 5, unless reduced by mortality or isolation. The cages were suspended, stainless steel and wire mesh.
- Diet: A04CP1-10 (formerly A04C-10P1) from S.A.F.E. (Scientific Animal Food and Engineering, Augy (formerly Epinay-sur-Orge), France, ad libitum except at designated time periods.
- Water: Filtered and softened tap water from the municipal water supply, ad libitum.
- Acclimation period: 13 days

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

IN-LIFE DATES: From: 19 Nov 2003 To 11 Dec 2006

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Approx. at 4 weekly periods dietary requirements with two occasions of minor exceptions (second formulation at 200 ppm covered 5 weeks and the last formulation which covered the dietary needs until the end of the study).
- Mixing appropriate amounts with (Type of food): The test substance was incorporated into the diet (A04CP1-10 from S.A.F.E. (Scientific Animal Food and Engineering, Augy, France)) to provide the required dietary concentrations of 200, 1000, 4000 or 8000 ppm.
- Storage temperature of food: Ambient temperature

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in the diet was demonstrated in a previous study (SA03277). The stability of the test substance at 100 and 15000 ppm in the diet was verified for at least 104 days at room temperature and over 70 days frozen storage followed by 10 days at room temperature (covers the period of storage and usage for the study which covered the period of storage and usage for this study.
Fourteen formulations (F1 to F14) were prepared during the study at a concentration of 200 ppm and twenty-seven formulations (F1 to F27) were prepared during the study at all other concentrations. In addition, an additional formulation (F3bis) was prepared at 1000 ppm to replace the formulation F3 which was found slightly out of the in-house target ranges.
Formulation F1 consisted of four loads of approximately 41 kg at concentrations of 1000, 4000 and 8000 ppm and one load of approximately 22 kg at a concentration of 200 ppm.
Formulation F2 consisted of four loads of approximately 41 kg at concentrations of 1000, 4000 and 8000 ppm and one load of approximately 40 kg at a concentration of 200 ppm. Formulations F3 (and F3 bis for 1000 ppm) to F14 consisted of four loads of approximately 35 kg at concentrations of 1000, 4000 and 8000 ppm and one load of approximately 35 kg at a concentration of 200 ppm. Formulations F15 to F27 consisted of two loads of approximately 62 kg at concentrations of 1000, 4000 and 8000 ppm.
The dietary levels of the test substance were verified by HPLC-UV.
The homogeneity of the test substance in diet was verified on the first loads at 200, 1000, 4000 and 8000 ppm of the first formulation (F1), on formulation F2 at 200 ppm, on the first loads at 200 and 8000 pprn of formulations F3 and F9, and on the first loads at 1000 and 8000 ppm of formulations F15, F21 and F27, to demonstrate adequate formulation procedures. Mean values obtained from the homogeneity check were taken as measured concentration.
The concentration was checked for all loads at all dose levels for formulations F1, F3, F3bis, F6, F9, F12, F15, F18, F21, F24 and F27.
Homogeneity and concentration in the diet were within the in-house target range of 85 to 115% of nominal concentration (homogeneity 92-104%; concentration 92-111%). Therefore all study mixes were considered acceptable for use on study.

Duration of treatment / exposure:

At least 104 weeks for carcinogenicity phase and 52 weeks for chronic toxicity phase.

Frequency of treatment:

Continously via the diet

Post exposure period:

None

Dose / conc.:

200 ppm

Remarks:

corresponding to 9 and 13 mg/kg bw/day actual dose ingested for males and females, respectively (week period 1 to 52).

Dose / conc.:

1 000 ppm

Remarks:

corresponding to 39 and 56 mg/kg bw/day actual dose ingested for males and females, respectively (week period 1 to 104).

Dose / conc.:

4 000 ppm

Remarks:

corresponding to 159 and 220 mg/kg bw/day actual dose ingested for males and females, respectively (week period 1 to 104).

Dose / conc.:

8 000 ppm

Remarks:

corresponding to 321 and 447 mg/kg bw/day actual dose ingested for males and females, respectively (week period 1 to 104).

No. of animals per sex per dose:

70 (60 + 10 for interim sacrifice): 1000, 4000, 8000 ppm
10: 200 ppm

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: dose levels were selected based on the results from a previous 90-day dietary study in the rat (41), where dietary administration of up to 12000 ppm in males and females resulted in sulfonamide-like crystals in urine of both sexes, hepatocellular hypertrophy in the liver of males, hyperplasia of the urothelium in the urinary bladder of both sexes, increased incidence of basophilic tubules, brown pigments in proximal tubules and hyperplasia of collecting ducts and pelvic epithelium in the kidney of both sexes, metaplasia in the collecting duct epithelium in one female and increased severity of decreased size of the cortex of thymus in males. In addition, two females prematurely died at 12000 ppm, showing a range of lesions with a number of similarities to those observed on target organs at terminal sacrifice. The No Observed Effect Level in the rat 90-day study was 1000 ppm, with a Low Observed Adverse Effect Level of 4000 ppm.

Positive control:

None

Observations and examinations performed and frequency:

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily for all animals. Detailed physical examinations including palpation for masses were performed weekly throughout the study. The onset, location and dimension of the masses were recorded

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly for the first 13 weeks of study and approximately every 4 weeks thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes.
- Food consumption was recorded twice weekly during the first 6 weeks of treatment, then weekly up to Week 13, and once approximately every 4 weeks thereafter.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes. The weekly mean achieved dosage intake in mg/kg body weight/day for Weeks 1 to 13, then 1 week per month thereafter was calculated as the product of the dose level (ppm) and the group mean food consumption (g/day) divided by the group mean body weight (g) at the end of the week. The monthly and overall mean achieved dosage intake for the 24 months treatment were derived from the weekly data.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: During acclimatization phase and after approximately 12 and 24 months
- Dose groups that were examined: All animals during the acclimatization phase and all surviving animals after 12 and 24 months

HAEMATOLOGY: Yes
- Time schedule for collection of blood: For the interim sacrifice groups on Weeks 16 to 18, 23 or 24 and 51 or 52, and for the first ten suitable surviving rats of each sex of the terminal sacrifice groups on Weeks 16 to 18, 23 or 24, 51 or 52, 75 and 105 or 106.
- Anaesthetic used for blood collection: Yes, Isoflurane.
- Animals fasted: Yes, overnight fasting.
- How many animals: All surviving animals of the interim sacrifice groups on Weeks 16 to 18, 23 or 24 and 51 or 52, and on the first ten suitable surviving rats of each sex of the terminal sacrifice groups on Weeks 16 to 18, 23 or 24, 51 or 52, 75 and 105 or 106.
- Parameters examined: Haematocrit, haemoglobin concentration, leukocyte count, erythrocyte count, platelet count, leukocyte differential time, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, mean corpuscular volume, reticulocyte count.
- At terminal sacrifice, blood smears were prepared for all animals not sampled for hematology. When possible, a blood smear was prepared for the moribund animals, just before sacrifice.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Months 4, 6, 12, 18 and 25
- Animals fasted: Yes, overnight
- How many animals: All surviving animals of the interim sacrifice groups on Weeks 16 to 18, 23 or 24 and 51 or 52, and on the first ten suitable surviving rats of each sex of the terminal sacrifice groups on Weeks 16 to 18, 23 or 24, 51 or 52, 75 and 105 or 106
- Parameters: Alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma glutamyltransferase, glutamate dehydrogenase, albumin, creatinine, urea, total cholesterol, glucose (fasting), total bilirubin, total protein, triglycerides, calcium, chloride, inorganic phosporus, potassium and sodium.

