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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
analytical grade water
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
Males were dosed for a period of four weeks, up to and including the day before scheduled sacrifice. Females were dosed for 50 to 60 days throughout the study. This included two weeks prior to mating, the variable time to conception, the duration of pregnancy and thirteen days after delivery, up to and including the day before scheduled sacrifice.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
80 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 males/dose group and 15 females/dose group
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
CLINICAL SIGNS AND MORTALITY: All signs of illness, together with any behavioural changes or reaction to treatment were observed (cage side observation) once a day for individual animals. Dated and signed records of appearance, change and disappearance of clinical signs were maintained on clinical history sheets.
Throughout the study, all animals were checked early on each working day and again in the afternoon to look for dead or moribund animals to allow necropsy examination to be carried out during the working hours of that day.
Once before the first exposure (to allow for within-subject comparisons), and once a week thereafter, detailed clinical observations were made in all parental animals. The rats were subjected to detailed clinical examinations before initiation of the treatment (to allow for within-subject comparisons) and weekly thereafter during the treatment period. These observations were made outside the home cage in a standard arena and preferably at the same time. Signs noted included changes in skin, fur, eyes and mucous membranes, occurrence of secretions, excretions and autonomic activity such as lacrimation, piloerection, pupil size and unusual respiratory pattern. Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies, difficult or prolonged parturition or bizarre behavior were also recorded.

FUNCTIONAL OBSERVATION BATTERY: Five males and five females, randomly selected from each group were examined for assessment of sensory reactivity, assessment of grip strength and motor activity. These included the functional observational battery suggested by Moser V. C. (1989). [Animal Behavioral Methods in Neurotoxicity Assessment: SGOMSEC Joint Report; Beverly Kulig et. al, Environ Health Perspect 104 (Suppl 2):193-204 (1996)]
In males, these functional observations were made towards the end of their dosing period, shortly before scheduled sacrifice but before blood sampling for haematology or clinical chemistry. In females these functional tests were made during the last week of lactation (e.g., LD 6-13), shortly before scheduled sacrifice.

BODY WEIGHT: Males and females were weighed on the first day of dosing, weekly thereafter, and at termination. During pregnancy, females were weighed on days 0, 7, 14 and 20 and then within 24 hours of parturition (day 1 post-partum), and at day 4 and day 13 post-partum. These observations were reported individually for each adult animal.

FOOD CONSUMPTION: During pre-mating, food consumption was measured weekly (on days 0, 7 and 14). During pregnancy it was measured on gestation days 0, 7, 14 and 20 and in lactation on days 4 and 13. Food consumption during mating period was not measured. Food consumption was not corrected for spillage.
Food consumption was computed as the amount of food consumed in grams per animal per day.
Sacrifice and pathology:
Haematology and Clinical Chemistry: Animals were fasted overnight prior to blood sampling but had access to water ad libitum. Blood sampling was performed, under light CO2 anaesthesia, through the orbital sinus of all males and females sacrificed at termination (day 29 for males and day 14 of lactation for females). Samples were collected separately in tubes containing EDTA (dipotassium salt), for haematology, and Heparin, for clinical chemistry, as anticoagulants.
For hormone analysis (serum total T4), blood samples were collected separately in plain tubes and serum was separated. Blood samples from the pups were collected by cardiac puncture. Pups were anesthetized by barbiturate anaesthetic (Sodium Thiopentone) agent prior to blood collection.
Baseline Sampling: The base-line values for haematological and clinical chemistry parameters including the hormone estimation (Total T4) were obtained by randomly selected five males and five females. Sampling was performed before start of treatment.

Haematology (During End of the Premating Period): After completion of the premating period (day 14 from start of treatment), haematological examination was made in five males and five females randomly selected from each group. The following haematological parameters were estimated on blood samples:
1. Haemoglobin (HGB)
2. Haematocrit (PCV)
3. Erythrocyte Count, Total (Total RBC)
4. Reticulocytes
5. Total Leukocyte Count (Total WBC)
6. Platelet Count, Total (Platelet)
7. Absolute RBC indices : Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH) and Mean corpuscular hemoglobin concentration (MCHC)
8. Prothrombin time as a measure of blood clotting (seconds)
9. Differential Leukocyte (WBC) Count
Leucocytes were differentiated as Neutrophils (N), Lymphocytes (L), Eosinophils (E), Monocytes (M) and Basophils (B);
10. General blood picture: Blood smear was assessed for abnormal and immature cells during microscopy.

