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

Carcinogenicity

Description of key information

Combined chronic toxicity and carcinogenicity study (OECD 453): oral, 2 years, rat
NOAEL (carcinogenicity), males/females = 159/220 mg/kg bw/day 
NOAEL (systemic toxicity), males/females = 39/56 mg/kg bw/day
Carcinogenicity study (OECD 451): oral, 1.5 years, mouse
NOAEL (carcinogenicity), male/females = 506/354 mg/kg bw/day 
NOAEL (systemic toxicity), males/females = 50/354 mg/kg bw/day 

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 Apr 2004 - 30 Nov 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Version / remarks:

adopted 12 May 1981

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Version / remarks:

adopted 25 Jul 2018

Deviations:

yes

Remarks:

no details on whether feed and water were analysed for contaminants

GLP compliance:

yes (incl. QA statement)

Remarks:

INTERMINISTERIAL GROUP FOR CHEMICAL PRODUCTS, Paris, France

Species:

mouse

Strain:

other: C57BL/6J

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories (St.-Germain-sur-1'Arbresle, France)
- Age at study initiation: 6 weeks
- Weight at study initiation: 19.9-20.4 g mean group weight males; 16.3-17.2 g mean group weight females
- Housing: Individually in stainless steel and wire mesh cages
- Diet : A04CP1-10 (formerly referenced as A04C-10 PI) 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: At least 9 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: 28 Apr 2004 - 30 Nov 2006

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): The test substance formulations were prepared to cover the dietary requirements over 8 weekly periods (exceptions; 1st and 10th formulations at 3500 ppm which covered approximately 2 and 15 weeks respectively and the last formulation which covered dietary needs until end of study.
- Mixing appropriate amounts with: Test substance was incorporated into the diet to provide the required dietary concentrations of 350, 2000, 3500 or 7000 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 has been demonstrated in a previous and recent 90-day rat study, SA02352. The stability of the test substance at 100 and 15000 ppm in the diet was verified for up to 104 days when kept at ambient temperature, which covered the period of storage and usage on this study. The dietary levels of the test substance were verified by HPLC-UV. The homogeneity of the test substance in diet was verified at each concentration on the first formulation (F1), on formulation F2 at 3500 and 7000 ppm, on formulation F6 at 350 and 7000 ppm and on formulation F9 at 3500 ppm, to demonstrate adequate formulation procedures. In addition, the concentration of test substance in the preparations at all dose levels was verified prior to administration to the animals for the following formulations : F3, F6 and F9
- Homogeneity Analysis: 94-103% of nominal concentration
- Concentration Analysis: 93-101% of nominal concentration
Results were within the in-house target range of 85 to 115% of nominal concentration and were therefore considered to be acceptable for use on the current study.

Duration of treatment / exposure:

Main study: 78 weeks
Interim Sacrifice: 52 weeks

Frequency of treatment:

Continously via the diet.

Post exposure period:

none

Dose / conc.:

350 ppm

Remarks:

corresponding to 50 and 63 mg/kg bw/day actual dose ingested for males and females, respectively.

Dose / conc.:

2 000 ppm

Remarks:

corresponding to 287 and 354 mg/kg bw/day actual dose ingested for males and females, respectively.

Dose / conc.:

3 500 ppm

Remarks:

corresponding to 506 and 616 mg/kg bw/day actual dose ingested for males and females, respectively.

Dose / conc.:

7 000 ppm

Remarks:

corresponding to 1041 and 1416 mg/kg bw/day actual dose ingested for males and females, respectively. This dose level exceeded the maximum tolerated dose (MTD), therefore, all surviving females were sacrificed during Week 6 and all surviving males were sacrificed during Week 46.

No. of animals per sex per dose:

60 (50 main study + 10 interim sacrifice)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results from a previous 90-day dietary study in the mouse (SA03005), where dietary administration of up to 7000 ppm resulted in changes in the thymus of females only. The incidence and severity of lymphocytolysis in the thymus was greater than in controls. However the toxicological relevance of this change was unclear as it 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". There were no treatment-related histopathological findings in males. The NOEL in the 90-day mouse study was 2000 ppm.

Positive control:

no

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 faeces.

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 approx. 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 weekly for up to Week 13, and once approx. 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 bw/day for Weeks 1 to 13, then 1 week per month thereafter, the mean achieved dosages were calculated as the product of the dose level (ppm) and the group mean food consumption (g/day) per week divided by the group mean body weight (g) at the end of the week. The monthly and overall mean achieved dosage intake for the 18 months treatment were derived from the weekly data.

OPHTHALMOSCOPIC EXAMINATION: No.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to necropsy at interim and at Weeks 53 or 55 and terminal sacrifice at Weeks 79, 80, 81 or 82.
- Anaesthetic used for blood collection: Yes, isoflurane.
- Animals fasted: Yes, overnight fasting
- How many animals: At approx. 12 months, 10 animals (interim sacrifice groups) and 10 animals (terminal sacrifice groups); at approx. 18 months, 20 animals of the terminal sacrifice groups, with the exception of the 3500 ppm dose group where 18 males were sampled, instead of 20.
- Parameters examined: Haematocrit, haemoglobin, leukocyte count, erythrocyte count, platelet count, leukocyte differential time, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration and mean corpuscular volume. 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: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
On Days 368, 369 or 370 for the 12-month interim kill, and on Days 551 to 568 for the carcinogenicity phase, all surviving animals dedicated to the interim sacrifice/chronic toxicity group and carcinogenicity phase group, respectively, were sacrificed by exsanguination under deep anesthesia (isoflurane). An approximately equal number of animals randomly distributed amongst all groups were sampled on each day. Animals were diet fasted overnight prior to sacrifice. All animals, including animals found dead, killed after accidental trauma, or killed for humane reasons, were necropsied. Necropsy included the examination of all major organs, tissues and body cavities. All significant macroscopic findings were recorded.
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, gallbladder 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 {retina}, optic nerve), 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.
Tissue 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. In addition, urinary bladder stones were retained from two males (OT3M0889, 2000 ppm and OT5M2628, 3500 ppm) and one female (OT5F2734, 3500 ppm) at terminal necropsy and were analyzed for the presence of test substance.
All the samples collected at necropsy. (except exorbital lachrymal gland, larynx/pharynx, nasal cavities and urinary bladder stones) from all animals in all groups were embedded in paraffin wax for the carcinogenicity phase. Liver, kidney, urinary bladder and macroscopic findings from all animals in all groups were embedded in paraffin wax for the interim sacrifice/chronic toxicity phase. Histological sections, stained with hematoxylin and eosin, were prepared from all organs and tissue samples for all animals allocated to the carcinogenicity phase, and from the liver, kidney and urinary bladder for animals allocated to the interim sacrifice/chronic toxicity phase. Additionally, histological slides and staining of significant macroscopic findings sampled from animals of the interim sacrifice groups were performed.

