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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:
Sep 2018 to Jul 2019
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 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
RccHan Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS Ltd
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 41 - 47 days
- Weight at study initiation: males 134-173 g, females 116-151 g
- Fasting period before study: no
- Housing: 3 or 4 animals per cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 12 days

DETAILS OF FOOD AND WATER QUALITY: Teklad 2014C diet; potable water from public supply

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 40-70%
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amounts of test material were added to 50% of the final volume of vehicle and stirred magnetically until the test item was uniformly mixed. Vehicle was added to achieve the final volume and mixed until homogeneous.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 0, 20, 60, 200 mg/ml
- Amount of vehicle (if gavage): 5 ml / kg bw
- Lot/batch no. (if required):
- Purity:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentrations of dose formulations were analysed by HPLC/MS. The mean concentrations of WS400123 in test formulations analyzed for the study were within +10/-15% of nominal concentrations, confirming accurate formulation. The difference from mean remained within 2%, confirming precise analysis.
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: In an OECD422 study in Sprague Dawley rats, oral administration of WS400123 at dose levels of 100, 300 and 1000 mg/kg/day was well tolerated.

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: once per week starting 1 week before first adminstration

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pre-treatment and in week 12
- Dose groups that were examined: all animals (pre-treatment), animals of high dose group and control group (week 12)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 13
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes
- How many animals: all animals

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 13
- Animals fasted: Yes
- How many animals: all animals

URINALYSIS: Yes
- Time schedule for collection of urine: week 13
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: weekly arena observations
- Dose groups that were examined: all animals of all dose groups
- Battery of functions tested: sensory activity / grip strength / motor activity / other: week 12, all animals
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Other examinations:
Estrous cycles by vaginal smear: for 14 days during weeks 12 and 13 of treatment, all groups

Sperm analysis: after scheduled sacrifice of each male, sperm motility (all groups), morphology and homogenisation-resistant spermatid count (groups 1 and 4)
Statistics:
The following sequence of statistical tests was used for grip strength, motor activity, body weight, sperm analysis, organ weight and clinical pathology data: A parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. The F1 approximate test was applied. This test is designed to detect significant departure from monotonicity of means when the main test for the comparison of the means is a parametric monotonic trend test, such as Williams’ test (Williams 1971, 1972). The test statistic compares the mean square, NMS, for the deviations of the observed means from the maximum likelihood means, calculated under a constraint of monotonicity with the usual error mean square, EMS. The null hypothesis is that the true means are monotonically ordered. The test statistic is F1 = NMS/EMS which can be compared with standard tables of the F distribution with 1 and error degrees of freedom. If the F1 approximate test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test (Dunnett 1955, 1964) was performed instead. Where there were only two groups, comparisons were made using t-tests.
A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. The H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied. This test is designed to be used when the main test for comparison of the means is a non-parametric monotonic trend test, such as Shirley's test (Shirley 1977).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Overall (Week 0 to 13) body weight gain was slightly low, when compared with controls, in males and females receiving 1000 mg/kg/day (87% of control in both sexes) and in females receiving 300 mg/kg/day with statistical significance attained in the high dose males only. Body weight gain was unaffected in males and females at 100 mg/kg/day and in males at 300 mg/kg/day
see "overall remarks, attachments" : Figure body weight
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
see "overall remarks, attachments" : Table food consumption
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The hematological examination performed after 13 weeks of treatment revealed, when compared with controls, statistically significantly higher lymphocyte counts in males and females receiving 100, 300 or 1000 mg/kg/day resulting in higher total leucocyte counts in all groups.
Basophil and large unstained cell counts were higher than control in females receiving 100, 300 or 1000 mg/kg/day. In males, basophil, monocyte and large unstained cell counts were higher than controls at 1000 mg/kg/day.
In males and females receiving 100, 300 or 1000 mg/kg/day there was a generally dose-related trend towards lower hematocrit concentration, hemoglobin concentration, erythrocyte count and reticulocyte counts when compared with controls. There was a corresponding slight reduction of mean cell hemoglobin, mean cell hemoglobin concentration and mean cell volume in both sexes.
Platelet counts were high, when compared with controls, in males and females receiving 100, 300 or 1000 mg/kg/day, with the variation from control being clearly dose-related in both sexes.
All other intergroup differences, including those attaining statistical significance, were minor or without dose-relationship and were attributed to normal biological variation.

see "overall remarks, attachments" : Table Haematology
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The biochemical examination of the blood plasma performed after 13 weeks of treatment revealed, when compared with controls, lower high and low density lipoprotein concentrations and total cholesterol in males and females receiving 100, 300 or 1000 mg/kg/day but without a clear dose-relationship.
Plasma creatinine concentrations were low, when compared with controls, in males and females receiving 100, 300 or 1000 mg/kg/day.
Alanine phosphatase and aspartate amino-transferase activity and bile acid concentrations were higher than controls in males receiving 1000 mg/kg/day and, in females, bile acid concentrations were high at 1000 mg/kg/day.
All other intergroup differences, including those attaining statistical significance, were minor or without dose-relationship and were attributed to normal biological variation. Such differences included the statistically significant variations to plasma sodium concentrations in males and females and the slightly higher plasma proteins in females, where the difference from control was marginal.

see "overall remarks, attachments" : Table Blood chemistry
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
The examination of urine performed in Week 13 revealed a treatment-related trend towards low urinary glucose and creatinine in females receiving 300 or 1000 mg/kg/day with statistical significance attained at the high dose. In males, urinary protein was statistically significantly low in males receiving 100, 300 or 1000 mg/kg/day but there was no effect upon glucose or creatinine. There was a slight increase in the incidence of the presence of urinary bilirubin in males and female receiving 1000 mg/kg/day.
All other inter-group differences from control, including those attaining statistical significant, were minor or without dose-response. Such differences including the statistically significant increase of specific gravity in males receiving 1000 mg/kg/day, where there was no dose-relationship and the difference from control was marginal.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No effects were observed upon sensory reactivity and grip strength. Motor activity was low in males and females receiving 1000 mg/kg/d and males receiving 300 mg/kg/d.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The analysis of organ weights performed after 13 weeks of treatment revealed a dose-related trend towards high kidney weights in males and females receiving 300 or 1000 mg/kg/day and females receiving 100 mg/kg/day.
Liver weights were high, when compared with controls, in males and females given 1000 mg/kg/day.
All other inter-group differences were minor or lacked dose-relationship and were therefore considered to be unrelated to treatment.

see "overall remarks, attachments" : Table Organ weights
Gross pathological findings:
no effects observed
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed
Description (incidence and severity):
There were no effects of treatment at any dose upon estrous cycles.

Sperm motility, testicular spermatid numbers and sperm morphology were unaffected by treatment with WS400123 up to and including the highest dose.

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:
no

Any other information on results incl. tables

Administration of WS400123 to Han Wistar rats by oral gavage for 13 weeks at doses up to 1000 mg/kg/day was generally well-tolerated, but caused lower body weight gain at 1000 mg/kg/day and lower motor activity at 1000 or 300 mg/kg/day, attributed to a non-specific toxic response. The liver and kidney were identified as target organs, since there was evidence of an effect upon hepatic and renal function, but there was no correlating histopathology and the findings were considered non-adverse. There were minor changes in the erythrocytes, though this did not elicit any compensatory reticulocytosis and there was no histopathological evidence of extramedullary hematopoiesis or findings in the bone marrow, and there was an increase of some leucocyte subpopulations but without correlating findings in the bone marrow, spleen or thymus and there was no microscopic evidence of an immune response.

Applicant's summary and conclusion

Executive summary:

Administration of WS400123 to Han Wistar rats by oral gavage for 13 weeks at doses up to 1000 mg/kg/day was generally well-tolerated, but caused lower body weight gain at 1000 mg/kg/day and lower motor activity at 1000 or 300 mg/kg/day, attributed to a non-specific toxic response. The liver and kidney were identified as target organs, since there was evidence of an effect upon hepatic and renal function, but without correlating histopathology and the findings were considered non-adverse. The NOAEL was derived at the highest dose level of 1000 mg/kg/day.