URINALYSIS: Yes
- Time schedule for collection of urine: All the surviving animals of the interim sacrifice groups on Weeks 15 or 16, 26 or 27 and 52 or 53, and on the first ten suitable surviving rats of the terminal sacrifice groups on Weeks 15 or 16, 26 or 27, 52 or 53, 78 and 103.
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes. Diet and water were withdrawn during the overnight (approx. 16 hours) collection period.
- Parameters examined: Appearance, volume, specific gravity / osmolality / refractive index, pH, sediment (microscopic), protein, glucose, ketones, bilirubin, blood/red blood cells, urobilinogen

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Month 12
- Dose groups that were examined: All surviving animals
- Battery of functions tested: Sensory activity / grip strength / motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, on Days 379 to 382 for the 12-month chronic phase, and on Days 732 to 746 for the 24-month carcinogenicity phase, all surviving animals dedicated to chronic phase and carcinogenicity phase groups, respectively, were sacrificed by exsanguination under deep anesthesia (inhalation of Isoflurane). At final sacrifice, due to the high mortality rate observed in males treated at 4000 and 8000 ppm, it was decided, in agreement with the sponsor representative, to sacrifice the males first, and then the females. Animals were diet fasted overnight prior to sacrifice. 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. The following organs or tissues were sampled and/or weighed at necropsy: digestive system (including tongue, submaxillary gland, oesophagus, stomach, duodenum, jejenum, illeum, caecum, colon, rectum, liver and pancreas), cardiovascular/haemopoietic system (including thoracic aorta, heart, bone marrow, sternum, mesenteric lymph node, submaxillary lymph node, spleen, thymus, neurologic (including brain, sciatic nerve, spinal cord, eyes, optic nerves), glandular systems (including pituitary gland, adrenal gland, exorbital lachrymal gland, parathyroid gland, thyroid gland and Harderian gland), respiratory system (including trachea, lung, nasal cavities, pharynx, larynx), urogenital system (including kidney, urinary bladder, testis, epididymis, prostate gland, seminal vesicle, ovary, uterus with cervix, mammary gland, vagina) and other tissues (including bone, skeletal muscle, skin, all gross lesions and masses and articular surface.

ORGAN WEIGHTS: mean organ weight, organ/terminal body weight and organ/brain weight of the brain, heart, kidney, thymus, epididymis, testis, prostate, adrenal gland, liver, pituitary gland, spleen and thyroid gland, uterus and ovary were recorded.

HISTOPATHOLOGY: Yes, all tissues and organs as in gross pathology (except exorbital lachrymal gland, larynx/pharynx and nasal cavities) were embedded in paraffin wax. For the 12-month chronic and 24-month carcinogenicity phase, histological sections, stained with hematoxylin and eosin, were prepared from all organs and tissue samples.
12-month interim sacrifice (52 weeks)
Histopathology examinations were performed as follows for the 12-month interim sacrifice:
- all organs and tissue samples from animals sacrificed or dying during the treatment period,
- all organs and tissue samples from animals of control and high dose groups,
- liver, lung, kidney and urinary bladder from animals of the intermediate dose groups,
- gross abnormalities from all animals.
For all unscheduled sacrificed or dead animals on study, a determination of the factors contributing to death was made.
Initial examinations were performed by the study pathologist. Following the initial examination, an in-house 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.
Carcinogenicity phase (104 weeks)
For the carcinogeniciy phase investigations, 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 a determination of the factors having contributed to death was made. Initial examinations were performed by the study pathologist. Following the initial examination, an external review pathologist), undertook an independent peer-review of representative slides and diagnoses according to standardized operating procedures. The diagnosis presented in the report represents the consensus opinion of the two pathologists.

Statistics:

Mean and standard deviation were calculated for each group. All statistical analyses were carried out separately for males and females.

For details on statistics performed please refer to "Any other information on materials and methods incl. tables".

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

See Attachment 1 for summary tables of clinical signs.

8000 ppm:
- Soiled anogenital region was noted in both sexes. Soiled anogenital region was also recorded as soiled fur (localized in the anogenital region) in males and a higher incidence of red genital discharge, reduced motor activity, general pallor, wasted appearance and hunched posture was noted in females.

4000 ppm:
- soiled anogenital region was noted in both sexes. Soiled anogenital region was also recorded as soiled fur (localized at the anogenital region) in males.

1000 ppm:
- a slightly higher incidence of soiled anogenital region was noted in males. No treatment-related clinical signs were observed in females.

After one and/or two years of treatment, dental abnormality and incisors cut in both sexes and ocular discharge in females occurred at a slightly higher incidence at 8000, 4000 and 1000 ppm, in comparison with the controls. However, in the absence of a dose-relationship and of associated treatment-related macroscopic or microscopic changes, dental abnormality and ocular discharge were considered not to be treatment-related. In addition, the apparent higher incidence of skin lesions occurring at 8000 ppm in males was considered not to be treatment-related since these lesions mainly consisted of tail lesions which were observed on animals dying from accidental trauma.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

Not applicable.

Mortality:

mortality observed, treatment-related

Description (incidence):

See Attachment 1 for summary tables of mortality data.

After at least 104 weeks of treatment the mortality rate was not affected by treatment in males and was slightly higher at 8000 ppm in females compared to controls, where 14 out of 31 unscheduled mortalities were attributed to treatment-related nephropathy. However, the difference to controls was not statistically significant.

In the males, statistically significance for higher mortality rate was achieved at 4000 ppm. As treatment-related nephropathy was not reported as a cause of death at this dose level and no increased mortality rate was observed at 8000 ppm, the statistically significant increase was considered incidental and not treatment-related.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

See Attachment 1 for summary tables of body weight and body weght gain data.

8000 ppm:
- Overall, mean body weight on study Day 708 and mean cumulative body weight gain between study Days 1 to 708 were similar to controls in both sexes.

4000 ppm
- The mean cumulative body weight loss of 68 g noted during the interval Weeks 78 to 101 was not considered treatment related since it was not dose-related and the values were within the historical control ranges (-25 to -73 g in a total of 6 studies). Overall, mean body weight on study Day 708 was reduced by 6% (not statistically significant), whilst mean cumulative body weight gain between study Days 1 to 708 was reduced by 9% (not statistically significant), compared to the controls. This effect was not considered treatment-related since it was not dose-related.

There was no treatment-related effect on mean body weight or body weight gain parameters at 4000 ppm in females or at 1000 and 200 ppm in either sex over the two years of treatment (one year only at 200 ppm).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

See Attachment 1 for compound intake.

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, non-treatment-related

Description (incidence and severity):

8000 ppm:
- At Month 25, slightly lower mean erythrocyte count (-8%, not statistically significant), haemoglobin concentration (-9%, p<0.05) and haematocrit (-9%, p<0.05) were noted in females only. These changes were considered to be incidental as only 2/9 animals were affected.

Any other statistically significant differences were considered to be chance findings in view of their low amplitude and/or their sporadic occurrence.

Clinical biochemistry findings:

no effects observed

Endocrine findings:

not examined

Description (incidence and severity):

Not applicable.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

See Attachment 2 for summary tables of urinalysis findings.

Throughout the urine sampling periods, sulfonamide-like crystals were observed at 8000 and 4000 ppm in both sexes. This finding was dose-related in terms of incidence and/or severity on most occasions, the effect being more pronounced in females than in males. No sulfonamide-like crystals were observed at 1000 ppm, 200 ppm or in control animals in either sex.

When compared to the control groups, a tendency towards lower urinary protein levels was noted throughout the study in both sexes at 8000 and 4000 ppm (except for females at Month 24). The same variation was seen in males at 1000 ppm at Month 6/7, 12 and 18. This change was considered not to be adverse in view of its low magnitude.

The other variations were judged to be incidental and not treatment-related.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Locomotor activity: There was no treatment-related effect on overall mean exploratory locomotor activity in either sex. The pattern of the locomotor activity over time was similar to the control at all dose levels in both sexes. The statistically significant increase observed in males at 200 ppm during interval 16-30 min was considered to be a chance finding as it was observed in isolation and with no dose-relationship.

Sensory reactivity: All reflexes and responses evaluated were unaffected by the treatment at any dose level in either sex. The few changes in the tail pinch response noted for males in all dose groups were considered to reflect inter-individual variations rather than any treatment-related effect.

Grip strength: The fore- and hindlimb grip strength were unaffected by the treatment at any dose level in either sex. The slight increase reaching statistical significance in hindlimb grip strength observed in the female high dose group (+19%, p<0,05) was mainly attributable to a particularly high value for animal NT5F5156 and was therefore considered to be incidental.