Clinical Chemistry (At Termination): Clinical Chemistry examinations were made in five males and five females randomly selected from each group. Plasma samples were analysed individually for determination of clinical chemistry parameters.

Hormone Analysis: Blood (Serum) samples were taken based on the following schedule:
1. two of the surplus pups, pooled, and used for determination of serum total T4 levels on day 4 after birth - (as mentioned in “3.4.3: Litter Size”)
2. at least two pups per litter at termination on Lactation day 13 - serum total T4
3. from all adult males at termination (day 29) - serum total T4
4. from all dams at termination on lactation day 14 - serum total T4 (dams were fasted over night on lactation day 13
Blood samples of pups (on day 4 after birth or at lactation day 13) and adults (at day 29 for the males and at lactation day 13 for the females) were assessed for serum levels of thyroid hormone (T4).
The hormone analysis was employed using commercially available ELISA kits manufactured by Elabscience, WuHan, P.R.C.

Gross Necropsy: All adult animals in the study were humanely sacrificed by exsanguination under deep CO2 anaesthesia and were subjected to a full, detailed gross necropsy which included careful examination of the external surface of the body, all orifices, the cranial, thoracic and abdominal cavities and their contents. Special attention was paid to the organs of the reproductive system. The number of implantation sites was recorded.
All the tissues listed in Appendix 16 from all adult male (day 29) and female (lactation day 14) rats were preserved in 10% neutral buffered formalin. Testes were collected in Modified Davidson's fluid. The tunica albuginea of the testes was gently punctured at both poles of the organ with a needle to permit rapid penetration of the fixative.
Vaginal smears were examined on the day of necropsy to determine the stage of the oestrous cycle. Dead pups and pups killed on day 13 post-partum were carefully examined externally for gross abnormalities. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development.

Organ Weights: The testes, epididymides, prostate, seminal vesicles with coagulating glands as a whole, levator ani plus bulbocavernosus muscle complex, Cowper’s glands and glans penis of all male adult animals were weighed at termination (on day 29). Uterus with cervix and ovaries from all adult females were weighed at termination on lactation day 14.
From all adult males and females, thyroid glands were preserved and weighed post fixation. At lactation day 13 the thyroid glands from 1 male and 1 female pup per litter were preserved and weighed post fixation.
Trimming was done very carefully and only after fixation to avoid tissue damage.
In addition, from five adult males and females, randomly selected from each group, the liver, kidneys, adrenals, thymus, spleen, brain and heart were trimmed of any adherent tissue, as appropriate and their weights were taken as soon as possible after dissection to avoid drying.

Histopathology: Full histopathology was carried out on the preserved organs and tissues of the randomly selected five adult males and females in the control (G1) and high dose group (G4) sacrificed at termination.
The tissues (listed in Appendix 16) fixed in 10% neutral buffered formalin, were embedded in paraffin wax, sectioned at 5 m thickness and stained with haematoxylin and eosin, for microscopic examination. These examinations were not extended to animals of other dosage groups, as treatment-related changes were not observed in the high dose group.
In absence of any microscopic alteration in the thyroid glands of adult male and female rats and in absence of any alterations in the values of total T4, microscopic evaluation of thyroid glands from the pups was not carried out.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment with W3803S04SOLSIV for males (28 days) and for females (50 to 60 days involving pregnancy and lactation period) did not induce any adverse clinical signs at any dose levels. No abortion was observed in any of the dose levels during the study period. One dam each from 80 mg/kg/d (G2) and 200 mg/kg/d (G3) delivered before term on gestation days 18 and 16 respectively. These incidences of premature parturition were not related to treatment and are considered as incidental as all pups were alive, normal in size and shape in comparison to full term pups delivered on gestational day 21-22.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There was no incidence of any treatment related mortality amongst the rats treated with the W3803S04SOLSIV at any of the dose levels in both males and females. All animals survived throughout the treatment period of the study.
One dam (ID. Rk1564) from G2 (200 mg/kg/d) was found dead on the second day of lactation. In this dam one dead pup in right uterine horn and one dead pup in vaginal tract were observed which were autolysed. This death is considered as incidental and not related with the treatment.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
W3803S04SOLSIV at and up to the dose of 500 mg/kg/d dose group did not have any remarkable and adverse effects on the weight gains by the treated male and female rats in this study. The values of mean body weights and mean body weight gain for rats treated with test item at and up to 500 mg/kg/d did not differ significantly (p>0.05) from those of the concurrent control group rats during the premating and mating period, gestation period and the lactation period.

