Registration Dossier

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:
June 2020 - January 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
This RSS includes data until Day 93 (end of the treatment period) only.
The data on reverse animals are not available at the mandatory deadline.
A new update of the dossier with complete results of the OECD 408 will be done in the next few months.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
Polysulfides, di-tert-dodecyl
EC Number:
270-335-7
EC Name:
Polysulfides, di-tert-dodecyl
Cas Number:
68425-15-0
Molecular formula:
not applicable
IUPAC Name:
di (alkyl C11-C13 Branched, C12 Rich), polysulfure S3-S5 rich

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Wistar Han, Crl:WI(Glx/BRL/Han) IGS BR.
Details on species / strain selection:
The rat is a rodent species accepted by Regulatory Authorities for this type of study and background data from previous studies are available at the laboratory.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Wistar Han, Crl:WI(Glx/BRL/Han) IGS BR.
- Females (if applicable) nulliparous and non-pregnant: [yes/no] yes
- Age at study initiation: 7 weeks old.
- Weight at study initiation: between 150 and 250 g for the males and between 110 and 190 g for the females.
- Fasting period before study: no
- Housing: roup-housed with a minimum of 2 and a maximum of 3 animals from the same sex and group, in polycarbonate cages with stainless steel lids
- Diet (e.g. ad libitum): free access to SSNIFF rat/mouse pelleted maintenance diet
- Water (e.g. ad libitum): free access to tap water
- Acclimation period: 13 days

DETAILS OF FOOD AND WATER QUALITY:
The batches of diet and sawdust are analyzed by the suppliers for composition and contaminant
levels.
Bacterial and chemical analyses of water are performed regularly by external laboratories. These
analyses include the detection of possible contaminants (pesticides and heavy metals).
It is expected that no contaminants that could interfere with, or prejudice, the outcome of the
study will be found in the diet, drinking water or sawdust.

ENVIRONMENTAL CONDITIONS
¿ temperature : 22 ± 2°C,
¿ relative humidity : 50 ± 20%,
¿ light/dark cycle : 12 h/12 h,
¿ ventilation : approximately 8 to 15 cycles/hour of filtered, non-recycled air.

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route has been selected since it is a route of administration which is requested by the
Regulatory Authorities for this type of test item.
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% (w/v) carboxymethylcellulose aqueous solution
Details on oral exposure:
The dose formulations will be administered by gavage, using a plastic syringe fitted with a plastic gavage tube, once a day, in the morning (treatment may be performed in the afternoon on the day of FOB tests).
Formulation type : suspension in the vehicle
A constant dosage-volume of 5 mL/kg/day will be used.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical method : High Performance Liquid Chromatography with UV detection (HPLC/UV).

Determination of test item concentrations in dose formulations: Once at the beginning of the treatment period, once during the course of the treatment period, and twice at the end of the treatment period (staggered dates for males and females) (total of 4)
Duration of treatment / exposure:
at least 13 weeks.
At the end of the treatment period, the principal animals in each group will be euthanized, except
for the first five group 1 and 4 animals per sex (recovery animals), which will be kept for a
6-week treatment-free period.
Day 1 corresponds to the first day of the treatment period.
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Control group (group 1): 15 males and 15 females
Low dose group (group 2) : 10 males and 10 females
Intermediate dose group (group 3) : 10 males and 10 females
High dose group (group 4): 15 males and 15 females
Control animals:
yes, concurrent vehicle
Details on study design:
The quantity of dose formulation administered to each animal will be adjusted according to the
most recently recorded body weight.
A constant dosage-volume of 5 mL/kg/day will be used.

Rationale for dose level selection
The dose levels have been selected in agreement with the Sponsor, based on the results of a previous 28-day repeated toxicity study (OECD 407), in which the test item was administered daily by oral route (gavage) to male and female Sprague-Dawley rats at dose levels of 50, 250 and 1000 mg/kg/day, followed by a two week recovery period (control and high dose groups). Ptyalism was observed in all animals of both sexes given 1000 mg/kg/day during the treatment period only. The mean food consumption and body weight gain were similar between treated and control animals. No abnormalities of toxicological importance were noted among haematological, blood biochemical, urinalysis, organ weights and pathological parameters.
Since no adverse effect was observed at any dose level, similar dose range was selected for the
present study.
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day before the beginning of the treatment period, at least once a day during the treatment period, after completion of treatment of the last animal in the animal room, at least once a day at approximately the same time of the day during the treatment-free period.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once a week before the beginning of the treatment period, at least once a week during the treatment period, at least once a week during the treatment-free period.
- Observations : Observations will include (but will not be limited to) changes in the skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling, as well as the presence of clonic and tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behavior (e.g. self-mutilation, walking backwards) will also be evaluated.