Immunological findings:

not examined

Description (incidence and severity):

Not applicable.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There was no treatment-related change in mean terminal body weights and organ weights when compared to controls. The few apparent organ weight differences observed were considered not to be treatment-related, even when statistically significantly higher in comparison to controls since they were not correlated with any microscopic findings.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

See Attachment 3 for summary tables of gross pathology data.

Unscheduled deaths:
- Two hundred and fifty-six animals were either found dead or killed for humane reasons before the end of the study.
- At 8000 ppm, 14/31 deaths in females were attributed to treatment-induced nephropathy.
- At 8000 ppm, treatment-related macroscopic findings consisted of abnormal shape, pelvic dilatation, irregular surface, gritty content and stones in the kidney in females.
- Treatment-related presence of stones was noted in the urinary bladder in males at 8000 ppm.

Terminal sacrifice:
- Treatment-related macroscopic findings were observed in the kidney and consisted of abnormal shape, pelvic dilatation and gritty content in both sexes at 8000 ppm, irregular surface and stones in females at 8000 ppm and pelvic dilatation, gritty content and stones in females at 4000 ppm.
- Stones were observed in the urinary bladder and were considered to be treatment-related in both sexes at 8000 ppm and in one female at 4000 ppm.

Neuropathological findings:

not examined

Description (incidence and severity):

Not applicable.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See Attachment 4 for summary tables of histopathology data.

Scheduled and unscheduled deaths:
- Treatment-related effects were observed in the urinary tract, i.e. kidney, urinary bladder and ureter, including a combination of hyperplastic and inflammatory changes associated with the presence of stones.

Kidney:
- Treatment-induced nephropathy was observed in both sexes in the kidney at 8000 ppm, with a higher severity in females, and at 4000 ppm in females only.
- Treatment-induced nephropathy is a general term which incorporates some or all of the following findings: stones, papillary necrosis/loss/scarring, collecting duct hyperplasia, bilateral cortico-medullary scarring, pelvic dilatation and bilateral cortex/medulla tubular dilatation.
- In addition, an increased incidence of pelvic dilatation was noted at 4000 ppm in males.
- A slight increase in severity of simple pelvic epithelium hyperplasia was observed in both sexes at 8000 and 4000 ppm.
- Nodular pelvic epithelium hyperplasia was observed in 2/60 females and a higher incidence of intrapelvic mineral deposit was observed in 14/60 females at 8000 ppm.

Urinary Bladder:
- Intraluminal stones and multifocal/diffuse urothelial hyperplasia were observed in the urinary bladder in both sexes at 8000 ppm and in females at 4000 ppm. These lesions were considered to be treatment-related.
- Suburothelial mononuclear cell infiltrate and mixed cell infiltrate were considered to be associated lesions.

Ureter:
- Intraluminal stones and multifocal/diffuse urothelial hyperplasia were observed in the ureter of a number of animals in both sexes at 8000 ppm. These lesions were considered to be treatment related.

Testes:
- An increased incidence of the commonly occurring lesion diffuse bilateral tubular degeneration of the testis (associated with diffuse interstitial oedema and/or focal/multifocal mineralization in most cases) and bilateral oligospermia of the epididymis (associated with epithelial degenerative change) was observed at 8000 ppm, which was considered to be treatment-related.

Indirect systemic changes commonly associated with induced nephropathy, including compensatory hyperplasia of the parathyroid glands associated with mineralization, especially involving lungs and the gastric mucosa, were observed at 8000 ppm mainly in females.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At the end of the 2-year treatment period, there was no evidence of a direct treatment-related increased incidence of tumors of any type in any organ.

At 8000 ppm, a transitional cell carcinoma was observed in the kidney in one male and a transitional cell carcinoma was observed in the urinary bladder in one female. Those two single tumors were considered to be secondary to the combination of hyperplastic and inflammatory changes associated with the presence of stones.

The very few additional tumors observed at 200, 1000, 4000 and 8000 ppm were considered incidental due to the rather isolated occurrence in single animals without being reproducible across the different dose groups (refer to Attachment 5: Table 19, attached background material).

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Relevance of carcinogenic effects / potential:

At the carcinogenicity phase (24-month) sacrifice, treatment-related effects were found in the urinary tract, i.e. kidney, urinary bladder, and ureters. These changes were due to treatment-induced nephropathy, characterized in the kidney by a combination of hyperplastic and inflammatory changes associated with the presence of stones. Urothelial hyperplasia was noted in the urinary bladder in both sexes at the 12-month interim sacrifice, whereas intraluminal stones and multifocal/diffuse urothelial hyperplasia were also observed in the urinary bladder and in a number of ureters in both sexes at the carcinogenicity phase. The neoplastic changes comprised of a high dose-related transitional cell carcinoma in the kidney of one male and a transitional cell carcinoma in the urinary bladder of one female. These findings, seen only at 8000 ppm, were considered to be secondary to the combination of hyperplastic and inflammatory changes associated with the presence of stones.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

1 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects seen at this dose level.

Remarks on result:

other: corresponding to 39 and 56 mg/kg bw/day actual dose ingested for males and females, respectively.

Key result

Dose descriptor:

LOAEL

Remarks:

systemic

Effect level:

4 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

gross pathology

histopathology: non-neoplastic

urinalysis

Remarks on result:

other: corresponding to 159 and 220 mg/kg bw/day actual dose ingested for males and females, respectively.

Key result

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Effect level:

4 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects seen at this dose level.

Remarks on result:

other: corresponding to 159 and 220 mg/kg bw/day actual dose ingested for males and females, respectively.

Key result

Dose descriptor:

LOAEL

Remarks:

carcinogenicity

Effect level:

8 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: neoplastic

Remarks on result:

other: corresponding to 321 and 447 mg/kg bw/day actual dose ingested for males and females, respectively.

Critical effects observed:

yes

Lowest effective dose / conc.:

4 000 ppm

System:

urinary

Organ:

bladder

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Critical effects observed:

yes

Lowest effective dose / conc.:

8 000 ppm

System:

urinary

Organ:

ureter

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Conclusions:

The study was performed in accordance to OECD TG 453 under GLP conditions and is considered reliable. In conclusion, dietary administration of the test substance over a 24-month period to the Wistar rat, at dose levels up to 8000 ppm (corresponding to 321 mg/kg/day in males and 447 mg/kg/day in females), produced a transitional cell carcinoma in the kidney of one male and a transitional cell carcinoma in the urinary bladder of one female. The No Observed Adverse Effect Level (NOAEL) for carcinogenicity was 4000 ppm (equivalent to 159 mg/kg bw/day in males and 220 mg/kg bw/day in females) . The incidence of these tumours was very low and was considered to be secondary to the chronic hyperplastic changes resulting from chronic irritation due to the presence of stones. Thus, the test substance was considered not to be directly carcinogenic in the rat.

The systemic NOAEL over a 12 month period of dietary administration to the Wistar rat was 4000 ppm in both sexes (equivalent to 181 mg/kg body weight/day in males and 249 mg/kg body weight/day in females). The systemic NOAEL over a 24 month period of dietary administration to the Wistar rat was 1000 ppm in both sexes (equivalent to 39 mg/kg/day in males and 56 mg/kg/day in females).