ORGAN WEIGHTS: Liver, heart, spleen, kidney, testis, epididymis, ovary, uterus, brain and adrenal gland.

HISTOPATHOLOGY: Yes
Twelve-month interim sacrifice (52 weeks of treatment):
Histopathological examinations were performed on liver, kidney and urinary bladder from all animals, including those prematurely sacrificed or found dead during the treatment period. Examination of significant macroscopic findings sampled from animals of the interim sacrifice groups were performed.

Carcinogenicity phase (78 weeks of treatment):
Histopathological examinations were performed on all organs and tissues embedded, including gross abnormalities, from all animals in all groups including decedents. Initial examinations were performed. For all unscheduled sacrificed or dead animals during the study, a determination of the factors having contributed to death was made. In addition, "haematopoietic system" was created as a miscellaneous organ in the module P of the data acquisition system Path/Tox, in order to enter the Peto code for malignant tumours which are specific to the hematopoietic system (e.g. lymphoma, histiocytic sarcoma). Following the initial examination, an external review undertook an independent peer-review of representative slides and diagnoses (according to standardized operating procedures). The diagnoses presented in the report represent the consensus opinion of the two pathologists

Statistics:

The variables analyzed and subjected to statistical analysis were body weight parameters, body weight change parameters calculated according to time intervals, average food consumption/day parameters calculated according to time intervals, motor activity parameters, grip strength parameters, haematology parameters (except eosinophils, basophils, monocytes and large unstained cells), clinical chemistry parameters, quantitative urinalysis, terminal body weight, absolute and relative organ weights parameters, survival data, neoplastic and non-neoplastic finding incidence. Statistical methods included means and standard deviation, Bartlett test for comaprison of homogeneity of variance, analysis of variance (ANOVA), square root transformation of data, Dunnett's test (2-sided), non-parametric Kruskal-Wallis test for body weight and food consumption parameters and haematology parameters as appropriate. Group means were compared at the 5% and 1% levels of significance. Statistical analyses were carried out using Path/Tox System V4.2.2. (Module Enhanced Statistics). Survival analysis was performed. Kaplan-Meier estimation procedures were followed. Statistical significance of differences in survival rates between treated and control groups and dose related trends in survival was assessed using Cox's and Tarone's tests on life table data. Neoplastic and non-neoplastic findngs were analyzed non-adjusted or survival adjusted. Methods included Cochran-Armitage method for trend (1-sided) and the Fisher's exact test (1-sided) for control versus treatment comparisons. Statistical analyses were conducted using SAS programs. Analyses were performed on all findings in the carcinogenicity phase and group incidences were compared at the 5% and 1% levels of significance.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

7000 ppm: See field Any other results on results incl. tables.

3500 ppm:
- Treatment-related clinical signs were confined to males and consisted of clinical observations which were, on most occasions, recorded on the days preceding the death or early sacrifice of the animals and were indicative of their general poor health condition.
- Other clinical signs observed were considered to be incidental as they occurred at a similar frequency in the control and treated groups, did not occur in a dose-related manner, or occurred in isolation.

Summary data of clinical signs can be found in Attachment 1.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

Not applicable.

Mortality:

mortality observed, treatment-related

Description (incidence):

7000 ppm: See field Any other results on results incl. tables.

3500 ppm:
- The mortality incidence in males at 3500 ppm was significantly increased compared with controls after 12 and 18 months of treatment (20.0% and 58.1%, respectively, compared to 5.0% and 24.4% in the respective control group). The higher mortality rate was largely due to the presence of stones within the urinary tract, causing acute renal failure (by urinary blockage) or chronic renal failure (by concomitant chronic kidney/urinary bladder irritation and inflammation) or due to secondary treatment-related nephropathy following product administration.

No treatment-related effect on mortality rate were observed at 3500 ppm in females and at 2000 and 350 ppm in either sex.

Animals which died by accidental trauma or at Study Director request were excluded from statistical analysis.

Summary data for mortality can be found in Attachment 2.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

7000 ppm: See field Any other results on results incl. tables.

3500 ppm:
Males
- During the first week of treatment, mean body weight of males was reduced by 4% (p<0.01), whilst mean body weight gain was reduced by 33% (p<0.01), compared to the controls.
- Thereafter, starting from Day 92 mean body weight was slightly reduced compared to the controls, the effect being statistically significant (p<0.01 or p<0.05) at most time points, resulting in a reduction of 5% at the end of the treatment period (Day 540).
- Mean cumulative body weight gain of males was slightly lower in comparison with the controls at several time points over the study period, resulting in an overall mean cumulative body weight gain reduction of 12% by Day 540, when compared to controls.

Females
- There was no treatment-related effect on mean body weight and mean cumulative body weight gain during the first year of treatment.
- Mean body weight was 5% lower than in the control group on Day 1 (p<0.01). This effect was attributed to the later starting date of the additional dose group at 3500ppm. As a consequence, mean body weight was lower than controls by between 1 and 5% during the first four weeks of treatment.
- During the last 6 months of treatment, mean cumulative body weight gain was statistically significantly reduced by between 29 and 71% at most time points in comparison to the controls, resulting in a mean body weight reduction of 3% (p<0.05) on Day 540, compared to controls.
- Overall mean cumulative body weight gain was similar to the controls over the study period.

