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Description of key information

Combined Repeated Dose Oral (Gavage) Toxicity Study with the Reproduction/Developmental Toxicity Screening Test with WS400152 in rats according to OECD 422:

Doses / Study Design:                                               0 (vehicle control), 100, 300, 1000 mg/kg bw/day, with 5 toxicity subgroup animals/sex/dose.

Treatment period, toxicity subgroup animals:      Daily, for five consecutive weeks

Sacrifice, toxicity subgroup animals:                      Week 6

Effects considered to represent mild toxicity of WS400152 were evident at 1000 mg/kg/day in males. These comprised:

- Increased incidence and severity of myocardial inflammation/degeneration/fibrosis; in 2 of the affected males associated with increased aspartate amino-transferase (AST) activity in blood plasma;

- Low scores of motor activity for both rearing and ambulatory activity during most of the 1-h recording period;

- Transiently (Treatment week 1 to 2) bodyweight gain lower than in concurrent controls.

Conclusion: NOAEL of 300 mg/kg/day was attained for subacute (5 weeks) repeated dose toxicity testing.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
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:
guideline study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
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. QA statement)
Limit test:
no
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: 70 days.
- Weight at treatment start: Males: minimum 328 g, maximum 380 g,
Females: minimum 224 g, maximum 271 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: 5 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_GAH0076".
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).
- For Treatment Week 1 the group mean chemically determined concentrations of WS400152 in test formulations administered to the animals were 115.0 to 119.5% of the corresponding nominal concentration, thus slightly passing the upper limit of the acceptable range of 85 to 110%. However, for Treatment Week 2 and the final treatment week, the chemically determined group mean concentrations were 93.5 to 105% of the corresponding nominal concentration, thus being within the acceptable range. Overall, these results confirmed accurate 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 48 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)
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:
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: 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.

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. Due to myocardial histopathology findings in males at 1000 mg/kg/day,
the heart was examined from all adult male animals of all dose groups. 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.

* As an exception the sternum was not retained from 1 toxicity subgroup male of Group 1 (verhicle control) and 3 toxicity subgroup males of Group 4 (1000 mg/kg bw/day). To ensure the required number of tissues were available for examination, this tissue was retained from 4 other males treated at the corresponding dose level.
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.
If this H1 test was significant, suggesting that the dose-response was not monotone, the Steel's test was performed instead.


-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
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
mainly at 1000 mg/kg/day and mainly transient
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
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
although most of them not of toxicological significance
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Motor activity reduced in males at 1000 mg/kg/day
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Increased incidence & severity of myocardial inflammation/degeneration/fibrosis in males at 1000 mg/kg/day
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS, NEUROBEHAVIOUR AND MORTALITY
There were no deaths and no toxicologically relevant findings, except that in males at 1000 mg/kg/day motor activity scores were low for both, rearing and ambulatory activity, during most of the 1-h recording period after 5 weeks of treatment. However, underactive behaviour was not observed as a clinical sign during the study.

Increased salivation at all dose levels and chin rubbing occasionally seen at 100 mg/kg/day and more commonly at 300 or 1000 mg/kg/day, as well as paddling of the forepaws transiently seen in females at 300 or 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
At 1000 mg/kg/day, bodyweight gain was lower than in concurrent controls in males from treatment week 1 to 2 and in main phase females (dams) at this dose level during the first week of gestation and during the first week of lactation. This was also the case in dams at 300 mg/kg/day during the first week of lactation. Food consumption was unaffected by treatment with the test material.

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 after 4 weeks at 1000 mg/kg/day:
- alanine amino-transferase (ALT) activity increased in both sexes,
- aspartate amino-transferase (AST) activity increased in 2 of the males showing also myocardial inflammation/degeneration/fibrosis
- total cholesterol concentration increased in females
- calcium concentration increased in females
- phosphorus concentration increased in females.
The increases in ALT and cholesterol were attributed to increased metabolic activity in the liver, those in calcium and phosporus to increased metabolic activity and/or excretion by the kidneys. The increased AST activity may have been related to the myocardial histopathology findings in the two affected animals.

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

GROSS PATHOLOGY
Macroscopic pathology findings attributable to treatment with the test material were not evident.

HISTOPATHOLOGY (NON-NEOPLASTIC)
An increase in incidence and severity of myocardial inflammation/degeneration/fibrosis in males at 1000 mg/kg/day (listed in Table 2) was attributed to treatment with WS400152 and considered to represent mild toxicity. Single incidences of this finding, minimal in degree, in males at 100 or 300 mg/kg/day were not attributed to treatment as this was also seen in one male animal of the concurrent vehicle control group.

OTHER RESULTS
Reproductive and developmental toxicity parameters are addressed in separate endpoints.
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: see 'Remark'
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: see 'Remark'
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
cardiovascular
Organ:
heart
Treatment related:
yes

Table 2: Summary of Treatment Related Findings in Males and Toxicity Phase Females Killed after 5 Treatment Weeks

Group/sex

1/M

2/M

3/M

4/M

1/F

2/F

3/F

4/F

Dose (mg/kg/day)

0

100

300

1000

0

100

300

1000

 

 

Heart

 

Myocardial Inflammation/ Degeneration/Fibrosis

 

Minimal

1

1

1

4

0

0

0

0

Slight

0

0

0

1

0

0

0

0

Total

1

1

1

5

0

0

0

0

 

 

 

 

 

 

 

 

 

Number of tissues examined

10

10

10

10

5

0

0

5

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
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

Repeated dose toxicity: via oral route - systemic effects (target organ) cardiovascular / hematological: heart

Justification for classification or non-classification

In the OECD 422 Study, the NOAELs for systemic toxicity were 300 mg/kg/day for adult male rats and 1000 mg/kg/day for adult female rats. These NOAELs do not necessitate any classification regarding repeated exposure according to European classification rules [REGULATION (EC) 1272/2008].

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