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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:
4 August 2020 - 28 December 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
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:
June 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:
Polysulfides, di-tert-dodecyl

Test animals

Species:
rat
Strain:
other: Crl:WI(Glx/BRL/Han) IGS BR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, l’Arbresle, France
- Females nulliparous and non-pregnant: Not reported
- Age at study initiation: 7 to 8 weeks old
- Weight at study initiation: 177.0 g to 219.6 g (males); 128.8 g to 182.1 g (females)
- Fasting period before blood sampling: No (except of blood collection on the day of necropsy)
- Housing: The animals were housed by two or three from the same sex and group, in polycarbonate cages with stainless steel lids containing autoclaved sawdust. Each cage contained rat hut and nylabone for environmental enrichment.
- Diet: SSNIFF rat/mouse pelleted maintenance diet (SSNIFF Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water (filtered with a 0.22 µm filter), ad libitum
- Contaminant analysis: No contaminants were present in the diet, drinking water or sawdust at levels which could have been expected to interfere with, or prejudice, the outcome of the study.
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS (set to maintain)
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: From: 4 August 2020 To: 28 December 2020

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
A plastic syringe fitted with a plastic gavage tube was used.
The oral route was selected since it is a route of administration which is requested by the Regulatory Authorities for this type of test item.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item dose formulations were prepared on the eve or on the days of treatment. Control dose formulation were prepared according to the vehicle expiry date. Control and test item formulations were delivered at room temperature and protected from light.
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 (As the stability of the test item dose formulations was determined to be = 24 hours, the 4-hour stability period was not used. Therefore, test item dose formulations were also prepared on the eve of the days of treatment instead of on the days of treatment only).

DOSAGE VOLUME:
- 5 mL/kg/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Once at the beginning of the treatment period, once during the course of the treatment period, and once at the end of the treatment period. Charles River Laboratories Den Bosch was informed prior to
study start in which weeks of the study concentration of formulation samples were analyzed. Accurately weighted formulation samples (~ 500 mg) were collected from control and test item dose formulations and were kept frozen at -20°C and protected from light until analyzed by Charles River Laboratories Den Bosch under the Test Facility Study No. 20259510, using High Performance Liquid Chromatography with ELSD detection (HPLC/ELSD).

Acceptance criterion:
Measured concentration = nominal concentration ± 10%
Duration of treatment / exposure:
91 days for females and 92 days for males followed by a six week recovery period (treatment-free period) for 5 animals/sex of the control and high dose groups.
Frequency of treatment:
Once 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:
Vehicle control group: 15 per sex
100 mg/kg bw/day group: 10 per sex
300 mg/kg bw/day group: 10 per sex
1000 mg/kg bw/day group: 15 per sex
Control animals:
yes, concurrent vehicle
Details on study design:
- The dose levels were selected in agreement with the Sponsor, based on the results of a previous 28-day repeated toxicity study (OECD 407, 12602-TSR), 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 2 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 hematological, 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.
- Fasting period before blood sampling for clinical biochemistry: Yes
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily

DETAILED CLINICAL EXAMINATIONS: Yes
- Time schedule: Once a week
- Observations included (but were not 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) were also evaluated.

FOB: Yes
- Week 12 before the daily treatment. This evaluation included a detailed clinical observation (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, the assessment of reactivity to manipulation or to different stimuli, (touch response, forelimb grip strength, pupillary reflex, visual stimulus response, auditory startle reflex, tail pinch response, righting reflex, landing foot splay, rectal temperature (at the end of the observation period), and motor activity (measured by automated infra-red sensor equipment over a 60-minute period).
- Parameters: touch response, forelimb grip strength, pupillary reflex, visual stimulus response, auditory startle reflex, tail pinch response, righting reflex, landing foot splay, rectal temperature (at the end of the observation period).
- in the cage "touch escape",
- in the hand: fur appearance, salivation, lacrimation, piloerection, exophthalmia, reactivity to handling, pupil size (presence of myosis or mydriasis)
- 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.

BODY WEIGHT: Yes
- Time schedule for examinations: 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.