BODY WEIGHT: Yes
- Time schedule for examinations: at least once a week before the beginning of the treatment period, on the first day of treatment, at least once a week until the end of the study.

FOOD CONSUMPTION : Yes
- Time schedule for examinations: The quantity of food consumed by the animals in each cage will be recorded at least once a week from the first day of treatment until the end of the study.
Food consumption will be calculated per animal and per day. If one of the animals in the same
cage dies, the number of days for which that animal has been present in the cage will be taken
into consideration for the calculation of food consumption.

WATER CONSUMPTION : No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: on all animals, before the beginning of the treatment period, on control and high-dose animals (including recovery animals) on one occasion during Week 13.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during Week 13.
- Anaesthetic used for blood collection: No
- Animals fasted: Yes, for an overnight period of at least 14 hours but not exceeding 18 hours.
- How many animals: all surviving animals
- Parameters checked : erythrocytes, mean cell volume, packed cell volume, hemoglobin, mean cell hemoglobin concentration, mean cell hemoglobin, thrombocytes, leucocytes, differential white cell count with cell morphology, reticulocyte count, prothrombin time.
A blood smear for possible determination of the differential white cell count (with cell
morphology) will be prepared for each animal and stained with May Grünwald Giemsa, then
archived. If the samples are not accepted by the ADVIA 120 Analyzer a microscopic evaluation
will be performed.
A blood smear (stained with cresyl blue) for possible determination of the reticulocyte count will
also be prepared for each animal, then archived. If the samples are not accepted by the
ADVIA 120 Analyzer, a microscopic evaluation will be performed.
Two bone marrow smears will be prepared from the femoral bone (at necropsy) of all animals
euthanized on completion of the treatment or treatment-free period. The bone marrow smears
will be stained with May Grünwald Giemsa.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during Week 13.
- Animals fasted: Yes, for an overnight period of at least 14 hours but not exceeding 18 hours.
- How many animals: all surviving animals
- Parameters checked : - sodium, potassium, chloride, calcium, inorganic phosphorus, glucose, urea, creatinine, total bilirubin, total protein, albumin, albumin/globulin ratio, total cholesterol, HDL, LDL, triglycerides, bile acids, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase.

PLASMA/SERUM HORMONES/LIPIDS: Yes, thyroid hormones.
The levels of the thyroid hormones (T3 and T4) will be determined by LC-MS/MS method and
thyroid stimulating hormone (TSH) will be determined by Luminex MAP® technology for all
surviving animals from control and high dose groups (groups 1 and 4) sacrificed at the end of the
treatment.

URINALYSIS: Yes
- Time schedule for collection of urine: during Week 13
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, for an overnight period of at least 14 hours but not exceeding 18 hours.
- Parameters checked : quantitative parameters (volume, pH, specific gravity), semi-quantitative parameters (proteins, glucose, ketones, bilirubin, nitrites, blood, cytology of sediment) and qualitative parameters (appearance, color).

NEUROBEHAVIOURAL EXAMINATION: Yes
Each animal will be evaluated once between Week 11 to 13 before the daily treatment. This evaluation will include a detailed clinical examination, the assessment of reactivity to manipulation and different stimuli, and motor activity.
The animals will be randomized and all animals will be observed in the cage, in the hand and in
the standard arena.
-Detailed Clinical Observation
The following parameters will be assessed and graded: in the cage “touch escape”, in the hand: fur appearance, salivation, lacrimation, piloerection, exophthalmia, reactivity to handling, pupil size (presence of myosis or mydriasis), and in the standard arena (two-minute recording): grooming, palpebral closure, defecation, urination, tremors, twitches, convulsions, gait, arousal (hypo- and hyper- activity), posture, stereotypy, behavior, breathing, ataxia and hypotonia.
-Reactivity to Manipulation or to Different Stimuli. The following measurements, reflexes and responses will be recorded: touch response, forelimb grip strength, pupil reflex, visual stimulus, auditory startle reflex, tail pinch response, righting reflex, landing foot splay, at the end of observation: rectal temperature.
-Motor Activity. For each animal motor activity will be measured by automated infra-red sensor equipment over a 60-minute period.

