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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented and reported study fully adequate for assessment. The study was conducted according to an internationally accepted technical guideline and in compliance with GLP in a recognized contract research organization.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
of 1996
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals 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: ca. 72 days.
- Weight at treatment start: Males: minimum 325 g, maximum 387 g,
Females: minimum 229 g, maximum 280 g.
- Housing Inside a barriered rodent facility:
all animals pre-pairing + toxicity subgroups: In groups up to 5 by sex in solid floor polycarbonate cages.
during pairing (1 male+1 female/cage): In RB3 modified polypropylene cages with stainless steel grid-floor over absorbent paper-lined trays.
males after pairing: In groups up to 5 in solid floor polycarbonate cages.
females during gestation and lactation: Females housed individually (+litter) in solid floor polycarbonate cages.
- Bedding material (in solid floor cages): Wood based bedding, sterilised by autoclaving before use.
- Cage enrichment: Aspen chew block + plastic shelter (except during pairing or post Gestation Day 20).
- Diet (ad libitum): Standard rodent diet (SDS VRF1 Certified) without antibiotic, chemotherapeutic or prophylactic agent.
- Fasting (diet withheld): Main phase males and Toxicity phase females overnight before blood sampling for clinical pathology.
- Water (ad libitum): Potable drinking water from the public supply.
- Acclimation period: 7 days before treatment start, under laboratory conditions.

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

IN-LIFE DATES:
- Duration of test, males & toxicity phase females: Five weeks
Duration of test, main phase females (i.e. reproductive subgroup): From 14 days prior to pairing to day 7 of lactation.
Duration of test, offspring: From birth to day 7 of lactation.

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): 22 ± 3°C
- Relative Humidity (%): 40 to 70%
- Photoperiod (artificial lighting): 12 hrs day / 12 hrs night
- Rate of air exchange: At least 15 changes/h
Deviations from the target ranges for temperature and relative humidity were not evident.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
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): 5 mL/kg bw/day.
Actual dose volumes were calculated at about weekly or shorter intervals accounting for the latest bodyweight. Litter animals were not dosed.

- For concentrations of test material in vehicle at different dose levels, see Table 1 in "Any other information on materials and methods incl. tables"

- Justification for choice of vehicle:
The suitability of propylene glycol as a vehicle was established during the 7-day range-finding study:
Endpoint study record "7.5.1 Repeated dose toxicity: oral - 7d_range-finding_gavage_HLS_GAH0116".
In addition, in the present main study, concentrations of dose formulations were chemically analysed.

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 coupled with a mass spectrometer (HPLC-MS/MS).
- Mean concentrations (verified for first and last treatment week) of the test material formulations were confirmed at each dose level.
Chemical analysis confirmed that the mean concentrations of WS400123 in prepared formulations were 97.0% to 112.9% of the corresponding
nominal concentration, thus confirming acceptable accuracy of formulation for dosing of the animals.
- Homogeneity and stability of test material formulations at 2 and 200 mg/L and at storage and handling conditions similar to those adopted for dosing of the animals were confirmed.
Duration of treatment / exposure:
- Treatment period, males & toxicity phase females: Daily, for five consecutive weeks, in males commencing 14 days prior to pairing
- Treatment period, main phase females (i.e. reproductive subgroup): 43 to 47 days (from 14 days prior to pairing to day 6 of lactation)
- Offspring were not dosed
Frequency of treatment:
Daily, 7 days/week (during parturition, dosing omitted as appropriate)
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:
Toxicity phase animals: */ 5 females
Main phase animals (i.e. reproductive subgroups): 10 males / 10 females
*Explanatory note by the notifier:
Examinations assigned to the toxicity phase females to meet the requirements of a 28-day repeat dose oral toxicity study were also assigned to 5 (for some examinations to 10) main phase males per dose group. Therefore, these 5 main phase males per dose group are called also "toxicity subgroup" in the present robust study summary for clarification. After pairing with main phase females, all males (F0) were killed at the same time (Week 6).
Control animals:
yes, concurrent vehicle
Details on study design:
This study was conducted to examine both repeated dose toxicity and  reproductive/developmental toxicity as an OECD screening combined study
(OECD 422 test guideline).  Therefore, animals initially entering the study were divided into toxicity subgroup animals (toxicity phase) and reproductive subgroup animals (main phase), whereby 5 of the 10 F0 males (used for pairing) per dose group formed the toxicity male subgroups.

