Registration Dossier

Administrative data

Description of key information

There are several studies available for evaluation of which the 90-day in dogs,rats and mice are the longest in duration.

The lowest NOAEL is derived from the 2 -generation study in rats, resulting to a NOAEL of 65 mg/kgbw/d (based on 1000 ppm in diet) due to transient effects on body weights at 3000 ppm.
NOAEL 90-d rat = 3000 ppm (214 mg/kg/day for males; 248 mg/kg/day for females).
NOAEL 90-d dog = 8000 ppm (260 mg/kg/day for males; 255 mg/kg/day for males).

NOAEL 90-d mice = 2500 ppm (420 mg/kg/day for males; 380 mg/kg/day for females).
NOAEL 90-d rat = 2500 ppm (200 mg/kg/day for males; 210 mg/kg/day for males).

In SIDS dossier, a NOAEL of < 120 mg/kg bw/d is reported from an OECD 422 study.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007 August 3 - 2007 November 30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to EC and OECD guidelines and according to GLP principles.
Qualifier:
according to
Guideline:
OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity in Non-Rodents)
Deviations:
yes
Remarks:
See below
Qualifier:
according to
Guideline:
EU Method B.27 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)
Deviations:
yes
Remarks:
See below
Qualifier:
according to
Guideline:
EPA OPPTS 870.3150 (90-Day Oral Toxicity in Non-rodents)
Deviations:
yes
Remarks:
See below
Principles of method if other than guideline:
Protocol deviations:
1 Temporary deviations from the maximum level of relative humidity occurred. Evaluation: Laboratory historical data do not indicate an effect of the deviations.
2 Diets prepared for use in weeks 1-2 and 3-4 were dried for approximately 2 and 3 days respectively, instead of approximately 24 hours.
Evaluation: Based on food intake and analytical results of diets prepared for use in week 3-4, this was considered to have no adverse effect on the quality of the diets.
3 Inadvertently, no food consumption was determined for animal no. 7 between days 16-17. Evaluation: deviation was of an incidental nature. Sufficient food consumption data were available for evaluation.
4 On the following days, the following dogs were inadvertently separated on the day following their group housing: day 16 (nos. 30 and 31), day 43 (nos. 29-30), day 60 (nos. 27-28) and day 70 (nos. 23 and 24). Evaluation: Food intake data on these days were excluded, since these were considered not to be representative. Sufficient food intake data were available for evaluation.
5 V3286 (treatment) or V3278 (pretest) was used as batch for SSNIFF® Hd Ereich, Extrudat. Evaluation: Both batches were valid at the time of use.
6 Lacrimal glands from animal no. 32 were not available for histopathology. Evaluation: Sufficient data was available for evaluation.
The study integrity was not adversely affected by the deviations.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
dog
Strain:
Beagle
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Marshall BioResources, Green Hill 2001, Via San Zeno, 25018 Montichiari (BS), Italy.
- Age at study initiation: Approximately 5-6 months
- Weight at study initiation: males 7.1 - 9.4 kg; females 6.1 - 8.2 kg
- Fasting period before study: not applicable
- Housing: Animals were housed in stainless steel cages (dimensions 143 x 88 x 106 cm) with a resting shelf (dimensions 61 x 46 cm) and Tenderfoot floors. The animals were group-housed per dose group/sex for at least 3 hours per day, on weekdays only.
- Diet (e.g. ad libitum): Animals were offered the test diet once daily at 0.300 kg/animal/day.
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: Approximately 3 weeks.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.0 - 22.6°C
- Humidity (%): 33 - 97%. Temporary deviations from the maximum level of relative humidity occurred. Laboratory historical data do not indicate an effect of the deviations.
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was dissolved in acetone (± 18-20 ml acetone per kg diet) and then mixed with some powder feed (premix). Subsequently, this premix was mixed with the bulk of the diet. Elix water (approximately 25% in total) was added to aid pelleting. The pellets were dried for approximately 24 hours at 35°C before storage.

DIET PREPARATION
- Rate of preparation of diet (frequency): Diets were prepared every two weeks, or sooner.
- Mixing appropriate amounts with (Type of food): Powder diet for dogs (SSNIFF® Hd Ereich, V3286 (treatment) or V3278 (pretest), Extrudat; SSNIFF® Spezialdiäten GmbH, Soest, Germany).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of the accuracy diet preparations revealed values within the range of 80-99% of nominal, which was considered to represent an acceptable level of accuracy for diets. Diet preparations were prepared homogenously.
Duration of treatment / exposure:
At least 90 days.
Frequency of treatment:
Once daily at 300 g/animal/day
Remarks:
Doses / Concentrations:
1000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
3500 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
8000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
4
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Dose levels were based on the results of a 28-day dietary toxicity study with Beagle dogs and on a palatability study with Beagle dogs. In the palatability study, one male and one female received the test substance at 5000 or 10.000 ppm in the diet for one week per concentration. At 10.000 ppm, food intake was notably reduced (with approximately 60%). At 5000 ppm, food intake levels recovered to normal levels after an initial reduction. No changes in body weight or clinical signs were noted that were considered to be an effect of treatment with the test substance.
In the 28-day study, two Beagle dogs per sex received the test substance at 1000, 3500 or 8000 ppm in diet. The highest dose of 8000 ppm was selected based on the results of the palatability study. At 8000 ppm, one female showed a reduced food intake during most of the treatment period, but variations in food intake were also noted during the pretest phase. This same female showed a reddish discolouration of the duodenum, which would be in line with the irritating properties of the test substance. Body weights appeared unaffected by treatment and no clinical signs of toxicity were observed. Haematology and clinical biochemistry revealed no toxicologically significant changes.
Based on the significantly reduced food intake observed at 10.000 ppm in the palatability study, it was decided in consultation with the sponsor to select 8000 ppm as the highest dose level for the 90-day main study.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes, mortality
- Time schedule: At least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly during pretest and treatment

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Daily during pretest and treatment.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during pretest and week 13
- Dose groups that were examined: All animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during pretest and week 13
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined: White blood cells, Red blood cells, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin,
Mean corpuscular haemoglobin concentration, Platelets, Red blood cell distribution width, Differential leucocyte count (Neutrophils, Lymphocytes, Monocytes, Eosinophils, Basophils), Reticulocytes, Prothrombin time, Activated Partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during pretest and week 13
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined: Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase, Gamma glutamyl transferase, Lactate dehydrogenase, Glutamate dehydrogenase, Total Bilirubin, Glucose, Creatinine, Urea, Total Protein, albumin, Total globulin, Albumin Globulin ratio, Cholesterol, Triglycerides, Phospholipids, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate.

URINALYSIS: Yes
- Time schedule for collection of urine: during pretest and week 13
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: Volume, Specific gravity, Clarity, Colour, pH, Blood, Leucocytes, Bilirubin, Urobilinogen, Protein, Ketones, Glucose, Nitrite
Sediment (Leucocytes, Erythrocytes, Casts, Epithelial cells, Crystals, Bacteria, Other)

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
Organ weights: Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Pituitary gland, Prostate, Spleen, Testes, Thymus, Thyroid with parathyroids, Uterus
GROSS PATHOLOGY: Yes (see table)
HISTOPATHOLOGY: Yes (see table)
Statistics:
The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The exact Fisher-test was applied to frequency data.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
Faeces with red particles were recorded for one male at 8000 ppm (no. 13).
Two males at 3500 ppm (nos. 9 and 11) showed a lean appearance from week 2 and 6 onwards, respectively.

BODY WEIGHT AND WEIGHT GAIN
A reduced body weight gain/weight loss was noted for 3 out of 4 males at 3500 ppm and all males at 8000 ppm, throughout most of the treatment period. Slight weight loss primarily occurred in the first five weeks of treatment. A slightly lower body weight gain/slight weight loss throughout treatment was also recorded for one female at 3500 ppm (no. 26) and one female at 8000 ppm (no. 30).

Body weights of males at 3500 and 8000 ppm were already slightly lower than control levels during the pretest phase. Body weights and body weight gain of other treated groups remained in the same range as controls over the study period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food consumption of males at 8000 ppm was slightly reduced during most of the treatment period (achieving a level of statistical significance on most occasions during the first 6 weeks), but slightly recovered towards control levels as treatment progressed. Food intake among females at 8000 ppm appeared slightly reduced in the first week only, achieving a level of statistical significance. No further differences in food intake of these females could be discerned.

A significant variation in individual food intake values was noted in primarily females. As these variations in food intake were also apparent among control animals, it was considered that this was due to palatability of the pelleted basal diet (derived from a ground, extruded diet).

CLINICAL CHEMISTRY
The following statistically significant changes in clinical biochemistry parameters distinguished treated animals from control animals:
- Decreased total protein levels in males at 8000 ppm,
- Decreased albumin levels in males at 3500 and 8000 ppm,
- Decreased potassium levels in males and females at 8000 ppm,
- Decreased calcium levels in males at 3500 and 8000 ppm,
- Increased sodium and chloride levels in females at 8000 ppm (not statistically significant for sodium levels),
- Increased inorganic phosphate levels in females at 8000 ppm.