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

chronic toxicity: oral

Remarks:

combined repeated dose and carcinogenicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 Nov 2003 to 11 Dec 2006

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Version / remarks:

adopted 12 May 1981

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Version / remarks:

adopted 25 Jun 2018

Deviations:

yes

Remarks:

no details on whether feed and water were analysed for contaminants

GLP compliance:

yes (incl. QA statement)

Remarks:

INTERMINISTERIAL GROUP FOR CHEMICAL PRODUCTS, Paris, France

Limit test:

no

Species:

rat

Strain:

other: Wistar Rj:WI (IOPS HAN)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: R. Janvier, Le Genest St Isle, France.
- Age at study initiation: Approx. 6 weeks old
- Weight at study initiation: 221-222 g mean group weight (males), 163-165 g mean group weight (females)
- Fasting period before study: Not applicable, feeding study
- Housing: By sex in groups of 5 unless reduced by mortality or isolation. The cages were suspended, stainless steel and wire mesh.
- Diet: A04CP1-10 from S.A.F.E. (Scientific Animal Food and Engineering, Augy, France), ad libitum except at designated time periods.
- Water: Filtered and softened tap water from the municipal water supply, ad libitum.
- Acclimation period: 13 days

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

IN-LIFE DATES: From: 19 Nov 2003 To: 14-17 Dec 2004

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Approximately 4 weekly periods, apart from a second formulation at 200 ppm which covered 5 weeks and the last formulation which covered the dietary needs until the end of the study.
- Mixing appropriate amounts with (Type of food): The test substance was incorporated into the diet (A04CP1-10 from S.A.F.E. (Scientific Animal Food and Engineering, Augy, France)) to provide the required dietary concentrations of 200, 1000, 4000 or 8000 ppm.
- Storage temperature of food: When not in use, the diet formulations were stored at ambient temperature.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in the diet was demonstrated in a previous study (SA03277). The stability of the test substance at 100 and 15000 ppm in the diet was verified for at least 104 days at room temperature and over 70 days frozen storage followed by 10 days at room temperature (covers the period of storage and usage for the study which covered the period of storage and usage for this study.
Fourteen formulations (F1 to F14) were prepared during the study at a concentration of 200 ppm and twenty-seven formulations (F1 to F27) were prepared during the study at all other concentrations. In addition, an additional formulation (F3bis) was prepared at 1000 ppm to replace the formulation F3 which was found slightly out of the in-house target ranges.
Formulation F1 consisted of four loads of approximately 41 kg at concentrations of 1000, 4000 and 8000 ppm and one load of approximately 22 kg at a concentration of 200 ppm.
Formulation F2 consisted of four loads of approximately 41 kg at concentrations of 1000, 4000 and 8000 ppm and one load of approximately 40 kg at a concentration of 200 ppm. Formulations F3 (and F3 bis for 1000 ppm) to F14 consisted of four loads of approximately 35 kg at concentrations of 1000, 4000 and 8000 ppm and one load of approximately 35 kg at a concentration of 200 ppm. Formulations F15 to F27 consisted of two loads of approximately 62 kg at concentrations of 1000, 4000 and 8000 ppm.
The dietary levels of the test substance were verified by HPLC-UV.
The homogeneity of the test substance in diet was verified on the first loads at 200, 1000, 4000 and 8000 ppm of the first formulation (F1), on formulation F2 at 200 ppm, on the first loads at 200 and 8000 pprn of formulations F3 and F9, and on the first loads at 1000 and 8000 ppm of formulations F15, F21 and F27, to demonstrate adequate formulation procedures. Mean values obtained from the homogeneity check were taken as measured concentration.
The concentration was checked for all loads at all dose levels for formulations F1, F3, F3bis, F6, F9, F12, F15, F18, F21, F24 and F27.
Homogeneity and concentration in the diet were within the in-house target range of 85 to 115% of nominal concentration (homogeneity 92-104%; concentration 92-111%). Therefore all study mixes were considered acceptable for use on study.

Duration of treatment / exposure:

52 weeks for chronic toxicity phase and 104 weeks for carcinogenicity phase

Frequency of treatment:

Continously via the diet.

Dose / conc.:

200 ppm

Remarks:

corresponding to 9 and 13 mg/kg bw/day actual dose ingested for males and females, respectively (week period 1 to 52).

Dose / conc.:

1 000 ppm

Remarks:

corresponding to 45 and 62 mg/kg bw/day actual dose ingested for males and females, respectively (week period 1 to 52).

Dose / conc.:

4 000 ppm

Remarks:

corresponding to 181 and 249 mg/kg bw/day actual dose ingested for males and females, respectively (week period 1 to 52).

Dose / conc.:

8 000 ppm

Remarks:

corresponding to 364 and 491 mg/kg bw/day actual dose ingested for males and females, respectively (week period 1 to 52).

No. of animals per sex per dose:

70 (60 + 10 for interim sacrifice): 1000, 4000, 8000 ppm
10: 200 ppm

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results from a previous 90-day dietary study in the rat, where dietary administration of up to 12000 ppm in males and females resulted in sulfonamide-like crystals in urine of both sexes, hepatocellular hypertrophy in the liver of males, hyperplasia of the urothelium in the urinary bladder of both sexes, increased incidence of basophilic tubules, brown pigments in proximal tubules and hyperplasia of collecting ducts and pelvic epithelium in the kidney of both sexes, metaplasia in the collecting duct epithelium in one female and increased severity of decreased size of the cortex of thymus in males. In addition, two females prematurely died at 12000 ppm, showing a range of lesions with a number of similarities to those observed on target organs at terminal sacrifice. The No Observed Effect Level in the rat 90-day study was 1000 ppm, with a Low Observed Adverse Effect Level of 4000 ppm.

Positive control:

None

Observations and examinations performed and frequency:

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least weekly, including palpitation for masses. Onset, location and dimension of the masses were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Each animal was weighed at least weekly during the acclimatisation period, then weekly for the first 13 weeks, approximately every 4 weeks thereafter. Additionally, diet fasted animals were weighed prior to scheduled necropsy. Body weights recorded prior to necropsy are referred to terminal body weights.

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: During the acclimatisation phase by indirect ophthalmoscopy; during the treatment period (after approximately 12 and 24 months) by funduscopic (indirect ophthalmoscopy) and biomicroscopic (slit lamp) examinations. Each eye was examined by direct ophthalmoscopy in the first instance, and then after instillation of an atropinic agent (Mydriaticum, Merck Sharp and Dohme), each eye was re-examined by means of a slit lamp and and an indirect ophthalmoscope.
- Dose groups that were examined: All animals during acclimatisation phase and all surviving animals were re-examined after approximately 12 and 24 months of treatment.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On Weeks 16 or 18, 23 or 24 and 51 or 52 from the interim sacrifice groups, and on Weeks 16 or 18, 23 or 24, 51 or 52, 78 and 105 or 106 from the terminal sacrifice groups. Blood (0.5 mL) was sampled by puncture of the retro-orbital plexus into tubes containing EDTA. When possible, a blood smear was prepared for the moribund animals, just before sacrifice. At terminal sacrifice blood smears were prepared for all animals.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight dietary fasting.
- How many animals: On all surviving animals of the 12-month interim sacrifice groups and on the first ten suitable surviving rats of the terminal sacrifice groups.
- Parameters examined: Haematocrit, haemoglobin concentration, leukocyte count, erythrocyte count, platelet count, prothrombin time, leukocyte differential count, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, mean corpuscular volume, reticulocyte count.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On Weeks 16 or 18, 23 or 24 and 51 or 52 from the interim sacrifice groups, and on Weeks 16 or 18, 23 or 24, 51 or 52, 78 and 105 or 106 from the terminal sacrifice groups. Blood was sampled by puncture of the retro-orbital plexus into tubes containing lithium heparin (for plasma), clot activator (for serum; 2.5 mL and 0.5 mL, respectively) and sodium citrate for coagulation (0.9 mL).
- Animals fasted: Yes, overnight.
- How many animals: On all surviving animals of the 12-month interim sacrifice groups and on the first ten suitable surviving rats of the terminal sacrifice groups.
- Parameters examined: Calcium, chloride, inorganic phosphorus, potassium, sodium, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma glutamyltransferase, albumin, creatinine, urea, total cholesterol, glucose (fasting), total bilirubin, total protein, triglycerides.

URINALYSIS: Yes
- Time schedule for collection of urine: On all the surviving animals of the 12-month interim sacrifice groups on Weeks 15 or 16, 26 or 27 and 52 or 53 and on the first ten suitable surviving rats of the terminal sacrifice groups on Weeks 15 or 16, 26 or 27, 52 or 53, 78 and 103.
- Metabolism cages used for collection of urine: No data.
- Animals fasted: Yes, diet and water were withdrawn during the overnight (approximately 16 hours) collection period.
- Parameters examined: Appearance, volume, specific gravity/ osmolality / refractive index, pH, sediment (microscopic), protein, glucose, ketones, bilirubin, blood / red blood cells, urobilinogen.

