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

Endpoint summary

Description of key information

Oral (OECD 408), subchronic, rat: NOEL (systemic), males/females = 58/70 mg/kg bw/day
Oral (OECD 409), subchronic, dog: NOAEL (systemic), males/females = 221/221 mg/kg bw/day
Oral (OECD 408), subchronic, mouse: NOEL (systemic) males = 1110 mg/kg bw/day; females = 398 mg/kg bw/day
Oral (OECD 452) chronic, dog: NOEL (systemic) males/females = 66/67 mg/kg bw/day
Oral (OECD 453) chronic, rat: NOAEL 12-month (systemic) males/females = 181/249 mg/kg bw/day; NOAEL 24-month (systemic) males/females = 39/56 mg/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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: 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 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.

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, 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:
- 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.

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.

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.

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.

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, 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.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 Feb - 29 Sep 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted 21 Sep 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted 25 Jun 2018

Deviations:

yes

Remarks:

See Principles of method if other than guideline

Principles of method if other than guideline:

No haematological examinations were performed. Only selected clinical chemistry parameters were analysed in plasma. No ophthalmological examinations were performed. No behavioural (functional observations) were performed. Mice were housed individually instead of in groups per sex. Phytoestrogen levels were not determined in the diet. T4, T3 and TSH levels were not assessed. Oestrus cycle not assessed. Only selected organ weights were determined.

GLP compliance:

yes (incl. QA statement)

Remarks:

INTERMINISTERIAL GROUP FOR CHEMICAL PRODUCTS, Paris, France

Limit test:

no

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
- Age at study initiation: 6 to 7 weeks
- Weight at study initiation: 16.4-20.8 g (males) and 14.9-18.0 g (females)
- Fasting period before study: Not applicable
- Housing: Individually in suspended stainless steel wire mesh cages
- Diet: Certified rodent powdered and irradiated diet A04C-10 P1 from S.A.F.E. (Scientific Animal Food and Engineering, route de Saint Bris, 89290 Augy, France), ad libitum. Except before blood collection prior to sacrifice when animals were diet fasted overnight
- Water: Filtered and softened water from the municipal water supply, ad libitum
- Acclimation period: 7 days

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

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Air changes (per hr): a target of 15 air changes per hour (average, not monitored)
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 26 Feb 2003 To: 05 Jun 2003

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Once (two loads) during the course of the study, for each concentration.
- Mixing appropriate amounts with (Type of food): Certified rodent powdered and irradiated diet A04C-10P1 from S.A.F.E. (Scientific Animal Food and Engineering, Epinay-sur-Orge, France)
- Storage temperature of food: At ambient temperature

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and concentration of two loads were checked according to analytical protocol: each sample that was taken for analytical purpose weighed approximately 40 g of which 10 g was analysed. The test substance was extracted from the diet by simple homogenisation with acetonitrile. The quantification was performed by High Performance Liquid Chromatography (HPLC) using UV detection at 250 nm and external standardisation.
Homogeneity of the preparations at 500 and 7000 ppm were checked (first load). The sampling was done at two levels, the surface and bottom. At each level, two samples were taken and analysed. Mean values obtained from the homogeneity checks were used as measured concentrations. For the intermediate level (2000 ppm, first load), and for all second loads, the concentration was checked on a single sample, sampling being done at three levels (surface, middle and bottom). A control sample was also taken for each sampling procedure and analysed.
Results for homogeneity of the test substance at the lowest and the highest concentration (500 and 7000 ppm) on the first load of diet preparation were within the range of 98-112%. Concentration checks at the three doses on the two loads were within the range of 100-104%. Hence all values were within the in-house target range of 85-115% of the nominal concentrations.

Duration of treatment / exposure:

91-93 days

Frequency of treatment:

Continously via the diet

Dose / conc.:

500 ppm

Remarks:

corresponding to 79 and 95 mg/kg bw/day actual dose ingested for males and females, respectively.

Dose / conc.:

2 000 ppm

Remarks:

corresponding to 321 and 398 mg/kg bw/day actual dose ingested for males and females, respectively.

Dose / conc.:

7 000 ppm

Remarks:

corresponding to 1110 and 1297 mg/kg bw/day actual dose ingested for males and females, respectively.

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The doses were set after evaluation of the results obtained from exploratory toxicity studies in mice. Groups of 10 male and 10 female mice were given control diet or the appropriate diet mixture.

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Moribundity and mortality twice daily (once daily on weekends or public holidays); clinical signs at least once daily for all animals
- Cage side observations included: Nature, onset, severity, reversibility and duration of clinical signs; signs of ill-health such as blood or loose feces

DETAILED CLINICAL OBSERVATIONS: Yes, detailed physical examinations
- Time schedule: At least weekly

BODY WEIGHT: Yes, body weights, body weight gains, absolute body weight gains.
- Time schedule for examinations: 2 times during the acclimatisation period, on Day 1 of test substance administration, then at weekly intervals and at necropsy

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 group mean achieved dosage for each sex from the consumption and body weight data, expressed as mg/kg bw/day: Yes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On Days 91, 92 or 93, in the morning, prior to necropsy, blood samples were collected from the retro-orbital venous plexus on lithium heparin for plasma chemistry determinations.
- Anaesthetic used for blood collection: Yes (by inhalation of isoflurane)
- Animals fasted: Yes, diet fasted overnight.
- How many animals: All surviving animals of all groups.
- Parameters checked examined: Total bilirubin, urea, total protein, albumin and total cholesterol concentrations and aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase activities, as well as any significant change in the appearance of the plasma.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, all surviving animals from all groups were sacrificed by exsanguination under deep anaesthesia (pentobarbital, intraperitoneal injection of 60 mg/kg bw). An approximately equal number of animals randomly distributed amongst all groups were sampled on each day (animals were diet fasted overnight prior to sacrifice). Furthermore, all animals either found dead or killed for animal welfare reasons were necropsied. Necropsy included the examination of all major organs, tissues and body cavities. Macroscopic abnormalities were recorded, sampled and examined microscopically. The following organs or tissues were sampled: Adrenal gland, Aorta, Articular surface (femoro-tibial), Bone (sternum), Bone marrow (sternum), Brain, Epididymis, Esophagus, Exorbital (lachrymal) gland, Eye, Gallbladder, Harderian gland, Heart, Intestine (duodenum, jejunum, ileum, cecum, colon, rectum), Kidney, Larynx/Pharynx, Liver, Lung, Lymph nodes (submaxillary, mesenteric), Mammary gland, Nasal cavities, Optic nerve, Ovary, Pancreas, Pituitary gland, Prostate, Sciatic nerve, Seminal vesicle, Skeletal muscle, Skin, Spinal cord (cervical, thoracic, lumbar), Spleen, Stomach, Submaxillary (salivary) gland, Testis, Thymus, Thyroid gland, Tongue, Trachea, Urinary bladder, Uterus (including cervix), Vagina.

ORGAN WEIGHTS: Adrenal glands, brain, heart, kidneys, liver, spleen, testis, thymus and uterus were weighed fresh at scheduled sacrifice only. Paired organs were weighed together. Organ to body weight and organ to brain weight ratios were determined.

HISTOPATHOLOGY: Yes, tissue samples were fixed with 10% neutral buffered formalin, except eye, epididymis, Harderian gland, optic nerve and testis that were fixed in Davidson's fixative. Histological sections, stained with hematoxylin and eosin, were prepared for all organs, except exorbital gland, larynx/pharynx and nasal cavities, from all animals. Histopathological examinations were performed on all tissues from all animals in the control and high dose groups; liver, lung, kidney and thyroid gland were examined in all animals of the study. Thymus was examined in the female intermediate dose group as necessary to identify the no-observed-effect-level.

Statistics:

See Any other information on material and methods incl. tables.

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no treatment-related mortalities during the study. One female (NT3F1229) from the 2000 ppm treated group was found dead on Day 25 after an accidental trauma. No change was found for this animal at the macroscopic and histological examinations.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

7000 ppm:
- Mean body weight gain/day was statistically significantly reduced on Weeks 8 and 13 in males, when compared to control values, but these differences were attributed to the particularly high control values noted over these two intervals rather than to any treatment-related effect.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Mean Achieved Doses:
500 ppm - 79 and 95 mg/kg bw/day actual dose ingested for males and females, respectively.
2000 ppm - 321 and 398 mg/kg bw/day actual dose ingested for males and females, respectively.
7000 ppm - 1110 and 1297 mg/kg bw/day actual dose ingested for males and females, respectively.

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:

not examined

Description (incidence and severity):

Not applicable.

Haematological findings:

not examined

Description (incidence and severity):

Not applicable.

Clinical biochemistry findings:

no effects observed

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:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Description (incidence and severity):

Not applicable.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

7000 ppm: Lymphocytolysis in the thymus was greater in females than in controls. This change may have arisen as a direct effect of the test substance or it may have resulted from increased levels of circulating cortisol caused by indirect stress.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Key result

Dose descriptor:

NOEL

Effect level:

7 000 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No effects observed at this dose level.