At 2000 and 350 ppm, there was no treatment-related effect on mean body weight and cumulative body weight gain in either sex over the 18 months of treatment. The few minor differences from controls which attained statistical significance were sporadic and were considered to be chance findings reflecting the number of comparisons made.

Summary data for body weights and body weight gains can be found in Attachment 3.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

7000 ppm: See field Any other results on results incl. tables.

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

During the last 6 months of treatment, mean food consumption was comparable to controls in males and reduced by between 2 and 10% in females at 3500 ppm compared to controls (p<0.01 at most intervals).

Food consumption was unaffected by treatment at 2000 and 350 ppm in either sex.

Summary data of food consumption and compound intake can be found in Attachment 4.

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:

effects observed, treatment-related

Description (incidence and severity):

3500 ppm:
- A tendency towards minimally lower red blood cell count was noted in both sexes. This change was associated with slightly lower haemoglobin concentration, haematocrit and mean corpuscular haemoglobin concentration and slightly higher mean corpuscular volume. The magnitude and statistical significance relative to the controls are presented in Attachment 5.
- Statistically significant differences were observed in relative neutrophil and lymphocyte counts (percentage) at 3500 ppm in females. However, in the absence of relevant variations in total leucocyte and absolute differential counts, the differences were considered incidental.

No treatment-related change was noted at 2000 and 350 ppm in either sex. The few statistically significant differences observed at 2000 and 350 ppm in females were considered not to be treatment-related in view of their low magnitude and the absence of a clear dose-effect relationship.

Clinical biochemistry findings:

not examined

Description (incidence and severity):

Not applicable.

Endocrine findings:

not examined

Description (incidence and severity):

Not applicable.

Urinalysis findings:

not examined

Description (incidence and severity):

Not applicable.

Behaviour (functional findings):

not examined

Description (incidence and severity):

Not applicable.

Immunological findings:

not examined

Description (incidence and severity):

Not applicable.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

3500 ppm:
12-month interim sacrifice
- In females, mean absolute and relative spleen and ovary weights were higher and statistically different when compared to controls. As these changes were not observed after 18 months of treatment and not correlated with any microscopic findings in the 18-month sacrificed animals, they were considered incidental.

18-month carcinogenicity phase
- Mean terminal body weight was decreased by 5% in males and females, the effect being statistically significantly different in females only (p<0.01) when compared to the controls.
- In males, mean absolute liver weight and mean liver to brain weight ratio were lower and statistically significantly different when compared to controls. This change was considered to be associated with the lower mean terminal body weight (-5%, not statistically significant) and was considered not to be treatment-related.
- In males, mean kidney to body weight ratio was statistically significantly higher compared to the controls. This significant change was attributed to the lower mean terminal body weight and was not correlated with any microscopic findings, it was therefore considered to be incidental.
- In females, mean absolute kidney weights were lower and statistically significantly different when compared to controls. In view of the low magnitude (-6%, p<0.05) this change was considered not to be treatment-related.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

12-month interim sacrifice

3500 ppm
Unscheduled deaths:
- Seven unscheduled deaths of animals belonging to the chronic toxicity phase were recorded. The macroscopic findings observed on six of these animals did not support a directly treatment-related death. Animal OT5M2624 (3500 ppm) was found dead on Day 305, with paleness and gritty content in the kidney, obviously small thymus and autolyzed organs. Pale kidney and gritty content were considered to be treatment-related as similar findings were noted in 18-month terminal sacrificed animals, but moderate autolysis made it difficult to consider these renal findings as a clear cause of death.

Terminal sacrifice:
- Thickened mucosa was found in the urinary bladder in 4/9 males, including animal OT5M2628 which had several urinary bladder stones.
- Enlarged spleen was found in 6/10 females. This change was considered not to be treatment-related since it was not correlated with any microscopic findings and not found during the carcinogenicity phase.

18-month carcinogenicity phase (summary data of the macroscopic findings is presented in Attachment 6)

3500 ppm
Unscheduled deaths:
- Stones or a higher incidence of gritty content were found in the kidney and urinary bladder of a number of males and of one female. They were retained at terminal necropsy from the urinary bladder of two males (OT3M0889, 2000 ppm and OT5M2628, 3500 ppm) and one female (OT5F2734, 3500 ppm) and found to consist of approximately 90 to 95% of test material.
- Additionally, a higher incidence of enlarged kidney, renal pelvic dilatation, pale kidney or kidney with irregular surface was observed in males and/or females than in the controls.
- Thickening of the mucosa in the urinary bladder and dilatation of the ureters were also observed in a number of males.
- A higher incidence of dark liver was found in males, 4/10 cases being associated with microscopic hepatocellular hypertrophy.

Terminal sacrifice:
- Stones were found in the kidney and urinary bladder of a number of males and in the urinary bladder of one female. Kidney with irregular surface was observed in males.

2000 ppm
Unscheduled deaths:
- Treatment-related macroscopic findings were observed in the urinary tract of males only.
- Gritty content was found in the kidney of two males and in the urinary bladder of one male.

Terminal sacrifice:
- Gritty content was found in the kidney of one male and stones were noted in the urinary bladder of one male.

Neuropathological findings:

not examined

Description (incidence and severity):

Not applicable.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Summary data of the microscopic findings is presented in Attachment 7.

12-month interim sacrifice

3500 ppm
Unscheduled deaths:
- Animal OT5M2624 was found dead on Day 305 with moderate, bilateral renal cortical basophilic tubules; slight hyaline casts and minimal bilateral pelvic dilatation. These findings were considered to be treatment-related as similar findings were noted in the 18-month terminal sacrificed animals.