GROSS PATHOLOGY
Necropsy findings distinguishing treated dogs from control dogs were essentially confined to the stomach and included:
- Irregular surface of the pylorus (2/4 females at 1000 ppm and 2/4 females at 8000 ppm), or cardia (1/4 females at 8000 ppm),
- Reddish foci on the pylorus and/or cardia (1/4 females at 1000 ppm and 1/4 females at 8000 ppm).
In addition, one male at 3500 ppm (no. 9) showed an emaciated appearance along with a reduced size of the prostate and thymus.

HISTOPATHOLOGY: NON-NEOPLASTIC
In the stomach, congestion was seen in one female at 1000 and 8000 ppm, correlating to the reddish foci at necropsy. In two females at 1000 and 8000 ppm, the lymphoid follicles in the stomach correlated to the irregular surface recorded at necropsy. These microscopic findings were however within background pathology, and hence were not clearly related to the necropsy findings.

Minor grades of diffuse hypertrophy of the zona fasciculata of the adrenals were recorded in 3/4 males at 1000 ppm) and 3/4 males at 3500 ppm, and in 3/4 males and 3/4 females at 8000 ppm (minimal-slight). This finding at these grades is a not uncommon non-specific response to stress in dogs and was considered to be a secondary physiological adaptive change of no toxicological significance.
Dose descriptor:
NOAEL
Effect level:
8 000 ppm
Sex:
male/female
Basis for effect level:
other: No toxicologically significant effects observed.
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 260 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: No toxicologically significant effects observed.
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 255 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: No toxicologically significant effects observed.
Critical effects observed:
not specified

Mean test article intake over the study period was as follows:

group

Nominal dietary inclusion level [ppm]

Average intake [mg test substance/kg body weight/day]

(range indicated within brackets)

males

females

2

1000

30

(27-35)

36

(30-43)

3

3500

133

(128-139)

114

(107-125)

4

8000

260

(229-275)

255

(229-278)

Conclusions:
NOAEL: 8000 ppm (260 mg/kg/day for males, or 255 mg/kg/day for females).
Executive summary:

Title

90-Day oral toxicity study with p-TSA by dietary administration in male and female Beagle dogs.

Guidelines

The study was based on the following guidelines:

- OECD 409, “Repeated Dose 90-day Oral Toxicity Study in Non-Rodents”, 1998.

- EC Directive 87/302/EEC, B.27: “90-days repeated Oral Dose Study using Non-rodent species”, 1988.

- OPPTS 870.3150, 90-day oral toxicity in nonrodents. Office of Prevention, Pesticides and Toxic Substances (7101), EPA 712-C-98-200, August 1998.

Rationale for dose levels

Dose levels were based on the results of a 28-day dietary toxicity study with Beagle dogs and on a palatability study with Beagle dogs. In the palatability study, one male and one female received the test substance at 5000 or 10.000 ppm in the diet for one week per concentration. At 10.000 ppm, food intake was notably reduced (with approximately 60%). At 5000 ppm, food intake levels recovered to normal levels after an initial reduction. No changes in body weight or clinical signs were noted that were considered to be an effect of treatment with the test substance.

In the 28-day study, two Beagle dogs per sex received the test substance at 1000, 3500 or 8000 ppm in diet. The highest dose of 8000 ppm was selected based on the results of the palatability study. At 8000 ppm, one female showed a reduced food intake during most of the treatment period, but variations in food intake were also noted during the pretest phase. This same female showed a reddish discolouration of the duodenum, which would be in line with the irritating properties of the test substance. Body weights appeared unaffected by treatment and no clinical signs of toxicity were observed. Haematology and clinical biochemistry revealed no toxicologically significant changes.

Based on the significantly reduced food intake observed at 10.000 ppm in the palatability study, it was decided in consultation with the sponsor to select 8000 ppm as the highest dose level for the 90-day main study.

Study outline

Beagle dogs received the test substance by dietary intake for at least 90 days. One control group and three treated groups were tested, each consisting of 4 males and 4 females.

Evaluated parameters

Chemical analysis of prepared diets was conducted to assess homogeneity and accuracy of preparations.

The following parameters were evaluated: clinical signs (daily), body weight (weekly), food consumption (daily), ophthalmoscopic examination (at pretest and end of treatment), clinical pathology (pretest and end of treatment), macroscopy and organ weights at termination, and histopathology on selected tissues.

Results

Dietary analyses confirmed that diets were prepared accurately and homogenously.

Mean test article intake over the study period was as follows:

group

Nominal dietary inclusion level [ppm]

Average intake [mg test substance/kg body weight/day]

(range indicated within brackets)

males

females

2

1000

30

(27-35)

36

(30-43)

3

3500

133

(128-139)

114

(107-125)

4

8000

260

(229-275)

255

(229-278)

Treatment at 3500 and 8000 ppm resulted in a reduced weight gain (or slight weight loss on some occasions), primarily among males. Food intake at 8000 ppm was also reduced, again primarily among males.

Blood analyses revealed a number of changes at 3500 and 8000 ppm including lower total protein, albumin, potassium and calcium levels, and increased sodium, chloride and inorganic phosphate levels. In the absence of any signs of specific target organ toxicity, these changes were considered to have occurred secondary to the lower body weight/food intake.

Necropsy showed stomach effects in a few animals, consisting of an irregular surface of the pylorus (two females at 1000 and 8000 ppm each), or cardia (one female at 8000 ppm), or reddish foci on the pylorus and/or cardia (one female at 1000 and 8000 ppm each). No treatment-related supportive histopathological changes were observed. Also, since the incidence of these findings was not clearly related to the dose, it was considered that there was no adverse effect on the functional integrity of the stomach.

Faeces with red particles were recorded for one male at 8000 ppm. No correlating macro- or microscopic findings were noted to support this finding. Considering its very incidental nature (on a single day throughout treatment), this was considered not indicative of toxicity.

No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. ophthalmoscopy, haematological investigations, urinalysis, organ weights and histopathology).

Conclusion

In the absence of adverse effects on the gastro-intestinal tract or any other organ system, the lower food intake (and lower body weight gain/weight loss) was considered to be related to palatability of the test diet, rather than being indicative of primary systemic toxicity. Therefore, a No Observed Adverse Effect Level (NOAEL) for p-TSA of 8000 ppm was established (corresponding to 260 and 255 mg p-TSA/kg/day for males and females respectively).

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2006 October 25 - 2007 January 30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to EC and OECD guidelines and according to GLP principles.
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
See below.
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
See below.
Principles of method if other than guideline:
Deviations:
1. One adrenal from animal no. 67 and from animal no. 4 was not available for histopathology. Reasons for this included that these tissues were not discernable at necropsy or trimming, or were erroneously not collected at necropsy. Tissues are listed in raw data and pathology report. Evaluation: Sufficient tissues were available for evaluation.
2. No terminal body weight was determined from animal no. 55, and no adrenal weight was recorded for animal no. 4. Evaluation: Sufficient data were available for evaluation.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Wistar Han, Crl:WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: Approximately 6 weeks
- Housing: 5 animals/sex in Macrolon cages with sterilised saw dust as bedding material and paper as cage-enrichment.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.0-23.2
- Humidity (%): 35-87
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): diets were prepared for weeks 1-4, 5-8 and 9-13.
- Mixing appropriate amounts with (Type of food): Standard powder rodent diet

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations analysed in the diets were between 83 and 113% of target. In the control diet, no test substance was detected.
Duration of treatment / exposure:
At least 90 days.
Frequency of treatment:
Ad libitum
Remarks:
Doses / Concentrations:
1000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
3000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
10000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Based on results of a 28-day range finding study
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes, mortality
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

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
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pre-test and week 13
- Dose groups that were examined: all animals, animals from groups 1 and 4, respectively

HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 13
- Anaesthetic used for blood collection: Yes, iso-flurane
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined: Erythrocytes count, haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelet count, Red cell distribution width, Total leucocytes count, Differential leucocyte count, Prothrombin time, Partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 13
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined: Alanine aminotransferase, Alkaline phosphatase, Aspartate aminotransferase, Bilirubin, total, Chloride, Cholesterol, total, Creatinine, Glucose, Phosphorus, Protein, total, Protein, albumin, Urea, Calcium, Potassium, Sodium.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: week 12-13
- Dose groups that were examined: all
- Battery of functions tested: grip strength / motor activity / other: hearing ability, pupillary reflex, static righting reflex.
Sacrifice and pathology:
Organ weights: Adrenal glands, Ovaries, Brain, Spleen, Epididymides, Testes, Heart, Thymus, Kidneys, Uterus, Liver.
GROSS PATHOLOGY: Yes (see table)
HISTOPATHOLOGY: Yes (see table)
Other examinations:
None.
Statistics:
None.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
BODY WEIGHT AND WEIGHT GAIN
Body weights and body weight gain were reduced in males and females at 10.000 ppm, achieving a level of statistical significance on most occasions. Total weight gain deficit over the treatment period compared to controls was 21% and 11% for males and females, respectively.
Body weights and body weight gain of animals at 1000 or 3000 ppm remained in the same range as controls over the study period.

HISTOPATHOLOGY: NON-NEOPLASTIC
In 2/10 males at 10.000 ppm, a minimal degree of hyperplasia of the urothelium of the urinary bladder was recorded.

