Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-04-28 until 2008-07-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Storage Conditions: ambient temperature, in the dark

Test animals

Species:
rat
Strain:
other: Crl:CD®(SD)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, UK
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 184-293g
- Housing: The animals were housed one per cage in solid-bottomed polypropylene cages (ca 42 x 27 x 20cm); sterilized wood shavings were provided as bedding. A stainless steel food hopper and polycarbonate water bottle were provided for each cage. The cages were suspended on racks.
Cages, bedding and water bottles were changed at regular intervals, as appropriate.
- Diet (e.g. ad libitum): Rat and Mouse Breeder Diet No. 3 (Expanded) SQC, supplied by Special Diets Services Ltd., Stepfield, Witham, Essex, UK was available to the animals ad libitum. The diet was supplied with a batch analysis for nutritive constituents and a range of significant contaminants. The analytical certificate for a batch of diet used during this study is retained in the study archive.
- Water (e.g. ad libitum): The animals had access to domestic quality mains water ad libitum. The supply is analyzed regularly for dissolved and suspended materials, including a range of significant contaminants. The analytical certificate for a typical recent analysis is retained in the study archive.
- Acclimation period: The animals were acclimatized in the Charles River animal room from arrival until commencement of treatment on Day 6 of gestation (3-5 days)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C ± 2°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): minimum of 15 air changes per hour
- Photoperiod (hrs dark / hrs light): Light hours were 0700-1900 h.

IN-LIFE DATES:
Experimental Start Date: 02 May 2008
Experimental Completion Date: 11 July 2008

Environmental Enrichments:
To provide environmental enrichment, wooden chewsticks were made available to the animals, as appropriate. The chewsticks were not considered to contain any additional substances in sufficient concentrations to influence the outcome of the study.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
The vehicle used was 2% medium viscosity CMC containing 0.2% Tween 80.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For the first 5 days of the study, formulations were prepared daily and used within the established stability period of 24 hours. After 8 days stability had been established, formulations were prepared at approximately weekly intervals, and used within the 8 days stability period (established in Charles River Laboratories Study No. 424799, Method 2479).
An appropriate amount of test item was weighed and placed in a suitably sized glass container, and then the appropriate amount of vehicle was added and mixed by magnetic stirring until a visibly homogenous suspension was obtained.

VEHICLE
The vehicle used was 2% medium viscosity CMC containing 0.2% Tween 80. Full details of the vehicle preparation are retained in the study raw data.

Amount:
10 ml/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of dosing formulations was undertaken with regard to concentration and homogeneity on two formulation occasions during the study; from formulations prepared for use on Day 1 of the study and Week 2 of the study. Triplicate samples were withdrawn from each formulation, including Control, and were assayed at Charles River, using a method supplied by the Sponsor and previously validated under a separate protocol and contract (Charles River Laboratories Study No. 424799, Method 2479).
The results of the analysis of the dosing formulations were within ±8% of the nominal concentration indicating acceptable accuracy of formulation. The low coefficients of variation (<6.5%) indicated that the formulations were homogenous.
Details on mating procedure:
- Impregnation procedure: cohoused
- Length of cohabitation: 3 successive days
- Proof of pregnancy: day of detection of a vaginal plug or sperm in a vaginal smear = Day 0 of gestation
- further information:
Ninety eight time-mated female rats were obtained in one delivery. The delivery consisted of 3 subbatches, mated over 3 successive days. On delivery, one batch was on Day 1 of gestation, the second on Day 2 and the third on Day 3 (day of detection of a vaginal plug or sperm in a vaginal smear = Day 0 of gestation). No more than two females were mated by any one male.
Duration of treatment / exposure:
over Days 6-19 inclusive of gestation (Day 0 = day of detection of mating).
Frequency of treatment:
animals were dosed once daily, at approximately the same time each day.
Doses / concentrationsopen allclose all
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
500 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:
24, The spare animals were numbered 97 and 98. These animals were not used and were not regarded as part of the study.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were agreed with the Sponsor after evaluation of existing relevant toxicological data.
- Animal Identification: Each animal received a subcutaneously implanted electronic identification chip which identified it individually within the study and which corresponded to that animal’s number. It was also given a cage card which was color coded for treatment group and marked with the study number, animal number, cage number and treatment group.

Examinations

Maternal examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
All the animals were checked for viability early in the morning and again as late as possible each working day throughout the period of the study. In addition, all the animals were examined for reaction to treatment during each day from commencement of dosing. The onset, intensity and duration of any signs were recorded, particular attention being paid to the first 1-2 hours after dosing on each day, then as required thereafter. All the animals were also given a detailed clinical examination, once prior to the start of dosing and daily from commencement of dosing.

BODY WEIGHT: Yes
Body weights were recorded on Days 4 and 6-20 of gestation. For clarity/brevity of reporting, only values on Days 4, 6, 9, 13, 17 and 20 are presented.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
The weight of food consumed by each animal was recorded daily, commencing on Day 4 of gestation (weighed quantity first offered on Day 3).