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Organ Weight : The body weight of each animal will be recorded before euthanasia at the end of the treatment or treatment-free period. The ratio of organ weight to body weight (recorded immediately before euthanasia) will be calculated.
A complete macroscopic post-mortem examination will be performed on all animals. This will
include examination of the external surfaces, all orifices, the cranial cavity, the external surfaces
of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated
organs and tissues and the neck with its associated organs and tissues.

HISTOPATHOLOGY: Yes, A microscopic examination will be performed on:
¿ all tissues for the control and high-dose animals (groups 1 and 4) euthanized at the end of the treatment period and for all animals that die or are euthanized prematurely,
¿ for all macroscopic lesions from all low- and intermediate-dose animals (groups 2 and 3)
euthanized on completion of the treatment period,
¿ immunostained kidneys from all males from the control and high dose-level groups
euthanized at the end of the treatment period.
Optional endpoint(s):
Estrous cycle :
The estrous cycle stage will be determined from daily vaginal smears during 3 weeks of the
treatment period for all females (including recovery animals).

Sperm parameters :
Sperm sampling will be performed on all surviving animals at the end of the treatment period
(except recovery animals) during euthanasia, after intraperitoneal injection of sodium
pentobarbital.
-Epididymal Sperm Motility (all males, except recovery animals),
-Epididymal Sperm Morphology (for control and high-dose groups, except recovery animals),
-Epididymal Sperm Count (all males, except recovery animals),
-Testicular Sperm (all males, except recovery animals)
Statistics:
Provantis software will be used to perform the statistical analysis of body weight, food
consumption, sperm parameters, motor activity, hematology, coagulation, blood biochemistry,
urinalysis, and thyroid hormone data.
PathData software will be used to perform the statistical analysis of organ weight data (level of
significance of 0.05 or 0.01).