NEUROBEHAVIOURAL EXAMINATION: During study Month 12, a neurotoxicity assessment was performed on all surviving animals at all dose levels by observers who were blind with respect to the dose level.
Sensory reactivity
The following reflexes and responses were recorded:
- Pupillary reflex (by covering the eyes of the animal for a few seconds and then observing pupillary constriction by focusing a narrow beam of light in the eyes).
- Surface righting reflex (by putting the animal on its back and measuring its ability/rapidity to reassume a normal standing position).
- Corneal reflex (by touching the medial canthus with a fine object and observing the quick and complete close of the eyelids).
- Flexor reflex (by pinching the toes and measuring the presence/strength of the flexor response of each hindlimb).
- Auditory startle response (by measuring the response to an auditory stimulus).
- Tail pinch response (by pinching the tail with forceps and measuring the animal's reaction).
Grip strength
The fore- and hindlimb grip strength of all animals were measured quantitatively using a grip strength tester equipped with one pull or one push strain gauge (Bioseb, Chaville, France). The mean of three successive measurements was noted for both fore- and hindlimb grip strength.

Sacrifice and pathology:

GROSS PATHOLOGY:
Yes, on Days 379 to 382 for the 12-month chronic phase, and on Days 732 to 746 for the 24-month carcinogenicity phase, all surviving animals dedicated to chronic phase and carcinogenicity phase groups, respectively, were sacrificed by exsanguination under deep anesthesia (inhalation of isoflurane). At final sacrifice, due to the high mortality rate observed in males treated at 4000 and 8000 ppm, it was decided, in agreement with the Sponsor Representative, to sacrifice the males first, and then the females. Animals were diet fasted overnight prior to sacrifice. All animals were necropsied. The 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. The following organs or tissues were sampled and/or weighed at necropsy: Tongue, submaxillary gland, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, liver*, pancreas*, trachea*, lung*, nasal cavities*, pharynx*, larynx*, thoracic aorta, heart*, bone marrow* (sternum), mesenteric lymph node, submaxillary lymph node, spleen*, thymus, kidney*, urinary bladder, testis*, epididymis*, prostate gland, seminal vesicle, ovary*, uterus (with cervix)*, mammary gland*, vagina, brain*, sciatic nerve, spinal cord (cervical, thoracic, lumbar), eyes (retina), optic nerves, pituitary gland, adrenal gland*, lachrymal exorbital gland, parathyroid gland*, Harderian gland, bone (sternum), skeletal muscle, skin, all gross lesions and masses (incl. their lymph nodes if possible), articular surface (femorotibial joint) (* organs were weighed). Samples were fixed by immersion in neutral buffered 10% formalin with the exception of the eye and optic nerve, Harderian gland, epididymis and testis that were fixed in Davidson's fixative.

HISTOPATHOLOGY:
Yes, all the above samples listed (except exorbital lachrymal gland, larynx/pharynx and nasal cavities) were embedded in paraffin wax. For the 12-month chronic and 24-month carcinogenicity phase, histological sections, stained with hematoxylin and eosin, were prepared from all organs and tissue samples. Histopathology was performed in-house for the chronic toxicity phase. Histopathology examinations for the 12-month interim sacrifice were performed as follows:
- all organs and tissue samples from animals sacrificed or dying during the treatment period
- all organs and tissue samples from animals of control and high-dose groups
- liver, lung, kidney and urinary bladder from animals of the intermediate-dose groups
- gross abnormalities from all animals.
For all unscheduled sacrificed or dead animals on study, a determination of the factors having contributed to death was made. Initial examinations were performed by the study pathologist. Following the initial examination, an in-house review pathologist undertook an independent peer-review of representative slides and diagnoses according to standardised operating procedures. The diagnoses presented in this report represent the consensus opinion of the two pathologists.

Other examinations:

None

Statistics:

Mean and standard deviation were calculated for each group. All statistical analyses were carried out separately for males and females.

For details on statistics performed please refer to "Any other information on materials and methods incl. tables".

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

See Attachment 1 for summary tables of clinical signs.

8000 ppm:
- Soiled anogenital region was noted in both sexes. Soiled anogenital region was also recorded as soiled fur (localized in the anogenital region) in a number of females. In addition, red coloured urine and genital discharge were noted in a few females.

4000 ppm:
- Soiled anogenital region was noted in both sexes

Mortality:

mortality observed, non-treatment-related

Description (incidence):

See Attachment 1 for summary tables of mortality.

During the first year of treatment the mortality rate was not affected by treatment in either sex. One male from the control group and two males at 8000 ppm (allocated to the carcinogenicity phase) died due to accidental trauma, and one male at 1000 ppm died during anesthesia. The animals that died of accidental trauma are not included in the tables presented in Attachment 1.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See Attachment 1 for summary tables of body weight and body weight gains.

8000 ppm:
- Mean cumulative body weight gain was reduced during the first week of treatment by 7% (p<0.01) in males and by 12% (p<0.01) in females, when compared to controls, whilst mean body weight was unaffected in both sexes. Thereafter, mean body weight and body weight gain parameters were not significantly different from controls in both sexes throughout the study.

4000 ppm:
- In males, mean cumulative body weight gain was reduced during the first week of treatment by 5% (p<0.05) when compared to controls. Thereafter, mean body weight and body weight gain parameters were comparable to controls throughout the study.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

For compound intake, see Attachment 1.

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:

no effects observed

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Throughout the first year of the study, lower mean total protein and/or globulin concentrations were observed at 8000 and 4000 ppm in males, resulting in a higher albumin/globulin ratio than in the controls. These differences were slight and were not observed during the second year of the study, therefore they were considered to be incidental. The other statistically significant differences were considered to be chance findings in view of their sporadic occurrence and/or their low amplitude.

Endocrine findings:

not examined

Description (incidence and severity):

Not applicable.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

See Attachment 2 for summary tables of urinalysis findings.

- Throughout the urine sampling periods, sulfonamide-like crystals were observed at 8000 and 4000 ppm in both sexes. This finding was dose-related in terms of incidence and/or severity on most occasions, the effect being more pronounced in females than in males. No sulfonamide-like crystals were observed at 1000 ppm, 200 ppm or in control animals in either sex.
- When compared to the control groups, a tendency towards lower urinary protein levels was noted throughout the study in both sexes at 8000 and 4000 ppm (except for females at Month 24). The same variation was seen in males at 1000 ppm at Month 6/7, 12 and 18. This change was considered not to be adverse in view of its low magnitude.
- Additionally, at Month 12, a tendency towards higher urinary volume was noted at 8000 ppm in females (+59%, p<0.05). This change was considered incidental as it was not observed during the second year of the study.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Locomotor activity: There was no treatment-related effect on overall mean exploratory locomotor activity in either sex. The pattern of the locomotor activity over time was similar to the control at all dose levels in both sexes. The statistically significant increase observed in males at 200 ppm during interval 16-30 min was considered to be a chance finding as it was observed in isolation and with no dose-relationship.

Sensory reactivity: All reflexes and responses evaluated were unaffected by the treatment at any dose level in either sex. The few changes in the tail pinch response noted for males in all dose groups were considered to reflect inter-individual variations rather than any treatment-related effect.

Grip strength: The fore- and hindlimb grip strength were unaffected by the treatment at any dose level in either sex. The slight increase reaching statistical significance in hindlimb grip strength observed in the female high dose group (+19%, p<0,05) was mainly attributable to a particularly high value for animal NT5F5156 and was therefore considered to be incidental.

Immunological findings:

not examined

Description (incidence and severity):

Not applicable.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There was no treatment-related change in mean terminal body weights and organ weights when compared to controls. The few apparent organ weight differences observed were considered not to be treatment-related, even when statistically significantly higher in comparison to controls since they were not correlated with any microscopic findings.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Unscheduled deaths:
- Four animals were either found dead or killed for humane reasons before the end of the 12-month chronic phase. Female NT5F5155 (found dead on study Day 364, 8000 ppm) had mottled bilateral kidney with gritty content. These observations were considered to be treatment related since they were correlated with treatment-related histological changes.
- No treatment-related changes were identified in the other decedents.

Terminal sacrifice (12-months):
- All gross pathology changes were considered to be incidental and not treatment related.