Dose descriptor:
NOAEL
Effect level:
3 000 ppm
Basis for effect level:
other: Reduced body weight gain, histopathology (minimal urethelial hyperplasia in two animals)
Key result
Dose descriptor:
NOAEL
Effect level:
214 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: Reduced body weight gain, histopathology (minimal urethelial hyperplasia in two animals)
Key result
Dose descriptor:
NOAEL
Effect level:
248 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: Reduced body weight gain
Dose descriptor:
LOAEL
Effect level:
10 000 ppm
Basis for effect level:
other: Reduced body weight gain, histopathology (minimal urethelial hyperplasia in two animals)
Dose descriptor:
LOAEL
Effect level:
738 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: Reduced body weight gain, histopathology (minimal urethelial hyperplasia in two animals)
Dose descriptor:
LOAEL
Effect level:
795 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: Reduced body weight gain.
Critical effects observed:
not specified
Conclusions:
LOAEL: 10.000 ppm (738 mg/kg/day for males, or 795 mg/kg/day for females).
NOAEL: 3000 ppm (214 mg/kg/day for males, or 248 mg/kg/day for females).
Executive summary:

The study was based on the following guidelines.

- EC Directive 67/548/EEC, B Repeated Dose (90 days) Toxicity (oral), 2001.

- OECD 408, Repeated Dose 90-day Oral Toxicity Study in Rodents, 1998.

- EPA 712-C-98-199, 90-Day Oral Toxicity, 1998.

Based on a 28-day dietary range finding study (NOTOX Project 474053) and in consultation with the sponsor, the dose levels for this 90-day dietary study were selected to be 0, 1000, 3000 and 10.000 ppm. SPF-bred Wistar Han rats received the test substance by dietary intake for at least 90 days. One control group and three treated groups were tested, each consisting of 10 males and 10 females.

Chemical analyses of diet preparations were conducted to assess accuracy and homogeneity of diet preparations.

The following parameters were evaluated: Clinical signs (daily), functional observations (week 13), body weight and food consumption (weekly), and ophthalmoscopy (pretest and week 13). At termination: clinical pathology, macroscopy, organ weights and histopathology on a selection of tissues.

RESULTS

Homogeneity and accuracy of diet preparations were demonstrated by analyses.

Dietary inclusion levels of 0, 1000, 3000 and 10.000 ppm were equivalent to an average test article intake of 0, 70, 214 and 738 mg/kg body weight/day for males, and 0, 80, 248 and 795 mg/kg body weight/day for females.

At 10.000 ppm, a significant and consistent reduction of body weight gain was recorded (up to 21% compared to control animals).

Histopathology revealed a minimal degree of hyperplasia of the urothelium of the urinary bladder in two males at 10.000 ppm.

No other treatment-related toxicologically significant changes were noted in any of the remaining parameters examined/determined in this study (i.e. clinical appearance, functional observations, food consumption, clinical laboratory investigations, macroscopic examination and organ weights).

The observed minimal urethelial hyperplasia in two animals seems to be caused by local irritation by the chronic availability of p-TSA metabolite (p-sulphamonylbenzoic acid) in the urine, following the rapid and complete excretion of p-TSA. Such effect is local and fully reversible when exposure ends. As the hyperplasia is observed in only two animals, is minimal and likely a fully reversible temporal effect upon exposure, is not an adverse effect persé, the reduction of the body weight gain of 21% in males is considered serious enough to assign the 10000 ppm a LOAEL. Especially as a relation to palatability is not completely clear.

LOAEL: 10.000 ppm (738 mg/kg/day for males, or 795 mg/kg/day for females).

NOAEL: 3000 ppm (214 mg/kg/day for males, or 248 mg/kg/day for females).

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2006 October 19 - 2007 July 02
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study performed according to OECD and EU guidelines and according to GLP principles. The two-generation study design is of similar duration as the 90-day study, but lacks some parameters parameters that are commonly evaluated in repeated dose studies.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: (P) 5-6 wks; (F1) 3 wks
- Housing: Pre-mating: Animals were housed in groups of 4 animals/sex/cage in Macrolon plastic cages.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages
Post-mating: Males were housed in groups of 4 animals/sex/cage in Macrolon plastic cages. Females were individually housed in Macrolon plastic cages.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.6-24.2
- Humidity (%): 27-95
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Diets were prepared for one week during the first week of study and every two weeks from Week 2 of study onwards. For the last weeks of the study, diets were prepared for a maximum of 36 days.
- Mixing appropriate amounts with Standard powder rodent diet
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Sampling and analysis of diet preparations were performed on four occasions (week 1, 11, 22 and 33) according to the following scheme:
Group 1: accuracy (middle position of container)
Group 2: accuracy and homogeneity (top, middle and bottom position of container)
Group 3: accuracy (middle position of container)
Group 4: accuracy and homogeneity (top, middle and bottom position of container)
Duration of treatment / exposure:
F0-generation: 10 weeks prior to mating and continuing until euthanasia.
F1-generation (F0-offspring): The F1-generation was potentially exposed to the test substance in utero, through nursing during lactation and directly following weaning. After weaning, pups were treated for a minimum of 10 weeks prior to mating and continuing until euthanasia.
F2-generation (F1-offspring): The F2-generation was potentially exposed to the test substance in utero and through nursing during lactation.
Frequency of treatment:
Ad libitum
Remarks:
Doses / Concentrations:
0, 1000, 3000 and 10000
Basis:
nominal in diet
No. of animals per sex per dose:
24
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: based on results of a 28-day toxicity study in Wistar Han rats at dose levels of 0, 1000, 5000 and 25000 ppm.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Details on results:
BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
ORGAN WEIGHTS (PARENTAL ANIMALS)
Males P generation:
At 3000 ppm:
Decreased body weights and body weight gain. Terminal body weights at necropsy (92% of control) were decreased which resulted in relative organ weight changes for the brain and kidneys (105% and 110% of control, respectively).
At 10000 ppm:
Decreased body weights and body weight gain. Terminal body weights at necropsy (91% of control) were decreased which resulted in relative organ weight changes for the brain, liver, kidneys and seminal vesicles (108%, 108%, 118% and 116% of control, respectively).

Females P generation:
At 3000 ppm:
Decreased body weights and body weight gain. Terminal body weights at necropsy (94% of control) were decreased which resulted in relative organ weight changes for the brain (107% of control).
At 10000 ppm:
Decreased body weights and body weight gain. Terminal body weights at necropsy (91% of control) were decreased which resulted in relative organ weight changes for the brain, kidneys, adrenals and spleen (109%, 121%, 112% and 110% of control, respectively).

Males F1 generation:
At 3000 ppm:
Decreased body weights and body weight gain. Terminal body weights at necropsy (93% of control) were decreased which resulted in absolute organ weight changes for the spleen (90% of control) and relative organ weight changes for the brain (108% of control).
At 10000 ppm:
Decreased body weights and body weight gain. Terminal body weights at necropsy (87% of control) were decreased which resulted in absolute organ weight changes for the brain, liver, and spleen (95%, 89% and 89% of control, respectively), and relative organ weight changes for the brain, kidneys, seminal vesicles, testes and prostate (108%, 111%, 124%, 109% and 118% of control, respectively).

Females F1 generation:
At 3000 ppm:
Decreased body weights, body weight gain and terminal body weights at necropsy (93% of control).
At 10000 ppm:
Decreased body weights and body weight gain. Decreased food consumption during the last week of pregnancy. Terminal body weights at necropsy (85% of control) were decreased which resulted in absolute organ weight changes for the brain (93% of control) and relative organ weight changes for the brain, kidneys, adrenals, spleen and pituitary gland (110%, 118%, 112%, 113% and 120% of control, respectively).

BODY WEIGHT (OFFSPRING)
ORGAN WEIGHTS (OFFSPRING)
Male pups F1 generation:
At 10000 ppm:
Lower body weights for pups (terminal body weight at necropsy was 85% of control) which resulted in absolute organ weight changes for the brain, spleen and thymus (95%, 78% and 74% of control, respectively), relative organ weight changes for the brain (111% of control), and a delay in balanopreputial separation (108% of control).

Female pups F1 generation:
At 10000 ppm:
Lower body weights for pups (terminal body weight at necropsy was 84% of control) which resulted in absolute organ weight changes for the brain, spleen and thymus (94%, 78% and 73% of control, respectively), relative organ weight changes for the brain (112% of control), and a delay in vaginal opening (114% of control).

Male pups F2 generation:
At 10000 ppm:
Lower body weights for pups (terminal body weight at necropsy was 81% of control) which resulted in absolute organ weight changes for the brain, spleen and thymus (95%, 75% and 70% of control, respectively) and relative organ weight changes for the brain (117% of control).