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Ptyalism was transiently observed in 1/15 control females in Week 11, 1/10 females at 100 mg/kg/day from Week 08, 3/10 males at 300 mg/kg/day from Week 07 and in all males and females at 1000 mg/kg/day from Week 2 at the earliest. Although this finding was noted in a single control female, this clinical sign was attributed to the test item. As this clinical sign is commonly observed when a test item is administered by gavage, it was considered not to represent an adverse effect.
Reflux at dosing was noted in 1/15 control males, 2/10 males given 300 mg/kg/day and in 1/15 males and 1/15 females given 1000 mg/kg/day on one to seven occasions. This sign is commonly observed when a test item is administered by gavage, was observed sporadically and was therefore not considered to be test item-related.
The other clinical signs recorded during the study, i.e. alopecia, thin fur, scabs, bent/shortened tail, wound, hunched posture, thin appearance, erected fur and/or fast breathing, were transient, of isolated occurrence, probably resulted from reflux at dosing, observed both in control and test item-treated animals, and/or had no dose-relationship. They were therefore considered to be unrelated to the test item treatment.
Mortality:
no mortality observed
Description (incidence):
No unscheduled deaths occurred during the treatment period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In females, the body weight and body weight evolution were not affected by the test item.
At 1000 mg/kg/day in males, when compared with controls, statistically significant, lower mean body weight gain was recorded throughout the treatment period (-17.5% vs. controls), leading to a statistically significant lower mean body weight from Week 09 (-7.9% vs. controls on Day 57) until the end of the treatment period (-9.8% vs. controls on Day 91, Week 13). The differences in body weight gain were particularly noticeable from Week 06 until the end of the treatment period (Days 36 to 91). This effect was attributed to the test item treatment.
At 300 mg/kg/day, statistically significant lower mean body weight gain (p<0.01) was observed in Week 09 (i.e. between Days 57 and 64). As this variation was sporadic and did not affect the mean body weight, it was considered to be incidental.
Body weight of males given 100 mg/kg/day was not affected.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Mean food consumption was not affected by the test item during the treatment period.
Instances of statistically significantly higher or lower mean food consumption were noted at all dose levels in males (i.e. for the following intervals: Days 1-8, 29-36, 78-85 and 85-91) and in females (i.e. for the following intervals: Days 43-50, 50-57, 57-64 and 78-85). As these differences were poorly dose-related, transient, of opposite trends, resulted from low control values and/or did not correlate with effects on mean body weight/body weight gain, a test item relationship could be considered unlikely.
Food efficiency:
not examined
Description (incidence and severity):
n/a
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
n/a
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No findings were observed at the end of the treatment period.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
When compared with controls, a trend towards a higher mean white blood cell count was noted in males at all dose levels and in females from 300 mg/kg/day. This included a slightly increase in the neutrophil counts of both sexes. As this trend was not statistically significant, poorly dose-related and/or did not correlate with any histopathological findings, it was not considered to represent an adverse effect.
As variations observed in the lymphocyte counts were of opposite trend in males (-9.1 % vs. controls) and females (+26.6 % vs. controls) at the high dose level, these differences were not considered to be test item-related.
There were no test item-related changes in coagulation parameters at any dose level at the end of the treatment period.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
When compared with mean control values, the main blood biochemistry changes were observed at 1000 mg/kg/day as follows:
¿ higher mean urea level in males and females (+12.2% and +19.5%, respectively; not statistically significant) along with lower mean protein level and higher mean A/G ratio in males (-4.4% and +10.4%, respectively; p<0.01) and lower mean protein level in females (-4.2%; not statistically significant).
¿ higher mean low density lipoprotein (LDL) level in in males (+44.3%; p<0.05),
The findings described above were of minimal magnitude and/or within the range of the historical control data and without histopathological correlates. They were therefore not considered to represent adverse effects.
The other statistically significant differences observed between control and test item-treated animals, namely in the potassium level (males at 100 and 300 mg/kg/day), the creatinine level (females from 300 mg/kg/day and males at 1000 mg/kg/day), the biliary acid level and the aspartate aminase activity (males at 1000 mg/kg/day) were considered to be incidental and not test item-related based on the direction of the variation (i.e. creatinine level) and/or as they were noted with no dose-relationship, resulted from isolated individual values, were not associated with any other blood biochemistry parameter changes (i.e. aspartate aminotransferase activity) and/or were without any link to microscopic findings.
Endocrine findings:
effects observed, non-treatment-related
Description (incidence and severity):
thyroid hormone :
At 1000 mg/kg/day and when compared with controls, there was an increase in mean TSH concentration (+40.5% vs. controls) along with decrease in mean T4 concentrations (-12.2% vs. controls). As these differences were of minimal magnitude, not statistically significant, were not associated with mean T3 level or effects at pathology, as there were high intervariabilitly and/or no correlates between individual TSH and T4 values, and/or as individual values were within or near the control range values, the relationship with the test item is unlikely.
