Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Apr 2018 to Jan 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:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Han Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS Ltd
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Age at study initiation: 43 - 49 days
- Weight at study initiation: males 142-179 g, females 125-161 g
- Fasting period before study: no
- Housing: 5 animals per cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 14 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, warmed in a water bath to 50°C for approx. 10 min, 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, 120 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 with UV detector. The mean concentrations of WS400104 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.:
600 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 Charles River Wistar rat, oral administration of WS400104 at dose levels of 100, 300 and 1000 mg/kg/day was well tolerated. At 1000 mg/kg/d there was minimal or mild hyperkeratosis/parakeratosis of the non-glandular stomach in 4/5 males and 2/5 females. No effects were observed at lower doses.

The stomach findings at 1000 mg/kg/day suggested a response to the irritancy of the test item, rather than a systemic response to treatment. There was also no evidence of erosions, ulcerations, squamous cell hyperplasia or increased inflammation but there was the possibility that more severe stomach damage may develop in a study of longer duration and, consequently, the highest dose for this 13-week study was selected at 600 mg/kg/day. The low and intermediate dose levels of 100 and 300 mg/kg/day were selected to allow evaluation of a dose response.
The change of strain of rat from the Wistar to the Han Wistar was requested by the Sponsor. The Han Wistar is the strain of choice at Envigo for which there is a greater extent of historical control data. This change of strain was not considered to result in a different toxicological profile to that seen in the previous study.

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 10 and 11 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:
no effects observed
Description (incidence and severity):
see "overall remarks, attachments" : Figure body weight
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The haematological investigation performed in Week 13 revealed, when compared to controls, slightly but statistically significantly low haematocrit in females receiving 600 mg/kg/day. This associated with a trend towards low haemoglobin concentration and erythrocyte count but these differences from controls were not statistically significant and did not elicit any compensatory increase of reticulocyte numbers. Males were unaffected.
Females receiving 600 mg/kg/day had higher than control lymphocyte count, resulting in an increased total leucocyte count. Males were unaffected.
All other inter-group differences from controls, including those that attained statistical significance, were minor, lacked dose-relationship or were confined to one sex and were therefore attributed to normal biological variation. Such differences included the slightly low mean cell volume in males receiving 600 mg/kg/day since there were no alterations of any of the other erythrocyte indices. They also included a small but statistically significant prolongation of activated partial thromboplastin time in males receiving 600 mg/kg/day which was due to particularly high values in two animals (Nos. 21 and 22; 31.8 and 32.8 seconds); all other values were within the range of values reported for the controls (20.3 to 26.1 seconds).

see "overall remarks, attachments" : Table Haematology
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The biochemical examination of the blood plasma in Week 13 revealed, when compared to the controls, slightly high aspartate amino-transferase activity in males receiving 300 or 600 mg/kg/day, though the extent of the difference from controls was not dose related, and a small increase of alanine amino-transferase activity in females receiving 600 mg/kg/day.
Females receiving 600 mg/kg/day had slightly high plasma creatinine concentrations but there was no similar finding in males.
Glucose concentrations were slightly higher than controls and total cholesterol concentrations were slightly lower than controls in males receiving 300 or 600 mg/kg/day, but these differences from controls were statistically significant only at 600 mg/kg/day. Females were unaffected.
Plasma potassium concentrations were higher than controls in males receiving 300 or 600 mg/kg/day; females were unaffected.
All other inter-group differences from controls, including those that attained statistical significance, were minor, lacked dose-relationship or were confined to one sex and were therefore attributed to normal biological variation.
see "overall remarks, attachments" : Table Blood chemistry
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
There was an indication of an effect on renal function at 300 and 600 mg/kg/day. Urinary pH was low in both sexes and there was an increase in urinary protein output and plasma potassium concentration in males and plasma creatinine in high dose females. There was, however, no treatment-related histopathological finding in the kidneys. Consequently, these findings may have been due to the renal excretion of the test item and/or metabolites and, as such, were considered to be of no toxicological significance.
Behaviour (functional findings):
no effects observed
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Slightly high absolute and relative liver weights for females at 600 mg/kg/day were observed.
see "overall remarks, attachments" : Table Organ weights
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
High incidence of thickening and depression in the non-glandular mucosa of the stomach of males and females of the 300 mg and 600 mg/kg/day dose groups was observed.
see "overall remarks, attachments" : Table macropathology
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The majority of animals given 300 or 600 mg/kg/day had minimal to moderate epithelial hyperplasia and hyperkeratosis within the non-glandular stomach, correlating with the thickened non-glandular mucosa identified macroscopically. Ulceration or erosion, with some associated inflammation, was reported in two males given 600 mg/kg/day and one female given 300 mg/kg/day, respectively, and these correlated with the macropathology finding of depressions within the non-glandular stomach mucosa.
see "overall remarks, attachments" : Table histopathology
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
All other findings were considered incidental and unrelated to treatment, attributed to normal biological variation.

During weeks 10 and 11 of treatment, vaginal smears performed in females of all dose groups did not indicate any effects of dosing on estrous cycle. Sperm parameters determined after 13 weeks of dosing did not show effects in high dose males compared to control males.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
> 300 - < 600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical biochemistry
organ weights and organ / body weight ratios
Key result
Dose descriptor:
NOAEL
Effect level:
>= 600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
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.:
600 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes

Applicant's summary and conclusion