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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Sprague Dawley rats, strain: Crl:CD(SD) with appropriate range of bodyweight at study start.
- Source: Charles River (UK) Ltd.
- Age at treatment start: 43 to 49 days.
- Weight at treatment start: Males: minimum 216 g, maximum 278 g,
Females: minimum 175 g, maximum 229 g.
- Housing Inside a barriered rodent facility:
main study and recovery animals In groups of 5 by sex in solid floor polycarbonate cages.
- Bedding material (in solid floor cages): Wood based bedding.
- Cage enrichment: Aspen chew block + plastic shelter.
- Diet (ad libitum): Rat and Mouse No. 1 Maintenance Diet.
- Fasting (diet withheld): overnight before blood sampling for hematology/clinical blood chemistry and during urine collection.
- Water (ad libitum): Potable drinking water from the public supply.
- Acclimation period: 14 days before treatment start.

Routine analysis of the batch of diet used and water, chew blocks and wood bedding did not provide evidence of contamination that might have prejudiced the study.

ENVIRONMENTAL CONDITIONS

Air conditioned room kept at positve pressure without re-circulation of the filtered fresh air supplied to the room.
Controlled environment, environmental conditions were set at:
- Temperature (°C): 21 ± 2°C
- Relative Humidity (%): 40 to 70%
- Photoperiod (artificial lighting): 12 hrs day / 12 hrs night
Deviations from these target ranges were not evident.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
- Concentration in vehicle: The concentration of the test material in vehicle varied between dose groups thus allowing constant dosage volume in terms of mL/kg bw/day.
- Amount (dose volume by gavage): 2 mL/kg bw/day.
Actual dose volumes were calculated at about weekly intervals accounting for the latest body weight.

CONCENTRATION OF TEST MATERIAL IN VEHICLE

Group Concentration of Test Substance Treatment Volume Dose Level of Test Substance
(mg/mL) (mL/kg bw/day) (mg/kg bw/day)
Vehicle Control 0 2 0
Low Dose 50 2 100
Mid Dose 150 2 300
High Dose 500 2 1000
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Chemical analysis of test material formulations by high performance liquid chromatography (HPLC/UV) using an external standard technique.
- Concentrations (verified at treatment week 1 and 12) of the test material formulations were confirmed at each dose level.
- Chemical analysis confirmed that the prepared formulations were within 4% of the corresponding nominal concentration.
Duration of treatment / exposure:
13 weeks followed by a four week recovery period
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0 (vehicle control), 100, 300, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10 animals per sex per dose group plus 5 animals per sex in vehicle control and high dose groups for the recovery period.
Control animals:
yes, concurrent vehicle
Positive control:
not applicable

Examinations

Observations and examinations performed and frequency:
Clinical observations performed and frequency:
- Clinical signs : At least twice a day (before and after administration)
- Detailed physical examination
and arena observations: Before treatment start and at least once a treatment week.
- Functional Observation Battery:* During treatment week 12 (before dosing) on the first five main study and all recovery animals in groups 1 and 4 and all main study animals from groups 2 and 3.
- Body weight, all animals: Weekly from one week before treatment start till end of the study.
- Food consumption, all animals: Weekly from one week before treatment start till end of the study.
- Water consumption: During week -1, 1, 4, 8, and 12 and week 1 and 4 of the recovery period.

* (FOB) including sensory reactivity tests (approach, touch, auditory startle reflex, tail pinching), grip strength and motor activity.

- Oestrous cycles: For the first 15 days (Day 1 to 15) of treatment and the last 15 days (Day 77 to 91) of treatment from all females.

- Ophthalmic examination of all animals in pretreatment period and in week 12 all animals of groups 1 and 4.

- Hematological examinations during treatment week 13 of all main study animals, and in recovery week 4 of all recovery study animals:
Red blood cell count, white blood cell count, platelet count, hemoglobin concentration, hematocrit value, differential leukocyte counts,
protrombin time, activated partial thromboplastin time, mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration, morphology.

- Blood (plasma) chemical examinations during treatment week 13 of all main study animals, and in recovery week 4 of all recovery study animals:
Total protein, albumin, A/G ratio, urea, blood urea nitrogen, creatinine, glucose, total cholesterol, sodium, potassium, aspartate aminotransferase, alanine aminotransferase.