Female pups F2 generation:
At 10000 ppm:
Lower body weights for pups (terminal body weight at necropsy was 82% of control) which resulted in absolute organ weight changes for the spleen and thymus (76% and 73% of control, respectively) and relative organ weight changes for the brain and thymus (118% and 90% of control, respectively).
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 ppm
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
body weight and weight gain
other: Corresponds to 52-78 mg/kg bw/day for males and 75-161 mg/kg bw/day for females.
Remarks on result:
other: 1000 ppm based on decreased body weights and body weight gain from 3000 ppm and higher
Critical effects observed:
not specified

Table 1. Mean test article intake when corrected for mean value of recovery

Nominal dose

1000 ppm

3000 ppm

10000 ppm

Mean value of recovery

930 ppm

2820 ppm

9600 ppm

MALES F0

Pre mating

75

236

787

Post mating

52

165

568

FEMALES F0

Premating

85

264

862

Postcoitum

75

238

739

Lactation

161

499

1631

MALES F1

Premating

78

237

832

Post mating

53

165

566

FEMALES F1

Premating

86

264

887

Postcoitum

75

232

733

Lactation

160

487

1582

Table 2. Reproductive toxicity

Dose(ppm)

0

1000

3000

10000

dr

m

f

m

f

m

f

m

f

F0animals

Mortality

no treatment-related findings

Clinical signs

no treatment-related findings

Body weight gain

dc

dc

dc

dc

mf

Food consumption

no treatment-related findings

Mating/fertility/gestation

no treatment-related findings

Oestrus cycle

no treatment-related findings

Sperm evaluation

no treatment-related findings

Organ weight

- terminal body weight

dc(94%)

dc(92%)

dc(91%)

dc(91%)

- brain

icr

(107%)

icr

(105%)

icr

(109%)

icr

(108%)

- liver

icr

(108%)

- kidneys

icr

(110%)

icr

(121%)

icr

(118%)

f

- adrenals

icr

(112%)

- spleen

icr

(110%)

- seminal vesicles

icr

(116%)

Pathology

Macroscopy

no treatment-related findings

Microscopy

no treatment-related findings

F1pups

Litter size

no treatment-related findings

Post implantation loss

no treatment-related findings

Live birth index

no treatment-related findings

Viability index

no treatment-related findings

Lactation index

no treatment-related findings

Sex ratio

no treatment-related findings

Clinical signs

no treatment-related findings

Body weight

dc

dc

Sexual maturation

ic

ic

Pup development

no treatment-related findings

Organ weight

- terminal body weight

dc(84%)

dc(85%)

- brain

dca(94%)

icr

(112%)

dca(95%)

icr

(111%)

- spleen

dca(78%)

dca(78%)

- thymus

dca(73%)

dca(74%)

Pathology

Macroscopy

no treatment-related findings

Microscopy

no treatment-related findings

F1animals

Mortality

no treatment-related findings

Clinical signs

no treatment-related findings

Body weight gain

dc

dc

dc

dc

mf

Food consumption

dc

Mating/fertility/gestation

no treatment-related findings

Oestrus cycle

no treatment-related findings

Sperm evaluation

no treatment-related findings

Organ weight

- terminal body weight

dc(93%)

dc(93%)

dc(87%)

dc(85%)

mf

- brain

icr(108%)

dca(95%)

icr(108%)

dca(93%)

icr(110%)

- pituitary

icr(120%)

- liver

dca(89%)

- kidneys

icr(111%)

icr(118%)

- adrenals

icr(112%)

- spleen

dca(90%)

dca(89%)

icr(113%)

- testes

icr(109%)

- prostate

icr(118%)

- seminal vesicles

icr(124%)

Pathology

Macroscopy

no treatment-related findings

Microscopy

no treatment-related findings

F2pups

Litter size

no treatment-related findings

Post implantation loss

no treatment-related findings

Live birth index

no treatment-related findings

Viability index

no treatment-related findings

Lactation index

no treatment-related findings

Sex ratio

no treatment-related findings

Clinical signs

no treatment-related findings

Body weight

dc

dc

Pup development

no treatment-related findings

Auditory and visual response

not performed

Organ weight

- terminal body weight

dc(81%)

dc(82%)

- brain

dca(95%)

icr

(117%)

icr

(118%)

- spleen

dca(75%)

dca(76%)

- thymus

dca(70%)

dca(73%)

dcr

(90%)

 Pathology

no treatment-related findings

dc/ic    statistically significantly decreased/increased compared to the controls

a/r       absolute/relative organ weight

(%)      relative to control

dr        dose-related

Conclusions:
The definitive parental NOAEL was established as being 1000 ppm based on decreased body weights and body weight gain from 3000 ppm and higher.
When corrected for mean test article intake the NOAEL of 1000 ppm corresponds to 52 -78 mg/kg bw/day for males and 75-161 mg/kg bw/day for females.
Executive summary:

Full evaluation see 7.8.1 Toxicity to reproduction

A two-generation reproduction toxicity study of p-TSA in rats by dietary administration.

The study was based on the following guidelines:

- OECD 416, Two-Generation Reproduction Toxicity Study, January 2001.

- OPPTS 870.3800, Reproduction and Fertility Effects, August 1998.

- EC, Commission directive 2004/73/EC, B.35:"Two-generation reproduction toxicity study", April 2004.

 

After acclimatisation, male and female Wistar rats of the Fe-generation were exposed by dietary inclusion to graduated doses of the test substance. The dose levels for the F0-generation and for the F1-generation were 1000, 3000 and 10000 ppm.

At regular intervals, prepared diets were analysed for accuracy of preparation and homogeneity.

F0-males and F0-females were exposed to the test substance 10 weeks prior to mating and exposure ended at termination. F0-females were allowed to produce and rear a litter until day 21 of lactation. On day 4 of lactation litters were reduced in size to eight pups by random culling of F1-pups. After weaning, one F1-male and one F1-female of each litter were selected for cross mating with a pup of another litter of the same dose group to produce a F2-generation. Mating commenced at least 10 weeks after weaning. F1-offspring selected for mating were dosed from weaning until they were killed after weaning of the F2-offspring on day 21 of lactation. On day 4 of lactation a selection of F2-pups was culled. Pups of the F2-offspring were killed shortly after weaning.

 

All animals were subjected to daily clinical observation. Body weight and food consumption were measured over the treatment period. The regularity and duration of the estrous cycle was examined. At necropsy, macroscopic observations and organ weights were recorded. Sperm morphology, motility and count were assessed. A histopathological examination was performed on all reproduction organs and tissues. Reproduction parameters, breeding data and pup development were assessed. Blood samples were collection from F1 females (10/group) for possible measurement of thyroid hormones. These samples were discarded without analyses.

 

RESULTS

Chemical analysis revealed that the diets were prepared properly and were considered to be homogeneous.

 

The following changes were considered to be related to treatment:

F0-GENERATION

at 1000 ppm (group 2):

•      No treatment-related findings.

 at 3000 ppm (group 3):

•      Decreased body weights and body weight gain.

Terminal body weights at necropsy were decreased which resulted in relative organ weight changes for the brain and kidneys.

at 10000 ppm (group 4):

•      Decreased body weights and body weight gain.

Terminal body weights at necropsy were decreased which resulted in relative organ weight changes for the brain, liver, kidneys, seminal vesicles, adrenals, and spleen.

Lower body weights for male and female pups which resulted in organ weight changes for the brain, spleen and thymus and a delay in vaginal opening and balanopreputial separation.

 

F1-GENERATION

At 1000ppm (group2):

•      No treatment-related findings.

At 3000ppm (group3):

•      Decreased body weights and body weight gain.

Terminal body weights at necropsy were decreased which resulted in organ weight changes for the brain and spleen.

At 10000ppm (group4):

•      Decreased body weights and body weight gain.

Decreased food consumption for females during the last week of pregnancy.

 

Terminal body weights at necropsy were decreased which resulted in organ weight changes for the brain, liver, kidneys, seminal vesicles, adrenals, spleen, testes, prostate, and pituitary gland. Lower body weights for male and female pups which resulted in organ weight changes for the brain, spleen and thymus.

 

CONCLUSION

Treatment with p-TSA by dietary inclusion in male and female Wistar rats at dose levels of 1000, 3000 and 10000 ppm revealed Fo-and F1-parental toxicity at 3000 and 10000 ppm and developmental toxicity (related to decreased body weights of dams) at 10000 ppm.

Reproduction and breeding parameters were unaffected for both generations for treatment up to 10000 ppm.

 

Based on these findings, the definitive parental No Observed Adverse Effect Level (NOAEL) was established as being 1000 ppm.

When corrected for mean test article intake the NOAEL of 1000 ppm corresponds to 52 -78 mg/kg bw/day for males and 75-161mg/kg bw/day for females.

