Registration Dossier

Administrative data

Description of key information

Repeated oral dosing (gavage) to rats in the frame of a sub-acute and a sub-chronic study did not lead to any adverse or toxic effects. In both studies the NOAEL was derived at the highest dose level, i.e. 1000 mg/kg/d.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
supporting 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.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
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
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: 70 days.
- Weight at treatment start: Males: minimum 357 g, maximum 403 g,
Females: minimum 225 g, maximum 267 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 of 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: 6 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): 21 ± 2°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.
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 body weight. 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_GAH0091".
In addition, in the present main study, concentrations of dose formulations were determined by chemical analysis.

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).
- Concentrations (verified at first and last treatment week) of the test material formulations were confirmed at each dose level.
- Chemical analysis confirmed that the mean concentrations of WS400130 in prepared formulations were 93.5% to 99.5% of the corresponding
nominal concentration, thus confirming accuracy of formulation.
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)
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 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 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) necessitating any reduction of target dose levels in the present OECD 422 combined repeat dose toxicity and reprotoxic/develpmental toxicity screening study.
Positive control:
Not included in the study.
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 5 (before dosing) on all toxicity subgroup animals (5 males + 5 females/group).
- Body weight, all males: Weekly throughout the study.
Body weight, Toxicity Females: Weekly throughout the study.
Body weight, Repro. Females: Weekly for pre-pairing period; on gestation days 0, 6, 13, 20; on lactation days 1 & 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.

Hematological 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, total bilirubin, bile acids, sodium, potassium, chloride,
calcium, inorganic phosphorus, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transpeptidase.

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 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 low dose female was killed on treatment day 6 in poor health condition resulting from an intubation error)
(1 mid dose female without viable litter was killed on lactation day 1)
(1 high dose female was killed on gestation day 19 in poor health condition resulting from an intubation error)

Gross pathology:
- adult/parental animals: Full macroscopic examination with tissue collection.
- offspring: Full 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).

Organ Weights:
- main phase and tox. subgr. adults: Adrenals, brain, epididymides, heart, kidneys, liver, lungs & bronchi, ovaries, pituitary, prostate, seminal vesicles
& coagulation gland, spleen, testes, thymus, thyroid with parathyroids, uterus with cervix & oviducts.

Histopathology:
- toxicity subgroups: The following organs were microscopically observed for the control and 1000 mg/kg bw/day groups:
Brain, eyes, Harderian glands, optic nerves, pituitary gland, thyroid with parathyroids, heart, thymus, liver, spleen,
adrenals, kidneys, testes, epididymides, ovaries, lung, trachea, esophagus, stomach, duodenum, jejunum, ileum,
caecum, colon, rectum, Peyer's patch, lymph node (axillary, mesenteric), urinary bladder, uterus (with cervix &
oviducts), vagina, spinal cord, sciatic nerve, skeletal muscle, skin with mammary glands, 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.
- reproductive subgroups 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:
Grip strength, motor activity, bodyweight, food consumption, organ weight, gestation length, litter size, survival indices & clinical pathology data were statistically analysed by adoption of the following sequence of tests:

- 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.

-For 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.

-For 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).
Each treated group was compared to control using a Wald chi-square test.

Gestation length
- Exact (or asymptotic) two-tailed Linear-by-linear test (Cytel 1995), with equally spaced scores. If the test was statistically significant (p<0.05), the highest
dose group was excluded and the test re-applied until the test was no longer statistically significant (p≥0.05).

For statistical references, see next field.
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:
no effects observed
Description (incidence and severity):
attributable to treatment with the test material
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
of toxicological significance
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
Description (incidence and severity):
attributable to treatment with the test material
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
There were no deaths attributable to treatment with the test material. One low dose female was killed on treatment day 6 in poor health condition resulting from an intubation error, one mid dose female without viable litter was killed on lactation day 1 and one high dose female was killed on gestation day 19 in poor health condition resulting from an intubation error.

Toxicologically relevant clinical signs or effects on sensory reactivity, grip strength or motor activity were not evident. A low incidence of chin rubbing occurring in females at 0 (vehicle control), 100 and 300 mg/kg/day, and a low incidence of salivation in females at 100, 300 and 1000 mg/kg/day may have been related to palatability of the formulations and therefore were considered to be of no toxicological importance.

BODYWEIGHT, WEIGHT GAIN AND FOOD CONSUMPTION
There were no adverse effects on bodyweight, weight gain and food consumption.

CLINICAL PATHOLOGY
Haematology parameters were not affected by treatment with the test material. Intergroup comparison of clinical biochemistry parameters in blood plasma revealed the following treatment-related changes: low plasma calcium levels in both sexes at 1000 mg/kg/day and low albumin and total protein levels in males at this dose level. In the absence of any correlating in life or histopathology findings these differences were considered not to be of toxicological significance.