Neuropathological findings:

not examined

Description (incidence and severity):

Not applicable.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See Attachment 3 for summary tables of histopathology data.

Unscheduled deaths:
8000 ppm:
- Female NT5F5155 had a treatment-induced nephropathy characterized by bilateral papillary necrosis/loss/scarring, collecting duct hyperplasia, bilateral cortico-medullary scarring and bilateral cortex/medulla tubular dilatation, which was considered to be the cause of death.

Terminal sacrifice:
8000 ppm:
- In the kidney, a slightly higher incidence and/or a slight increase in severity of treatment-related findings, also observed but with a higher incidence and severity in the carcinogenicity phase, i.e. unilateral pelvic dilatation, bilateral papillary necrosis/loss/scarring, bilateral cortical/medullary scarring and pelvic epithelium hyperplasia, was noted in the kidney in one or both sexes.
- Treatment-related urothelial hyperplasia was noted in the urinary bladder in a few animals of both sexes.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There was no evidence of a treatment-related effect on the incidence of neoplastic findings. The very few tumors observed (males: 200ppm: N-histiocytic sarcoma of the haemopoetic system, the bone marrow sternum, the liver and spleen; malignant N-schwannoma and B lipoma of the abdominal cavity; B lipoma subcutis; females: 200 ppm: B-fibroadenoma of the mammary gland; females, 4000 and 8000 ppm: B-endometrial stomal polyp of the uterus; females: 8000 ppm: B-lipoma subcutis;) were considered incidental.

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Key result

Dose descriptor:

NOAEL

Effect level:

4 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at this dose level.

Remarks on result:

other: corresponding to 181 and 249 mg/kg bw/day actual dose ingested for males and females, respectively (week period 1 to 52).

Key result

Dose descriptor:

LOAEL

Effect level:

8 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

urinalysis

Remarks on result:

other: corresponding to 364 and 491 mg/kg bw/day actual dose ingested for males and females, respectively (week period 1 to 52).

Critical effects observed:

yes

Lowest effective dose / conc.:

8 000 ppm

System:

urinary

Organ:

bladder

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Conclusions:

The study was performed in accordance to OECD TG 453 under GLP conditions and is considered reliable. In conclusion, the NOAEL over a 12 month period of dietary administration to the Wistar rat was 4000 ppm in both sexes (equivalent to 181 mg/kg bw/day in males and 249 mg/kg bw/day in females).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Version / remarks:

adopted 12 May 1981

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Version / remarks:

adopted 25 Jun 2018

Deviations:

yes

Remarks:

no details on whether feed and water were analysed for contaminants

GLP compliance:

yes (incl. QA statement)

Remarks:

INTERMINISTERIAL GROUP FOR CHEMICAL PRODUCTS, Paris, France

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

485-320-2

EC Name:

-

Cas Number:

221667-31-8

Molecular formula:

C18H18N205S

IUPAC Name:

N-[4-(cyclopropylcarbamoyl)benzenesulfonyl]-2-methoxybenzamide

Test animals

Species:

rat

Strain:

other: Wistar Rj:WI (IOPS HAN)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: R. Janvier, Le Genest St Isle, France
- Age at study initiation: 6 weeks
- Weight at study initiation: 221 - 222 g mean group weight males; 163 - 165 g mean group weight
females
- Housing: By sex in groups of 5, unless reduced by mortality or isolation. The cages were suspended, stainless steel and wire mesh.
- Diet: A04CP1-10 (formerly A04C-10P1) from S.A.F.E. (Scientific Animal Food and Engineering, Augy (formerly Epinay-sur-Orge), France, ad libitum except at designated time periods.
- Water: Filtered and softened tap water from the municipal water supply, ad libitum.
- Acclimation period: 13 days

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

IN-LIFE DATES: From: 19 Nov 2003 To 11 Dec 2006

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Approx. at 4 weekly periods dietary requirements with two occasions of minor exceptions (second formulation at 200 ppm covered 5 weeks and the last formulation which covered the dietary needs until the end of the study).
- Mixing appropriate amounts with (Type of food): The test substance was incorporated into the diet (A04CP1-10 from S.A.F.E. (Scientific Animal Food and Engineering, Augy, France)) to provide the required dietary concentrations of 200, 1000, 4000 or 8000 ppm.
- Storage temperature of food: Ambient temperature.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in the diet was demonstrated in a previous study (SA03277). The stability of the test substance at 100 and 15000 ppm in the diet was verified for at least 104 days at room temperature and over 70 days frozen storage followed by 10 days at room temperature (covers the period of storage and usage for the study which covered the period of storage and usage for this study.
Fourteen formulations (F1 to F14) were prepared during the study at a concentration of 200 ppm and twenty-seven formulations (F1 to F27) were prepared during the study at all other concentrations. In addition, an additional formulation (F3bis) was prepared at 1000 ppm to replace the formulation F3 which was found slightly out of the in-house target ranges.
Formulation F1 consisted of four loads of approximately 41 kg at concentrations of 1000, 4000 and 8000 ppm and one load of approximately 22 kg at a concentration of 200 ppm.
Formulation F2 consisted of four loads of approximately 41 kg at concentrations of 1000, 4000 and 8000 ppm and one load of approximately 40 kg at a concentration of 200 ppm. Formulations F3 (and F3 bis for 1000 ppm) to F14 consisted of four loads of approximately 35 kg at concentrations of 1000, 4000 and 8000 ppm and one load of approximately 35 kg at a concentration of 200 ppm. Formulations F15 to F27 consisted of two loads of approximately 62 kg at concentrations of 1000, 4000 and 8000 ppm.
The dietary levels of the test substance were verified by HPLC-UV.
The homogeneity of the test substance in diet was verified on the first loads at 200, 1000, 4000 and 8000 ppm of the first formulation (F1), on formulation F2 at 200 ppm, on the first loads at 200 and 8000 pprn of formulations F3 and F9, and on the first loads at 1000 and 8000 ppm of formulations F15, F21 and F27, to demonstrate adequate formulation procedures. Mean values obtained from the homogeneity check were taken as measured concentration.
The concentration was checked for all loads at all dose levels for formulations F1, F3, F3bis, F6, F9, F12, F15, F18, F21, F24 and F27.
Homogeneity and concentration in the diet were within the in-house target range of 85 to 115% of nominal concentration (homogeneity 92-104%; concentration 92-111%). Therefore all study mixes were considered acceptable for use on study.

Duration of treatment / exposure:

At least 104 weeks for carcinogenicity phase and 52 weeks for chronic toxicity phase.

Frequency of treatment:

Continously via the diet.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 000 ppm

Remarks:

corresponding to 39 and 56 mg/kg bw/day actual dose ingested for males and females, respectively (week period 1 to 104).

Doses / concentrations 2

Dose / conc.:

4 000 ppm

Remarks:

corresponding to 159 and 220 mg/kg bw/day actual dose ingested for males and females, respectively (week period 1 to 104).

Doses / concentrations 3

Dose / conc.:

8 000 ppm

Remarks:

corresponding to 321 and 447 mg/kg bw/day actual dose ingested for males and females, respectively (week period 1 to 104).

No. of animals per sex per dose:

70 (60 + 10 for interim sacrifice): 1000, 4000, 8000 ppm
10: 200 ppm

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: dose levels were selected based on the results from a previous 90-day dietary study in the rat, where dietary administration of up to 12000 ppm in males and females resulted in sulfonamide-like crystals in urine of both sexes, hepatocellular hypertrophy in the liver of males, hyperplasia of the urothelium in the urinary bladder of both sexes, increased incidence of basophilic tubules, brown pigments in proximal tubules and hyperplasia of collecting ducts and pelvic epithelium in the kidney of both sexes, metaplasia in the collecting duct epithelium in one female and increased severity of decreased size of the cortex of thymus in males. In addition, two females prematurely died at 12000 ppm, showing a range of lesions with a number of similarities to those observed on target organs at terminal sacrifice. The No Observed Effect Level in the rat 90-day study was 1000 ppm, with a Low Observed Adverse Effect Level of 4000 ppm.

Positive control:

None

Examinations

Observations and examinations performed and frequency:

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily for all animals. Detailed physical examinations including palpation for masses were performed weekly throughout the study. The onset, location and dimension of the masses were recorded

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly for the first 13 weeks of study and approximately every 4 weeks thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes.
- Food consumption was recorded twice weekly during the first 6 weeks of treatment, then weekly up to Week 13, and once approximately every 4 weeks thereafter.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes. The weekly mean achieved dosage intake in mg/kg body weight/day for Weeks 1 to 13, then 1 week per month thereafter was calculated as the product of the dose level (ppm) and the group mean food consumption (g/day) divided by the group mean body weight (g) at the end of the week. The monthly and overall mean achieved dosage intake for the 24 months treatment were derived from the weekly data.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: During acclimatization phase and after approximately 12 and 24 months
- Dose groups that were examined: All animals during the acclimatization phase and all surviving animals after 12 and 24 months

HAEMATOLOGY: Yes
- Time schedule for collection of blood: For the interim sacrifice groups on Weeks 16 to 18, 23 or 24 and 51 or 52, and for the first ten suitable surviving rats of each sex of the terminal sacrifice groups on Weeks 16 to 18, 23 or 24, 51 or 52, 75 and 105 or 106.
- Anaesthetic used for blood collection: Yes, Isoflurane.
- Animals fasted: Yes, overnight fasting.
- How many animals: All surviving animals of the interim sacrifice groups on Weeks 16 to 18, 23 or 24 and 51 or 52, and on the first ten suitable surviving rats of each sex of the terminal sacrifice groups on Weeks 16 to 18, 23 or 24, 51 or 52, 75 and 105 or 106.
- Parameters examined: Haematocrit, haemoglobin concentration, leukocyte count, erythrocyte count, platelet count, leukocyte differential time, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, mean corpuscular volume, reticulocyte count.
- At terminal sacrifice, blood smears were prepared for all animals not sampled for hematology. When possible, a blood smear was prepared for the moribund animals, just before sacrifice.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Months 4, 6, 12, 18 and 25
- Animals fasted: Yes, overnight
- How many animals: All surviving animals of the interim sacrifice groups on Weeks 16 to 18, 23 or 24 and 51 or 52, and on the first ten suitable surviving rats of each sex of the terminal sacrifice groups on Weeks 16 to 18, 23 or 24, 51 or 52, 75 and 105 or 106
- Parameters: Alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma glutamyltransferase, glutamate dehydrogenase, albumin, creatinine, urea, total cholesterol, glucose (fasting), total bilirubin, total protein, triglycerides, calcium, chloride, inorganic phosporus, potassium and sodium.

URINALYSIS: Yes
- Time schedule for collection of urine: All the surviving animals of the interim sacrifice groups on Weeks 15 or 16, 26 or 27 and 52 or 53, and on the first ten suitable surviving rats of the terminal sacrifice groups on Weeks 15 or 16, 26 or 27, 52 or 53, 78 and 103.
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes. Diet and water were withdrawn during the overnight (approx. 16 hours) collection period.
- Parameters examined: Appearance, volume, specific gravity / osmolality / refractive index, pH, sediment (microscopic), protein, glucose, ketones, bilirubin, blood/red blood cells, urobilinogen

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Month 12
- Dose groups that were examined: All surviving animals
- Battery of functions tested: Sensory activity / grip strength / motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, on Days 379 to 382 for the 12-month chronic phase, and on Days 732 to 746 for the 24-month carcinogenicity phase, all surviving animals dedicated to chronic phase and carcinogenicity phase groups, respectively, were sacrificed by exsanguination under deep anesthesia (inhalation of Isoflurane). At final sacrifice, due to the high mortality rate observed in males treated at 4000 and 8000 ppm, it was decided, in agreement with the sponsor representative, to sacrifice the males first, and then the females. Animals were diet fasted overnight prior to sacrifice. 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. The following organs or tissues were sampled and/or weighed at necropsy: digestive system (including tongue, submaxillary gland, oesophagus, stomach, duodenum, jejenum, illeum, caecum, colon, rectum, liver and pancreas), cardiovascular/haemopoietic system (including thoracic aorta, heart, bone marrow, sternum, mesenteric lymph node, submaxillary lymph node, spleen, thymus, neurologic (including brain, sciatic nerve, spinal cord, eyes, optic nerves), glandular systems (including pituitary gland, adrenal gland, exorbital lachrymal gland, parathyroid gland, thyroid gland and Harderian gland), respiratory system (including trachea, lung, nasal cavities, pharynx, larynx), urogenital system (including kidney, urinary bladder, testis, epididymis, prostate gland, seminal vesicle, ovary, uterus with cervix, mammary gland, vagina) and other tissues (including bone, skeletal muscle, skin, all gross lesions and masses and articular surface.

ORGAN WEIGHTS: mean organ weight, organ/terminal body weight and organ/brain weight of the brain, heart, kidney, thymus, epididymis, testis, prostate, adrenal gland, liver, pituitary gland, spleen and thyroid gland, uterus and ovary were recorded.

HISTOPATHOLOGY: Yes, all tissues and organs as in gross pathology (except exorbital lachrymal gland, larynx/pharynx and nasal cavities) were embedded in paraffin wax. For the 12-month chronic and 24-month carcinogenicity phase, histological sections, stained with hematoxylin and eosin, were prepared from all organs and tissue samples.
12-month interim sacrifice (52 weeks)
Histopathology examinations were performed as follows for the 12-month interim sacrifice:
- all organs and tissue samples from animals sacrificed or dying during the treatment period,
- all organs and tissue samples from animals of control and high dose groups,
- liver, lung, kidney and urinary bladder from animals of the intermediate dose groups,
- gross abnormalities from all animals.
For all unscheduled sacrificed or dead animals on study, a determination of the factors contributing to death was made.
Initial examinations were performed by the study pathologist. Following the initial examination, an in-house 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.
Carcinogenicity phase (104 weeks)
For the carcinogeniciy phase investigations, 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 a determination of the factors having contributed to death was made. Initial examinations were performed by the study pathologist. Following the initial examination, an external review pathologist), undertook an independent peer-review of representative slides and diagnoses according to standardized operating procedures. The diagnosis presented in the report represents the consensus opinion of the two pathologists.

Statistics:

Mean and standard deviation were calculated for each group. All statistical analyses were carried out separately for males and females.

For details on statistics performed please refer to "Any other information on materials and methods incl. tables".

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

See Attachment 1 for summary tables of clinical signs.

8000 ppm:
- Soiled anogenital region was noted in both sexes. Soiled anogenital region was also recorded as soiled fur (localized in the anogenital region) in males and a higher incidence of red genital discharge, reduced motor activity, general pallor, wasted appearance and hunched posture was noted in females.

4000 ppm:
- Soiled anogenital region was noted in both sexes. Soiled anogenital region was also recorded as soiled fur (localized at the anogenital region) in males.

1000 ppm:
- A slightly higher incidence of soiled anogenital region was noted in males. No treatment-related clinical signs were observed in females.

After one and/or two years of treatment, dental abnormality and incisors cut in both sexes and ocular discharge in females occurred at a slightly higher incidence at 8000, 4000 and 1000 ppm, in comparison with the controls. However, in the absence of a dose-relationship and of associated treatment-related macroscopic or microscopic changes, dental abnormality and ocular discharge were considered not to be treatment-related. In addition, the apparent higher incidence of skin lesions occurring at 8000 ppm in males was considered not to be treatment-related since these lesions mainly consisted of tail lesions which were observed on animals dying from accidental trauma.

Mortality:

mortality observed, treatment-related

Description (incidence):

See Attachment 1 for summary tables of mortality data.

After at least 104 weeks of treatment the mortality rate was not affected by treatment in males and was slightly higher at 8000 ppm in females compared to controls, where 14 out of 31 unscheduled mortalities were attributed to treatment-related nephropathy. However, the difference to controls was not statistically significant.

In the males, statistically significance for higher mortality rate was achieved at 4000 ppm. As treatment-related nephropathy was not reported as a cause of death at this dose level and no increased mortality rate was observed at 8000 ppm, the statistically significant increase was considered incidental and not treatment-related.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

See Attachment 1 for summary tables of body weight and body weght gain data.

8000 ppm:
- Overall, mean body weight on study Day 708 and mean cumulative body weight gain between study Days 1 to 708 were similar to controls in both sexes.

4000 ppm
- The mean cumulative body weight loss of 68 g noted during the interval Weeks 78 to 101 was not considered treatment related since it was not dose-related and the values were within the historical control ranges (-25 to -73 g in a total of 6 studies). Overall, mean body weight on study Day 708 was reduced by 6% (not statistically significant), whilst mean cumulative body weight gain between study Days 1 to 708 was reduced by 9% (not statistically significant), compared to the controls. This effect was not considered treatment-related since it was not dose-related.

There was no treatment-related effect on mean body weight or body weight gain parameters at 4000 ppm in females or at 1000 and 200 ppm in either sex over the two years of treatment (one year only at 200 ppm).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

See Attachment 1 for compound intake.

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, non-treatment-related

Description (incidence and severity):

8000 ppm:
- At Month 25, slightly lower mean erythrocyte count (-8%, not statistically significant), haemoglobin concentration (-9%, p<0.05) and haematocrit (-9%, p<0.05) were noted in females only. These changes were considered to be incidental as only 2/9 animals were affected.

Any other statistically significant differences were considered to be chance findings in view of their low amplitude and/or their sporadic occurrence.

Clinical biochemistry findings:

no effects observed

Endocrine findings:

not examined

Description (incidence and severity):

Not applicable.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

See Attachment 2 for summary tables of urinalysis findings.

Throughout the urine sampling periods, sulfonamide-like crystals were observed at 8000 and 4000 ppm in both sexes. This finding was dose-related in terms of incidence and/or severity on most occasions, the effect being more pronounced in females than in males. No sulfonamide-like crystals were observed at 1000 ppm, 200 ppm or in control animals in either sex.

When compared to the control groups, a tendency towards lower urinary protein levels was noted throughout the study in both sexes at 8000 and 4000 ppm (except for females at Month 24). The same variation was seen in males at 1000 ppm at Month 6/7, 12 and 18. This change was considered not to be adverse in view of its low magnitude.

The other variations were judged to be incidental and not treatment-related.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Locomotor activity: There was no treatment-related effect on overall mean exploratory locomotor activity in either sex. The pattern of the locomotor activity over time was similar to the control at all dose levels in both sexes. The statistically significant increase observed in males at 200 ppm during interval 16-30 min was considered to be a chance finding as it was observed in isolation and with no dose-relationship.

Sensory reactivity: All reflexes and responses evaluated were unaffected by the treatment at any dose level in either sex. The few changes in the tail pinch response noted for males in all dose groups were considered to reflect inter-individual variations rather than any treatment-related effect.

Grip strength: The fore- and hindlimb grip strength were unaffected by the treatment at any dose level in either sex. The slight increase reaching statistical significance in hindlimb grip strength observed in the female high dose group (+19%, p<0,05) was mainly attributable to a particularly high value for animal NT5F5156 and was therefore considered to be incidental.

Immunological findings:

not examined

Description (incidence and severity):

Not applicable.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There was no treatment-related change in mean terminal body weights and organ weights when compared to controls. The few apparent organ weight differences observed were considered not to be treatment-related, even when statistically significantly higher in comparison to controls since they were not correlated with any microscopic findings.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

See Attachment 3 for summary tables of gross pathology data.

Unscheduled deaths:
- Two hundred and fifty-six animals were either found dead or killed for humane reasons before the end of the study.
- At 8000 ppm, 14/31 deaths in females were attributed to treatment-induced nephropathy.
- At 8000 ppm, treatment-related macroscopic findings consisted of abnormal shape, pelvic dilatation, irregular surface, gritty content and stones in the kidney in females.
- Treatment-related presence of stones was noted in the urinary bladder in males at 8000 ppm.

Terminal sacrifice:
- Treatment-related macroscopic findings were observed in the kidney and consisted of abnormal shape, pelvic dilatation and gritty content in both sexes at 8000 ppm, irregular surface and stones in females at 8000 ppm and pelvic dilatation, gritty content and stones in females at 4000 ppm.
- Stones were observed in the urinary bladder and were considered to be treatment-related in both sexes at 8000 ppm and in one female at 4000 ppm.

Neuropathological findings:

not examined

Description (incidence and severity):

Not applicable.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See Attachment 4 for summary tables of histopathology data.

Scheduled and unscheduled deaths:
- Treatment-related effects were observed in the urinary tract, i.e. kidney, urinary bladder and ureter, including a combination of hyperplastic and inflammatory changes associated with the presence of stones.

Kidney:
- Treatment-induced nephropathy was observed in both sexes in the kidney at 8000 ppm, with a higher severity in females, and at 4000 ppm in females only.
- Treatment-induced nephropathy is a general term which incorporates some or all of the following findings: stones, papillary necrosis/loss/scarring, collecting duct hyperplasia, bilateral cortico-medullary scarring, pelvic dilatation and bilateral cortex/medulla tubular dilatation.
- In addition, an increased incidence of pelvic dilatation was noted at 4000 ppm in males.
- A slight increase in severity of simple pelvic epithelium hyperplasia was observed in both sexes at 8000 and 4000 ppm.
- Nodular pelvic epithelium hyperplasia was observed in 2/60 females and a higher incidence of intrapelvic mineral deposit was observed in 14/60 females at 8000 ppm.

Urinary Bladder:
- Intraluminal stones and multifocal/diffuse urothelial hyperplasia were observed in the urinary bladder in both sexes at 8000 ppm and in females at 4000 ppm. These lesions were considered to be treatment-related.
- Suburothelial mononuclear cell infiltrate and mixed cell infiltrate were considered to be associated lesions.

Ureter:
- Intraluminal stones and multifocal/diffuse urothelial hyperplasia were observed in the ureter of a number of animals in both sexes at 8000 ppm. These lesions were considered to be treatment related.

Testes:
- An increased incidence of the commonly occurring lesion diffuse bilateral tubular degeneration of the testis (associated with diffuse interstitial oedema and/or focal/multifocal mineralization in most cases) and bilateral oligospermia of the epididymis (associated with epithelial degenerative change) was observed at 8000 ppm, which was considered to be treatment-related.

Indirect systemic changes commonly associated with induced nephropathy, including compensatory hyperplasia of the parathyroid glands associated with mineralization, especially involving lungs and the gastric mucosa, were observed at 8000 ppm mainly in females.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At the end of the 2-year treatment period, there was no evidence of a direct treatment-related increased incidence of tumors of any type in any organ.

At 8000 ppm, a transitional cell carcinoma was observed in the kidney in one male and a transitional cell carcinoma was observed in the urinary bladder in one female. Those two single tumors were considered to be secondary to the combination of hyperplastic and inflammatory changes associated with the presence of stones.

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects seen at this dose level.

Remarks on result:

other: corresponding to 39 and 56 mg/kg bw/day actual dose ingested for males and females, respectively.

2

Key result

Dose descriptor:

LOAEL

Effect level:

4 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

gross pathology

histopathology: non-neoplastic

urinalysis

Remarks on result:

other: corresponding to 159 and 220 mg/kg bw/day actual dose ingested for males and females, respectively.

Target system / organ toxicity

1

Critical effects observed:

yes

Lowest effective dose / conc.:

4 000 ppm

System:

urinary

Organ:

bladder

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

2

Critical effects observed:

yes

Lowest effective dose / conc.:

8 000 ppm

System:

urinary

Organ:

ureter

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Applicant's summary and conclusion

Conclusions:

The study was performed in accordance to OECD TG 453 under GLP conditions and is considered reliable. In conclusion, the systemic NOAEL over a 24 month period of dietary administration to the Wistar rat was 1000 ppm in both sexes (equivalent to 39 mg/kg/day in males and 56 mg/kg/day in females). At 8000 ppm (equivalent to 321 mg/kg/day in males and 447 mg/kg/day in females), the neoplastic changes of transitional cell carcinoma in the kidney of one male and a transitional cell carcinoma in the urinary bladder of one female were considered to be secondary to the combination of hyperplastic and inflammatory changes associated with the presence of stones.