FOOD CONSUMPTION: Yes
- 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.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before the beginning of the treatment period and on control and high-dose animals (including recovery animals) on one occasion during Week 13.
- As no relevant changes were detected in control and high-dose animals at the end of the treatment period, the animals in the other groups were not examined and examinations were not
performed at the end of the treatment-free period.
- The pupils of the animals were dilated with tropicamide. After assessment of the corneal reflex (at instillation of the tropicamide), the appendages, optic media and fundus were examined by indirect ophthalmoscopy.
- When necessary, a slit-lamp biomicroscope was used to investigate abnormalities of the anterior
segment and the lens.

MONITORING OF ESTROUS CYCLE: Yes
- Time schedule: daily vaginal smears during 3 weeks of the treatment period

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Completion of treatment period and at the end of the treatment-free period (control and high dose animals)
- Animals fasted: Yes o/n
- Parameters: See table 1
- Bone marrow: Two bone marrow smears were prepared from the femoral bone (at necropsy) of all animals euthanized on completion of the treatment or treatment-free period and stained with May Grünwald Giemsa.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Completion of treatment period and at the end of the treatment-free period (control and high dose animals).
- Animals fasted: Yes o/n
- Parameters: See table 2

Thyroid hormones: Yes
- Time of blood sample collection: Between 7.5 and 10 a.m. 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 and treatment-free period.
- Animals fasted: Yes o/n

URINALYSIS: Yes
- Time schedule for collection of urine: Completion of treatment period (except recovery animals), and at the end of the treatment-free period.
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes
- Parameters: See table 3

COAGULATION: Yes
- Time schedule for blood collection: Completion of treatment period
- Animals fasted: Yes
- Parameters: Prothrombin Time (PT)

SPERM PARAMETERS: Yes
- Sperm sampling was performed on all animals at the end of the treatment period at necropsy (except recovery animals) during euthanasia, after intraperitoneal injection of sodium pentobarbital.
- Parameters: Epididymal sperm motility (proportion of motile and non-motile spermatozoa), Epididymal sperm morphology, Epididymal sperm count (number of spermatozoa per cauda and per gram of cauda), Testicular sperm (number of sperm heads per gram of testis and the daily sperm production rate)
Sacrifice and pathology:
EUTHANASIA:
- On completion of the treatment or treatment-free periods, after at least 14 hours fasting, all animals were euthanized by an intraperitoneal injection of sodium pentobarbital followed by exsanguination.

ORGAN WEIGHT: Yes
- The body weight of each animal was recorded before euthanasia at the end of the treatment or treatment-free periods. The organs specified in the Table 4 were weighed wet as soon as possible after dissection.
- The ratio of organ weight to body weight (recorded immediately before euthanasia) was calculated.

MACROSCOPIC EXAMINATION: Ye
- A complete macroscopic post-mortem examination was performed on all animals. This included 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.

PRESERVATION OF TISSUES:
- See table 4

MICROSCOPIC EXAMINATION: Yes
Micorscopic examination was performed on:
- all tissues listed in Table 4 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,
- immunostained kidneys in all males from the control and high dose groups euthanized at the end of the treatment period.In addition, a detailed examination of the testes was performed for control and high-dose males (groups 1 and 4, except recovery animals).