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: An external examination was followed by a macroscopic examination of the contents of the thoracic and abdominal cavities; any findings were recorded and representative samples of abnormal tissues were fixed in neutral buffered 10% formalin.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No data
- Number of late resorptions: No data
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- skeletal examinations: Yes: half per litter
- Head examinations: No data

Each implant was classified as being live, or a dead fetus (dead full term fetus that showed no evidence of maceration), or a late embryonic death (macerated tissue identifiable as an embryo or fetus, with recognizable external features such as tail, limbs, mouth and nares present; attached to
distinct, identifiable placenta), or an early embryonic death (discrete, formless, discolored tissue mass attached to the internal uterine wall; may be of varying size).
The foetuses were examined for externally visible abnormalities, each live fetus was individually identified within the litter and its weight was recorded. The fetuses were sexed during subsequent dissection procedures. Half of the viable fetuses from each uterus were fixed in methylated ethyl alcohol, the remaining half in Bouin’s fluid.
Following initial fixation, the fetuses fixed in alcohol were examined by open dissection for abnormalities of the thoracic and abdominal viscera. These viscera were then discarded. The eviscerated carcasses were macerated in potassium hydroxide, the skeletons stained with Alizarin Red S, then the fetuses cleared with aqueous glycerol solutions. These preparations were examined for the presence of skeletal abnormalities and for the extent of ossification. The fetuses fixed in Bouin’s fluid were examined for soft tissue abnormalities by a technique derived from that of Wilson (1965).
Statistics:
Statistical analysis was performed on body weight gain over Days 6-20 of gestation and on mean fetal weight. Body weight gain was subjected to analysis of variance and fetal weight was analysed by the Kruskal-Wallis test. These tests were two-sided and performed at the 5% significance level. Pairwise comparisons were only performed against the Control group.
For the other parameters, no formal statistical analyses were considered necessary; interpretation of the data being by inspection of the individual and group values.
Historical control data:
The rat is a standard rodent species for the developmental toxicity testing in animals required by the regulatory authorities. The normal processes of gestation in rats and the specific background foetal pathology are well documented in this laboratory.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical observations that were considered to be related to treatment with the test item.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Group mean body weight gains performance in all treated groups were similar to Control throughout the study.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Group mean food consumption performance in all treated groups were similar to Control throughout the study.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no necropsy findings that were considered to be related to treatment with the test item.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Details on maternal toxic effects:
Intergroup differences in pregnancy performance and foetal weights were negligible due to the slight degree and the absence of a dose-relationship.
At 150 mg/kg/day, a slight increase in the early embryonic deaths was mainly due to Animal 36 that had 13 early deaths; this animal also had a low number of live implants and a low uterus weight. In the context of this study, this isolated finding was not considered to be toxicologically relevant.

Effect levels (maternal animals)

Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
External malformations:
no effects observed
Description (incidence and severity):
The type and distribution of the major foetal abnormalities did not suggest any obvious association with treatment.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The type and distribution of the skeletal abnormalities did not suggest any obvious association with treatment. Slight intergroup differences in the distribution of skeletal ossification parameters were not obviously associated with treatment. At 1000 mg/kg/day, there was a greater incidence of foetuses with incomplete ossification of the thoracic centrum (19 foetuses in 9 litters) and an increase in the number of foetuses with ossified sacrocaudal vertebra with connection between centrum and arches at 150 mg/kg/day (72 foetuses in 20 litters). Although these values were outside the current Control background range of 3 foetuses in 2 litters to 12 foetuses in 6 litters for the first finding and 18 foetuses in 11 litters to 56 foetuses in 17 litters for the latter, the type and distribution of these findings did not indicate any obvious association with treatment.
Visceral malformations:
no effects observed
Description (incidence and severity):
The type and distribution of the minor visceral did not suggest any obvious association with treatment.

Effect levels (fetuses)

Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Basis for effect level:
other: fetotoxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
From the findings in this study, the maternal and fetal no observed effect level (NOEL) were both considered to be 1000 mg/kg/day.
Executive summary:

In a prenatal development toxicity study in rats (according to GLP and OECD guideline 414), the test item was orally administered to groups of 24 pregnant Sprague-Dawley rats. These animals were dosed by gavage once daily over Days 6-19 of gestation, where the day of detection of mating was designated as Day 0. Dose levels were 0 (2% medium viscosity CMC containing 0.2% Tween 80), 150, 500, and 1000 mg/kg/day. The dose volume applied was 10 mL/kg body weight. Animals were regularly monitored during gestation for clinical signs of toxicity and for body weight and food consumption performance. Animals were killed on Day 20 of gestation. The status of each implantation was recorded and the fetuses were weighed and examined for visceral and skeletal abnormalities, including the state of skeletal ossification. At levels up to 1000 mg/kg/day, there were no obvious effects of treatment on clinical observations, necropsy findings, body weight gain or food consumption performance. Intergroup differences in pregnancy performance and fetal weights were of a slight degree and did not follow a dose-relationship; therefore these variations are not attributable to treatment. The type and distribution of fetal abnormalities and variants did not suggest any obvious effect of treatment. From the findings in this study, the no observed effect level for both maternal and fetal toxicity was considered to be 1000 mg/kg/day.