Terminal sacrifice:
- A slightly higher incidence and severity of renal cortical basophilic tubules was found in males.
- One male (OT5M2628) had some findings in the urinary bladder that were considered to be treatment-related as they were also observed in 18-month terminal sacrificed animals: simple urothelial hyperplasia, interstitial oedema, induced arteritis/periarteritis, suburothelial mixed cell infiltrate and intramuscular inflammatory cell infiltrate.

No treatment-related changes were seen at 3500 ppm in females and at 2000 and 350 ppm in either sex.

18-month carcinogenicity phase

3500 ppm
Scheduled and unscheduled deaths:
Liver
- Minimal to slight centrilobular to panlobular hepatocellular hypertrophy was observed in males.
- Increased incidence of hepatocellular single cell necrosis was observed in males.
- A loss of diffuse hepatocellular vacuolation was observed in unscheduled male and female deaths. This effect was considered to be incidental as it was not observed in terminal sacrificed animals.

Kidney
- Stones were found in the pelvis in both sexes and in the collecting ducts in males only.
- A higher incidence of concretions in the collecting ducts was noted in males.
- Other treatment-related findings were seen mainly in males and in a few females, and consisted of induced hyperplastic changes (collecting duct hyperplasia and papillary/pelvic epithelium hyperplasia) and changes resulting from chronic inflammation or urinary blockage (atrophy/fibrosis/scar, perirenal inflammation, glomerular chamber and cortical or cortex/medulla tubular dilatation, parenchymal cysts and pelvic dilatation).
- In addition in males, a higher incidence and severity of bilateral cortical basophilic tubules was noted and a higher incidence of cortical and medullar tubular mineralization was found.
- The incidence of cellular casts was also increased.

Urinary bladder
- Stones were observed in both sexes.
- Other treatment-related findings were seen in males and/or females and consisted of hyperplastic/metaplastic changes (simple, nodular/glandular, atypical hyperplasia and squamous metaplasia, and atypical mesenchymal proliferation) and secondary changes due to stone induced irritation or urinary blockage (muscular degeneration/haemorrhage(s) and interstitial oedema, intramuscular or serosal inflammatory cell infiltrate; intraluminal or suburothelial mixed cell infiltrate and arteritis/periarteritis).

Urethra (prostate)
- Stones and interstitial mixed cell infiltrate were noted.
- Urothelial hyperplasia was also observed.

Ureters (sampled when abnormalities were detected macroscopically)
- Stones and urothelial hyperplasia were noted in males.

2000 ppm
Scheduled and unscheduled deaths:
Liver
- Minimal to slight centrilobular to panlobular hepatocellular hypertrophy was observed in males.

Kidney
- Stones were found in the pelvis and collecting ducts at in males.
- Other treatment-related findings were seen in males only, and consisted of induced hyperplastic changes (collecting duct hyperplasia and papillary/pelvic epithelium hyperplasia) and changes resulting from chronic inflammation or urinary blockage (atrophy/fibrosis/scar, perirenal inflammation, glomerular chamber and cortical or cortex/medulla tubular dilatation, parenchymal cysts and pelvic dilatation).
- In addition in males, a higher incidence and severity of bilateral cortical basophilic tubules was noted and a higher incidence of cortical and medullar tubular mineralization was found.
- The incidence of cellular casts was also increased.

Urinary bladder
- Stones were observed in a few males.

Urethra (prostate)
- Urothelial hyperplasia was observed

All the effects seen in the urinary tract (combination of neoplastic/hyperplastic and inflammatory changes) were considered to be due to a single and chronic irritative mechanism, resulting from the formation of stones (which were observed on a number of animals) or crystals.

A number of indirect treatment-related effects was noted in various organs and were considered to be secondary to the stress induced by the stone deposition and/or the treatment-related nephropathy:
- In the spleen a higher incidence of apoptotic bodies was noted in males at 3500 ppm (9/50), almost all in unscheduled animals (8/30), when compared to controls (3/47). A higher incidence of lymphoid (white pulp) atrophy was noted in males at 3500 ppm (6/50), all in unscheduled animals (6/30), when compared to controls (3/47).
- In the mesenteric lymph node a higher incidence of apoptotic bodies was noted in males at 3500 ppm (10/49, p<0.05), almost all in unscheduled animals (8/29), when compared to controls (3/48).

In the heart, a higher incidence of epicardial mixed cell infiltrate was noted in males dosed at 3500 ppm (5/50), when compared to controls (0/50) (p<0.05). This minimal change only observed in males at 3500 ppm was considered to be an indirect and non-adverse response to the treatment.

In the epididymis, a higher incidence of bilateral oligospermia was noted in animals dosed at 3500 ppm (9/50), 2000 ppm (14/50, p<0.01) or 350 ppm (7/50), when compared to controls (3/50). Since this change was not dose-related and since there was no concomitant higher incidence of bilateral testicular degeneration when compared to controls, this finding was judged to be incidental and not treatment-related.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

3500 ppm (see Attachment 8 for a summary of neoplastic findings, incl. historical control data):
- A treatment-related slightly higher incidence of transitional cell papillomas was noted in the urinary bladder in females. This finding was considered to be secondary to chronic hyperplastic changes resulting from the chronic irritation caused by stones.
- A slightly higher incidence of histiocytic sarcoma was noted in females. This type of tumour is common in the aged C57BL/6J mouse. This change was considered not to be treatment-related since it was observed in females only and it was in agreement with the average incidence of such a type of hematopoietic tumour in the C57BL/6J female mouse reported in the literature (4.8% between 13-18 months of age and 10% at 24 months of age, up to 42% incidence after 24 months of study duration according to the Registry of Industrial Toxicology Animal data (RITA group).

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Relevance of carcinogenic effects / potential:

Neoplastic changes comprised transitional cell papilloma in the urinary bladder of 2/49 females at 3500 ppm. This finding was considered to be secondary to the chronic hyperplastic changes due to the presence of calculi. Thus the test substance was considered not to be directly carcinogenic in the mouse.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

350 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No adverse effects observed at this dose level.

Remarks on result:

other: corresponding to 50 mg/kg bw/day actual dose ingested for males.