During Premating and Mating Period: The percent body weight gain by the male and female rats treated with the test item at and up to 500 mg/kg/d of body weight was found to be comparable to that of the control rats during premating and mating period.
During Gestation Period: Test item did not have any adverse effect on the body weight gain during the gestation period in female rats.
There was no significant difference (p>0.05) in the mean body weights of control and treated groups of females during the period of gestation. The mean body weight of female rats as measured on gestation days 0, 7, 14 and 20 and the mean body weight changes computed between gestation days 0-7, 7-14 and 14-20 by rats treated with the test item did not differ significantly (p>0.05) from those of the control group rats except that a slight reduction was observed in body weight changes computed between gestation days 0-7 in 200 mg/kg/d dose group when compared to control group. Since there was no dose dependency, this was considered as incidental.
During Lactation Period: The mean body weight of female rats as measured on lactation days 0, 4 and 13 and the mean body weight changes computed between lactation days 0-4 and 4-13 by rats treated with the test item did not differ significantly (p>0.05) from those of the control group rats. Slight reduction was observed in body weight gain in 80 and 200 mg/kg/d dose group females when compared to control group. Since there was no dose dependency, this was considered as incidental.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Treatment of male and female rats at and up to the dose of 500 mg/kg/d of W3803S04SOLSIV did not induce any remarkable and adverse effects on the daily food intake of animals. The values of mean daily food intake by rats treated with the test item did not differ significantly (p>0.05) from those of the concurrent control group females during the premating period, gestation period and the lactation period.

During Premating Period: Mean food consumption of male and female rats from control group and treated dose groups was found to be comparable during the two weeks of the premating period.
During Gestation Period: The average amount of food consumed per rat per day during gestation period did not differ significantly (p>0.05). Similarly the mean values of food consumption per rat per day during gestational days 0-7, 7-14 and 14-20 by rats treated with the test item did not differ significantly (p>0.05) from those of the control group females. Slight reduction was observed in food consumption in 80 mg/kg/d dose group females when compared to control group. Since there was no dose dependency this was considered as incidental.
During Lactation Period: The average amount of food consumed per rat per day during lactation period did not differ significantly (p>0.05) from the values of the control group. Similarly the mean values of food consumption per rat per day during lactation days 0-4 and 4-13 by rats treated with test item did not differ significantly (p>0.05) from those of the control group rats.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Baseline Sampling was performed before start of treatment on randomly selected five male and five female rats. The mean values of haematological parameters such as haemoglobin, haematocrit (PCV), total and differential leucocyte counts, total RBC count, RBC indices, platelet count, reticulocyte count and prothrombin time of male and female rats, were obtained. General blood picture was evaluated on stained blood smear slides.

During Premating Period: After completion of premating treatment period (day 14 from start of treatment), haematological examinations were made on five males and five females randomly selected from each group. There were no treatment related changes in the above list of haematological parameters as the mean values were comparable to the same from concurrent control animals. Few instances of statistically significant (p<0.05) changes observed, but were found not treatment related because of following reasons.
In males, the mean Neutrophil count was significantly higher (p<0.05) in low dose group (28.04%) and in mid dose group (30.24%) compared to the concurrent control group (19.40%). The mean lymphocyte count was significantly lower (p<0.05) in low dose group (68.92%) and in mid dose group (67.44%) compared to the concurrent control group (78.48%). These changes were considered incidental as there was no dose relationship and also the observed values were very well within the historical control range of INTOX (N=20.9 to 37% and L= 48.9 to 86.4%). The mean Monocytes count was significantly lower (p<0.05) in the high dose group (0.25%) compared to the concurrent control data (0.96%), however, this change was considered incidental as the observed value was very well within the historical control range of INTOX (M= 0.0 to 3.7%).
In females, the Mean reticulocyte count was significantly lower (p<0.05) in low dose group (3.41%) compared to the concurrent control data (5.43 %) and this change was considered incidental as the observed value was very well within the historical control range of INTOX (RTC = 1.3 to 6.2 %)
General blood picture evaluation performed on stained blood smear slides revealed no morphological abnormalities and immature cells in red blood cells, white blood cells and platelets in any of the treated rats including the control animals.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Baseline Sampling was performed before the start of treatment on randomly selected five male and five female rats. The mean values of clinical chemistry parameters such as total protein, albumin, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, glucose, urea, creatinine, blood urea nitrogen, total bile acids, total cholesterol, sodium, potassium and total T4 of male and female rats, were obtained.
During termination period:
At termination of treatment, the above listed clinical chemistry parameters were determined in all males (on day 29) and all females (on lactation day 14).
Treatment of male and female rats at and up to the dose of 500 mg/kg/d of W3803S04SOLSIV did not induce any changes in the plasma levels of above parameters at termination of the treatment period. Few instances of statistically significant (p<0.05) changes were observed but were considered incidental as there was no dose relationship and the values were within historical control range of the laboratoy.
Estimation of Thyroid Hormones (Total T4):
Serum samples from all dams sacrificed at termination on lactation day 14 were assessed for total T4. Similarly, serum samples from all males sacrificed at termination on day 29 were assessed for total T4.
The test item, up to the dose level of 500 mg/kg/d of body weight, did not induce any change in the total T4 levels.
Urinalysis findings:
not specified
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
The functional observations (neurological examinations) conducted for males towards the end of their dosing period and for females during the last week of lactation (e.g., lactation day 6-13), shortly before scheduled sacrifice, did not reveal any remarkable and treatment related incidence of neurological abnormalities. Also no findings, indicative of a neurotoxic potential of the test item, were encountered during these examinations.
Immunological findings:
no effects observed
Description (incidence and severity):
Based on the results of haematology especially Total and Differential Leucocyte Counts and general blood picture along with the results of organ weights and histopathology of thymus and spleen, there was no evidence of immunological effects.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The values of absolute and relative organ weights were found to be comparable between the treated and control group of male and female rats except for the following few changes.
A few instances of statistically significant (p<0.05) changes when compared to that of control organ weights were noticed and were considered incidental as they were within historical control ranges. They were;
G4 Male-Mean absolute weight of Glans penis 0.179 ± 0.034; Historical data range 0.070 to 0.201 g
G3 and G4 Male - Mean absolute weight of Thyroid gland 0.026 ± 0.00 (G3) and 0.027 ± 0.00 (G4); Historical data range 0.012 to 0.038 g.
G2 Female - Mean absolute weight of ovaries 0.09 ± 0.01; Historical data range 0.08 to 0.15 g.
G2 and G3 Female - Mean absolute weight of adrenals 0.070 ± 0.010 (G2) and 0.068 ± 0.006(G3); Historical data range 0.039 to 0.091 g.
G2 and G3 Male - Mean relative weight of Epididymides 0.41 ± 0.05 in G2 and 0.42 ± 0.03 in G3; Historical data range 0.314 to 0.495.
G3 and G4 Male - Mean relative weight of Thyroid gland 0.007 ± 0.00 (G3) and 0.008 ± 0.00 (G4); Historical data range 0.005 to 0.010.
G3 Female - Mean relative weight of adrenals 0.03 ± 0.00; Historical data range 0.022 to 0.037.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The test item, at and up to the dose level of 500 mg/kg/d, did not induce any remarkable and treatment related gross pathological alterations in any of the tissues of treated rats, as evidenced by the detailed necropsy examination carried out at termination of the study period. Gross pathological examination of the reproductive organs of male and female rats did not reveal any treatment related morphological alterations. Few incidental findings of gross pathology were noted but were not of toxicological significance because of the low incidence and the absence of dose dependency. They were as follows:
Slight reddening and multiple abscesses in the lungs - 2/10 in G4 males,
Small size of Cowper's glands -1/10 in G3 males,
One dead pup in right uterine horn and one dead pup in vaginal tract-which were autolysed-1/15 in G2 females.
The gross pathology of undersized Cowper's glands and lungs reddening and abscesses were examined microscopically and were found to be atrophy of cowper's glands and moderate acute bronchioalveolar inflammation of lungs respectively as described in 4.1.10 section.
Neuropathological findings:
no effects observed
Description (incidence and severity):
The functional observations (neurological examinations) conducted for males towards the end of their dosing period and for females during the last week of lactation (e.g., lactation day 6-13), shortly before scheduled sacrifice, did not reveal any remarkable and treatment related incidence of neurological abnormalities. Also no findings, indicative of a neurotoxic potential of the test item, were encountered during these examinations.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Histopathological examinations of the tissues (Appendix 16) from five adult male and female rats randomly selected from control group and high dose treated groups did not reveal any significant treatment related histopathological alterations.