The thyroid hormone levels (i.e. T3, T4 and TSH) were considered to be unaffected by the test item treatment in females at 1000 mg/kg/day although increased TSH level was recorded (+13.4%; not statistically significant).
Based on the slight magnitude of this variation, the absence of statistical significance and absence of association with T3 or T4 variations, and/or as this was mainly due to one isolated female (female 4509: 2264 pg/mL), this finding was considered to be incidental.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
When compared with controls, increased mean urine volumes were recorded in both sexes at all dose levels and a statistically significant, lower mean pH value was observed in males at 1000 mg/kg/day (-13.8%; p<0.05). As these differences were of low magnitude and/or not statistically significant, poorly dose-related, did not impact the specific gravity, were within the range of the Historical Control Data and/or and did not correlate with any with any adverse histopathological findings, they were not considered to represent an adverse effect.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no test item-related effects on FOB tests or motor activity in any male or female group at the end of the treatment period.
At 1000 mg/kg/day and when compared with controls, higher mean numbers of horizontal movements and rearing were noted in males (+37% and +32%, respectively). As this difference was of slight magnitude, not statistically significant and did not correlate with any other findings, it was not considered to be test item-related.
Higher (males at 100 mg/kg/day) or lower (females at 300 mg/kg/day) mean numbers of horizontal movements were noted in other groups. These findings were not considered to be test item-related as they were not dose-related, did not correlate with rearing and/or as values remained within the standard deviation of control values.
Urination was noted in 9/10 males at 1000 mg/kg/day versus 4/10 control males. In view of the incidence and as this correlated with a higher, but not statistically significant, mean urine volume (see § Urinalysis), a test item-related effect could not be ruled out. As the grading of urination was of slight magnitude and did not correspond to any adverse findings, this finding was not considered to represent an adverse effect.
Differences from controls in fur appearance were noted in isolated males at all dose-levels. Differences from controls in landing foot splay were noted in males and females at 1000 mg/kg/day (93 mm and 94 mm vs. 83 mm and 85 mm in control males and females, respectively). In view of the very slight severity and incidence or magnitude, in the absence of correlating clinical signs during the study, and/or in the absence of a dose-relationship at lower dose levels, these findings were considered to be unrelated to the test item treatment.
Immunological findings:
not examined
Description (incidence and severity):
n/a
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
No organ weight changes occurred that were considered to be related to TPS 32 administration.
The statistically significant changes in relative (to body weight) organ weights for the adrenal glands, brain, kidneys, liver, spleen, and testes in males at 1000 mg/kg/day were considered to be a reflection of reductions in terminal body weight
(-17% when compared with controls) and not due to test-item-related organ toxicity. There were no microscopic correlates.
Other organ weight changes were not considered to be related to the test item as they were small in amplitude, had no gross or microscopic correlates, were not dose-related in magnitude, and/or were not consistent for the sexes.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No macroscopic findings were observed that were considered to be related to TPS 32 administration.
In one male at 1000 mg/kg/day (4007), a yellow mass correlating microscopically with a malignant tumor (adenocarcinoma) was observed in the prostate. Although prostatic adenocarcinoma is rare in rats, particularly at this age (Creasy et al., 2012; Bosland and Prinsen 1990), this isolated finding was considered to be most probably incidental and unrelated to TPS 32 administration in the absence of pre-neoplastic or neoplastic change in the prostate of other males of this group.
The other macroscopic findings had no histologic correlates or correlated with common histologic findings in control rats, and were considered to be incidental.
Neuropathological findings:
not examined
Description (incidence and severity):
n/a
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Administration of TPS 32 at 1000 mg/kg/day for 13 weeks induced increased cellularity and aggregates of macrophages within the mesenteric lymph node. This finding was not considered to be adverse in the absence of macroscopic correlates and microscopic degenerative changes.
TPS 32 administration induced hyaline droplet nephropathy at 1000 mg/kg/day in males only. This was characterized by the presence of dense eosinophilic droplets in proximal tubular epithelium. These hyaline droplets, occasionally seen in untreated male rats, are consistent with a2u globulin and are known to increase after treatment with a wide range of drugs or chemicals (Greaves, 2012). Although human excrete proteins of a similar nature, they are found in only trace amounts and therefore this finding was considered to be non-relevant for human. This finding was not considered to be adverse in the current study in the absence of associated degenerative features.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
n/a
Other effects:
no effects observed
Description (incidence and severity):
Estrous cycle :
There were no test item-related effects on mean estrous cycle length or mean number of cycles during the treatment period.
When compared with controls, a statistically significant lower mean number of cycles (p<0.05) and a higher mean cycle length (p<0.01) were noted in females at 300 mg/kg/day. As these differences were not dose-related, they were considered to be incidental.