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
During the treatment period, ptyalism (recorded as hypersalivation) was transiently observed in 1/15 control females in Week 11, 1/10 females at 100 mg/kg bw/day from Week 8, 3/10 males at 300 mg/kg bw/day from Week 7 and in almost all males and females at 1000 mg/kg bw/day from Week 2 at the earliest. Although this finding was also noted in one control female, it was attributed to the test item. As this clinical sign is commonly observed when a test item is administered by gavage, it was not considered to be adverse.
Treatment-related clinical signs were no longer observed during the treatment-free period.
Reflux at dosing was noted in 1/15 control males, 2/10 males at 300 mg/kg bw/day and 1/15 males and 1/15 females at 1000 mg/kg bw/day on isolated occasions. This sign is commonly observed when a test item is administered by gavage and was observed only sporadically. It 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 or treatment-free periods.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight and body weight evolution were not affected by the test item in males at 100 and 300 mg/kg bw/day or in females (all dose levels).
At 1000 mg/kg bw/day in males, when compared with controls, statistically significant, lower mean body weight gain was recorded throughout the treatment period (-18% vs. controls), leading to a statistically significant lower mean body weight from Week 9 (-8% vs. controls on Day 57) until the end of the treatment period (-10% vs. controls on Day 91, Week 13). The differences in body weight gain were particularly noticeable from Week 6 until the end of the treatment period (Days 36 to 91). This effect was attributed to the test item treatment.
During the treatment-free period, a statistically significant, higher mean body weight gain was observed in previously treated males, leading to a terminal mean body weight similar to that of the control animals (+4% vs. controls).
Other variations of body weight gain were sporadic and did not affect the mean body weight, it was considered to be incidental.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Mean food consumption was not affected by the test item during the treatment period in males and females.
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). The same sporadic variations were noted in previously treated males during the treatment-free period (i.e. on Days 91-96 and Days 96-103).
As these differences were poorly dose-related, transient, of opposite trends, resulted from low or high 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 specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No findings were observed at the end of the treatment period.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related variations in hematological parameters.
No statistically differences were observed at the end of treatment period.
At the end of the treatment period and when compared with controls, a trend towards a marginally higher mean white blood cell count in males at all dose levels and in females from 300 mg/kg/day and a trend towards a minimal increase in the neutrophil counts of both sexes were noted.
Increased white blood cell count in males (+50%) and increased neutrophil count in males (2.2-fold; p < 0.01) and females (+69%) were still observed at the end of the 6-week treatment-free period.
As these differences were minimal, not statistically significantly different from controls and/or poorly dose-related at the end of the 13-week treatment period, no relationship with the test-item treatment could be concluded.

Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
When compared with mean control values, the main relevant blood biochemistry changes were observed at 1000 mg/kg/day as follows:
• lower mean protein level in both sexes (males: -4% with p < 0.01 and females: -4%, not statistically significant)
• higher mean A/G ratio in males (+10%; p < 0.01),
• higher mean Low-Density Lipoprotein (LDL) level in males (+44%; p < 0.05),
The findings described above were of minimal magnitude and/or within the range of the Historical Control Data, without histopathological correlates and reversible. They were therefore not considered to represent adverse effects.
The other statistically significant differences between control and test item-treated animals at the end of the treatment period, namely in potassium level (increased in males at 100 and 300 mg/kg/day), creatinine level (decreased in females from 300 mg/kg/day and males at 1000 mg/kg/day), biliary acid level (increased in females at 1000 mg/kg/day) and aspartate aminotransferase activity (increased in 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.
At the end of the treatment-free period, there were no test item-related changes in blood biochemistry parameters at any dose level.
Endocrine findings:
effects observed, non-treatment-related
Description (incidence and severity):
Thyroid hormone levels (i.e. T3, T4 and TSH) were considered to be unaffected by the test item treatment in males and females at 1000 mg/kg/day.
At the end of the treatment period, in males, at 1000 mg/kg/day and when compared with controls, there was an increase in mean TSH concentration (+41% vs. controls) along with a decrease in mean T4 concentration ( 12% vs. controls). As these differences were of minimal magnitude, were not statistically significant, were not associated with changes in mean T3 level or effects at pathology, and as there were high individual inter-variability and/or no correlates between individual TSH and T4 values, and/or as individual values were within or near the control range values, they were considered to be not test item-related treatment.
The increased TSH level recorded in females at 1000 mg/kg/day was considered to be incidental based on the slight magnitude of this variation, the absence of statistical significance, the absence of associated T3 or T4 variations, and/or as this was mainly due to one isolated female (female No. 4509: 2264 pg/mL).