Histopathological examination of the reproductive organs of male and female rats did not reveal any treatment related morphological alterations.
Some incidental and spontaneous lesions observed in animals from the control and high dose group (500 mg/kg/d) included-
 Minimal mononuclear cells infiltration (3/10 in G1), Minimal focal necrosis (1/10 in G1), Minimal bile duct hyperplasia (2/10 each in G1 and G4) in liver;
 Minimal peribronchial mononuclear cell infiltration (4/10 in G1, 2/10 in G4), Moderate acute bronchioalveolar inflammation (2/10 in G4), Minimal foam cell infiltration (2/10in G4) in lungs;
 Minimal myocardial mononuclear cell infiltration (1/10 in G1) in heart;
 Minimal calcification (1/10 in G1), Minimal nephroblastematosis (1/10 in G1 and G4) , Minimal dilated tubules (2/10 in G4) in kidneys;
 Minimal cortical vacuolation (3/10 in G1 and G4), Minimal accessory cortical tissue (1/10 in G4) ; in adrenals;
 Minimal sinus dilation (1/10 in G1) in Axillary lymph node;
 Minimal /mild tubular degeneration (1/5 in G1 and G4) in testes;
 Minimal interstitial mononuclear cell infiltration (2/5 in G4) in prostate;
 Minimal spermatocele (1/5 in G4) in epididymides;
 Minimal lymphoid hyperplasia (1/5 in G4) in mesenteric lymph node;
 Glandular cyst (2/10 in G4) and minimal submucosal mononuclear cell infiltration (1/10 in G1) in glandular stomach;
 Minimal inflammatory cells infiltration (1/10 in G4) in skeletal muscles;
 Minimal decidual reaction (1/5 in G4) in uterus;
 Minimal lymphoid hyperplasia (1/10 in G1) in colon.
All above listed findings are expected background lesions commonly observed in conventionally housed rats of this age. These findings were hence considered to be incidental in nature and not related to treatment with W3803S04SOLSIV (Ref. 1).
Isolated incidence of minimal spermatocele in epididymides of one high dose male was considered as incidental and not of toxicological significance and the same has been reported as background lesion (Ref. 2).
(Ref. 1: McInnes EF, ed, Background lesions in laboratory animals: A colour Atlas, Saunders Elsevier, Edinburgh, 28-31.
Ref. 2: Proliferative and non proliferative lesions of the rat and mouse male reproductive system, 2012, Toxicologic Pathology, 40, 40s -121s)
Histopathological findings: neoplastic:
not specified

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
>= 500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
gross pathology
haematology
histopathology: non-neoplastic
immunology
mortality
neuropathology
organ weights and organ / body weight ratios

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on the findings of this study, it is concluded that, the No-Observed-Adverse-Effect-Level (NOAEL) of W3803S04SOLSIV in Wistar rats, following oral administration for a period of four weeks for males and 50 to 60 days for females for systemic toxicity was found to be greater than or equal to 500 mg/kg/d body weight.