Sperm parameters :
No test item-related effects were noted in testicular sperm count, epididymal sperm count, motility or morphology.
Details on results:
Chemical analyses of the dose formulations
The test item concentrations in the administered dose formulations analyzed in Weeks 1, 7 and 13 remained within the acceptable range of ± 10% of the nominal concentrations except for the low dose level in Week 07 (-12%).
No test item was observed in the control dose formulation.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg diet
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Daily administration of TPS32 by oral gavage for 13 weeks to Wistar Han rats at 10, 300 or 1000 mg/kg/day was clinically well tolerated. No adverse effects were observed up to 1000 mg/kg/day
Executive summary:

The objective of this study was to evaluate the potential toxicity of the test item, TPS 32, following daily oral administration (gavage) to rats for 13 weeks. On completion of the treatment period, designated animals were held for a 6-week treatment-free period in order to evaluate the reversibility of any findings.


One group of 15 male and 15 female Wistar Han rats was treated daily by the oral route (gavage) with the test item, TPS32 (batch No. J03LB58B10), at the dose level of 1000 mg/kg/day (group 4) for 13 weeks (i.e. 91 days for females and 92 days for males). Two other groups of 10 males and 10 females were treated with the test item at the dose level of 100 or 300 mg/kg/day (groups 2 and 3, respectively). One control group of 15 males and 15 females received the vehicle only (corn oil) under the same experimental conditions and acted as a control group (group 1).
The test item was administered as a solution in the vehicle under a constant dosage volume of 5 mL/kg/day.
At the end of the treatment period, the animals were euthanized, except for the first five group 1 and 4 animals per sex, which are kept for a 6-week treatment-free period.


Test item concentrations in dose formulations were determined once at the beginning, during and at the end of the treatment period. The dose formulations were administered by gavage, using a plastic syringe fitted with a plastic gavage tube, once a day, in the morning, except on the day of FOB tests when treatment was performed in the afternoon.
The quantity of dose formulation administered to each animal was adjusted according to the most recently recorded body weight. The dose formulations were maintained under delivery conditions (at room temperature and protected from light) throughout the administration procedure. The control and test item dose formulations were stirred just before administration, maintained under continuous magnetic stirring throughout the administration procedure and administered within 4 hours after the end of their preparation.
Each animal was checked for mortality and morbidity once a day during the acclimation period and at least once a day during the treatment period, including weekends and public holidays.


Each animal was observed once a day before the beginning of the treatment period and at least once a day during the treatment period, after completion of treatment of the last animal. Detailed clinical examinations were performed on all animals once a week before the beginning of the treatment period and then at least once a week until the end of the study.
Each animal (except recovery animals) was evaluated for functional observation battery (FOB) once in Week 12 before the daily treatment.
The body weight of each animal was recorded at least once a week before the beginning of the treatment period, on Day 1 and then once a week until the end of the treatment period.
The quantity of food consumed by the animals in each cage was recorded once a week from the first day of treatment until the end of the treatment period. Food consumption was calculated per animal and per day.
Ophthalmological examinations were performed on all animals before the beginning of the treatment period and on control and high-dose animals (including recovery animals) on one occasion during Week 13.
The estrous cycle stage was determined from daily vaginal smears for 3 weeks of the treatment period in all females (including recovery animals).


Clinical pathology parameters (hematology, coagulation, blood biochemistry and urinalysis) were investigated for all animals (except recovery animals) on completion of the treatment period.
Blood samples for thyroid hormone levels (T3, T4 and TSH) were collected from group 1 and 4 animals (except recovery animals) at the end of the treatment period.
The body weight of each animal was recorded before euthanasia at the end of the treatment period.
Organs were weighed wet as soon as possible after dissection. The ratio of organ weight to body weight (recorded immediately before euthanasia) was calculated.
A complete macroscopic post-mortem examination was performed on all animals (except recovery animals). This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues.
Sperm sampling for the determination of sperm parameters was performed on all animals during euthanasia at necropsy (except recovery animals).
Microscopic examination was performed on selected organs as follows: all tissues listed in the Tissue Procedure Table for the control and high-dose animals (groups 1 and 4) euthanized at the end of the treatment period, all macroscopic lesions from all low- and intermediate-dose animals (groups 2 and 3) euthanized on completion of the treatment period, on immunostained kidneys in all males from the control and high dose groups euthanized at the end of the treatment period.


Daily administration of TPS32 by oral gavage for 13 weeks to Wistar Han rats at 10, 300 or 1000 mg/kg/day was clinically well tolerated.


At the end of the treatment period, the test item induced non-adverse changes in body weight and body weight gain in males at 1000 mg/kg/day, hematological parameters (i.e. white blood cell count along with neutrophil count) in males at all dose levels and in females from 300 mg/kg/day, blood biochemistry parameters (i.e. urea, protein and LDL levels and/or A/G ratio) in males and females at 1000 mg/kg/day and urinalysis parameters (volume and/or decreased pH value) in both sexes at all dose levels and at microscopic examination at 1000 mg/kg/day (increased cellularity and aggregates of macrophages within the mesenteric lymph node in both sexes and hyaline droplet nephropathy along with α2u globulin in males only).