At the end of the treatment-free period, statistically significant variations from controls were observed in TSH and T4 levels in females (i.e. -47% and +29% vs. controls, respectively; p < 0.05). These differences were not considered to be test item-related as no changes had been observed at the end of the treatment period.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
When compared with controls, increased mean urine volumes were recorded in males from 300 mg/kg/day (up to +57% at 1000 mg/kg/day) and a statistically significant, lower mean pH value was observed in males at 1000 mg/kg/day (-14%; p < 0.05). This correlated with a higher incidence of urination at FOB (see Section 7.4) and accumulation of hyaline droplets in proximal tubular cells of kidneys at microscopic examination (see Section 7.17.1) that were observed in males at the high dose level.
Although higher mean urine volumes were still observed in males and were therefore not reversible (+55% vs. controls; p < 0.05) at the end of the treatment-free period, these findings were not considered to be adverse as they were of low magnitude and/or were not statistically significant, did not impact specific gravity, reversible (pH) and/or and did not correlate with any adverse histopathological findings.
As other variations observed in the mean urine volumes of females at all dose levels were poorly dose-related and/or within the range of the historical control data and did not correlate with any other findings, these differences were not considered to be test item-related.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related effects on FOB tests or motor activity in males or females in any group during the study period.
At 1000 mg/kg bw/day and when compared with controls, higher mean numbers of horizontal and/or rearing movements were noted in males (+38% and +32%, respectively) at the end of the treatment period or at the end of the treatment-free period (+30% for horizontal movements only). As these differences were of slight magnitude, close to the mean values of the HCD, not statistically significant, not consistent with each other and did not correlate with any other clinical findings, they were not considered to be test item-related.
Higher (males at 100 mg/kg bw/day) or lower (females at 300 mg/kg bw/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 the number of rearing movements and/or as values remained within the standard deviation of control values.
Urination was noted in 9/10 males at 1000 mg/kg bw/day vs. 4/10 control males. In view of the incidence and as this correlated with a higher, but not statistically significant, mean urine volume, a test item-related effect could not be ruled out. As the grading of urination was of slight magnitude, did not correspond to any adverse findings and was no longer observed at the end of the treatment-free period, this finding was not considered to be adverse.
At the end of the treatment period, differences from controls in fur appearance were noted in isolated males at all dose levels and in landing foot splay in males and females at 1000 mg/kg bw/day (93 mm and 94 mm vs. 83 mm and 85 mm in control males and females, respectively). This trend was still present in males at the end of the treatment-free period (110 mm vs. 98 mm in controls). 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
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the treatment period: The statistically significant increases 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). There were no microscopic correlates. These variations in relative organ weights were therefore not considered to be toxicologically significant.
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.


At the end of the treatment-free period: No organ weight changes were considered to be related to TPS 32 administration.
The statistically significant changes in absolute and/or relative organ weights for the spleen in recovery males, and heart and liver in recovery females at 1000 mg/kg bw/day were not considered to be toxicologically significant in the absence of test item-related microscopic changes and/or similar weight variations in these organs at the end of the treatment period.
Other differences in organ weights were minor and reflected the usual range of individual variations.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the treatment period: No macroscopic findings were considered to be related to TPS 32 administration.
In one male at 1000 mg/kg bw/day, 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.

At the end of the treatment-free period: There were no TPS 32-related macroscopic observations.
The few other macroscopic findings noted at the end of the treatment-free period were among those commonly recorded spontaneously in the rat.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the treatment period: TPS 32-related microscopic findings were observed in the mesenteric lymph node and kidneys at 1000 mg/kg/day.
In the mesenteric lymph node, increased cellularity of macrophages, increased incidence and severity of macrophage aggregates were found in both sexes at 1000 mg/kg bw/day when compared with controls. Macrophages cellularity was increased in the medullary sinuses and paracortex mainly. Within aggregates, macrophages were enlarged and have small clear intracytoplasmic vacuoles in high-dose animals.
In the kidneys from males at 1000 mg/kg bw/day, accumulation of hyaline droplets in proximal tubular cells was increased in incidence and severity compared to controls. This was characterized by dense eosinophilic droplets which were positive for alpha 2u globulin at immunohistochemistry.
In the Gut-Associated Lymphoid Tissue (GALT), minimal large clear vacuoles suggestive of lipids were noted with a low incidence in control males and females and in high-dose males. They were considered to be related to the vehicle, corn oil.
Other microscopic findings noted in treated animals were considered incidental changes, as they also occurred in controls, were of low incidence, had no dose-relationship in incidence or severity, and/or are common background findings for the rat.

