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Diss Factsheets

Administrative data

Endpoint:
developmental toxicity
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-reference
Reason / purpose for cross-reference:
reference to same study

Data source

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

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

Constituent 1
Details on test material:
- Name of test material (as cited in study report): WS400109

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. 71 days.
- Weight at treatment start: Males: minimum 327 g, maximum 411 g,
Females: minimum 230 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_GAH0134".
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).
- 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 WS400109 in prepared formulations were 94% to 104% 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.
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): 42 to 46 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)
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 / 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 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.

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-pairing period; on gestation days 0, 6, 13, 20; on lactation days 1, 4 & 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, thyroids 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.




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, 4-7 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_GAH0135"
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:
For gestation length included in the study report.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
WS400109 was tested in the rat in a Combined Repeated Dose Oral (Gavage) Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD 422) at dose levels of 0 (vehicle control), 100, 300 and 1000 mg/kg bw/day. The study was terminated on Lactation Day 7. Effects on reproductive and developmental endpoints were not evident. Therefore, in this screening study the no-observed-effect-level (NOEL) regarding reproductive and developmental toxicity was the highest dose level applied, i.e. 1000 mg/kg bodyweight/day. The study results do not warrant the classification of WS400109 regarding reproductive or developmental toxicity according to European classification rules [DIRECTIVE 67/548/EEC and REGULATION (EC) 1272/2008].