Key result

Dose descriptor:

LOAEL

Remarks:

systemic

Effect level:

2 000 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

gross pathology

histopathology: non-neoplastic

Remarks on result:

other: corresponding to 287 mg/kg bw/day actual dose ingested for males.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

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 354 mg/kg bw/day actual dose ingested for females.

Key result

Dose descriptor:

LOAEL

Remarks:

systemic

Effect level:

3 500 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

Remarks on result:

other: corresponding to 616 mg/kg bw/day actual dose ingested for females.

Key result

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Effect level:

3 500 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No adverse effects observed at this dose level

Remarks on result:

other: corresponding to 506 mg/kg bw/day actual dose ingested in males.

Key result

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Effect level:

2 000 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse effects observed at this dose level

Remarks on result:

other: corresponding to 354 mg/kg bw/day actual dose ingested for females.

Key result

Dose descriptor:

LOAEL

Remarks:

carcinogenicity

Effect level:

3 500 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: corresponding to 616 mg/kg bw/day actual dose ingested for females.

Critical effects observed:

yes

Lowest effective dose / conc.:

2 000 ppm

System:

urinary

Organ:

bladder

kidney

urethra

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Critical effects observed:

yes

Lowest effective dose / conc.:

2 000 ppm

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Critical effects observed:

yes

Lowest effective dose / conc.:

3 500 ppm

System:

urinary

Organ:

ureter

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

General Condition of the High Dose Group (7000 ppm):

The dietary level of 7000 ppm clearly exceeded the Maximum Tolerated Dose in both males and females (marked individual body weight loss and mortality in both sexes), leading to early sacrifice of the males and females on Days 320 and 40, respectively.

Four females were found dead between Days 16 and 34. Eleven females were killed for humane reasons between Days 11 and 35. Clear evidence of toxicity was noted on these latter animals: clinical signs indicative of poor health condition were recorded on one or more occasions prior to death, which included wasted appearance, reduced motor activity, piloerection, laboured or rapid respiration, coldness to touch, hunched posture and/or soiled anogenital region. Most animals showed a severe body weight loss on the days preceding death, corresponding to an overall cumulative body weight loss of between 1 and 4 g, associated with a markedly reduced food consumption, compared to the controls. The remaining females of this dose group (45 animals) were sacrificed on Day 40.

In males, 5 animals were either found dead or killed for humane reasons during the first three months of treatment and 26 animals were either found dead or killed for humane reasons between Days 218 and 316. Most of these animals showed a severe body weight loss on the days preceding death, they had a wasted appearance and exhibited clinical signs related to poor health condition on one or more occasions prior to death, which included reduced motor activity, uncoordinated movements, prostration, general pallor, piloerection, slow respiration, coldness to touch, hunched posture and/or generalized soiled fur. During the first week of treatment, mean body weight was reduced by 3% (p<0.01) in males, whilst mean cumulative body weight gain was reduced by 47% (p<0.01), compared to the controls. Thereafter, the mean body weight gain was lower than in controls on most intervals up to Day 316, resulting in a lower cumulative body weight gain of between 8 and 30%, the effect being statistically significant on most occasions up to Day 71 and on all occasions from Day 78 onwards (p<0.05 or p<0.01). Food consumption was reduced by 7 to 12% between Days 253 and 316 (p<0.01 on the two last intervals), when compared to controls. The remaining males of this dose group (29 animals) were sacrificed on Day 320.

Although histopathological examination was performed on the liver, kidney, urinary bladder, heart (including aorta), thymus and macroscopic findings from unscheduled deaths in females and from the first ten surviving females sacrificed on Day 40, the results were not used for assessing any compound-related toxicity due to the confounding effects of the overall poor condition of these animals. Therefore, for animals at 7000 ppm, only data pertaining to mortality, clinical signs, body weight parameters and food consumption (in-life phase), which reflected the poor condition of the animals, were included in the report.

 

Conclusions:

The study was performed in accordance to OECD TG 451 under GLP conditions and is considered reliable. In conclusion, dietary administration of the test substance over an 18-month period to the mouse, at dose levels up to 3500 ppm (corresponding to 506 mg/kg/day in males and 616 mg/kg/day in females), produced transitional cell papilloma in the urinary bladder of females. The incidence of these tumours was very low and was considered to be secondary to the chronic hyperplastic changes resulting from chronic irritation due to the presence of calculi. Thus the test substance was considered not to be directly carcinogenic in the mouse. The No Observed Adverse Effect Level (NOAEL) for systemic toxicity was 350 ppm for males (equivalent to 50 mg/kg/day) and 2000 ppm for females (equivalent to 354 mg/kg/day). In regard to carcinogenicity, a NOAEL of 3500 ppm (equivalent to 506 mg/kg bw/day) is derived for males and a NOAEL of 2000 ppm (equivalent to 354 mg/kg bw/day) for females.

Reference 2

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 Nov 2003 to 11 Dec 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Version / remarks:

adopted 12 May 1981

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Version / remarks:

adopted 25 Jun 2018

Deviations:

yes

Remarks:

no details on whether feed and water were analysed for contaminants

GLP compliance:

yes (incl. QA statement)

Remarks:

INTERMINISTERIAL GROUP FOR CHEMICAL PRODUCTS, Paris, France

Species:

rat

Strain:

other: Wistar Rj:WI (IOPS HAN)

Sex:

male/female

Details on test animals or test system and environmental conditions:

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

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

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

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

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

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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

Duration of treatment / exposure:

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

Frequency of treatment:

Continously via the diet

Post exposure period:

None

Dose / conc.:

200 ppm

Remarks:

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

Dose / conc.:

1 000 ppm

Remarks:

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

Dose / conc.:

4 000 ppm

Remarks:

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

Dose / conc.:

8 000 ppm

Remarks:

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

No. of animals per sex per dose:

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

Control animals:

yes, plain diet

Details on study design:

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

Positive control:

None

Observations and examinations performed and frequency:

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

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

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

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

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

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

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

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

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

Sacrifice and pathology:

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

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

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

Statistics:

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

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

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

See Attachment 1 for summary tables of clinical signs.