Endpoint:
repeated dose toxicity: oral, other
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Jul 2006 - Jan 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
1998, September 21
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: p-Toluenesulfonamide obtained from Acros Organics (Geel, Belgium) in one lot (A009615201). Lot A009615201 was purified by Battelle’s Organic Synthesis Group (
Columbus, OH) and was renamed lot 112003. Lot 112003 was used in the 2-week and 3-month studies.
- Purity test date: not specified

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: under a headspace of inert gas at room temperature, protected from light
- Stability under test conditions: stable

OTHER SPECIFICS: The dose formulations were prepared once during the 2-week studies and eight times during the 3-month studies. A premix was prepared by hand by mixing p-toluenesulfonamide with
feed and then blended with additional feed in a Patterson-Kelly twin-shell blender. Formulations were stored in doubled polyethylene bags sealed with twist ties protected from light at room temperature for up
to 42 days.
Species:
rat
Strain:
other: B6C3F1/N; F344/N (rats 2-week) / F344/NTac (rats 3-month)
Details on species / strain selection:
The rationale for change of rat strain from F344/N to F344/NTac was a programmatic decision. For many years, the NTP used the inbred F344/N rat for its toxicity and carcinogenicity studies. Over a period of time, the F344/N rat exhibited sporadic seizures and idiopathic chylothorax, and consistently high rates of mononuclear cell leukemia and testicular neoplasia. Because of these issues in the F344/N rat and the NTP’s desire to find a more fecund rat model that could be used in both reproductive and carcinogenesis studies for comparative purposes, a change in the rat model was explored.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: 2-week studies: Male and female F344/N rats were obtained from the NTP colony maintained at Taconic Farms, Inc. (Germantown, NY). 3-Month studies: male and female F344/
NTac rats were obtained from the commercial colony at Taconic Farms, Inc.
- Age at study initiation: 2-week studies: F344/N rats were 5 weeks old. 3-Month studies: F344/NTac rats were 5 to 6 weeks old.
- Weight at study initiation: See annex
- Housing: Five per cage. Cages: Polycarbonate (Lab Products, Inc., Seaford, DE), changed twice per week for F344/N rats. Bedding: Irradiated heat-treated Sani-Chips® hardwood chips (P.J. Murphy Forest Products Corp., Montville, NJ), changed at least twice per week. Cage filters: Omnishield Paper, Remay 2024 (Dupont), Harlan Teklad, (Indianapolis, IN), changed every 2 weeks. Racks: Stainless steel (Lab Products, Inc., Seaford, DE); changed every 2 weeks.
- Diet (e.g. ad libitum): ad libitum, Irradiated NTP-2000 open formula meal diet (Zeigler Brothers, Inc.,Gardners, PA).
- Water (e.g. ad libitum): ad libitum, Tap water
- Acclimation period: 12-17 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72° ± 3° F
- Humidity (%): 50% ± 15%
- Air changes (per hr): at least 10/hour
- Photoperiod (hrs dark / hrs light):12 hours/day
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): once during the 2-week studies and eight times during the 3-month studies
- Mixing appropriate amounts with (Type of food): mixing p-toluenesulfonamide with feed. A premix was prepared by hand and then blended with additional feed in a Patterson-Kelly twin-shell blender.
- Storage temperature of food: Formulations were stored in doubled polyethylene bags sealed with twist ties protected from light at room temperature for up to 42 days.

OTHER: Dates of exposure: 2-week studies Rats: Jul 5 - Jul 19 2006. 3-month studies Rats: Oct 17 (males) or 18 (females), 2006 to Jan 16 (males) or 17 (females), 2007
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Periodic analyses of the dose formulations of p-toluenesulfonamide were conducted by the study laboratory using HPLC/UV.
Duration of treatment / exposure:
2 week studies: 15 days
3 months studies: Core study F344/NTac rats: 14 weeks, Clinical pathology study F344/NTac rats: 22 days
Frequency of treatment:
5 days per week
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Male, 3 month studies
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Males, 3-month studies
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Males, 3-month studies
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
Males, 3-month studies
Dose / conc.:
380 mg/kg bw/day (nominal)
Remarks:
Males, 3-month studies
Dose / conc.:
725 mg/kg bw/day (nominal)
Remarks:
Males, 3-month studies
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Females, 3-month studies
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
Females, 3-month studies
Dose / conc.:
110 mg/kg bw/day (nominal)
Remarks:
Females, 3-month studies
Dose / conc.:
210 mg/kg bw/day (nominal)
Remarks:
Females, 3-month studies
Dose / conc.:
400 mg/kg bw/day (nominal)
Remarks:
Females, 3-month studies
Dose / conc.:
780 mg/kg bw/day (nominal)
Remarks:
Females, 3-month studies
No. of animals per sex per dose:
2 week studies: 5 animals per sex per dose
3 month studies: 10 animals per sex per dose
Control animals:
yes, plain diet
Details on study design:
In the 2-week studies, groups of five male and five female rats were fed diets containing 0, 750, 1,500, 3,000, 10,000, or 30,000 ppm p-toluenesulfonamide (equivalent to average daily doses of approximately 95, 185, 370, 1,170, or 3,135 mg p-toluenesulfonamide/kg body weight to male F344/N rats, 80, 170, 335, 1,050, or 2,645 mg/kg to female F344/N rats. In the 3-month studies, groups of 10 male and 10 female F344/NTac rats and mice were fed diets containing, 0, 625, 1,250, 2,500, 5,000, or 10,000 ppm p-toluenesulfonamide (equivalent to average daily doses of approximately 50, 100, 200, 380, or 725 mg/kg to male F344/NTac rats, 30, 110, 210, 400, or 780 mg/kg to female F344/NTac rats.
Positive control:
No
Observations and examinations performed and frequency:
2-week studies:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Initially, day 8 and at end of studies

BODY WEIGHT: Yes
- Time schedule for examinations: Initially, day 8 and at end of study

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal recorded on day 8 and at end of studies.

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

3-month studies:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Initially, day 8, weekly thereafter and at end of studies

BODY WEIGHT: Yes / No / Not specified
- Time schedule for examinations:

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal recorded on day 8, weekly thereafter and at end of studies.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was collected from the retroorbital sinus of clinical pathology F344/NTac rats on days 3 and 22 and from core study animals at the end of the studies for h
ematology and clinical chemistry (F344/NTac rats only).
- Anaesthetic used for blood collection: Yes, a 70%:30% CO2:O2 mixture
- Animals fasted: No
- Parameters checked : Hematocrit; hemoglobin; erythrocyte, reticulocyte, and platelet counts; mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; and leukocyte counts and diffe
rentials.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was collected from the retroorbital sinus of clinical pathology F344/NTac rats on days 3 and 22 and from core study animals at the end of the studies for hemat
ology and clinical chemistry (F344/NTac rats only).
- Animals fasted: No
- Parameters checked: Urea nitrogen, creatinine, total protein, albumin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, and bile acids
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Sacrifice and pathology:
2 –week studies:
GROSS PATHOLOGY: Yes 2 week studies: Necropsies were performed on all animals. 3 month studies: Necropsies were performed on all core study animals. Organs weighed were heart, right kidney, liver, lu
ng, right testis, and thymus.
HISTOPATHOLOGY: Yes. Histopathology was performed on 0, 10,000, and 30,000 ppm F344/N rats. In addition to gross lesions and tissue masses the heart, right kidney, liver, lung, right testis, and thymus were examined.

3-month studies:
GROSS PATHOLOGY: Yes. Necropsies were performed on all core study animals. Organs weighed were heart, right kidney, liver, lung, right testis, and thymus.
HISTOPATHOLOGY: Yes. Complete histopathology was performed on 0 and 10,000 ppm core study F344/NTac rats. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, eyes, Harderian gland, heart and aorta, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung and mainstem bronchi, lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, seminal vesicle, skin, spleen, stomach (forestomach and glandular), testis with epididymis, thymus, thyroid gland, tongue, trachea, urinary bladder, and uterus.
Other examinations:
Sperm Motility and Vaginal Cytology: At the end of the 3-month studies, spermatid and sperm samples were collected from male animals in the 0, 2,500, 5,000, and 10,000 ppm groups. The following param
eters were evaluated: spermatid heads per testis and per gram testis, sperm motility, and sperm per cauda epididymis and per gram cauda epididymis. The left cauda, left epididymis, and left testis were weighed. Vaginal samples were collected for up to 12 consecutive days prior to the end of the studies from females exposed to 0, 2,500, 5,000, or 10,000 ppm for vaginal cytology evaluations.
Statistics:
The Fisher exact test (Gart et al., 1979), a procedure based on the overall proportion of affected animals,
was used to determine significance of the incidences of nonneoplastic lesions. Continuous variables,
Organ and body weight data, which historically have approximately normal distributions, were anal
yzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972)
. Hematology, clinical chemistry, spermatid, and epididymal spermatozoal data, which have typically
skewed distributions, were analyzed using the nonparametric multiple comparison methods of Shirley
(1977) (as modified by Williams, 1986) and Dunn (1964). Jonckheere’s test (Jonckheere, 1954) was
used to assess the significance of the dose-related trends and to determine whether a trend-sensitive
test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not
assume a monotonic dose-related trend (Dunnett’s or Dunn’s test).
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
2-WEEK STUDY IN F344/N RATS
Final mean body weights and mean body weight gains of 10,000 and 30,000 ppm males and 30,000 ppm females were significantly less than those of the controls; the mean body weight gain of 10,000 ppm f
emales was significantly less than that of the controls.

3-MONTH STUDY IN F344/NTAC RATS
The final mean body weights and the mean body weight gains of 10,000 ppm males and females were significantly less than those of the controls; in addition, the mean body weight gains of 2,500 ppm males and 5,000 ppm males and females were significantly decreased.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
2-WEEK STUDY IN F344/N RATS
Feed consumption by 10,000 and 30,000 ppm males and 30,000 ppm females was less than that by the controls throughout the study.