At the end of the treatment-free period: No microscopic examination was performed.
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
estrous cycle: There were no test item-related effects on mean estrous cycle length or mean number of cycles at the end of the treatment period.
When compared with controls, a statistically significantly 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 bw/day. As these differences were not dose-related and mainly due to the contribution of isolated females (i.e. Nos 3503, 3508 and 3509), they were considered to be incidental.
A statistically significantly higher number of females cycling normally was also noted at 500 mg/kg/day. This was not considered to be of toxicological importance due to the direction of the variation.

Sperm parameters: At the end of the treatment period, no test item-related effects were noted for testicular sperm count, epididymal sperm count, motility or morphology.
Details on results:
Discussion of pathology :
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 effect could be associated with higher urinary volumes and lower mean body weight gains observed in these animals. This was characterized by the presence of dense eosinophilic droplets in proximal tubular epithelium. These hyaline droplets, occasionally seen in untreated male rats, were positive for alpha 2u globulin at immunohistochemistry. Alpha 2u globulin hyaline droplets 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 is 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.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no

Any other information on results incl. tables

Dose formulation analysis: 


The test item concentrations in the administered dose formulations (i.e. mean accuracy between 90% and 110%) 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 7 (-12%). No re-analysis was performed as the stability period was already expired.


No test item was measured in the control dose formulation.


 

Applicant's summary and conclusion

Conclusions:
The toxicity of the test item, TPS 32, was evaluated after daily oral administration (gavage) to Wistar Han rats at dose levels of 100, 300 or 1000 mg/kg/day for 13 weeks followed by a 6-week treatment-free period.
At 1000 mg/kg/day, non-adverse treatment-related changes were restricted to ptyalism in both sexes during the treatment period, a reversible increased incidence of urination in males at FOB , and lower body weight and body weight gain in males that were no longer observed at the end of the treatment-free period. Slightly lower protein levels in both sexes, higher A/G ratio and LDL level in males, lower urinary pH values and higher urinary volumes in males were also observed at the end of the treatment and/or treatment-free periods. These laboratory findings were considered to be non-adverse.
Administration of TPS 32 resulted in non-adverse treatment-related findings in the mesenteric lymph node (increased cellularity and aggregates of macrophages in both sexes) and kidneys (non-adverse alpha 2u globulin nephropathy in males) at 1000 mg/kg/day.
At 300 mg/kg/day, non-adverse treatment-related changes consisted of ptyalism in males during the treatment period and non-adverse higher urinary volumes in males at the end of the treatment period.
At 100 mg/kg/day, non-adverse treatment-related changes were limited to ptyalism in females during the treatment period.
Consequently, the NOAEL (No Observed Adverse Effect Level) for oral administration of TPS 32 was established at 1000 mg/kg/day in male and female Wistar Han rats.
Executive summary:

Potential toxicity of TPS 32 was investigated in rat a 13 weeks oral (gavage) toxicity study (OECD, GLP) followed by a 6 week treatment-free period (investigation revisibility of findings).


One group of 15 Wistar Han rats/sex was treated daily by the oral route (gavage) with the test item TPS 32 at the dose level of 1000 mg/kgbw/day (group 4) for 13 weeks. Two other groups of 10 rats/sex  were treated with the test item at the dose level of 100 or 300 mg/kg bw/day (groups 2 and 3, respectively). One control group of 15 rats/sex received the vehicle only (corn oil) under the same experimental conditions (group 1, control). At the end of the treatment period, the animals were euthanized, except for the first 5 groups 1 and 4 animals per sex, which were kept for a 6-week treatment-free period.


Actual concentrations of TPS 32 in the dose formulations administered to the animals during the study remained within the acceptable range of ± 10% of the nominal concentrations, except for the low-dose formulation in Week 7 (-12%).
No test item was observed in the control dose formulation.
There were no unscheduled deaths during the treatment or treatment-free period.
Test-item related clinical signs of toxicity were restricted to non-adverse hypersalivation in both sexes at 1000 mg/kg bw/day during the treatment period only.


