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Toxicological information

Repeated dose toxicity: oral

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Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): p-TSA
- Substance type: white crystalline powder
- Physical state: solid
- Analytical purity: 99.9%
- Impurities (identity and concentrations): 0.1% water
- Purity test date: 2006 July 28
- Lot/batch No.: 0603514009
- Expiration date of the lot/batch: 2008 March 31
- Stability under test conditions: Stable in diet for at least 25 days at room temperature
- Storage condition of test material: At room temperature in the dark in tightly closed containers. Keep dry.
- Other: Hygroscopic. Reacts with acids with formation of heat.

Test animals

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

Administration / exposure

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
Doses / concentrationsopen allclose all
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.

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

open allclose all
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.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

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)

Applicant's summary and conclusion

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).