3-MONTH STUDY IN F344/NTAC RATS
Feed consumption by 5,000 ppm males and 10,000 ppm males and females was less than that by the controls early in the study but generally recovered to near control values later in the study.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
3-MONTH STUDY IN F344/NTAC RATS
F344/NTac rats, no clinical pathology changes detected statistically for male or female F344/NTac rats were considered biologically significant or toxicologically relevant to the administration of p-toluenesulfon
amide.
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
2-WEEK STUDY IN F344/N RATS
The absolute kidney weights were decreased in 30,000 ppm males by approximately 19%; the relative kidney weights were increased in 3,000 ppm or greater males and in 10,000 and 30,000 ppm females (Ta ble C1). There were no histologic findings that correlated with these organ weights changes. Other organ weight changes in male F344/N rats included decreases in the absolute heart, liver, and thymus weights at 30,000 ppm; the absolute lung weights at 1,500 ppm or greater; and the relative lung weight at 1,500 ppm (Table C1). The relative testis weight of 30,000 ppm males was increased. Absolute heart and liver weights were decreased in 30,000 ppm female rats.

3-MONTH STUDY IN F344/NTAC RATS
Absolute and relative thymus weights of 10,000 ppm males were significantly less by approximately 22% compared to those of the controls (Tables 4 and C2). Relative kidney weights were increased in 2,500 ppm or greater males, and were up to approximately 14% greater than the controls in the 10,000 ppm group. Corresponding histologic lesions were not observed in the kidney or thymus. The mean absolute heart weight of 10,000 ppm females was decreased by approximately 8%, which was attributed to as imilar decrease in mean body weight of that group compared to controls
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
See: Organ weight findings
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 210 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Conclusions:
Under the conditions of this study, when p-toluenesulfonamide was administered in the feed at concentrations up to 725 mg/kg bw to male rats and at concentrations up to 780 mg/kg bw to female rats. There was no evidence for treatment-related mortality or treatment-related lesions.
Executive summary:

In this 2-week and 90-day repeated exposure study, groups of male and female rats top-toluenesulfonamide by the oral route 5 days per week for 3 months. There were 10 rodents in each dose group. Doses for the 2-week studies were 0, 750, 1500, 3000, 10000 or 30000 parts per million (ppm) in feed. This approximately correspond to 0, 95, 185, 370, 1170 or 3135 mg p-toluenesulfonamide/kg body weight to male rats, and 0, 80, 170, 335, 1050 or 2645 mg/kg to female rats. Doses for the 3-month studies were 0, 625, 1,250, 2,500, 5,000, or 10,000 parts per million (ppm) in feed. These doses approximately correspond to 0, 50, 100, 200, 380 or 725 mg p-toluenesulfonamide/kg body weight to male mice, and 0, 30, 110, 210, 400 or 780 mg/kg to female mice. Groups of rats that received untreated feed alone served as the control group for dose groups that were exposed to 625 to 10,000 ppmp-toluenesulfonamide in feed. Samples collected were subject to hematological and clinical chemistry analysis and sperm mortality and vaginal cytology were assessed (3-month studies). Gross pathology and histopathology were performed for the presence of any lesions that might be signs of disease. There were no treatment-related clinical findings or effects on survival. The final mean body weights of the treated groups were within 10% of the final mean body weights in the control group. There were no treatment-related lesions in male or female rats. There were no treatment-related findings from the clinical chemistry or genetic toxicology evaluations. There were some changes in organ weights in the treated groups, and it is uncertain if this would result in any treatment-related effects after longer exposures.

Endpoint:
repeated dose toxicity: oral, other
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Jul 2006 - Jan 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
1998, September 21
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: p-Toluenesulfonamide obtained from Acros Organics (Geel, Belgium) in one lot (A009615201). Lot A009615201 was purified by Battelle’s Organic Synthesis Group (Columbus, OH) and was renamed lot 112003. Lot 112003 was used in the 2-week and 3-month studies.
- Purity test date: not specified

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: under a headspace of inert gas at room temperature, protected from light
- Stability under test conditions: stable

OTHER SPECIFICS: The dose formulations were prepared once during the 2-week studies and eight times during the 3-month studies. A premix was prepared by hand by mixing p-toluenesulfonamide with feed and then blended with additional feed in a Patterson-Kelly twin-shell blender. Formulations were stored in doubled polyethylene bags sealed with twist ties protected from light at room temperature for up to 42 days.
Species:
mouse
Strain:
other: B6C3F1/N
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: 2-week studies: Male and female B6C3F1/N mice were obtained from the NTP colony maintained at Taconic Farms, Inc. (Germantown, NY). 3-Month studies: male and female B6C3F1/N mice were obtained from the NTP colony maintained at Taconic Farms, Inc.
- Age at study initiation: 2-week studies: mice were 5 to 6 weeks old. 3-Month studies: mice were 7 to 8 weeks old.
- Weight at study initiation: See annex
- Housing: Five per cage; male mice were housed individually. Cages: Polycarbonate (Lab Products, Inc., Seaford, DE), changed twice per week for female mice and once weekly for male mice. bedding: Irradiated heat-treated Sani-Chips® hardwood chips (P.J. Murphy Forest Products Corp., Montville, NJ), changed at least twice per week for F344/N rats and female mice and once weekly for male mice. Cage filters: Omnishield Paper, Remay 2024 (Dupont), Harlan Teklad, (Indianapolis, IN), changed every 2 weeks. Racks: Stainless steel (Lab Products, Inc., Seaford, DE); changed every 2 weeks.
- Diet (e.g. ad libitum): ad libitum, Irradiated NTP-2000 open formula meal diet (Zeigler Brothers, Inc., Gardners, PA),
- Water (e.g. ad libitum): ad libitum, Tap water
- Acclimation period: 12-17 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72° ± 3° F
- Humidity (%): 50% ± 15%
- Air changes (per hr): at least 10/hour
- Photoperiod (hrs dark / hrs light):12 hours/day
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): once during the 2-week studies and eight times during the 3-month studies
- Mixing appropriate amounts with (Type of food): mixing p-toluenesulfonamide with feed. A premix was prepared by hand and then blended with additional feed in a Patterson-Kelly twin-shell blender.
- Storage temperature of food: Formulations were stored in doubled polyethylene bags sealed with twist ties protected from light at room temperature for up to 42 days.

OTHER: Dates of exposure: 2-week studies Mice: Jul 6 - Jul 20, 2006. 3-month studies Mice: Oct 19 (males) or 20 (females), 2006 to Jan18 (males) or 19 (females), 2007.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Periodic analyses of the dose formulations of p-toluenesulfonamide were conducted by the study laboratory using HPLC/UV.
Duration of treatment / exposure:
2 week studies: 15 days
3 months studies: 14 weeks
Frequency of treatment:
5 days per week
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Males, 3-month studies
Dose / conc.:
120 mg/kg bw/day (nominal)
Remarks:
Males, 3-month studies
Dose / conc.:
230 mg/kg bw/day (nominal)
Remarks:
Males, 3-month studies
Dose / conc.:
420 mg/kg bw/day (nominal)
Remarks:
Males, 3-month studies
Dose / conc.:
770 mg/kg bw/day (nominal)
Remarks:
Males, 3-month studies
Dose / conc.:
1 760 mg/kg bw/day (nominal)
Remarks:
Males, 3-month studies
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Females, 3-month studies
Dose / conc.:
90 mg/kg bw/day (nominal)
Remarks:
Females, 3-month studies
Dose / conc.:
210 mg/kg bw/day (nominal)
Remarks:
Females, 3-month studies
Dose / conc.:
380 mg/kg bw/day (nominal)
Remarks:
Females, 3-month studies
Dose / conc.:
780 mg/kg bw/day (nominal)
Remarks:
Females, 3-month studies
Dose / conc.:
1 890 mg/kg bw/day (nominal)
Remarks:
Females, 3-month studies
No. of animals per sex per dose:
2 week studies: 5 animals per sex per dose
3 month studies: 10 animals per sex per dose
Control animals:
yes, plain diet
Details on study design:
Additional information on doses:
In the 2-week dose range finding studies, groups of five male and five female mice were fed diets containing 0, 750, 1,500, 3,000, 10,000, or 30,000 ppm p-toluenesulfonamide (equivalent to average daily doses of approximately 0, 150, 300, 700, 2,035, or 7,690 mg p-toluenesulfonamide/kg body weight to male mice, and 0, 125, 280, 635, 2,410, or 6,000 mg/kg to female mice.

In the 3-month studies, groups of 10 male and 10 female mice were fed diets containing, 0, 625, 1,250, 2,500, 5,000, or 10,000 ppm p-toluenesulfonamide (equivalent to average daily doses of approximately 0, 120, 230, 420, 770, or 1,760 mg p-toluenesulfonamide/kg body weight to male mice, and 0, 90, 210, 380, 780, or 1,890 mg/kg to female mice.
Positive control:
no
Observations and examinations performed and frequency:
2-week studies:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Initially, day 8 and at end of studies

BODY WEIGHT: Yes
- Time schedule for examinations: Initially, day 8 and at end of study

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal recorded on day 8 and at end of studies.