Dose selection was based on the results of a 7-day preliminary oral (gavage) toxicity study in the rat in which dose levels of 100, 300 or 1000 mg/kg/day did not have any overt treatment-related effects on young adult animals (females nulliparous and non-pregnant).
Positive control:
Not included in the study.

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 (except 1 or 2 females taking 5 to 12 days for pairing).
- Functional Observation Battery:* During treatment week 5 (before dosing) on all toxicity subgroup animals (5 males + 5 females/group).
- Body weight, all males: About weekly throughout the study.
Body weight, Toxicity Females: About weekly throughout the study.
Body weight, Repro. Females: Weekly for pre-pairing period; on gestation days 0, 6, 13, 20; on lactation days 1, 4 & 7.
- Food consumption, all males: Weekly for pre-pairing period and for the period after mating.
Food cons., Toxicity Females: About weekly throughout the study.
Food cons., Repro. Females: Weekly for pre-pairing period, during gestation for days 0-6, 6-13, 13-20, during lactation for days 1-4 & 4-7.

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

Haematological examinations (only for toxicity subgroup animals) during treatment week 5 after functional observation battery:
Red blood cell count, reticulocyte 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.

Blood (plasma) chemical examinations (only for toxicity subgroup animals) during treatment week 5 after functional observation battery:
Total protein, albumin, A/G ratio, urea, creatinine, glucose, total cholesterol, triglycerides, total bilirubin, bile acids, sodium, potassium, chloride,
calcium, inorganic phosphorus, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase.

This study was conducted to examine both repeated dose toxicity and  reproductive/developmental toxicity as an OECD screening combined study
(OECD 422 test guideline).  Therefore, some of the examinations were confined to toxicity subgroup animals, as indicated above.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, see below
WEIGHING OF ORGANS: Yes, see below
HISTOPATHOLOGY: Yes, see below

Terminal sacrifice
- all males (F0) and toxicity subgr. females: Killed in Week 6, after completion of the Treatment Week 5 investigations.
- reproductive subgr. females & offspring: Killed on Day 7 post partum.
(1 control & 1 mid dose female were killed on the respective Day 25 after mating, as they had failed to litter)
(1 mid dose female was found dead on Treatment Day 8 due to an intubation error)
(1 high dose female was found dead on Gestation Day 6 due to an intubation error)

Gross pathology:
- adult/parental animals: Full macroscopic examination.
- offspring, Lactation Day 7: Careful external macroscopic examination of all survivors for gross abnormalities.
- externally abnormal offspring
and premature deaths: Internal macroscopic examination including assessment of the presence of milk in the stomach, where possible. (Missing or grossly autolysed or cannibalised offspring could not be examined).
Organs Weights:
- all males (F0) +
toxicity subgroup females: Adrenals, brain, epididymides, heart, kidneys, liver, ovaries, prostate, seminal vesicles with coagulation gland,
spleen, testes, thymus, uterus with cervix & oviducts.
- dams: Ovaries

Histopathology:
- toxicity subgroups*: The following organs were microscopically observed for the control and 1000 mg/kg bw/day groups:
Brain, eyes, pituitary gland, thyroid with parathyroids, heart, thymus, liver, spleen, adrenals, kidneys, testes,
epididymides, ovaries, lung, trachea, oesophagus, stomach, duodenum, jejunum, ileum, caecum, rectum, colon,
Peyer's patch, lymph node (axillary, mesenteric), urinary bladder, uterus (with cervix & oviducts), vagina,
spinal cord, sciatic nerve, skeletal muscle, sternum with marrow, seminal vesicle & coagulation gland, prostate.
In addition, any gross lesions for all adult animals from all dose groups were examined by light microscopy.