Remarks on result:

other: corresponding to 1110 mg/kg bw/day actual dose ingested for males.

Key result

Dose descriptor:

NOEL

Effect level:

2 000 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No effects observed at this dose level.

Remarks on result:

other: corresponding to 398 mg/kg bw/day actual dose ingested for females.

Key result

Dose descriptor:

LOAEL

Effect level:

7 000 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: corresponding to 1297 mg/kg bw/day actual dose ingested for females.

Critical effects observed:

no

Lowest effective dose / conc.:

7 000 ppm

System:

haematopoietic

Organ:

thymus

Conclusions:

The study was performed in accordance to OECD TG 408 under GLP conditions and is considered reliable. Lymphocytolysis in the thymus of females given 7000 ppm was the only treatment related effect observed in the study. Therefore, in females the LOAEL and NOEL were 7000 ppm (1297 mg/kg bw/day) and 2000 ppm (398 mg/kg bw/day), respectively. In males the NOEL was 7000 ppm (1110 mg/kg bw/day).

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 Nov 2002 - 30 Jul 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted 21 Sep 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted 25 Jun 2018

Deviations:

yes

Remarks:

Rats were housed individually. Phytoestrogen levels were not determined in the diet. Levels of HDL/LDL were not measured. T4, T3 and TSH levels were not assessed. No vaginal smears taken for oestrus cycle assessment. No seminology performed.

GLP compliance:

yes

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. 7 weeks
- Weight at study initiation: Males 258-296 g, females 179-218 g
- Fasting period before study: Not applicable
- Housing: Individually
- Diet: Certified rodent powdered and irradiated diet A04C-10 PI from S.A.F.E. (Scientific Animal Food and Engineering, Route de Saint Bris, 89290 Augy, France), ad libitum. Except before blood and urine collections and prior to scheduled sacrifice, when animals were diet fasted overnight.
- Water: Filtered and softened water from the municipal water supply, ad libitum. Except during urine collection, when animals were water fasted.
- Acclimation period: Approximately 15 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: 06 Nov 2002 To: 21 Feb 2003

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
The test substance was ground to a fine powder before being incorporated into the diet by dry mixing.
- Rate of preparation of diet (frequency): there were two preparations for each concentration during the study.
- Mixing appropriate amounts with (Type of food): Certified rodent powdered and irradiated diet A04C-10 PI from S.A.F.E. (Scientific Animal Food and Engineering, Route de Saint Bris, 89290 Augy, France)
- 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:

Homogeneity and stability were checked in a pre-study at concentrations of 100 and 15000 ppm from surface, middle and bottom sample levels. The test substance was extracted from diet by simple homogenisation with acetonitrile. The dietary levels of the test substance were verified by HPLC-UV. All results for homogeneity and concentration were within the range of 96 - 102% of the nominal concentrations. Therefore, all values were within the target range of 85 - 115% of the nominal concentrations. The test substance was also found to be stable in the rodent diet at concentrations of 100 and 15 000 ppm after 104 days at ambient temperature and for periods of up to 70 days frozen storage following 10 days at ambient temperature.

Duration of treatment / exposure:

90 days

Frequency of treatment:

Continously via the diet.

Dose / conc.:

1 000 ppm

Remarks:

corresponding to 58 and 70 mg/kg bw/day actual dose ingested for males and females, respectively.

Dose / conc.:

4 000 ppm

Remarks:

corresponding to 240 and 281 mg/kg bw/day actual dose ingested for males and females, respectively.

Dose / conc.:

12 000 ppm

Remarks:

corresponding to 720 and 859 mg/kg bw/day actual dose ingested for males and females, respectively.

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dose levels were set after evaluation of the results of a subacute toxicity study with this substance.
- Fasting period before blood sampling for clinical biochemistry: Yes, diet fasted overnight prior to bleeding.

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were checked twice daily for moribundity and mortality throughout the study except on weekends and public holidays when they were checked once daily.
- Cage side observations included: Nature, onset, severity, reversibility and duration of clinical signs were recorded. Cages and cage-trays were inspected daily for evidence of ill-health such as blood or loose faeces.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed physical examinations were performed during the acclimatisation phase and at least weekly during the treatment and recovery period.

BODY WEIGHT: Yes
- Time schedule for examinations: Each animal was weighed three times during the acclimatisation period, on the first day of test substance administration, then at least weekly throughout the treatment and recovery periods.

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, the weight of food supplied and of that remaining at the end of the food consumption period was recorded weekly for all animals during the treatment period. From these records the mean daily consumption was calculated. Food spillage was also noted. The weekly mean achieved dosage intake in mg/kg/day for each week and for Weeks 1 to 13 was calculated for each sex.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: During the acclimatisation period and Week 13 of the treatment period.
- Dose groups that were examined: All animals during acclimatisation, and all surviving animals from control and high-dose group during Week 13. After instillation of an atropinic agent (Mydriaticum, Merck Sharp and Dohme) each eye was examined by means of an indirect ophthalmoscope.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: An approximately equal number of animals randomly distributed amongst all groups were sampled on Days 91, 92 or 93. Blood samples were taken from all surviving animals in all groups by puncture of the retro-orbital venous plexus prior to sacrifice. Blood (0.5 mL) was collected on EDTA for hematology.
- Anaesthetic used for blood collection: Yes (Isoflurane)
- Animals fasted: Yes, diet fasted overnight prior to bleeding.
- How many animals: All surviving animals
- Parameters examined: Red blood cell count, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, reticulocyte count, white blood cell count and differential count evaluation and platelet count (assayed using an Advia 120: Bayer Diagnostics, Puteaux, France); prothrombin time (assayed using ACL3000, Instrumentation Laboratory, Paris, France). A blood smear was prepared and stained with Wright stain. It was examined if the results of Advia 120 determinations were abnormal.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: An approximately equal number of animals randomly distributed amongst all groups were sampled on study Days 91, 92 or 93. Blood samples were taken from all surviving animals in all groups by puncture of the retro-orbital venous plexus prior to sacrifice. Blood was collected on lithium heparin for plasma (2.5 mL), on clot activator for serum (0.5 mL) and on sodium citrate for coagulation parameters (0.9 mL).
- Animals fasted: Yes, diet fasted overnight prior to bleeding.
- How many animals: All surviving animals
- Parameters examined: Total bilirubin, glucose, urea, creatinine, total cholesterol, triglycerides, chloride, sodium, potassium, calcium and inorganic phosphorus concentrations; aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and gamma-glutamyltransferase activities were assayed on plasma samples, total protein and albumin concentrations were assayed on serum samples using an Hitachi 911 (Roche Diagnostics, Meylan, France). Globulin concentrations and albumin/globulin ratio values were calculated. Any significant change in the general appearance of plasma and serum was recorded.

URINALYSIS: Yes
- Time schedule for collection of urine: Days 86 or 87 in the morning, overnight urine samples were collected.
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes, food and water were not accessible during urine collection.
- How many animals: All surviving animals
- Parameters examined: Any significant change in the general appearance of the urine was recorded. Quantitative parameters: urinary volume, pH, urinary refractive index; semi-quantitative parameters: glucose, bilirubin, ketone bodies, occult blood, protein, urobilinogen; microscopic examination of the sediment: presence of red blood cells, white blood cells, epithelial cells, bacteria, casts and crystals

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during the acclimatisation phase and during Week 12
- Dose groups that were examined: All animals
- Battery of functions tested: Grasping reflex (by pulling the animal across a textured surface), righting reflex (by putting the animal on its back), corneal reflex (following touching of the cornea with a fine nylon string), pupillary reflex (by covering the eyes of the animal for a few seconds and then observing pupillary reflex by focusing a light in the eyes), auditory startle reflex (by observation of the animal response to a clap of the hands), head shaking reflex (head shaking response of the animal due to blowing on the ear).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, on Days 91, 92 or 93, all surviving animals were sacrificed by exsanguination under deep anesthesia (pentobarbital, intraperitoneal injection of approximatley 60 mg/kg bw). An approximately equal number of animals randomly distributed amongst all groups were sampled on each day at the dosing phase and recovery sacrifice. Animals were diet fasted overnight prior to sacrifice. All animals, found dead or killed for humane reasons, were necropsied. The necropsy included the examination of all major organs, tissues and body cavities. Macroscopic abnormalities were recorded, sampled and examined microscopically. The following tissues were sampled: adrenal gland, aorta, articular surface (femorotibial), bone (sternum), bone marrow (sternum), brain, epididymis, esophagus, exorbital (lachrymal) gland, eye and optic nerve, harderian gland, heart, intestine (duodenum, jejunum, ileum, cecum, colon, rectum), kidney, larynx/pharynx, liver, lung, lymph nodes (submaxillary, mesenteric), mammary gland, nasal cavities, ovary, pancreas, pituitary gland, prostate gland, sciatic nerve, seminal vesicle, skeletal muscle, skin, spinal cord (cervical, thoracic, lumbar), spleen, stomach, submaxillary (salivary) gland, testis, thymus, thyroid gland (with parathyroid gland), tongue, trachea, urinary bladder, uterus (including cervix), vagina. Samples were fixed by immersion in neutral buffered 10% formalin with the exception of the eye, optic nerve, harderian gland, epididymis and testis that were fixed in Davidson's fixative.

ORGAN WEIGHT: Adrenal gland, brain, epididymis, heart, kidneys, liver, ovary, pituitary gland, prostate gland, spleen, testis, thymus, thyroid gland (with parathyroid gland) and uterus (including cervix) were weighed fresh at scheduled sacrifice only. Paired organs were weighed together.

HISTOPATHOLOGY: Yes, all of the above mentioned samples (except exorbital lachrymal gland, larynx/pharynx and nasal cavities) were embedded in paraffin wax. Histological sections, stained with hematoxylin and eosin, were prepared from all organs and tissue samples of the 90-day treatment phase. Histological sections were prepared from all the organs from all the animals in the control and high dose groups as well as from all decedent animals in intermediate dose group. Additionally, sections from the liver, lung, spleen, thymus, urinary bladder, thyroid gland and kidney and from significant gross pathology findings observed at necropsy were prepared for all the animals in all intermediate group. Significant macroscopic findings were also examined in all intermediate dose groups.

Statistics:

Mean and standard deviation were calculated for each group. The Bartlett test was performed to compare the homogeneity of group variances.
Body weight changes, terminal body weight, abosolute/relative organ weight, haematology:
- If Bartlett test not significant (α=0.05): means compared using ANOVA. If ANOVA not significant (α=0.05), group means considered homogeneous, no further analysis. If ANOVA significant (α=0.05): means of exposed groups compared to mean of control group using the Dunnett test (2-sided).
- If Bartlett test significant (α=0.05): group means compared using the non-parametric Kruskal-Wallis test. If Kruskal-Wallis test not significant (α=0.05): group means considered homogeneous, no further analysis performed. If Kruskal-Wallis test significant (α=0.05): means of exposed groups compared to the mean of control group using the Dunn test (2-sided) (only Kruskal-Wallis was performed for urine analysis (pH)).

Body weight, food consumption parameters: Log transformed if the Bartlett test was significant (α=0.05).
- If Bartlett test not significant (α=0.05) after log transformation: means compared using the ANOVA on log transformed data. If ANOVA on log transformed data not significant (α=0.05): group means considered homogeneous, no further analsis. If ANOVA on log transformed data significant (α=0.05): means of exposed groups compared to mean of control group using the Dunnett test (2-sided) on log transformed data.
- If Bartlett test significant (α=0.05) even after log transformation: group means compared using non-parametrical Kruskal-Wallis test. If Kruskal-Wallis test not significant (α=0.05): group means considered homogeneous, no further analysis performed. If Kruskal-Wallis test significant (α=0.05): means of exposed groups compared to the mean of control group using the Dunn test (2-sided).

If one or more group variance(s) equaled 0, means were compared using non-parametric procedures (group means compared at the 5 and 1% levels of sign)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

12000 ppm:
- Two females during Week 5 died. One female was found dead on Day 33, clinical signs observed prior to death (on Day 33 only) were soiled fur on the muzzle, neck and forelimbs. A second female (MT4F5257) was sacrificed for humane reasons on Day 36 of the study. Clinical signs observed for this animal prior to death (on Days 35 and 36) were soiled fur on the nose, forelimbs and anogenital region, wasted appearance and clonic convulsions. A body weight loss of 60 g was recorded for this animal between Weeks 4 and 5.
- Treatment-related clinical signs in animals surviving to terminal sacrifice consisted of soiled fur in the anogenital region observed on Day 15 and 71, respectively for males MT4M5244 and MT4M5245. In addition hair loss was observed on several occasions for males MT4M5241 and MT4M5245 and female MT4F5253.

Mortality:

mortality observed, treatment-related

Description (incidence):

12000 ppm:
- Two females died during Week 5 (see clinical signs field for details).

1000 ppm:
-One male animal in the 1000 ppm group (MT2M5200) was sacrificed for humane reasons on Day 71 of the study when it was observed that this animal had a misaligned jaw and abnormal dentition. This condition was considered to be due to an accidental trauma and not as a result of treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

12000 ppm:
Males:
- Absolute body weight in males was reduced by 5-7% between Weeks 5 and 13.
- Body weight gain was also reduced at a number of time intervals the effect being statistically significant (p<0.01) between Weeks 4 and 5 where there was a 64% reduction in body weight gain compared with the control group.

Females:
- The only effect on body weight was between Weeks 4 and 5. where there was a 6% reduction in body weight compared to the controls.
- In terms of body weight change, there was an actual loss of body weight of 0.7 g/day during this interval. This effect was mainly attributable to one female (MT4F5257) which lost 60 g between Weeks 4 and 5 and was sent for necropsy on Day 36.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

12000 ppm:
- Food consumption in males was reduced by 9% during Week 1 and by 13% during Week 5. the effect being statistically significant (p<0.01) during this last period.
- In females, food consumption was reduced by 17% during Week 5. the effect being statistically significant (p<0.01).
- At other intervals food consumption for both sexes was comparable or slightly higher than the control group.

Compound intake:
1000 ppm - corresponding to 58 and 70 mg/kg bw/day actual dose ingested for males and females, respectively.
4000 ppm - corresponding to 240 and 281 mg/kg bw/day actual dose ingested for males and females, respectively.
12000 ppm - corresponding to 720 and 859 mg/kg bw/day actual dose ingested for males and females, respectively.

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):

No toxicologically significant variation was noted. The statistically significantly lower mean calcium concentration seen in males at 4000 ppm was considered to be incidental.

Endocrine findings:

not examined

Description (incidence and severity):

Not applicable.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

12000 and 4000 ppm:
Sulfonamide-like crystals were observed in the urine from treated rats at 12000 ppm (8/10 males and 7/8 females) and at 4000 ppm (1/10 males and 6/10 females). In addition, in males at 12000 ppm, the number of usual urinary crystals observed was lower than in the control group.

Behaviour (functional findings):

no effects observed

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):

12000 ppm:
- In males, kidney to body weight ratio was increased by 12% (p<0.01). Absolute kidney weight and relative kidney/brain weight were also slightly increased by 4-5%. In female animals, absolute and relative kidney weights were increased by 11-13%, although none of these values were statistically significant.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Description (incidence and severity):

Not applicable.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Unscheduled Deaths

12000 ppm:
- The likely cause of death for female MT4F5254 was considered to be myocardial degeneration/necrosis. No clear cause of death was found for female except for lesions related to poor clinical condition. It was therefore unclear whether these deaths were directly related to treatment. However, both females presented a range of lesions with a number of similarities to those observed on target organs at terminal sacrifice.

1000 ppm:
- Male MT2M5200 developed an inflammatory lesion in the periodontal tissue which was unrelated to treatment and was induced by an accidental trauma.

Terminal sacrifice

12000 ppm:
- In the liver, slight centrilobular hepatocellular hypertrophy was noted in 8/10 males.
- In the urinary bladder, simple hyperplasia of the urothelium was noted in 2/10 males and 5/8 females.
- In the kidney, basophilic tubules and hyperplasia of collecting ducts and pelvic epithelium were seen in animals from all groups but the incidence and/or severity were slightly greater than controls in animals dosed at 12000 ppm. Marked metaplasia of the collecting duct epithelium was seen in 1/8 females. Marked metaplasia of the collecting duct epithelium was seen in 1/8 females. The findings are consistent with the presence of a component in the urine of treated animals leading to hyperplasia in distal collecting ducts without appreciable inflammation.
- In the spleen, brown pigment in the red pulp was seen in animals from all groups but the severity was marginally greater in a few animals dosed at 12000 ppm compared to controls. The effect was minor but may reflect a marginal increase in the turnover of red cells in affected animals.
- In the thymus. decreased size of the cortex (involution) was seen in animals from all groups but the severity was marginally greater in a few males dosed at 12000 ppm compared to the controls. In the absence of effects in other lymphoid tissues, this lesion is likely to represent a non-specific reaction to stress rather than an effect on the immune system.

4000 ppm:
- In the urinary bladder, simple hyperplasia of the urothelium was noted in 1/10 males and 1/10 females. This change is consistent with the presence of a component in the urine leading to hyperplasia in the absence of discernible inflammatory change.

Summary data of histopathological findings are presented in Table 1 (Any other information on results, incl. tables).

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

Not applicable.

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed at this dose level.

Remarks on result:

other: corresponding to 58 and 70 mg/kg bw/day actual dose ingested for males and females, respectively.

Key result

Dose descriptor:

LOAEL

Effect level:

4 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

urinalysis

Remarks on result:

other: corresponding to 240 and 281 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

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Critical effects observed:

yes

Lowest effective dose / conc.:

12 000 ppm

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Table 1: Histopathological findings at terminal sacrifice

Incidence of hyperplasia, urothelial, simple of urinary bladder
sex	male				female
group	control	1000	4000	12000	control	1000	4000	12000
number examined	10	9	10	10	10	8	10	8
slight	0	0	1	2	0	0	1	3
mild	0	0	0	0	0	0	0	1
moderate	0	0	0	0	0	0	0	1
total	0	0	1	2	0	0	1	5

 

Incidence of lesions in the kidney
sex	male				female
group	control	1000	4000	12000	control	1000	4000	12000
number examined	10	9	10	10	10	10	10	8
basophilic tubules
slight	3	6	4	1	1	0	2	2
mild	0	0	0	1	0	0	0	0
moderate	0	0	0	1	0	0	0	0
marked	0	0	0	0	0	0	0	1
total	3	6	4	3	1	0	2	3
hyperplasia, collecting ducts
slight	0	1	0	3	1	3	3	1
mild	0	0	0	0	0	0	0	2
moderate	0	0	0	1	0	0	0	0
marked	0	0	0	0	0	0	0	1
total	0	1	0	4	1	3	3	4
hyperplasia, pelvic epithelium, bilateral
slight	0	0	0	1	1	5	1	1
mild	0	0	0	0	1	0	0	1
moderate	0	1	0	0	0	0	0	2
total	0	1	0	1	2	5	1	4
hyperplasia, pelvic epithelium, unilateral
slight	2	0	1	5	4	3	2	2
Incidence of brown pigment in the red pulp of the spleen
slight	8	9	9	6	2	2	2	0
mild	0	0	1	4	8	8	8	6
moderate	0	0	0	0	0	0	0	2
total	8	9	10	10	10	10	10	8
Incidence of decreased size, cortex, (involution) of the thymus
number examined	10	9	9	10	10	10	10	8
slight	10	8	7	6	8	9	9	6
mild	0	1	1	3	0	0	0	1
moderate	0	0	0	1	0	0	0	0
total	10	9	8	10	8	9	9	7

Conclusions:

The study was performed in accordance to OECD TG 408 under GLP conditions and is considered reliable. In conclusion, the NOAEL in the Wistar rat when administered the test substance in the diet over a 90-day period was 1000 ppm (equivalent to 57.9 mg/kg/day for males and 70.1 mg/kg/day for females).

Reference 4

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 Apr - 11 Aug 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)

Version / remarks:

adopted 21 Sep 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Marshal Farms USA, Inc, North Rose, New York, USA
- Age at study initiation: 9 months
- Weight at study initiation: 7.5 - 10.3 kg (males), 6.2 - 8.3 (females)
- Fasting period before study: not applicable
- Housing: Individually housed in stainless steel runs
- Diet: Purina Certified Canine Diet Etts 5006-3; presented to the animals for 3-6 hours/day, beginning 11 days prior to study initiation and continuing throughout the study. Prior to this time, food was vailable for ad libitum consumption.
- Water: Tap water, ad libitum.
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY:
The feed was analysed for impurities by the supplier prior to the feed being received. Tap water and corn oil were periodically sampled and analysed for a variety of potential impurities (e.g., aflatoxins, chlorinated hydrocarbons, heavy metals, etc.). The contaminant concentration levels outlined in the "Certification Profile" for Purina Mills Certified Lab Chows was used as a standard of comparison for judging the acceptability of contaminant concentrations in the samples analysed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 29
- Humidity (%): 30 - 70
- Air changes (per hr): averaged 12.08
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 29 Apr 2004 To: 11 Aug 2004

Route of administration:

oral: feed

Vehicle:

other: corn oil and acteone

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet: Weekly
- Mixing appropriate amounts with: The test substance was mixed in the feed at the designated nominal concentrations.
- Storage temperature of food: Under freezer conditions, until presented to the animals. A sample of each batch of feed mixed was taken and retained in the freezer until the study was completed.

VEHICLE
- Amount of vehicle: Corn oil, at 1% by weight of the diet, along with acetone, was used as a vehicle to suspend the test substance prior to being mixed in the diet. The control diet was prepared the same as the treated diet, excluding only the test substance.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

High performance liquid chromatographic/mass spectrometer/mass spectrometer method was used to measure the test substance concentrations in dog ration for the study. The homogeneity of the test substance in the diet and the stability of the active ingredient of the test substance in feed, stored at room temperature for 7 days and freezer temperature for 35 days, was determined: the test substance was found to be homogenously distributed in the feed and was stable at both room temperature and freezer conditions. The concentration of the active ingredient in the feed was verified for study Weeks 1, 2, 3, 7, and 11 for all dose groups. The mean concentrations for the study were 97 to 98% of the nominal levels. %RSD (Relative standard deviation) values ranged from 3.65 to 5.98%.

Duration of treatment / exposure:

90 - 92 days

Frequency of treatment:

Continously via the diet.

Dose / conc.:

2 500 ppm

Remarks:

corresponding to 75 and 79 mg/kg bw/day actual dose ingested for males and females, respectively.

Dose / conc.:

7 500 ppm

Remarks:

corresponding to 221 mg/kg bw/day actual dose ingested for males and females.

Dose / conc.:

15 000 ppm

Remarks:

corresponding to 416 and 341 mg/kg bw/day actual dose ingested for males and females, respectively.

No. of animals per sex per dose:

4

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Selection of these dose levels was based on a 28 day feeding study in the Beagle dog (M-254188-01-1, 2005), which used doses of 3200, 10000, and 30000 ppm. In this previous study, there were microscopic lesions in the kidney of males and females in the 30000 ppm dose group and in males in the 10000 ppm dose group.

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily
- Cage side observations included: Clinical signs of toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At study initiation and weekly thereafter on all animals

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were measured weekly throughout the study. Body weights were also taken immediately prior to necropsy to allow for calculation of organ-to-body weight ratios.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes, individual food consumption measured daily throughout the study.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Following the acclimation period and prior to initiation of dosing.
- Dose groups that were examined: Ophthalmic examinations were conducted on all animals, and also on all animals sacrificed just prior to termination of the study.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Pre-exposure and during study Weeks 5, 9, and 12.
- Anaesthetic used for blood collection: No, via jugular venipuncture
- Animals fasted: Yes, animals were fasted overnight prior to the collection of blood.
- How many animals: All animals.
- Parameters examined: Haematocrit (HCT), Haemoglobin (HGB), Leukocyte count (WBC), Erythrocyte count (RBC), Platelet count, Blood clotting measurements (Thromboplastin time, Prothrombin time), Leukocyte differential count, Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin conc. (MCHC), Mean corpuscular volume (MCV), Reticulocyte count, Blood cell morphology, Red Blood Cell Distribution (RDW), Hemoglobin Distribution Width (HDW).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Pre-exposure and during study Weeks 5, 9, and 12.
- Animals fasted: Yes, animals were fasted overnight prior to the collection of blood.
- How many animals: All animals.
- Parameters examined: Electrolytes: Calcium (calc), Chloride (Cl), Phosphorus (Phos), Potassium (K), Sodium (Na); Enzymes: Alkaline Phosphatase (ALK), Creatine phosphokinase (CK), Lactic acid dehydrogenase (LD), Alanine aminotransferase (ALT/SGPT), Gamma Glutamyl transferase (GGT); Other: Albumin (ALB), Creatinine (Creat), Urea nitrogen (Urea-N), Total Cholesterol (Chol), Globulins (Glob), Glucose (gluc), Total bilirubin (T-Bili), Total protein (TP), Triglycerides (Trig), Uric Acid (Uric-A), Albumin/Globulin ratio (A/G)

URINALYSIS: Yes
- Time schedule for collection of urine: Pre-exposure and during study Weeks 5, 9, and 12
- Metabolism cages used for collection of urine: No data
- Animals fasted: No
- Parameters examined: Appearance, Volume (UVol), Specific gravity / osmolality (Sp.Gr.), pH, Sediment (microscopic), Protein (Pro), Glucose (Glu), Ketones (Ket), Bilirubin (Bil), Blood (Bld), Nitrite (Nit), Urobilinogen (Uro), Leukocytes (U-Leu)

Sacrifice and pathology:

Animals were euthanised at the end of the study by intravenous injection of Fatal-Plus (Vortech Pharamceuticals, Dearborn, Michigan, USA).

GROSS PATHOLOGY: Yes. A complete gross examination was performed on all animals, consisting of a systematic gross examination of each animal's general physical condition, body orifices, external and internal organs and tissues. Organs/tissues examined: Cecum, Colon, Duodenum, Esophagus, Gall bladder, Ileum, Jejunum, Liver, Pancreas, Rectum, Salivary glands, Stomach, Larynx, Lung, Nasopharynx, Nose, Pharynx, Trachea, Aorta, Bone marrow, Heart, Lymph node (mesenteric, retropharyngeal), Spleen, Thymus, Cervix, Epididymides, Fallopian tube (oviduct), Kidneys, Mammary gland, Ovaries, Prostate, Testes, Ureter, Urinary bladder, Uterus, Vagina, Adrenal gland, Thyroid (with parathyroid), Brain (multiple sections), Eyes, Nerve (optic, sciatic), Pituitary, Sciatic nerve, Spinal cord (cervical, thoracic, lumbar), Bone (rib/cc jct, sternum), Gross lesions, Muscle, Physical Identifier, Skin.

ORGAN WEIGHT: Liver, Lung, Heart, Spleen, Thymus, Epididymides, Kidneys, Ovaries, Prostate, Testes, Uterus, Adrenal gland, Thyroid, Brain, Pituitary.

HISTOPATHOLOGY: Yes, target organs (i.e., kidney, ureter, and urinary bladder) and gross lesions from all dose groups were examined histologically, while all other tissues listed above were examined histologically only from the control and high-dose groups. The physical identifier (i.e., ear tattoos) and vagina were not examined histologically. Tissues were processed routinely and stained with hematoxylin and eosin.

Statistics:

Statistical significance was determined at p≤0.05 for all tests with the exception of Bartlett's test, in which a probability value of p≤0.001 was used. All tests were two-tailed, except for gross and histopathologic lesion evaluations which were one-tailed. Continuous data were analyzed by Bartlett's test for homogeneity. If the data were homogeneous an ANOVA was performed, followed by Dunnett's t-test on parameters showing a significant effect by ANOVA. If the data were non-homogeneous a Kruskal-Wallis ANOVA was performed, followed by the Mann-Whitney U-test to identify statistical significance between groups. Frequency data, that were examined statistically, were initially analysed by a Chi-Square procedure. If there was statistical significance using the Chi-square test, each treatment group was compared to the control group using a Fisher's Exact test.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

15000 ppm:
- The only compound-related clinical findings was "thin", which was noted near the end of the study in females, and yellow powder in the urine of males.

Mortality:

mortality observed, treatment-related

Description (incidence):

15000 ppm:
- One early death, female VG3102, occurred on Day 35 of the study. This female was sacrificed-in-extremis due to clinical signs of impending death. This death was considered to be treatment-related due to the kidney calculi and abscessation.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

15000 ppm:
- The term body weights were statistically decreased only in the high-dose group females and considered to be compound-related (decreased 27% compared to controls). No effect on the body weight of the males.

See attachment 1 (attached background material) for tabulated body weight data.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

15000 ppm:
- A compound-related reduction in animals of both sexes were observed.

See Attachment 1 (attached background material) for tabulated food consumption data.
See Table 1 (Any other information on results incl. tables) 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, treatment-related

Description (incidence and severity):

15000 ppm:
- High WBC in early death female animal, which was thought to be due to kidney changes described microscopically (abscess).
- The mild anemia characterized by statistically significant decrease in erythrocyte counts, haemoglobin, and haematocrit in the high-dose group females at termination were thought to be compound-related (see Table 2: Any other information on results incl. tables).
- There were no compound-related changes in male dogs in any treatment group.
- There were some statistically significant differences in MCV, MCHC, RDW, and HDW, but these findings were not considered to be biologically significant.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

15000 ppm:
- Elevated urea nitrogen in high-dose group males and females at one month, two month, and termination (non-statistically significant at one and two months).
- Elevated serum creatinine in high-dose group males and females at one month, two month, and termination (non-statistically significant at one and two months, but statistically significant in females at termination).
- Elevated serum phosphorus in high-dose group females at termination.

See Attachment 2 (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):

15000 ppm:
- Elevated urine volume (non-statistical) at one-month, two-month and term intervals in male high-dose group dogs.
- Decrease in urine specific gravity in high-dose group male and female dogs.
- Decreased pH in the high-dose group females at one- and two-month intervals.

7500 ppm:
- Decrease in urine specific gravity in the mid-dose group females at termination.

See Attachment 3 (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):

15000 ppm:
- The term body weights were statistically decreased only in the high-dose group females and considered to be compound-related (decreased 27% compared to controls).
- The absolute brain and heart weights were statistically decreased in the high-dose group females but the relative organ weights were not affected, so this was thought to be due to the decreased body weights and therefore not a specific compound-related effect.

See Attachment 4 (attached background material) for tabulated terminal body weights and absolute and relative organ weights.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

15000 ppm:
- Calculi in the renal pelvis in three males and four females.
- The renal pelvis was dilated in one high-dose group dog of each sex.
- Calculi in the ureter and urinary bladder of one male.
- Dilated ureters in one female.

See Attachment 5 (attached background material) for tabulated gross pathology of the urinary findings.

Neuropathological findings:

not examined

Description (incidence and severity):

Not applicable.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

15000 ppm:
- The only microscopic observations that were thought to be compound-related were those noted in the urinary system of both male and female high-dose group dogs. The inflammatory changes, renal pelvis epithelial hyperplasia and necrosis and associated changes in the ureters and urinary bladder of high-dose group males and females correlated with the gross observations at necropsy of calculi in the kidneys, ureters and urinary bladder.

See Attachment 6 (attached background material) for tabulated histopathology data.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Key result

Dose descriptor:

NOAEL

Effect level:

7 500 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 221 mg/kg bw/day in males and females.

Key result

Dose descriptor:

LOAEL

Effect level:

15 000 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

urinalysis

Remarks on result:

other: corresponding to 416 mg/kg bw/day actual dose ingested for males.

Key result

Dose descriptor:

LOAEL

Effect level:

15 000 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

gross pathology

haematology

histopathology: non-neoplastic

mortality

urinalysis

Remarks on result:

other: corresponding to 341 mg/kg bw/day actual dose ingested for females.

Critical effects observed:

yes

Lowest effective dose / conc.:

15 000 ppm

System:

urinary

Organ:

bladder

kidney

ureter

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Table 1: Compound intake in mg/kg bw/day

Dose Group (ppm)	Males (mg/kg bw/day)	Females (mg/kg bw/day)
2500	75	79
7500	221	221
15000	416	341

Table 2: Erythrocyte count, haemoglobin and haematocrit in high dose females at study termination

Dose Group	Erythrocyte Count (10E6 / mm3)	Haemoglobin (g/dL)	Haematocrit (%)
Control	7.05	16.9	48.1
15000 ppm	5.18	12.5	38.0

Conclusions:

The study was performed in accordance to OECD TG 409 under GLP conditions and is considered reliable. In conclusion, the NOAEL in the Beagle dog was 7500 ppm (221 mg/kg for males and females) as there were no microscopic pathology observations to correlate with the decrease in urine specific gravity in the 7500 ppm females.

Reference 5

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).

Reference 6

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Jan 2005 - 25 Jan 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

Version / remarks:

adopted 12 May 1981

Deviations:

yes

Remarks:

Housing temperature range (18 - 29 °C) outside of the guideline range (22 °C ± 3 °C).

Qualifier:

according to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

Version / remarks:

adopted 25 Jun 2018

Deviations:

yes

Remarks:

Housing temperature range (18 - 29 °C) outside of the guideline range (22 °C ± 3 °C). No histopathological examination of vagina.

GLP compliance:

yes

Limit test:

no

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Marshall BioResources, Inc, North Rose, New York, USA.
- Age at study initiation: 5-6 months.
- Weight at study initiation: 7.1-9.2 kg (males), 4.8-7.2 (females).
- Fasting period before study: Not applicable.
- Housing: Individually housed in stainless steel runs.
- Diet: Purina Certified Canine Diet Etts 5006-3; presented to the animals for 3-6 hours/day, beginning 5 days prior to study initiation and continuing throughout the study. Prior to this time, food was available for ad libitum consumption except when the animals were fasted overnight for pre-clinical blood analyses.
- Water: Tap water, ab libitum.
- Acclimation period: 7 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-29
- Humidity (%): 30-70
- Air changes (per hr): averaged at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 13 Jan 2005 To: 25 Jan 2006

Route of administration:

oral: feed

Vehicle:

other: corn oil and acetone

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- The test substance was a suspension of acetone and corn oil (1% of the total batch by weight). The solution was slowly applied to the food ration with the mixer stirring at concentrations of 100 and 15000 ppm. The flask was rinsed with acetone and the rinse added to the ration. Total mixing time was ten minutes.

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): The test substance was mixed in the feed at the designated nominal concentrations.
- Storage temperature of food: Under freezer conditions, until presented to the animals A sample of each batch of feed mixed was taken and retained in the freezer until the study was completed.

VEHICLE
- Amount of vehicle: Corn oil, at 1% by weight of the diet, along with acetone, was used as a vehicle to suspend the test substance prior to being mixed in the diet. The control diet was prepared the same as the treated diet, excluding only the test substance.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The homogeneity of the test substance in the diet and the stability of the active ingredient of the test substance in feed, stored at room temperature and freezer temperature was determined by liquid chromatographic/mass spectrometer/mass spectrometer method. Therefore, samples were taken from the freshly prepared mixtures (from top, middle and bottom layer).
The test substance was found to be homogeneously distributed in the diet and was stable in the feed stored at room temperature for 7 days and freezer temperature for 35 days. The concentration of the active ingredient in the feed was verified for the first three study weeks of administration and then every 4 or 5 weeks until the end of the study. The mean concentrations for the study were 89 - 101% of the nominal levels. %RSD (Relative standard deviation) values ranged from 2.22 to 3.52%.