Motor activity was unaffected by the test item treatment. A higher incidence of urination was noted in males at 1000 mg/kg bw/day (9/10 males vs. 4/10 males in controls) at the end of the treatment period. This finding, that was no longer observed at the end of the treatment-free period, was not considered to be an adverse effect.
Lower body weight gain was recorded throughout the treatment period in males at 1000 mg/kg bw/day (-18% vs. controls; p < 0.001) leading to a lower body weight from Week 9 until the end of the treatment period (-10% vs. controls; p < 0.01). As these differences were of low magnitude, not associated with any other relevant findings and reversible at the end of the treatment-free period, they were considered to be non-adverse. No test item-related changes were noted in the body weight or body weight change of females.


Food consumption was not affected by the test item treatment.
No ophthalmological findings were observed at the end of the treatment period.
No test item-related variations were observed on the estrous cycle length or number of cycles at the end of the treatment period.
The epididymal sperm motility and morphology, and the testicular and epididymal spermatozoa count were unaffected by the test item treatment.
There were no test item-related variations in hematological parameters.
Coagulation parameters were not affected by the test item.


Decreased protein levels were observed in both sexes (-4% in males with p< 0.01 and females with no statistically significance) at 1000 mg/kg/day as well as an increased A/G ratio in males (+10% vs. controls; p<0.01). Increased LDL level was also noted in males at 1000 mg/kg/day (+44% vs. controls; p < 0.05). As these changes were of minimal magnitude and/or within the range of the Historical Control Data, without histopathological correlates and reversible, they were not considered to represent adverse effects.


Thyroid hormone levels (i.e. T3, T4 and TSH) were considered to be unaffected by the test item treatment in males and females at 1000 mg/kg/day.


Lower pH values (6.3 vs. 7.3 in controls; p < 0.05) were noted in males at 1000 mg/kg bw/day and higher, not statistically significant, urinary volumes were noted in males from 300 mg/kg bw/day (up to +57 % vs. controls at 1000 mg/kg bw/day). Increased volume was still observed in males (+55% vs. controls; p < 0.05) at the end of the treatment-free period whereas pH changes were reversible. These findings were not considered to represent an adverse effect as they were of low magnitude and/or not statistically significant, reversible, did not impact the specific gravity, and/or and did not correlate with any adverse histopathology findings.


The statistically significant increases 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). These changes were considered to be test item-related treatment but not considered to be toxicologically significant in the absence of changes in absolute organ weights and in the absence of microscopic correlates.


Non-adverse increased cellularity and aggregates of macrophages within the mesenteric lymph node were observed in both sexes at 1000 mg/kg bw/day. In addition, non-adverse alpha 2u globulin nephropathy, which is non-relevant for humans, was observed at 1000 mg/kg bw/day in males only. There were no test item-related macroscopic or organ weight changes at the end of the treatment or treatment-free period at any dose level.


In conclusion :


At 1000 mg/kg/day, non-adverse treatment-related changes were restricted to ptyalism in both sexes during the treatment period, a reversible increased incidence of urination in males at FOB, and lower body weight and body weight gain in males that were no longer observed at the end of the treatment-free period. Slightly lower protein levels in both sexes, higher A/G ratio and LDL level in males, lower urinary pH values and higher urinary volumes in males were also observed at the end of the treatment and/or treatment-free periods. These laboratory findings were considered to be non-adverse.


Administration of TPS 32 resulted in non-adverse treatment-related findings in the mesenteric lymph node (increased cellularity and aggregates of macrophages in both sexes) and kidneys (non-adverse alpha 2u globulin nephropathy in males) at 1000 mg/kg/day.


At 300 mg/kg/day, non-adverse treatment-related changes consisted of ptyalism in males during the treatment period and non-adverse higher urinary volumes in males at the end of the treatment period.


At 100 mg/kg/day, non-adverse treatment-related changes were limited to ptyalism in females during the treatment period.


Consequently, the NOAEL (No Observed Adverse Effect Level) for oral administration of TPS 32 was established at 1000 mg/kg/day in male and female Wistar Han rats.


 


 


 

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