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.
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: no adverse effects observed
Key result
Critical effects observed:
no
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
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI Wistar rats
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 7 weeks at start of treatment
- Weight at study initiation: males 229-261 g, females 177-215 g
- Fasting period before study: no
- Housing: 2 to 3 animals per cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 7 days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.0 - 25.6°C
- Humidity (%):28 - 67%
- Air changes (per hr): 15 - 20 per hour
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

IN-LIFE DATES: From: To:
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on oral exposure:
VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 20, 60, 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test substance was dispersed in propyleneglycol. This dispersion was diluted with tetrahydrofuran (1:2) and directly measured by near infrared (NIR) spectroscopy between 908 and 1676 nm (transmission).
Frequency of treatment:
once daily
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
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes /
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least weekly

BODY WEIGHT: Yes
- Time schedule for examinations: at least weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before start of treatment and in week 13
- Dose groups that were examined: all animals before treatment start, animals of control and high dose group in week 13

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of necropsy
- Anaesthetic used for blood collection: Yes pentobarbital
- Animals fasted: Yes
- How many animals: all
- Parameters checked in Table 1 below were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of necropsy
- Animals fasted: Yes
- How many animals: all
- Parameters checked in Table 2 below were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: the night before necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in Table 3 below were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 13
- Dose groups that were examined: all dose groups
- Battery of functions tested: sensory activity / grip strength / motor activity / other: meassurement of landing foot splay

IMMUNOLOGY: No

OTHER: oestrous cycles and sperm parameters
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see Table 4 below)

HISTOPATHOLOGY: Yes (see Table 5 below)
Other examinations:
Determination of the oestrous cycles in females of all dose groups during the last two weeks of dosing.

Evaluation of sperm parameters in males of the control and high dose groups at necropsy.
Statistics:
Group mean and standard deviation were calculated for numerical data. Statistical analysis was performed using SAS 9.2 (built in Provantis System). The following decision tree was automatically applied within the validated Provantis system for statistical evaluation of numeric data:
The normality and heterogeneity of variance between groups were checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunett (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis is the better option when the normality and heterogeneity assumptions implicit in the tests are adequate.

If either of the Shapiro-Wilk or Levene tests showed significance on the data, then the ANOVA type approach was not valid and a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons were performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
few incidental findings in animals of all dose groups
Mortality:
mortality observed, treatment-related
Description (incidence):
one animal of the low dose group died after misgavage on Day 90.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
During the treatment period, there were no adverse changes in the mean body weights or body weight gains between the dose groups either sex. However, slightly reduced body weight gain was noted in males of all dose groups reaching statistical significance compared to the control group. Reduction of body weight gain was not dose dependent and is not considered biologically relevant. see Table 1 Body weight (attached)
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There were no consistent changes measured in the food consumption during the treatment period between the dose groups. However there was sporadic lower food consumption measurements in Mid dose males and females compared to the Control group with statistical significance, but these were considered unrelated with treatment. see Table 2 Food consumption (attached)
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
see Table 3 Haematology (attached)
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
see Table 4 Clinical Chemistry (attached)
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no toxicologically significant changes in the animal behaviour, general physical condition, in the reactions to different type of stimuli, grip strength or motor activity in the control or treated groups, at the evaluation performed on Week 13.

When compared to the control, lower values were noted in grip strength tests for hind limb in males 1000 mg/kg bw (High dose) however it was not statistically significant.

There was no effect of treatment noted during the assessment of landing foot splay or motor activity.

During evaluation of motor activity, the total travelled distance and the pattern of activity was comparable to the control in both sexes.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
see Table 5 Organ weights (attached)
Gross pathological findings:
no effects observed
Description (incidence and severity):
Few findings were observed which were considered incidental or background as these findings were distributed at very low incidence in all dose group animals as well as in control animals.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Few findings were observed which were considered incidental or background as these findings were distributed at very low incidence in all dose group animals as well as in control animals.
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed
Description (incidence and severity):
The oestrous cycles of all females were determined during the last two weeks of dosing. All animals showed the normal distribution of the oestrous phases. There were no differences between dosed and control females.

Sperm parameters were determined after necropsy in control and high dose males. There were no statistically significnat differences in sperm count, sperm motility and not normal sperms.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Key result
Critical effects observed:
no
Conclusions:
In this subchronic oral toxicity study the test substance did not exhibit any adverse toxic effects to rats up to and including the highest dose level of 1000 mg/kg/day.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

In both repeat-dose studies, sub-acute and sub-chronic, the NOAEL for systemic toxicity in adult male and female rats after oral treatment with WS400130 was 1000 mg/kg bw/day. This does not necessitate any classification regarding repeated exposure according to European classification rules [REGULATION (EC) 1272/2008].