Duration of treatment / exposure:

370 - 372 days

Frequency of treatment:

Continously via the diet.

Dose / conc.:

1 000 ppm

Remarks:

corresponding to 29 and 28 mg/kg bw/day actual dose ingested for males and females, respectively.

Dose / conc.:

2 500 ppm

Remarks:

corresponding to 66 and 67 mg/kg bw/day actual dose ingested for males and females, respectively.

Dose / conc.:

8 000 ppm

Remarks:

corresponding to 226 and 242 mg/kg bw/day actual dose ingested for males and females, respectively.

No. of animals per sex per dose:

4

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Selection of these dose levels was based on a 28-day and a 90-day feeding study in the Beagle dog (M-254188-01-1 & M-263605-01-1). The 28-day study used doses of 3200, 10000, and 30000 ppm and the following compound-related findings were observed: 1. There was a compound-related increase in urine output in the high-dose group males and females. 2. There was a slight compound-related increase in kidney weight for the high-dose group males. 3. Gross pathology revealed compound-related renal calculi in the high-dose group males and females. 4. Compound-related microscopic lesions were observed in the kidney of mid- and high-dose group males and in high-dose group females. The calculi observed grossly in some kidneys were considered to have led to these microscopic changes. The 90-day study used doses of 2500, 7500, and 15000 ppm in the diet. In this study, one high-dose group female was sacrificed moribund, with kidney lesions observed grossly and microscopically. The following compound-related findings were observed: 1. "body thin" was noted near the end of the study in females, and yellow powder in the urine of males. 2. Decreased body weight for the females. 3. A reduction in food consumption for males and females. 4. Clinical chemistry: Elevated urea nitrogen in males and females, elevated serum creatinine in males and females, and elevated serum phosphorus in females. 5. Urinalysis: Elevated urine volume in males, a decrease in urine specific gravity in males and females and decreased urine pH in females. 6. Gross necropsy: Calculi in the renal pelvis of males and females, renal pelvis dilatation in males and females, calculi in the ureter and urinary bladder of males and dilated ureters in females. 7. Microscopic: Inflammatory changes in the urinary tract, renal pelvis epithelial hyperplasia and necrosis and associated changes in the ureters and urinary bladder of males and females which correlated with the gross observations at necropsy of calculi in the kidneys, ureters, and urinary bladder.

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily, except once daily on weekends and holidays.
- Cage side observations included: Clinical signs of toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At study initiation and weekly thereafter on all animals.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were measured weekly throughout the study. Body weights were also taken immediately prior to necropsy to allow for calculation of organ-to-body weight ratios.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes, individual food consumption measured daily throughout the study, with a few exceptions: when dogs were found together, the feed was discarded prior to obtaining the post food consumption weight, fresh feed was added to a food bowl prior obtaining the post food consumption weight, and food was spilled prior to obtaining the post food consumption weight.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Following the acclimation period and prior to initiation of dosing.
- Dose groups that were examined: Ophthalmic examinations were conducted on all animals, and also on all animals sacrificed just prior to termination of the study.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Pre-exposure and during study Weeks 12, 26, 39 and 52.
- Anaesthetic used for blood collection: No, via jugular venipuncture
- Animals fasted: Yes, animals were fasted overnight prior to the collection of blood.
- How many animals: All animals.
- Parameters examined: Haematocrit (HCT), Haemoglobin (HGB), Leukocyte count (WBC), Erythrocyte count (RBC), Platelet count, Blood clotting measurements (Thromboplastin time, Prothrombin time), Leukocyte differential count, Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin conc. (MCHC), Mean corpuscular volume (MCV), Reticulocyte count, Blood cell morphology, Red Blood Cell Distribution Width (RDW), Haemoglobin Distribution Width (HDW)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Pre-exposure and during study Weeks 12, 26, 39 and 52.
- Animals fasted: Yes, animals were fasted overnight prior to the collection of blood.
- How many animals: All animals.
- Parameters examined: Electrolytes: Calcium (calc), Chloride (Cl), Phosphorus (Phos), Potassium (K), Sodium (Na); Enzymes: Alkaline Phosphatase (ALK), Creatine phosphokinase (CK), Lactic acid dehydrogenase (LD), Alanine aminotransferase (ALT/SGPT), Aspartate aminotransferase (AST/SGOT), Gamma Glutamyl transferase (GGT); Other: Albumin (ALB), Creatine (Creat), Urea nitrogen (Urea-N), Total Cholesterol (Chol), Globulins (Glob), Glucose (gluc), Total bilirubin (T-Bili), Total protein (TP), Triglycerides (Trig), Uric Acid (Uric-A), Albumin/Globulin ratio (A/G)

URINALYSIS: Yes
- Time schedule for collection of urine: Pre-exposure and during study Weeks 13, 26, 39 and 53
- Metabolism cages used for collection of urine: No data
- Animals fasted: No
- Parameters examined: Appearance, Volume (UVol), Specific gravity / osmolality (Sp.Gr.), pH, Sediment (microscopic), Protein (Pro), Glucose (Glu), Ketones (Ket), Bilirubin (Bil), Blood (Bld), Nitrite (Nit), Urobilinogen (Uro), Leukocytes (U-Leu).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. Animals were euthanised at the end of the study by intravenous injection of Fatal-Plus (Vortech Pharamceuticals, Dearborn, Michigan, USA). A complete gross examination was performed on all animals, consisting of a systematic gross examination of each animal's general physical condition, body orifices, external and internal organs and tissues. Organs/tissues examined: Cecum, Colon, Duodenum, Esophagus, Gall bladder, Ileum, Jejunum, Liver, Pancreas, Rectum, Salivary glands, Stomach, Larynx, Lung, Nasopharynx, Trachea, Aorta, Bone marrow, Heart, Lymph node (mesenteric, retropharyngeal), Spleen, Thymus, Cervix, Epididymides, Fallopian tube (oviduct), Kidneys, Mammary gland, Ovary, Prostate, Testicle, Ureter, Urinary bladder, Uterus, Vagina, Adrenal gland, Thyroid (with parathyroid), Brain (Cerebellum, Cerebrum-Midbrain, Medulla/Pons), Eyes, Nerve (optic, sciatic), Pituitary, Spinal cord (cervical, thoracic, lumbar), Bone (rib/cc jct, sternum), Gross lesions, Muscle, Physical Identifier, Skin.

ORGAN WEIGHTS: Liver, Lung, Heart, Spleen, Thymus, Epididymides, Kidneys, Ovaries, Testicle, Uterus, Adrenal gland, Thyroids, Brain, Pituitary.

HISTOPATHOLOGY: Yes, all tissues were processed, embedded in paraffin, sectioned, mounted, and stained with hematoxylin and eosin for examination under the light microscope. Histopathologic evaluation was conducted on all protocol-required tissues from the control and high-dose group animals and from the kidneys and urinary bladder of low- and mid-dose group males.

Statistics:

Statistical significance was determined at p≤0.05 for all tests with the exception of Bartlett's test, in which a probability value of p≤0.001 was used. All tests were two-tailed, except for gross and histopathological lesion evaluations which were one-tailed. Continuous data were analyzed by Bartlett's test for homogeneity. If the data were homogeneous an ANOVA was performed, followed by Student's t-test on parameters showing a significant effect by ANOVA. If the data were non-homogeneous a Kruskal-Wallis ANOVA was performed, followed by the Mann-Whitney U-test to identify statistical significance between groups. Frequency data, that were examined statistically, were initially analyzed by a Chi-Square procedure. If there was statistical significance using the Chi-square test, each treatment group was compared to the control group using a Fisher's Exact test.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

8000 ppm:
- Yellow granular material in the urine on Days 175 and 179 in one male animal

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Compound Intake:
1000 ppm - 29 and 28 mg/kg bw/day actual dose ingested for males and females, respectively.
2500 ppm - 66 and 67 mg/kg bw/day actual dose ingested for males and females, respectively.
8000 ppm - 226 and 242 mg/kg bw/day actual dose ingested for males and females, respectively.

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):

There were no compound-related haematological findings.

8000 and 2500 ppm:
For males, there were statistically significant decreases in RBC, haemoglobin, and haematocrit in the mid- and high-dose groups at 6, 9, and 12 months (see Table 1 Any other information on results incl .tables). These statistically significant haematology parameters were not considered to be compound-related for the following reasons:
(1) the values were comparable to the pre-treatment time interval;
(2) the lower values for HCT were only present at the 3 month interval;
(3) the lower values were not dose-dependent;
(4) the values were well within historical control data (see Attachment 1, Attached background material). Furthermore, the lower values were not considered to be biologically significant.

