Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): WS400152

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: 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.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
Treatment of parental animals by oral gavage administration. Test substance was not directly administered to F1 animals.

- 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.

- 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.

Details on mating procedure:
- Male/female ratio per cage: 1/1
- Length of cohabitation: At the most 14 days, until proof of pregnancy was confirmed. 
- Proof of successful mating: Formation of at least one copulation plug and a sperm positive vaginal smear.
The day this was found was referred to as day 0 of gestation.
(During cohabitation, females were checked every morning for pregnancy).

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)
Details on study schedule:
- Age at mating of the mated animals in the study: 12 to 13 weeks
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.:
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.

Examinations

Parental animals: Observations and examinations:
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 parental males/group inclusively)*.
- 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.
- Hematology: During treatment week 5 after functional observation battery*
- Blood (plasma) chemistry: During treatment week 5 after functional observation battery*

* Examinations confined to toxicity subgroup animals are marked above with an asterisk*
and are detailed in the separate endpoint study record "7.5.1 Repeated dose toxicity: oral - Repeat dose tox combined_gavage_rat_HLS_GAH0077"

Explanatory note
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.
Oestrous cyclicity (parental animals):
Frequency of vaginal oestrus was determined by examination of vaginal smears taken daily from all main phase (i.e. reproductive subgroup) females from the beginning of the treatment period to the day of confirmed copulation.
- Regular: All observed cycles of 4 or 5 days
- Irregular: At least one cycle of 2, 3 or 6 to 10 days
- Acyclic: At least 10 days without oestrus
Sperm parameters (parental animals):
Parameters examined in male parental animals:
- testis weight,
- epididymis weight
- detailed qualitative histopathology examination of the testes taking into account the tubular stages of the spermatogenic cycle. This was to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was noted.
Litter observations:
STANDARDISATION OF LITTERS: Not performed. The study ended on lactation day 7.

LITTER PARAMETERS EXAMINED
- From Day 20 post copulation 3 times a day checks for evidence of parturition, any difficulties and numbers of live and dead offspring.
- Total litter size on day 1 of age and mortality/live litter size on each day until 7 days after littering.
- Sex ratio expressed as percentage males and calculated for total offspring on Day 1 and for live offspring on Days 1 & 7
(No. of male pups in litter/No. of offspring in litter) x 100
- Gestation index (No. of live litters born on day 0/No. of living pregnant females) x 100
- Clinical signs, recorded daily
- Body weight of live pups (on days 1, 4 and 7 after littering) and weight change from days 1-4, 4-7 and 1-7.



Postmortem examinations (parental animals):
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.

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.
Postmortem examinations (offspring):
Pup survivors were killed on Day 7 post partum.
Full macroscopic examination of decedent and surviving pups including assessment of the presence of milk in the stomach, where possible.
(Missing or grossly autolysed or cannibalised pups could not be examined).
Statistics:
As detailed in Endpoint study record "7.5.1 Repeated dose toxicity: oral - Repeat dose tox combined_gavage_rat_HLS_GAH0077"
Reproductive indices:
- Pre-coital interval (pairing days until detection of mating)
- No. of animals mating (evidence of successful copulation, i.e. at least one copulation plug and a sperm positive vaginal smear)
- No. of animals achieving pregnancy
- Percentage mating (No. of animals mating/No. of animals paired) x 100
- Fertility index (No. of animals achieving pregnancy/ No. or animals paired) x 100
- Conception rate (No. of animals achieving pregnancy/No. of animals mated) x 100
- Gestation length (time elapsing between detection of mating and commencement of parturition)
- No. of living pregnant females
- For further reproductive parameters, see also the above section "Litter observations" and section "Offspring viability indices" below.
Offspring viability indices:
- Post-implantation survival index  (Total no. of pups born/Total no. of uterine implantation sites) x 100
- Live birth index (No. of live pups on day 1 after littering/Total no. of pups born) x 100
- Viability index (No. of live pups on day 4 after littering /No. of live pups on day 1 after littering) x 100
- Lactation index (No. of live pups on day 7 after littering /No. of live pups on day 1 after littering) x 100
- For further parameters indicative of the viability of the offspring, see also the above section "Litter observations"

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
but specific measurement revealed motor activity reduced in males at 1000 mg/kg/day
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
effects on bodyweight gain mainly at 1000 mg/kg/day and mainly transient. Food consumption was unaffected by treatment
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Increased incidence & severity of myocardial inflammation/degeneration/fibrosis in parental males at 1000 mg/kg/day. In parental females only gross lesions were histopathologically examined. Nulliparous females were unaffected by treatment.
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
see Table8 8 to 12 on reproductive performance in attached background material

Details on results (P0)

CLINICAL SIGNS 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.

REPRODUCTIVE ENDPOINTS
Oestrous cycles, pre-coital interval, mating performance, fertility, gestation length, gestation index and litter size were unaffected by treatment.

GROSS PATHOLOGY
Macroscopic findings attributable to treatment with the test material were not evident. The mean numbers of uterine implantation sites in main phase females recorded on Lactation Day 7 were unaffected by treatment with WS400152.

ORGAN WEIGHTS
Toxicologically significant effects on organ weights were not evident in males and females following 5 treatment weeks or in dams on lactation Day 7. In dams of the high dose group (1000 mg/kg/day), ovary weights adjusted to terminal bodyweight were marginally higher (statistically significant at p<0.05) than concurrent controls. In the absence of confirmatory findings in nulliparous females after 5 treatment weeks, this finding was toxicologically not important.

HISTOPATHOLOGY
There were no histopathology findings indicative of adverse effects on reproductive organs in parental males and toxicity subgroup females (nulliparous and non-pregnant) after five weeks of treatment. In addition, evaluation of the seminiferous testicular tubules regarding their stage in the spermatogenic cycle and regarding the integrity of the various cell types present within the different stages did not reveal any cell or stage abnormalities. Main phase females (reproductive subgroup) killed on lactation day 7, in general, were not histopathologically examined.

Further details of histopathology findings in adult animals (main phase males and toxicity subgroup females) of the present study are given in separate endpoint study record "7.5.1 Repeated dose toxicity: oral - Repeat dose tox combined_gavage_rat_HLS_GAH0077".

Effect levels (P0)

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: see 'Remark'

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
attributable to parental treatment with the test material
Histopathological findings:
not examined

Details on results (F1)

There was no effect of parental treatment with WS400152 on offspring survival up to Day 7 of age, clinical signs, sex ratio, bodyweight, development or macropathology findings.

Effect levels (F1)

Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: NOEL = highest dose tested. Offspring development up to Day 7 of age.

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Categories Display