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

3-month studies:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Initially, day 8, weekly thereafter and at end of studies

BODY WEIGHT: Yes / No / Not specified
- Time schedule for examinations:

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal recorded on day 8, weekly thereafter and at end of studies.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was collected at the end of the studies for hematology.
- Anaesthetic used for blood collection: Yes, a 70%:30% CO2:O2 mixture
- Animals fasted: No
- Parameters checked : Hematocrit; hemoglobin; erythrocyte, reticulocyte, and platelet counts; mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; and leukocyte counts and differentials.

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
2 –week studies:
GROSS PATHOLOGY: Yes 2 week studies: Necropsies were performed on all animals. 3 month studies: Necropsies were performed on all core study animals. Organs weighed were heart, right kidney, liver, lung, right testis, and thymus.

HISTOPATHOLOGY: Yes. Histopathology was performed on 0, 10,000, and 30,000 ppm F344/N rats and mice. In addition to gross lesions and tissue masses the heart, right kidney, liver, lung, right testis, and thymus were examined; the right kidney was also examined in 1,500 and 3,000 ppm female mice.

3-month studies:
GROSS PATHOLOGY: Yes. Necropsies were performed on all core study animals. Organs weighed were heart, right kidney, liver, lung, right testis, and thymus.

HISTOPATHOLOGY: Yes. Complete histopathology was performed on 0 and 10,000 ppm core study F344/NTac rats and mice. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, eyes, gallbladder (mice), Harderian gland, heart and aorta, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, larynx (mice), liver, lung and mainstem bronchi, lymph nodes (mandibular and mesenteric), mammary gland, nose, pharynx (mice), ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, seminal vesicle, skin, spleen, stomach (forestomach and glandular), testis with epididymis, thymus, thyroid gland, tongue, trachea, urinary bladder, and uterus.
Other examinations:
Sperm Motility and Vaginal Cytology: At the end of the 3-month studies, spermatid and sperm samples were collected from male animals in the 0, 2,500, 5,000, and 10,000 ppm groups. The following parameters were evaluated: spermatid heads per testis and per gram testis, sperm motility, and sperm per cauda epididymis and per gram cauda epididymis. The left cauda, left epididymis, and left testis were weighed. Vaginal samples were collected for up to 12 consecutive days prior to the end of the studies from females exposed to 0, 2,500, 5,000, or 10,000 ppm for vaginal cytology evaluations.
Statistics:
The Fisher exact test (Gart et al., 1979), a procedure based on the overall proportion of affected animals, was used to determine significance of the incidences of nonneoplastic lesions. Continuous variables, Organ and body weight data, which historically have approximately normal distributions, were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972). Hematology, clinical chemistry, spermatid, and epididymal spermatozoal data, which have typically skewed distributions, were analyzed using the nonparametric multiple comparison methods of Shirley (1977) (as modified by Williams, 1986) and Dunn (1964). Jonckheere’s test (Jonckheere, 1954) was used to assess the significance of the dose-related trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett’s or Dunn’s test).
Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
3-MONTH STUDY IN MICE: One 10,000 ppm female mouse died during week 6 of an accidental death.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
2-WEEK STUDY IN MICE
Final mean body weights and mean body weight gains of 30,000 ppm males and females were significantly less than those of the controls; these groups lost weight during the study. The mean body weight gains of all remaining exposed groups of females were significantly less than those of the controls.

3-MONTH STUDY IN MICE
The mean body weight gains of 5,000 and 10,000 ppm males were significantly less than those of the controls; the final mean body weight and mean body weight gain of 1,250 ppm females were significantly greater than those of the controls.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
2-WEEK STUDY IN MICE
Feed consumption by exposed groups of mice was generally similar to that by the controls throughout the study.

3-MONTH STUDY IN MICE
Feed consumption by 625 and 1,250 ppm males was greater than that by the controls early in the study but returned to near control values later in the study; feed consumption by exposed groups of females was generally similar to that by the controls throughout the study.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
3-MONTH STUDY IN MICE
No changes attributable to the administration of p-toluenesulfonamide occurred in the hematology data for male or female mice.
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
2-WEEK STUDY IN MICE
Absolute kidney weights of 10,000 and 30,000 ppm females were increased by approximately 15% and 8%, respectively, compared to those of the controls (Table C3). Relative kidney weights were increased in 1,500 ppm or greater females and in 10,000 and 30,000 ppm males compared to those in the controls. The 30,000 ppm males and females had mean body weight decreases of approximately 14% and 15%, respectively, compared to those of the controls. There were no histological findings that correlated with these changes in kidney weights. Other changes in organ weights included increases in relative heart weights of 10,000 ppm females and 30,000 ppm males and females; an increase in the relative lung weight of 30,000 ppm males; an increase in the relative testis weight of 30,000 ppm males; a decrease in the absolute liver weight of 30,000 ppm females; and decreases in the absolute thymus weights of 30,000 ppm males and females and relative thymus weight of 30,000 ppm males

3-MONTH STUDY IN MICE
Female mice exposed to 10,000 ppm had increased absolute (approximately 13%) and relative (approximately 14%) kidney weights compared to those of the controls (Tables 7 and C4). Relative lung weight was increased by approximately 27% in 10,000 ppm males, and relative liver weight was increased by approximately 10% in 10,000 ppm females.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
See: Organ weight findings
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 420 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 380 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
Conclusions:
Under the conditions of this study, when p-toluenesulfonamide was administered in the feed at concentrations up to 1760 mg/kg bw to male mice and at concentrations up to 1890 mg/kg bw to female mice. There was no evidence for treatment-related mortality or treatment-related lesions.
Executive summary:

In this 2-week and 90-day repeated exposure study, groups of male and female rats and mice top-toluenesulfonamide by the oral route 5 days per week for 3 months. There were 10 rodents in each dose group. Doses for the 2-week studies were 0, 750, 1500, 3000, 10000 or 30000 parts per million (ppm) in feed. this approximately correspond to 0, 150, 300, 700, 2,035, or 7,690 mg p-toluenesulfonamide/kg body weight to male mice, and 0, 125, 280, 635, 2,410, or 6,000 mg/kg to female mice. Doses for the 3-month studies were 0, 625, 1,250, 2,500, 5,000, or 10,000 parts per million (ppm) in feed. These doses approximately correspond to 0, 120, 230, 420, 770, or 1,760 mg p-toluenesulfonamide/kg body weight to male mice, and 0, 90, 210, 380, 780, or 1,890 mg/kg to female mice. Groups of mice that received untreated feed alone served as the control group for dose groups that were exposed to 625 to 10,000 ppmp-toluenesulfonamide in feed. Samples collected were subject to hematological analysis and sperm mortality and vaginal cytology were assessed (3-month studies). Gross pathology and histopathology were performed for the presence of any lesions that might be signs of disease. There were no treatment-related clinical findings or effects on survival. The final mean body weights of the treated groups were within 10% of the final mean body weights in the control group. There were no treatment-related lesions in male or female mice. There were no treatment-related findings from the clinical chemistry or genetic toxicology evaluations. There were some changes in organ weights in the treated groups, and it is uncertain if this would result in any treatment-related effects after longer exposures.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
65 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Multiple high quality studies showing a consistent patterns of effects.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Repeated dose – by oral route

Available data:

Repeated dose toxicity: oral-90d-rat, 2007

Following a 28-day range finding study a 90-day oral study in rat with dosing by dietary admixture (OECD 408) was performed, applying dose levels of 0, 1000, 3000 and 10.000 ppm were, equivalent to an average intake of 0, 70, 214 and 738 mg/kg body weight/day for males, and 0, 80, 248 and 795 mg/kg body weight/day for females.

Results: At 10.000 ppm, a significant and consistent reduction of body weight gain was recorded (up to 21% compared to control animals). Histopathology revealed a minimal degree of hyperplasia of the urothelium of the urinary bladder in two males at 10.000 ppm.

No other treatment-related toxicologically significant changes were noted in any of the remaining parameters examined/determined in this study (i.e. clinical appearance, functional observations, food consumption, clinical laboratory investigations, macroscopic examination and organ weights).

 

The observed minimal urethelial hyperplasia in two animals seems to be caused by local irritation by the chronic of p-TSA metabolite (p-sulphamonylbenzoic acid) in the urine, following the rapid and complete excretion of p-TSA. Such effect is local and fully reversible when exposure ends. As the hyperplasia is observed in only two animals, is minimal and likely a fully reversible temporal effect upon exposure, it is not considered an adverse effect persé. The reduction of the body weight gain of 21% in males is considered serious enough to assign the 10000 ppm a LOAEL, especially as a relation to palatability is not completely clear.

LOAEL: 10.000 ppm (738 mg/kg/day for males, or 795 mg/kg/day for females).

NOAEL: 3000 ppm (214 mg/kg/day for males, or 248 mg/kg/day for females).

 

Repeated dose toxicity: oral-90d-dog, 2008

Additionally a 90-day oral repeated dose toxicity study was performed in a second species. p-TSA was dose by dietary administration in groups of four male and four female Beagle dogs at levels of 0, 1000, 3500 and 8000 ppm in the diet, equivalent to an average intake of 0, 30, 133 and 260 mg/kg body weight/day for males, and 0, 36, 114 and 255 mg/kg body weight/day for females.