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

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

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

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

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

Not applicable.

Mortality:

mortality observed, treatment-related

Description (incidence):

See Attachment 1 for summary tables of mortality data.

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

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

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

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

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

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

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

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

See Attachment 1 for compound intake.

Food efficiency:

not examined

Description (incidence and severity):

Not applicable.

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

not examined

Description (incidence and severity):

Not applicable.

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

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

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

Clinical biochemistry findings:

no effects observed

Endocrine findings:

not examined

Description (incidence and severity):

Not applicable.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

See Attachment 2 for summary tables of urinalysis findings.

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

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

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

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

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

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

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

Immunological findings:

not examined

Description (incidence and severity):

Not applicable.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

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

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

See Attachment 3 for summary tables of gross pathology data.

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

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

Neuropathological findings:

not examined

Description (incidence and severity):

Not applicable.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See Attachment 4 for summary tables of histopathology data.

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

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

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

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

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

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

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

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

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

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

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Relevance of carcinogenic effects / potential:

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

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

1 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects seen at this dose level.

Remarks on result:

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

Key result

Dose descriptor:

LOAEL

Remarks:

systemic

Effect level:

4 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

gross pathology

histopathology: non-neoplastic

urinalysis

Remarks on result:

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

Key result

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Effect level:

4 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects seen at this dose level.

Remarks on result:

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

Key result

Dose descriptor:

LOAEL

Remarks:

carcinogenicity

Effect level:

8 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: neoplastic

Remarks on result:

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

Critical effects observed:

yes

Lowest effective dose / conc.:

4 000 ppm

System:

urinary

Organ:

bladder

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Critical effects observed:

yes

Lowest effective dose / conc.:

8 000 ppm

System:

urinary

Organ:

ureter

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Conclusions:

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

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

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

159 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

The quality of the database is good comprising two carcinogenicity studies in rodent species (rat and mouse), according to OECD TG 453 and 451, respectively, under GLP conditions. Both studies are considered of reliable quality and validity, fulfilling the criteria of a key study. Thus, both are suitable for assessment of the present endpoint.

System:

urinary

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

Please refer to the discussion in the respective endpoint summary.

Justification for classification or non-classification

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

Additional information

Rat

A 2-year 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.

The test substance was administered to groups of 60 male and 60 female Wistar rats by continuous dietary treatment at 1000, 4000 and 8000 ppm, corresponding to 39, 159 and 321 mg/kg bw/day in males and 56, 220 and 447 mg/kg bw/day in females, respectively, over a 24-month period. Additionally, groups of 10 male and 10 female rats 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.

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.

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. 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. The very few additional tumors observed at 200, 1000, 4000 and 8000 ppm were considered incidental due to the rather isolated occurrence in single animals without being reproducible across the different dose groups.

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

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

Mouse

Additionally, an 18-month carcinogenicity study (M-281276-01-1) was performed in C57BL/6J mice. The study was performed under GLP conditions and in accordance with OECD TG 451 (adopted 1981). Deviations to the current version of the guideline (adopted 2018) are only minor and not considered to have an impact on the outcome of this study. The test substance was administered continuously via the diet to groups of mice following continuous dietary treatment at 350, 2000 and 3500 ppm, corresponding to 50, 287 and 506 mg/kg bw/day in males and 63, 354 and 616 mg/kg bw/day in females for 18 months. In addition, an interim sacrifice was performed after 52 weeks of treatment to assess chronic toxicity.

Groups of 60 male and 60 female mice were initially intended to be fed diet containing 0, 350, 2000 or 7000 ppm for at least 18 months. Since the Maximum Tolerated Dose was exceeded in females at 7000 ppm during the first weeks of treatment, an additional treatment group (60 animals/sex) at 3500 ppm was added to the study approximately one month after the start of treatment. The high rate of mortality observed in both sexes at 7000 ppm led to the early termination of this treatment group, with all surviving females being sacrificed during Week 6 and all males being sacrificed during Week 46.

The mortality incidence in males was higher at 3500 ppm than in the control group after 12 and 18 months of treatment, largely due to the presence of stones within the urinary tract, causing acute or chronic renal failure or due to secondary treatment-related nephropathy following product administration.

At the 18-month haematology determination, a tendency towards lower red blood cell count was noted in both sexes. This change was associated with slightly lower haemoglobin concentration, haematocrit and mean corpuscular haemoglobin concentration and slightly higher mean corpuscular volume. Histopathological treatment-related changes were mainly attributed to the presence of urinary stones or to treatment-related nephropathy. These changes were located in the urinary bladder and to a lesser extent in the kidney at the 12-month interim sacrifice.

At the carcinogenicity phase (18-month) sacrifice, changes due to treatment-related nephropathy were seen in the kidney, urinary bladder, prostatic urethra and ureters. In the liver, minimal to slight centrilobular to panlobular hepatocellular hypertrophy and an increased incidence of hepatocellular single cell necrosis were observed in males. All the effects seen in the urinary tract were considered to be due to a chronic irritative mechanism, resulting from the formation of stones or crystals. In addition, indirect treatment-related findings were observed in the lymphoid system in males and were considered to be secondary to the stress induced by the stone deposition and/or the treatment-related nephropathy. In the heart, a higher incidence of epicardial mixed cell infiltrates was noted in 5/50 males at 3500 ppm, compared to no cases in controls. This minimal change only observed in males was considered to be an indirect and non-adverse response to the treatment.

Neoplastic changes comprised transitional cell papilloma in the urinary bladder of 2/49 females at 3500 ppm. This finding was considered to be secondary to the chronic hyperplastic changes due to the presence of calculi. Analysis of urinary bladder stones taken from one male and one female at terminal sacrifice revealed that the composition of the stones consisted of approximately 90 to 95% of test material.

In conclusion, dietary administration of the test substance over an 18-month period to the mouse, at dose levels up to 3500 ppm (corresponding to 506 mg/kg bw/day in males and 616 mg/kg bw/day in females), produced transitional cell papilloma in the urinary bladder of females. The incidence of these tumours was very low and was considered to be secondary to the chronic hyperplastic changes resulting from chronic irritation due to the presence of calculi. Thus the test substance was considered not to be directly carcinogenic in the mouse. The No Observed Adverse Effect Level (NOAEL) for systemic toxicity was 350 ppm for males (equivalent to 50 mg/kg bw/day) and 2000 ppm for females (equivalent to 354 mg/kg bw/day). In regard to carcinogenicity, a NOAEL of 3500 ppm (equivalent to 506 mg/kg bw/day) is derived for males and a NOAEL of 2000 ppm (equivalent to 354 mg/kg bw/day) for females.

Evaluation on Carcinogenicity for the Registered Substance

An evaluation on carcinogenicity was conducted in order to assess the relevance to humans for the findings in the urinary tract in the respective carcinogenicity studies on the registered substance (M-414116-01-1). This evaluation involved an assessment of the available data on the substance, as well as assessing relevant literature sources for other substances (including sulfonamides) that are known to induce urinary tract calculi.

Data on the Substance:

In the in vitro and in vivo genotoxicity assays, the substance was negative with and without metabolic activation.

The substance is rapidly absorbed and excreted, predominantly in the urine, with most of the chemical excreted within 48 hours with approximately 70%-90% excreted in the urine. Metabolites have been identified, and some of these have also been evaluated in genotoxicity assays and have been uniformly negative.

Shorter-term toxicological studies are available in rats, mice, and dogs, and show 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. In dogs, the toxic dose was 8000 ppm in the one-year dog study, but was negative at 7500 in the 90-day dog study and 2500 ppm in the one-year dog study. In rats, toxicity was seen at 4000 ppm in short-term studies and at 4000 and 8000 ppm in the carcinogenicity study. Neither crystalluria nor toxicity was seen in the rat at a dose of 1000 ppm. In mice in long-term studies, crystalluria and toxicity were seen at 2000 and 3500 ppm, and a dose of 7000 ppm exceeded the maximum tolerated dose level, leading to early sacrifice of the animals. Stones from one male and one female mouse in the carcinogenicity assay were evaluated for composition and shown to consist of approximately 90%-95% of the test chemical. Thus, the crystals and calculi are typical of those seen with other sulfonamides, being composed predominantly of the parent chemical rather than a metabolite or normal constituents of the urine.

No skin irritation was observed on single dermal application of the substance in studies on guinea pigs, rabbits or rats. This demonstrates that the substance is not directly irritating

Although the incidences of bladder tumours were small in the rat and mouse long term studies, the high incidences of bladder hyperplasia justifies the conclusion that these were treatment related and did not occur by chance. It is also clear from the carcinogenicity studies that the carcinogenic effect is weak and occurs only at the highest dose. The carcinogenic stimulus is not the chemical itself, but rather, tumours are associated with the toxicity and regenerative proliferation associated with the formation of urinary tract calculi and crystals. These toxic manifestations of the compound were also observed in short-term studies in rats, mice, and in dogs. This is a common mode of action for bladder carcinogenesis in rodents for non-DNA reactive (non-genotoxic) chemicals (1-4*).

In summary, the substance induced a very low incidence of bladder tumours in rats and mice. This occurred only at high doses and was associated with the formation of urinary tract calculi. Urinary tract calculi represented a toxicological response only at high doses in rats, mice, and dogs, and is consistent with what is known chemically, metabolically and toxicologically for sulfonamide and related compounds (5-7).

In assessing potential carcinogenic risk of the substance to humans, not only are the findings from studies with the chemical valuable, but the vast literature available for the effects of urinary tract calculi and specifically for sulfonamides provides extensive and valuable information to lead to the conclusion that not only does the substance not pose a carcinogenic risk to humans, but it does not represent a carcinogenic hazard for humans. 

Examples of other compounds causing urinary tract calculi:

Chemicals that produce urinary tract calculi in rodents can act as urinary bladder carcinogens, and occasionally produce tumours also in the ureters and kidney pelves (1-4, 8-10). The mode of action is well-characterized, involving administration of doses of chemicals that are high enough to generate a concentration of the material in the urine that can precipitate to form urinary tract crystals and/or calculi. These urinary solids produce a toxic effect on the urothelial mucosa (the urothelium), primarily in the bladder since that is where most of the calculi accumulate (11, 12). Depending on the coarseness of the calculi, their size and number, variations in extent of damage and proliferation occur (13). This can also be influenced by dietary manipulations, which can alter the chemical composition of the urine affecting the formation of the urinary solid, such as altering pH or concentration of the chemicals involved in the formation of the crystals (4, 14).

Urinary solids can be formed from the parent compound or their metabolite, or solids can indirectly be formed from normal constituents of the urine because of dramatic alterations in the composition of the urine (10). An excellent example of the former that has been studied extensively is uracil (11, 12). An indirect mechanism has been well described with the example of muraglitazar, a PPAR agonist, which produces hypocitraturia (15). Since citrate is the major chelating substance in the urine for calcium, a reduction in the level of citrate leads to the ready precipitation of calcium salts, such as calcium phosphate and calcium oxalate. 

In the case of the registered substance, it appears that the urinary tract solids are formed primarily of the parent chemical itself rather than metabolites or from normal constituents of the urine. It is clear that it is not the chemical that is toxic, but rather, it is the urinary solid formed from the chemical that produces the toxicity.

Regardless of whether formed directly from the chemical or metabolite or indirectly from normal urinary constituents, urinary tract calculi represent a high dose phenomenon dependent on basic physical chemical properties of the solubility products of the chemical involved (1-4). This is the clearest example of a threshold phenomenon for carcinogenesis. In rodents, urinary solids can readily produce toxicity. This is especially true in male rats and mice compared to females, but both can be affected dependent on the toxicokinetics of the chemical in the specific gender and species.