* Histopathology examination only on control & high dose groups, 5 males and 5 females per group

- reproductive subgroups All above organs/tissues from premature deaths (1 mid dose & 1 high dose females) and any gross lesions from all adult animals from all dose groups were examined by light microscopy.
Other examinations:
Reproductive and developmental toxicity parameters (addressed in separate endpoints).
Statistics:
Statistical analysis of grip strength, motor activity, bodyweight, food consumption, organ weight, litter size, survival indices & clinical pathology data:

- Parametric analysis, if Bartlett's test for variance homogeneity was not significant at the 1% level.
F1 approximate test for monotonicity of dose-response. If this F1 test was not significant at the 1% level, Williams' test for a monotonic trend was applied.
If this F1 test was significant, suggesting that the dose-response was not monotone , the Dunnett's test was performed instead.

- Non-parametric analysis, if Bartlett's test was still significant at the 1% level following logarithmic and square-root transformations.
H1 approximate test for monotonicity of dose-response. If this H1 test was not significant at the 1% level, Shirley's test for a monotonic trend was applied.

Grip strength, motor activity, survival indices and clinical pathology data
if 75% of the data (across all groups) were the same value, pairwise comparison of each dose group against the control by Fisher’s Exact tests.

Organ weight data
Covariance analysis using terminal bodyweight as covariate (Angervall & Carlstrom, 1963)

Sex ratio
Analysis by generalised mixed linear model with binomial errors, a logit link function and litter as a random effect (Lipsitz et al 1991).
Comparison of each treated group with control using a Wald chi-square test

Pre-coital intervals
Exact (or asymptotic) 1-tailed (upper tail) Linear-by-linear test (Cytel 1995) using 4 different scores*

Gestation length
Exact (or asymptotic) two-tailed Linear-by-linear test (Cytel 1995), with equally spaced scores.*

- Live litters born
Exact 1-tailed (lower tail) Cochran-Armitage test (Cytel 1995)*

* If significant (p<0.05), exclusion of the highest dose group and re-testing until no longer significant (p≥0.05).

For statistical references, see next field.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
attributable to treatment with the test material
Mortality:
no mortality observed
Description (incidence):
attributable to treatment with the test material
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
reduction in haematocrit, haemoglobin concentration and erythrocyte count at 300 and 1000 mg/kg/day, but not considered to represent adverse effects
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional observation battery including sensory reactivity tests (approach, touch, auditory startle reflex, tail pinching), grip strength, motor activity and attention to clinical signs of neurotoxicity during animal observations.
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS, NEUROBEHAVIOUR AND MORTALITY
One mid dose female and one high dose female of the reproductive subgroups were found dead following intubation errors. One control and one mid dose females were killed on the respective Day 25 after evidence of mating, because they had failed to litter. Mortality, clinical signs or effects on sensory reactivity, grip strength or motor activity attributable to treatment with the test material were not evident. Forelimb grip strength lower in treated females than in concurrent controls and a statistically non-significant trend to reduced forelimb grip strength in high dose males after 5 weeks of treatment were attributed to natural variation, as the changes in females were not dose related and all values were within the historical control range. Hindlimb grip strength was unaffected in both sexes.

BODYWEIGHT, WEIGHT GAIN AND FOOD CONSUMPTION
Bodyweight and food consumption were unaffected by treatment with WS400123 in treated males and nulliparous, nonpregnant females. In dams receiving 1000 mg/kg bw/day, mean bodyweight gain was slightly lower than in concurrent controls during lactation Days 4-7, but did not differ statistically significantly from controls overall during Lactation Days 1-7 and therefore was considered to represent normal biological variation.

CLINICAL PATHOLOGY
Toxicologically significant changes in haematology or clinical chemistry (blood plasma) parameters were not evident. Haematocrit, haemoglobin concentration and erythrocyte counts statistically significantly lower after 5 weeks of treatment at 300 and 1000 mg/kg/day than in concurrent controls were not considered to represent adverse effects, because of the absence of adverse effects on clinical condition of animals, behaviour or reproductive performance and the absence of miccroscopic histopathology findings attributable to treatment with the test material.

ORGAN WEIGHTS
Toxicologically significant effects on organ weights were not evident in the present study.

GROSS PATHOLOGY AND HISTOPATHOLOGY (NON-NEOPLASTIC)
Macroscopic or microscopic pathology findings attributable to treatment with the test material were not evident.

OTHER RESULTS
Reproductive and developmental toxicity parameters are addressed in separate endpoints.

Effect levels

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: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion