Registration Dossier

Diss Factsheets

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Some information in this page has been claimed confidential.

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Remarks:
The experimental study was performed with a structural analogue; read-across from results of this study is justified (see below).
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: read-across from structural analogue
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010
Reference Type:
publication
Title:
Unnamed
Year:
2011

Materials and methods

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

Test material

Test animals

Species:
rat
Strain:
Sprague-Dawley
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: Ca. 70 days.
- Weight at treatment start: Males: minimum 341 g, maximum 390 g,
Females: minimum 212 g, maximum 277 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): 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 visually and by chemical analysis during the 7-day range-finding study:
Endpoint study record "7.5.1 Repeated dose toxicity: oral - 7d_range-finding_gavage_HLS_GAH0029".
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).
- Concentrations (verified at first, second and last treatment week) of the test material formulations were lower than expected from nominal concentrations (see Table 1).
- Mean concentrations of WS400151 in prepared formulations attained by chemical analysis were 70.5% to 87.2% of the corresponding nominal concentration. Evaluation of a number of aspects of the analytical and formulation procedures did not lead to identification of the cause for the lower concentrations achieved by chemical analysis. In order to take a conservative approach, it was concluded that the concentrations determined by chemical analysis reflected the concentrations of test substance formulation administered to the animals.
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).

Duration of treatment / exposure:
- Treatment period, males & toxicity phase females: Daily, for five consecutive weeks, in males commencing 14 days prior to mating
- Treatment period, main phase females (i.e. reproductive subgroup): 44 to 47 days (from 14 days prior to mating to day 6 of lactation)
- Offspring were not dosed
Frequency of treatment:
Daily, 7 days/week (during parturition, dosing omitted as appropriate)
Duration of test:
- Duration of test, all F0 males & toxicity subgroup females: Five weeks
Duration of test, reproductive subgroup females (F0): From 14 days prior to mating to day 7 of lactation.
Duration of test, offspring: From birth to day 7 of lactation.
Doses / concentrationsopen allclose all
Dose / conc.:
7.1 mg/kg bw/day (actual dose received)
Remarks:
dose levels derived from chemical determination of administered formulations
Dose / conc.:
21.9 mg/kg bw/day (actual dose received)
Dose / conc.:
75 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 young adult rats (females nulliparous and non-pregnant) in which a NOAEL could not be established at dose levels of 100, 300 or 1000 mg/kg/day.

Examinations

Maternal 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.
- Body weight: Weekly for pre-mating and mating period; on gestation days 0, 6, 13, 20; on lactation days 1 & 7.
- Body weight: Weekly for pre-pairing period; on gestation days 0, 6, 13, 20; on lactation days 1 & 7.
- Food consumption: Weekly for pre-pairing period, during gestation for days 0-6, 6-13, 13-20, during lactation for days 1-4 & 4-7.
- Frequency of vaginal estrus: Daily by examination of vaginal smears taken from the beginning of the treatment period to the day of confirmed copulation.
(During the pairing period, females were checked every morning for pregnancy).

Additional parameters examined:
- No. of animals mating (evidence of successful copulation, i.e. sperm positive vaginal smear and at least one copulation plug)
- Pre-coital interval (pairing days until detection of mating)
- No. of animals achieving pregnancy
- No. of living pregnant females
- From Day 20 post copulation 3 times a day checks for evidence of parturition, any difficulties and numbers of live and dead offspring.
- Gross pathology
- Organ weights
adrenals, brain, heart, kidneys, liver, lungs & bronchi, ovaries, pituitary, spleen, thymus, thyroid with parathyroids, uterus with cervix & oviducts.
- Histopathology (gross lesions).

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. Examinations confined to toxicity subgroup animals and/or to parental males (F0) are detailed in the separate endpoint study records,
"7.5.1 Repeated dose toxicity: oral - Repeat dose tox combined_gavage_rat_HLS_GAH0030" and
"7.8.1 Toxicity to reproduction - Tox to reproduction screening combined_gavage_rat_HLS_GAH0030"




Ovaries and uterine content:
The ovaries and uterine content were macroscopically examined after termination on day 7 of lactation, i.e. day 7 post partum.
Numbers of uterine implantation sites were recorded and post implantation survival determined.
In addition, gestation length (time elapsing between detection of mating and commencement of parturition) was recorded.
Fetal examinations:
Litters were examined post partum as follows (day of birth = day 0):
- Number:  Daily until day 7 post partum.
- Sex: In total litter: 1st day; in live litter 1st and 7th day
- Live births/mortality: Daily until day 7 post partum.
- Clinical signs: Daily until day 7 post partum
- Body weight of live pups: 1st, 4th, 7th day and weight change from days 1-4 and 1-7.
- Necropsy:  7th day (Full macroscopic examination of all 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_GAH0030"
Indices:
- Percentage mating (No. of animals mating/No. of animals paired) x 100
- Conception rate (No. of animals achieving pregnancy/No. of animals mated) x 100
- Fertility index (No. of animals achieving pregnancy/ No. or animals paired) x 100
- Gestation index (No. of live litters born on day 0/No. of living pregnant females) x 100
- 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
- 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
- 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





Historical control data:
Not included in the study report.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
two deaths, intubation error
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
reduced body weight gain, only at 75 mg/kg/day
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
reduced food consumption, only at 75 mg/kg/day
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
only at 75 mg/kg/day; counts of monocytes slightly high, of total white cells and neutrophils marginally high
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
only at 75 mg/kg/day: jejunum and duodenum thickened, mesenteric lymph nodes enlarged
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
at 21.9 and 75 mg/kg/day; in jejunum, mesenteric lymph node and/or duodenum
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
not examined
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: Based mainly on histopathology in nulliparous and non-pregnant females after 5 weeks of treatment

Details on maternal toxic effects:
CLINICAL SIGNS, NEUROBEHAVIOUR AND MORTALITY
Three high dose females (2 of the toxicity subgroup and 1 of the reproductive subgroup) were found dead following intubation errors. One low and one high dose females without viable litter were killed on Day 25 after evidence of mating. Mortality or clinical signs attributable to treatment with the test material or effects on sensory reactivity, grip strength or motor activity were not evident.

BODY WEIGHT AND WEIGHT GAIN
At 75 mg/kg/day, overall bodyweight gain was low in females during the first two treatment weeks and during gestation.

FOOD CONSUMPTION
At 75 mg/kg/day, food consumption was slightly low during the first two treatment weeks and lower than concurrent controls overall during gestation and lactation. At lower dose levels (7.1 and 21.9 mg/kg/day) foodconsumption 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 not affected by treatment.

GROSS PATHOLOGY
After five treatment weeks at 75 mg/kg/day, the jejunum of all surviving nulliparous non-pregnant toxicity phase females was thickened, the mesenteric lymph nodes were enlarged in 2 of these females, and the duodenum was thickened 2 females. At lower dose levels after 5 treatment weeks and at all dose levels in dams killed on Lactation Day 7, toxicologically relevant macroscopic lesions were not evident. In addition, mean numbers of uterine implantations were considered unaffected by treatment with the test substance.

ORGAN WEIGHTS
Following lactation, adjusted mean kidney weight was low with relationship to treatment. This was considered to be fortuitous and toxicologically not significant, as there was no such change in toxicity phase animals after 5 treatment weeks.

HISTOPATHOLOGY
In general, tissues from maternal animals were not histopathologically examined. In nulliparous, non-pregnant females histopathology findings indicative of adverse effects on reproductive organs were not evident. After five treatment weeks, the presence of foamy macrophages in the mucosa of the jejunum and duodenum at 75 mg/kg/day, and foamy histiocytosis in the sinuses of the mesenteric lymph nodes at 21.9 and 75 mg/kg/day were attributed to treatment with the test material and were considered potentially adverse, but were not considered to represent reproductive toxicity endpoints.

Further details of histopathology findings in adult animals (main phase males and nulliparous, non-pregnant 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_GAH0030".

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
> 7.1 - <= 21.9 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
histopathology: non-neoplastic

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
please note: fetuses were not examined in this reproduction and developmental toxicity screening study. Pups were examined on day 7 of lactation. No effects were observed on pup number, sex ratio, pup body weight, litter size and weight, postnatal survival, external malformations.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
no effects observed

Effect levels (fetuses)

Key result
Dose descriptor:
NOEL
Effect level:
> 75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
highest dose tested
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
changes in postnatal survival

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
In the present study, the no-adverse-effect-level (NOAEL) for maternal toxicity was 7.1 mg/kg/day. This NOAEL was derived mainly from histopathology findings in nulliparous, nonpregnant females after 5 weeks of treatment, because, in general, histopathology was not conducted on dams. The no-effect-level (NOEL) for developmental toxicity has been 75 mg/kg/day, i.e. the highest dose level tested. Dose levels substantially higher than this were not selected for the present study, because of general toxicity. Effects indicative of reproductive or developmental toxicity of WS400151 were not evident.