Relevance for humans:

The effects of urinary solids in humans are much less pronounced than in rodents (2-4). In contrast to rodents, urinary crystals do not appear to produce toxicity of the human urothelium (16-18). Crystals occur under a variety of circumstances in human urine, including normal human urine which contains magnesium ammonium phosphate (struvite) crystals and occasionally will also contain calcium phosphate or calcium oxalate crystals. Other crystals that form in human urine include uric acid and a variety of others that occur more rarely. Furthermore, a large number of drugs are also known to produce crystals in human urine, most notably, sulfonamides (5, 6, 8-10). However, other classes of drugs also can produce crystalluria including carbonic anhydrase inhibitors and anti-HIV protease inhibitors (8-10). Crystalluria in humans does not represent toxicity, but may indicate a propensity for the formation of calculi (such as calcium oxalate or calcium phosphate) or may represent a manifestation of systemic toxicity, such as in the instance of uric acid in patients with gout (16).

Calculi are rarely present in the human urinary tract for prolonged periods of time (17). The human urinary tract anatomy differs from that of rodents so that urinary tract calculi usually produce obstruction and consequent severe pain, requiring immediate removal, either by lithotripsy or by surgery for those not fortunate enough to have the calculi dissolve with increased liquid intake (17). Obstruction usually occurs 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 rodents, prolonged exposure to the calculus is required for tumours to occur (4, 11, and 12). If the calculus is removed prior to the formation of malignant tumours, the proliferative lesions regress rapidly, even to the extent that papillomas can reverse. In humans, prolonged exposure to urinary tract calculi is uncommon.

Interpretation of the association of calculi to bladder cancer in humans is complicated by a variety of confounding factors. To begin with, most studies do not find a statistically significant relationship between calculi and the development of urothelial tumours (5, 15, and 18). Secondly, many of the tumours that develop in patients with urinary tract calculi, like in other situations with prolonged urinary tract inflammation, are frequently squamous cell carcinomas rather than urothelial (transitional) cell carcinomas (19). In rodents, the tumours associated with calculi are preponderantly urothelial cell tumours. Lastly, and most importantly, prolonged urinary tract calculi in humans are uniformly associated with prolonged bacterial cystitis (20). Bacterial cystitis is a known risk factor for bladder cancer in humans, and the risk for developing tumours in patients with urinary tract calculi is somewhat less than in patients with bacterial cystitis without calculi, suggesting that the calculi do not add any additional risk to the development of urinary tract tumours compared to bacterial cystitis alone (20, 21).

Additional evidence that urinary tract calculi do not represent a carcinogenic hazard in species other than rodents includes the observation that urinary tract calculi are extremely common in dogs, whereas urothelial tumours in dogs are uncommon (22).

Taking this information together, it strongly suggests that urinary tract calculi do not pose a carcinogenic hazard for humans, similar to dogs. Furthermore, for the case of the registered substance, there is a long experience with the toxicology and biology in humans is known for sulfonamide-related chemicals.

Sulfonamides and related chemicals are known to have poor solubility in aqueous solutions, such as urine (5-7). When ingested orally, they are rapidly absorbed and excreted primarily in the urine, as is the case for registered substance, or in the urine and faeces. In the urinary tract, depending on solubility and various other factors affecting urinary chemical composition, urinary crystals are readily formed and in some species, also calculi (5-7). In rodents, as in the case for registered substance, crystals and calculi readily form (5-7). For some sulfonamides, such as the registered substance, this also is true for the dog. As mentioned above, urinary tract calculi do not represent a carcinogenic hazard in dogs.

In humans, sulfonamides have been used clinically for more than fifty years, and their toxicological effects are well known (23). This includes the usual formation of sulfonamide-containing crystals in the urine of patients on sulfonamides, without any adverse toxicological consequence (5). Calculi can form from sulfonamides in humans, but this is exceedingly rare and has not been associated with the formation of bladder tumours. In fact, many patients with long-standing bacterial cystitis with or without urinary tract calculi are treated with long-term sulfonamide antibiotic therapy (24). Based on this long clinical experience, sulfonamides are not considered carcinogens for humans, and are widely prescribed.

A similar non-DNA reactive consideration was given by the International Agency for Research on Cancer for another chemical, melamine, that produces urinary bladder tumours secondary to calculi, but in this instance in rats (25). Melamine is classified by IARC in their monograph series on evaluation of carcinogenic risk of chemicals as a class three substance, (the agent is not classifiable as to its carcinogenicity, based on mechanism). This was based on the fact that it is not only a high dose phenomenon, but exposure in humans is several orders of magnitude less than what is necessary for producing calculi and tumours in rats, and the likelihood that bladder tumours would result from calculi in humans is exceedingly low (as described above).

The lack of a relationship between urinary tract calculi and bladder cancer in humans is consistent with practical considerations of exposure to substances that can lead to the formation of calculi. For example, the most common calculi that occur in humans contain calcium, either as the oxalate or phosphate salt (26). In rodents, these produce urinary tract tumours. Calcium is an essential dietary ingredient, involved in many cellular, physiologic and structural functions (27). Calcium is present in many of the foods we consume, is used as a packaging ingredient for many food additives and drugs, and is an active ingredient in some drugs. There is no concern about its carcinogenic potential. In fact, there is accumulating evidence that it acts to prevent some types of cancer, such as colon carcinoma (28).

Summary and conclusion:

In summary, the registered substance, like many sulfonamides, when ingested at high exposure levels, produces urinary tract solids, including calculi, which leads to cytotoxicity, consequent inflammatory reaction and regenerative urothelial proliferation, and in the rat and mouse, rarely leads to bladder tumours. Based on the lack of genotoxicity, the well-known biology and toxicology of urinary tract calculi, and the lack of carcinogenic effect of sulfonamides in humans, even when significantly high doses leading to the formation of sulfonamide crystals in the urinary tract are formed, Substance, does not pose a carcinogenic hazard or risk to humans.

* The publicly available references included above (X) are cited in the original position paper (M-414116-01-1) and can be provided on request.