Other statistically significant findings were considered to be incidental due to the following reasons:
(1) there was no evidence of a dose-relationship;
(2) the statistically significant values were not biologically significantly different from the control group.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no compound-related clinical chemistry findings.

8000 ppm:
There was a non-statistically significant increase in ALP at the 6 and 9 month time points for males that was due to one animal (i.e., YM3003). This finding was considered not to be compound-related as:
(1) it only occurred in one animal in the high-dose group; and
(2) a similar increase in ALP was also observed in a low-dose group male (i.e., YM1004).

All other statistically significant findings were considered to be incidental due to the following reasons:
(1) there was no evidence of a dose relationship; and
(2) the statistically significant values were not biologically significantly different from the control group.

Endocrine findings:

not examined

Description (incidence and severity):

Not applicable.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no compound-related urinalysis findings.

Statistically significant findings were considered to be incidental due to the following reasons:
(1) there was no evidence of a dose relationship; and
(2) the statistically significant values were not biologically significantly different from the control group.

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, non-treatment-related

Description (incidence and severity):

Organ weights were not affected by compound-administration.

2500 and 1000 ppm:
The only statistically significantly change in organ weight was a decrease in the relative lung weight for low- and mid-dose group females. This change was not dose-dependent and not considered to be compound-related.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

8000 ppm:
The only compound-related gross pathology findings occurred in two high-dose group males:
(1) multiple raised black zones in the mucosa of the urinary bladder (YM3003 and YM3004);
(2) multiple yellow pinpoint calculi in the urinary bladder (YM3004); and
(3) urinary bladder - thick, abnormal consistency (YM30004).

All other gross findings were considered to be incidental.

Neuropathological findings:

not examined

Description (incidence and severity):

Not applicable.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

8000 ppm:
Urinary bladder
- Stone(s) were observed at the microscopic level in one male.
- In two males of this group, slight to moderate chronic mucosal hemorrhage and minimal to slight urothelial hyperplasia were noted.
- Stone(s) were also observed at the microscopic level in one female.
- Minimal suburothelial mononuclear cell infiltrate was observed in one male and one female.

Kidney
- One female had some microscopic intrapelvic stones. Associated with these stones, there were other minimal changes: pelvic epithelium hyperplasia, pelvic epithelial erosion and suburothelial mononuclear cell infiltrate.

See Table 2 (Any other information on results incl. tables) for histopathologic findings.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Key result

Dose descriptor:

NOAEL

Effect level:

2 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed at this dose level.

Remarks on result:

other: corresponding to 66 and 67 mg/kg bw/day actual dose ingested for males and females, respectively.

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

Remarks on result:

other: corresponding to 226 and 242 mg/kg bw/day actual dose ingested for males and females, respectively.

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

Table 1: Haematology Data for Males (control / mid- / high-dose group)

Study Day	RBC	HGB	HCT
-4	6.83 / 6.33 / 6.63	16.1 / 14.8 / 15.1	45.3 / 42.5 / 44.0
176 (6-month)	7.53 / 7.02* / 6.82*	17.5 / 16.7 / 16.1	48.7 / 46.7* / 44.6*
267 (9-month)	7.88 / 7.14* / 6.98	18.3 / 16.8* / 16.3*	53.5 / 49.5 / 48.1
363 (12-month)	7.69 / 7.23* / 7.12*	18.5 / 17.6* / 17.1*	50.6 / 48.6 / 47.4

Table 2: Histopathologic findings (urinary bladder and kidney)

Incidence and severity of microscopic changes in the urinary bladder, all animals, terminal sacrifice
Sex	Males				Females
Dose level (ppm)	0	1000	2500	8000	0	1000	2500	8000
Number of examined animals	4	4	4	4	4	4	4	4
Chronic mucosal hemorrhage
Minimal	0	0	0	0	0	0	0	0
Slight	0	0	0	1	0	0	0	0
Moderate	0	0	0	1	0	0	0	0
Total	0	0	0	2	0	0	0	0
Stone(s): focal/multifocal
Minimal	0	0	0	1	0	0	0	1
Total	0	0	0	1	0	0	0	1
Urothelial hyperplasia: simple: focal/multifocal
Minimal	0	0	0	1	0	0	0	0
Slight	0	0	0	1	0	0	0	0
Total	0	0	0	2	0	0	0	0
Suburothelial mononuclear cell infiltrate: focal/multifocal
Minimal	0	0	0	1	0	0	0	1
Total	0	0	0	1	0	0	0	1
Incidence and severity of microscopic changes in the kidney, all animals, terminal sacrifice
Intrapelvic stone(s)
Minimal	0	0	0	0	0	0	0	1
Total	0	0	0	0	0	0	0	1
Pelvic epithelium hyperplasia : focal/multifocal
Minimal	0	0	0	0	0	0	0	1
Total	0	0	0	0	0	0	0	1
Pelvic epithelial erosion
Minimal	0	0	0	0	0	0	0	1
Total	0	0	0	0	0	0	0	1
Suburothelial mononuclear cell infiltrate: focal/multifocal
Minimal	0	0	0	0	0	0	0	1
Total	0	0	0	0	0	0	0	1

Conclusions:

The study was performed in accordance to OECD TG 452 under GLP conditions and is considered reliable. In conclusion, the NOEL in the Beagle dog was 2500 ppm (66 and 67 mg/kg/day for males and females, respectively).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

39 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

The available information comprises adequate, reliable (Klimisch score 1) and consistent studies, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-X, 8.6, of Regulation (EC) No 1907/2006. The study with the lowest dose descriptor was selected for endpoint conclusion.

System:

urinary

Organ:

bladder

kidney

ureter

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

Please refer to the information provided in the carcinogenicity endpoint summary.

Additional information

Sub-acute

A sub-acute study is available addressing repeated dose toxicity via the oral route in the dog (M-254188-01-1). The purpose of this study was to determine the toxicity profile and palatability of the test substance for dose selection in the subsequent sub-chronic study. The study was performed under GLP conditions and in general accordance to OECD TG 409. The key differences were that the study duration was 28 days and only 2 animals/sex/dose were used. The study is considered a supporting study, due to deviations from the test guideline and because both sub-chronic and chronic studies are available in the dog.

Groups of 2 male and 2 female beagle dogs were treated at dose levels of 0, 3200, 10000, and 30000 ppm for 28 - 29 days, corresponding to 92, 274, and 880 mg/kg bw/day for males and 93, 314 and 677 mg/kg bw/day for females.

At 30000 ppm, there was a slight reduction in body weight in males and females. Urinary volume and blood urea nitrogen levels were increased in box sexes. At gross necropsy, calculi were observed in the kidneys of some males and females. Histopathology findings were observed in the kidneys of both sexes and were consistent with types of lesions one would anticipate with a physical insult in the region. These lesions included ulceration of the epithelium covering the papilla or pelvis, inflammation in the papilla or pelvis, hyperplasia of the epithelium covering the papilla or pelvis, and haemorrhage in the papilla or pelvis in males. Chronic interstitial inflammation in the kidney with or without haemorrhage was also observed in males. Similarly, in the kidneys of females, ulceration of the epithelium covering the papilla or pelvis, inflammation in the papilla or pelvis, and hyperplasia of the epithelium covering the papilla or pelvis were observed. At 10000 ppm, findings were limited to histopathology of the kidneys of males. Ulceration of the epithelium covering the papilla or pelvis, inflammation in the papilla or pelvis, hyperplasia of the epithelium covering the papilla or pelvis, and haemorrhage in the papilla or pelvis were observed in the kidneys. In addition, chronic interstitial inflammation in the kidney with or without haemorrhage was observed. No treatment-related effects were observed in females. At 3200 ppm, no treatment-related effects were observed in either males or females. In conclusion, the NOEL over a 28-day period of dietary administration to Beagle dogs was 3200 ppm (equivalent to 92 mg/kg bw/day) in males and 10000 ppm (equivalent to 314 mg/kg bw/day) in females.

Sub-chronic

There are four sub-chronic studies available addressing repeated dose toxicity via the oral route:

Rat

A 90-day repeated dose toxicity study (M-105691-01-1) was performed in Wistar rats. The study was performed under GLP condition and in accordance with OECD TG 408 (adopted 1998). Deviations to the current version of the guideline (adopted 2018) are considered minor and not to be expected to have an impact on reliability of this study.

The rats (10/sex/group) received the test substance in their feed for a period of 90 days. The following doses were administered: 1000, 4000 and 12000 ppm of the test substance, corresponding to 58, 240, and 720 mg/kg bw/day for males and 70, 281 and 859 mg/kg bw/day for females.

At 12000 ppm, the continuous administration of the test substance induced mortalities in two females during Week 5. Body weight gain decrease was most pronounced between Weeks 4 and 5 in both sexes with reduced food consumption. Urinalysis revealed sulfonamide-like crystals, radially striated spherical structures, in the urine of both sexes. Higher absolute and relative kidney weights were observed at terminal sacrifice in both sexes. Slight centrilobular hepatocyte hypertrophy and thymus involution were observed in males. Hyperplasia of the urothelium in the urinary bladder, of collecting ducts and pelvis epithelium in the kidneys and increased incidence and/or severity of basophilic tubules in the kidneys were observed in both sexes. In the spleen, brown pigment in the red pulp was seen in males and females. As sulfonamide-like crystals and hyperplasia of the urothelium in the urinary bladder were also seen in both sexes at 4000 ppm, this dose level was chosen as LOAEL, corresponding to 240 and 281 mg/kg bw/day for males and females, respectively. No adverse effects were observed at the dose level of 1000 ppm in either sex, therefore, this dose level, corresponding to 58 mg/kg bw/day for males and to 70 mg/kg bw/day for females, was determined to be the NOEL.

Mouse

A 90-day repeated dose toxicity study (M-104791-01-1) was performed in C57BL/6J Mice. The study was performed under GLP condition and in accordance with OECD TG 408 (adopted 1998). Deviations to the current version of the guideline (adopted 2018) are considered minor and not to be expected to have an impact on reliability of this study.

Ten mice/sex were fed a diet containing 500, 2000 and 7000 ppm of the test substance in their feed for a period of 91 to 93 days. The doses corresponded to 79, 321 and 1110 mg/kg bw/day for males and 95, 398 and 1297 mg/kg bw/day for females.

In the study, no effects were observed at the dose levels of 500 and 2000 ppm in either sex. Lymphocytolysis in the thymus of females given 7000 ppm was observed. No effects were observed in males at this dose level. Therefore, the dose of 7000 ppm (1297 mg/kg bw/day) was considered the LOAEL for females. The NOEL was 2000 ppm (398 mg/kg bw/day) for females and 7000 ppm (1110 mg/kg bw/day) for males.

Dog

A 90-day repeated dose toxicity study (M-263605-01-1) was performed in Beagle dogs. The study was performed under GLP condition and in accordance with OECD TG 409 (adopted 1998).

Four dogs/sex were fed a diet containing 2500, 7500, and 15000 ppm of the test substance (corresponding to 75, 221, and 416 mg/kg bw/day for males and 79, 221, 341 mg/kg bw /day for females) over a period of 90 to 92 days.

One female animal of the high-dose group was sacrificed in-extremis due to clinical signs of impending deaths. The death was considered to be compound-related due to kidney calculi and abscessation. Body weight loss was observed in females of the high-dose group and reduced food consumption in both sexes. Treatment-related urinalysis findings were elevated urine volume in males, a decrease in urine specific gravity in males and females and a decreased pH in females. At histopathology, calculi in the renal pelvis, ureter and/or urinary bladder were seen in most animals of the high-dose group. Correlated microscopic changes of the urinary tract were inflammatory changes, renal pelvis epithelium hyperplasia and necrosis as well as associated changes in the ureters and urinary bladder of males and females. At 7500 ppm, the only treatment-related effect was a decrease in urine specific gravity in females. As there were no microscopic pathology observations to correlate, the NOAEL was considered to be 7500 ppm (corresponding to 221 mg/kg bw/day for both sexes) and the LOAEL was 15000 ppm, corresponding to 416 and 341 mg/kg bw/day for males and females, respectively.

A 12-month toxicity study (M-283194-02-1) was performed in Beagle dogs. The study was performed under GLP condition and in accordance with OECD TG 452 (adopted 1981). Deviations to the current version of the guideline (adopted 2018) are considered minor and not to be expected to have an impact on reliability of this study.

Four dogs/sex received doses of 1000, 2500 and 8000 ppm in their feed for a period of 370 to 372 days, corresponding to 29, 66 and 226 mg/kg bw/day for males and 28, 67, and 242 mg/kg bw/day for females.

At the high-dose level of 8000 ppm, macroscopic and microscopic findings in the kidney and the urinary bladder were observed in both sexes. Like in the previous 90-day dog and the 90-day rat studies, the effects (haemorrhages, inflammation and hyperplasia) were consistent with the formation of stones in the urinary tract. Therefore, 8000 ppm, corresponding to 226 and 242 mg/kg bw/day for males and females, respectively, was determined as the LOAEL. No treatment-related effects were observed up to 2500 ppm, therefore, this dose level was determined to be the NOEL, corresponding to 66 and 67 mg/kg bw/day for males and females, respectively.

Chronic

There is one chronic toxicity study available addressing repeated dose toxicity via the oral route:

Rat

A combined chronic toxicity and carcinogenicity study (M-281767-01-1) was performed in Wistar rats. The study was performed under GLP condition and in accordance with OECD TG 453 (adopted 1981). Deviations to the current version of the guideline (adopted 2018) are considered minor and not to be expected to have an impact on reliability of this study.

Sixty rats/sex were fed a diet containing 1000, 4000 and 8000 ppm, corresponding to 39, 159 and 321 mg/kg/day in males and 56, 220 and 447 mg/kg/day in females, respectively, over a 24-month period. Additionally, groups of 10 rats/sex were treated at 200, 1000, 4000 and 8000 ppm, corresponding to 9, 45, 181 and 364 mg/kg bw/day in males and 13, 62, 249 and 491 mg/kg bw/day in females, over a 12-month period to investigate chronic toxicity only.

There were no treatment-related effects on food consumption, ophthalmological examination, neurotoxicity assessment, haematology and clinical chemistry evaluation at any dose level in either sex observed. The mortality rate was higher in females at 8000 ppm after 24 months of treatment and was largely due to secondary treatment-related nephropathy following product administration. Mean cumulative body weight gain was reduced during the first week of treatment by 7 and 12% in males and females treated at 8000 ppm, respectively, compared to the controls. Urinalysis revealed the presence of sulfonamide-like crystals throughout the study in both sexes, the effect being more pronounced in females than in males. At the 12-month interim sacrifice, treatment-related non-neoplastic findings were seen microscopically in the kidney and the urinary bladder.

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. A slightly higher incidence of the commonly occurring lesion diffuse bilateral tubular degeneration of the testis and bilateral oligospermia of the epididymis was observed at the carcinogenicity phase.

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.

In conclusion, dietary administration over a 24-month period to the Wistar rat, at dose levels up to 8000 ppm (corresponding to 321 mg/kg bw/day in males and 447 mg/kg bw/day in females) resulted in both non-neoplastic and neoplastic effects in the urinary tract. These effects were considered to be secondary to the presence of stones in the urinary organs. The NOAEL for systemic toxicity over a 12‑month period of dietary administration was 4000 ppm in both sexes (equivalent to 181 mg/kg bw/day in males and 249 mg/kg bw/day in females). The NOAEL for systemic toxicity over a 24‑month period was 1000 ppm in both sexes (equivalent to 39 mg/kg bw/day in males and 56 mg/kg bw/day in females).

Conclusions

The repeated dose toxicity studies with the registered substance were conducted in accordance to OECD testing guidelines and were fully compliant with GLP. Shorter term studies were performed in rats, mice, and dogs, and showed similar toxicity involving the lower urinary tract at high doses. At the highest doses in these three species, there was crystalluria typical of sulfonamides and calculus formation leading to cytotoxicity (necrosis), inflammation and regenerative hyperplasia. Acute haemorrhage occasionally was produced. Similar toxicity was also reported in the chronic/long-term studies, with neoplastic changes consisting of tumour formation in the urinary tract also observed at a low incidence at the highest dose levels tested in rats and mice. The major target organs in rats, mice and dogs were identified as the bladder, the ureter and the kidney. Analysis of the urinary calculi revealed that they were composed predominantly of the test substance, clearly indicating that the urinary solids are inducing the toxic effects, rather than the substance being directly toxic itself. The most sensitive study was the combined chronic toxicity and carcinogenicity study in rat, in which the NOAEL was 39 and 56 mg/kg bw/day in males and females, respectively. The NOAELs from this study were used as the starting point for the calculation of the derived no effects levels (DNELs) in the chemical safety assessment.

These effects in the urinary tract are not considered to be relevant to humans, especially the carcinogenic effects that were observed in long-term studies in rodents. This is due to anatomical differences between the urinary tract of humans and rodents. In humans, obstructions usually occur at the narrowing of the renal pelvic-ureteral junction, at the site where the ureter crosses the bony pelvic brim or at the bladder-urethral junction. In contrast, rats and mice can retain calculi in the bladder for long periods of time, primarily because the animal is horizontal and calculi can accumulate within the dome without producing obstruction. If obstruction does occur, it is most frequently partial and therefore not lethal to the animal. In humans, calculi are rarely present for prolonged periods of time, as obstructions lead to severe pain and require immediate removal. Rodents can retain calculi for prolonged periods of time, thus, inducing cytotoxicity, inflammation, and regenerative hyperplasia of the urothelial tissues, which can eventually lead to the formation of tumours. Please refer to the carcinogenicity endpoint summary for a more detailed discussion on the relevance to man of the urinary tract findings, especially in relation to formation of urinary tract tumours.

Justification for classification or non-classification

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