Results: Treatment at 3500 and 8000 ppm resulted in a reduced weight gain (or slight weight loss on some occasions), primarily among males. Food intake at 8000 ppm was also reduced, again primarily among males.

Blood analyses revealed a number of changes at 3500 and 8000 ppm including lower total protein, albumin, potassium and calcium levels, and increased sodium, chloride and inorganic phosphate levels. In the absence of any signs of specific target organ toxicity, these changes were considered to have occurred secondary to the lower body weight/food intake.

Necropsy and histopathological evaluations did not show effects indicative of toxicity. No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. ophthalmoscopy, haematological investigations, urinalysis, organ weights and histopathology).

In the absence of adverse effects on the gastro-intestinal tract or any other organ system, the lower food intake (and lower body weight gain/weight loss) was considered to be related to palatability of the test diet, rather than being indicative of primary systemic toxicity.

NOAEL: 8000 ppm (corresponding to 260 and 255 mg p-TSA/kg/day for males and females respectively).

 

Repeated dose toxicity: oral OECD 422 rat, 1994

Additional information is available from testing performed for OECD HPV evaluation by Japan, and reported in SIDS dossier on p-TSA.

OECD 422, Combined Repeat Dose and Reproductive/Developmental Toxicity Screening Test in rat by gavage under GLP. Dose levels: 0 (vehicle, 5% Arabic gum), 120, 300, 750 mg/kg/day, with 13 animals per dose group per sex. Dosing period: Females, from 14 days before mating to day 3 of lactation. Males 42 days.

750 mg group: decreased BW and food consumption. Also haematuria was observed in 4/13 animals within the first 3 days of dosing.

Decrease in the thymus weight in females (acceleration of involution), and

Slight, but statistically significant, increases in relative kidney and testicular weights were found in the males, and in relative kidney and liver weights in females likely related to the decreased BW.

Dark-coloured liver in 6/13 males at gross pathological evaluation

Increased WBC in males with increase in the proportion of neutrophils. Also in males increased BUN, ASAT, ALAT, chloride and potassium levels.

Histopathology showed thickened urinary bladder epithelium in 11/13 males, and 7/13 females.

300 mg: Decreased BW and food consumption females during gestation and lactation

Decrease in the thymus weight in females (acceleration of involution)

Increased WBC in males with increase in the proportion of neutrophils, and increased BUN, ASAT and chloride.

Histopathology showed thickened urinary bladder epithelium in 11/13 males, and 12/13 females.

120 mg: Only reportable effect observed is thickened urinary bladder epithelium in 6/13 males and 1/13 females at histopathological examination. Although this effect does not seem to show a clear dose response relation, it was not observed in any of the control animals. Therefore the NOEL was set at < 120 mg/kg bw/day.

Information available on benzenesulfonamides supports the common mechanism of action for toxicity to urinary tract observed in repeated dose studies with these compounds. The effect is indirect mediated by the appearence of calcium phosphate calculi caused by an indueced alkaline urine.

 

 

Additional relevant data from reproduction studies reported in this dossier:

2 -generation reproduction toxicity: OECD 416 rat, 2007

Animals were dosed 0, 1000, 3000 and 10,000 ppm in the diet. F0-parents were dose for 10 weeks prior to mating and continuing during mating until euthanasia. The F1-generation was potentially exposed to the test substance in utero, through nursing during lactation and directly following weaning, and then treated for a minimum of 10 weeks prior to mating and continuing until euthanasia.

Only observed effects at 3000 ppm consisted of decreased and body weight gain. In F0-parents, terminal body weights at necropsy were decreased (92% of control) which resulted in relative organ weight changes for the brain and kidneys (105% and 110% of control, respectively). In the F1-generation, terminal body weights at necropsy were also decreased (94% of control) which resulted in relative organ weight changes for the brain (107% of control).

Consequently, the NOAEL was set at 1000 ppm (corresponding to 52-78 mg/kg bw/day for males and 75-161mg/kg bw/day for females) based on decreased body weights and body weight gain, and due to this organ weight changes in both F0 and F1 parental animals.

 

Developmental toxicity: OECD 414 rabbit, 2008

Developmental toxicity was evaluated in an OECD 414 study in in which rabbits were dosed by dietary exposure to 0, 1000, 3000 and 11000 ppm (equivalent to 0, 41, 113 and 367 mg/kg body weight/day respectively) from days 7 to 29post-coitum.

Based on the effects on body weight, food and water consumption, the maternal NOAEL for p-TSA was established as being 3000 ppm (114 mg/kg body weight/day), although some transient effects on body weight were noted at 3000 ppm.

 

14-day repeated dose(NTP: http://tools.niehs.nih.gov/ntp_tox/index.cfm)

The National Toxicology Program (NTP) had conducted a 2-week study with p-TSA in rats and mice. Animals were administered the test article at 750, 1500, 3000, 10000 or 30000 ppm. No treatment related adverse effects were reported. A 90-day oral gavage study in rats is currently in progress.

 

 

Evaluation:

Based on the above data the overall NOAEL for systemic toxicity could be set at 231 mg/kg bw/day, representing the 90-day subchronic NOAEL in rat, being the most relevant among the reports of the highest quality (recent guideline, GLP, specifically address repeated dose toxicity).

Of the two available highest quality reports from studies with longest duration (90-days) one in rat and one in dog, the rat studies resulted to the lowest NOAEL of 3000 ppm (concentration in diet involves automatic correction for allometric scaling) equivalent to 214 and 248 mg p-TSA/kg/day for males and females respectively. The data from the rabbit study showing a NOAEL for maternal toxicity of 3000 ppm (114 mg/kgBW/day) is also fully in line. The 2-generation reproduction study is in agreement with these results as the effects reported for 3000 ppm concern effects in body weight which are mainly related to the gestation and lactation period, characterised by higher dietary intakes.

 

The results from the OECD 422 study with a NOEL < 120 mg/kg bw/day following about 43 days based solely on the presence of slight urothelial hyperplasia is not contradictory, but supports this assessment. In both studies in rat the most sensitive endpoint is urethelial hyperplasia is considered to be caused by local irritation by the availability of p-TSA metabolite (p-sulphamonylbenzoic acid) in the urine, following the rapid and complete uptake and excretion of p-TSA. Such effect is local and fully reversible when exposure ends. In the case of the gavage studies the dosing results to higher local concentrations. Supportive to this are the data from NTP on p-TSA, where a 90-day study in rats and mice by dietary administration op to 10,000 ppm were actually without any findings. (Tox-88, not in this dossier)

Although in the OECD 422 study this effect did not seem to show a clear dose response relation, and even at the highest dose did not show a highest severity beyond moderate, it was not observed in any of the control animals, and therefore the NOEL was set at < 120 mg/kg bw/day.

However, as in view of the lack of a clear dose relation in incidence and severity and the limited consequence regarding systemic toxicity 120 mg/kg bw/day can also be considered a NOAEL from this study.

The overall lowest NOAEL is coming from the 2-generation study indicating a NOAEL of 1000 ppm, which based on food intake, corresponds to52-78 mg/kg bw/day for males and 75-161 mg/kg bw/day for females. Based on all the above considerations, the lowest value of 65 mg/kg bw/day (average of males) represents a level that possibly could be considered overcautious, as it also provides a safe level for pregnant females for which the NOAEL actually is higher and developing young animals.

Repeated dose – by inhalation

Vp is (less than) 2.86*10-7 kPa (= 0.0003 Pa) at 20°C, and the respirable fraction (≤ 4 µm) of the crystalline solid is 0 %. In combination of low likelihood of exposures by aerosols from the use of the substance (also water solubility is low: 3.1 g/L at 20°C), the limited local effects, and the quick complete absorption, distribution and excretion which can also be expected for the inhalation route, do not make additional testing by inhalation of high priority.

 

Repeated dose – by dermal route

Substance is of low acute oral toxicity, not requiring classification. Also no classification is required for skin or eye irritation. Information on absorption indicates that p-TSA is rapidly and completely absorbed by oral route, but only 20% by dermal route. Further testing by dermal route is therefore scientifically unjustified.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Of the two available highest quality reports from studies involving the longest duration (90-days) one in rat and one in dog, the rat studies resulted to the lowest NOAEL of 3000 ppm (concentration in diet involves automatic correction for allometric scaling) equivalent to 214 and 248 mg p-TSA/kg/day for males and females respectively. However, a 2-generation study of similar quality indicates some effects of reduced body weight especially in pregnant and lactating animals from 3000 ppm, resulting to a NOAEL of 1000 ppm, equivalent to 52 -78 (average 65) mg/kg bw/day for males and 75-161 mg/kg bw/day for females. Also a recent developmental study mentions transient effects at 3000 ppm, and no effects at 1000 ppm.

Repeated dose toxicity: via oral route - systemic effects (target organ) urogenital: urinary bladder

Justification for classification or non-classification

Available studies do not indicate a need for classification for STOT-RE. p-TSA is of limited toxicity at levels of 100 mg/kg bw/day and below.