- Urinalysis during treatment week 13 of all main study animals, and in recovery week 4 of all recovery study animals:
clarity, colour, volume, pH, gravity, ketones, bilirubin/bile pigments, blood pigments, protein, creatinine, glucose.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, see below
WEIGHING OF ORGANS: Yes, see below
HISTOPATHOLOGY: Yes, see below

Terminal sacrifice
- main study animals: Killed following 13 weeks of treatment.
- recovery animals Killed following 13 weeks of treatment and 4 weeks of recovery.

Gross pathology: Full macroscopic examination with tissue collection.

Organs weights: Adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus, uterus with cervix.

Histopathology: The following organs were microscopically observed for the control and 1000 mg/kg bw/day groups:
Aorta, brain, eyes, femur, head,  pituitary gland, thyroid with parathyroids, heart, thymus, liver, spleen, adrenals, kidneys, 
epididymides, testes, ovaries, lung, trachea, esophagus, stomach, pancreas, duodenum, jejunum, ileum, caecum, colon,
lymph node (left axillary, mesenteric), salivary glands (submandibular, sublingual, parotid), urinary bladder, uterus (with cervix), vagina, spinal cord (transverse and longitudinal sections at the cervical, thoracic and lumbar levels), sciatic nerve, skin with mammary glands, sternum with marrow, seminal vesicle, prostate.

Sperm analysis: Immediately after sacrifice of each main study male, the left vas deferens, epididymis and testis were removed and epididymis and testis were weight.
Sperm motility was determined in all groups.
Sperm morphology, sperm count and homogenisation resistant spermatid count was determined in animals of groups 1 and 4
Statistics:
The following sequence of statistical tests was used for grip strength, motor activity, body weight, 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). The test statistic compares the non-monotonicity sums of squares, NRSS, for the deviations of the observed mean ranks from the maximum likelihood mean ranks with the non-parametric equivalent of the error sums of squares, ERSS = N(N+1)/12.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food efficiency:
not examined
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):
After 12 weeks of treatment males receiving 1000 mg/kg/day had group mean white blood cell, lymphocyte and basophil counts higher; after 4 weeks of recovery there was evidence of full recovery of these parameters. No such effect observed in females.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
After 12 weeks of treatment females receiving 300 or 1000 mg/kg/day showed higher plasma glucose concentrations when compared with controls; after 4 weeks of recovery there was evidence of recovery of this parameter. No such effect observed in males.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
After 12 weeks of treatment males and females receiving 1000 mg/kg/day had higher mean specific gravity when compared with controls; after 4 weeks of recovery there was evidence of recovery of this parameter.
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
After 13 weeks of treatment females receiving 1000 mg/kg/day had higher group mean adjusted kidney and liver weights; after 4 weeks of recovery there was evidence of recovery of this parameter. No such effect observed in males.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In each one male and female at 1000 mg and one female at 300 mg/kg/day in the non-glandular mucosa of the stomach the epithelium was thickened by minimal to moderate hyperplasia. No changes seen in the recovery animals that had received 1000 mg/kg/d.
Histopathological findings: neoplastic:
not specified
Details on results:
Assessment of the oestrous cycle in all females during the first 15 days and the last 15 days of treatment did not reveal any adverse effects of treatment.
Sperm motility, concentration and morphology were unaffected after 13 weeks of treatment at dose levels up to 1000 mg/kg/day.

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
Basis for effect level:
other: the small effects observed at the end of the 13 week dosing period showed clear recovery in the following 4 weeks without dosing.

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Oral administration of WS 400128 at dose levels up to 1000 mg/kg/day was well tolerated with no adverse effects on clinical/ophthalmic condition, sensory reaction, grip strength, motor activity, body weight performance, food/water consumption, oestrous cycles or sperm analysis.
Clinical pathology and organ weight investigations revealed some differences that could be associated with treatment at 1000 mg/kg/day but they were slight and/or restricted to a single sex and did not persist following cessation of treatment. Histopathological examinations revealed changes in the stomach that were indicative of irritation (epithelial hyperplasia, hyperkeratosis, erosion and inflammation) in the nonglandular mucosa of the stomach at 1000 mg/kg/day in both sexes and in one female that received 300 mg/kg/day. As these findings in the stomach were confined to the nonglandular mucosa which is not present in the human stomach, this finding is not considered relevant in terms of human toxicity.
It was therefore concluded that the no observed adverse effect level was 1000 mg/kg/day.