Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from September 29,1999 to October 28,1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant study conducted to recognised international test guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
2000

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:
- Name of test material (as cited in study report): D911P
- Physical state:yellow liquid
- Lot/batch No.:PLA/S20517/97
- Analytical purity: 99.2% m/m
- Storage: ambient temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Charles River (UK) Ltd, Margate, Kent
- Justification: the rat was chosen because of its use as a predictor of reproductive toxic change in man and the requirement for a rodent species by regulatory agencies. The CD strain was used because of the historical control data available in this laboratory.
- Age at study initiation:10 - 11 weeks
- Weight at study initiation:220-267 g
- Housing:barriered limit access, rodent facility. The facility was designed and operated to minimise the entry of external biological and chemical ag ents and to minimise the transference of such agents between rooms. Access was limited to authorised personnel who were required to shower and change into clean protective clothing. Where practicable, materials and equipment entered the facility through an autoclave or a chamber in which th eir external surfaces were treated with a bactericide. Before the study commenced the room was cleaned and disinfected with a bactericide.
- Diet :ad libitum
- Any other information about food:commercially available pelleted expanded rodent diet (DSD laboratory animal diet N°1) from special diet services Limited, periodical analysed by supplier.
- Water :ad libitum:
- Acclimation period:5 days
- Medical check: during the acclimatattion period , twice a day

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C +/- 4 °C
- Humidity (%): 55% (48% - 61%)
- Air changes (per hr):15 room air changed per hour
- Photoperiod :12 hour cycle dark/lightm on 6:00 GMT

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations were prapared on a weekly basis by hand mixing the test substance with the selected vehicle followed by homogenisation. The prepared formulations were divided into daily aliquots which were stored at ambient temperature

VEHICLE
- Justification for use and choice of vehicle:The oral (gavage) route was selected to simulate the conditions of possible human exposure
- Concentration in vehicle: 0, 50, 100, 200 mg/ml (to give dose levels of 0, 250, 500, 1000 mg/kg/day)
- Amount of vehicle (if gavage): 5 ml/kg b.w.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each formulation prepared for administration on one occasion during the first and last weeks of treatment were analysed for test material content. Four 1.0 ml sample were taken from each group on each occasion 2 samples per group were analysed for test material content and the
remaining 2 were retained as contingency samples.

Analysis showed concentrations to be within +3.8% / -1.8% of the nominal concentration.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: 3 x vaginal plugs or sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: no
Duration of treatment / exposure:
19 days (Days 1-19 of gestation)
Frequency of treatment:
Daily
Duration of test:
20 days (gestation)
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0
Basis:
nominal conc.
= dose level of 0mg substance / kg b.w. (control)
Remarks:
Doses / Concentrations:
50 mg/ml
Basis:
nominal conc.
= dose level of 250mg substance / kg b.w.
Remarks:
Doses / Concentrations:
100 mg/ml
Basis:
nominal conc.
= dose level of 500mg substance / kg b.w.
Remarks:
Doses / Concentrations:
200 mg/ml
Basis:
nominal conc.
= dose level of 1000mg substance / kg b.w.
No. of animals per sex per dose:
Three groups of 22 females + a control group (22 females)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on outcome of a preliminary study
- Rationale for animal assignment (if not random): Allocated to group/cage position in sequence of mating. Allocation adjusted to prevent to prevent any stock male providing more than one mated female in each treatment group. Allocation adjusted if Day 0 group mean body weights differed beyond acceptable limits.
- Other: Cage distribution arranged to minimise possible environmental variables

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes , Females were weighed and bodyweight recorded daily throughout the study. Bodyweights Days 0, 1, 3, 6, 9, 12, 15, 18 and 20 after mating were rported.

FOOD CONSUMPTION : Yes , food consumption recordings were made for the periods Days 1-2, 3-5, 6-8, 9-11, 12-14, 15-17 and 18-19 after mating.

POST-MORTEM EXAMINATIONS: Yes

- Organs examined: yes

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: Yes
- Number of distribution of foetuses in each uterine horn: yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter

Foetuses were identified within the litter with respect to their uterine position. Each foetus was weighed, sexed and examined for any external abnormalities. individual placental weights and placental abnormalities were recorded. Foetuses were then killed. Approximately half of each litter was allocated to fresh visceral examination, were eviscerated and fixed before processing staining with Alizarin Red, using a modification of the Dawson staining technique, for examination of their skeletons. The remaining foetuses were fixed in Bouin's fluid before examination by the Wilson free-hand serial sectioning technique.
Statistics:
Where appropriate, data was subjected to statistical analyses. Dependent on the heterogeneity of variance between groups, parametric tests (analysis of variance, Snedecor and Cochran 1967) followed by Williams' test (Williams' 1971/2) or non-parametric tests, (Kruskal-Wallis, Hollander and Wolfe 1973) followed by Shirley's test (Shirley 1977) were used to analyse these data, as appropriate. For litter data the basic sample unit was generally the litter and, due to preponderance of non-normal distributions, non-parametric analyses were routinely used.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

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

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
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

Any other information on results incl. tables

MATERNAL RESPONSAL:

Clinical signs

Increased levels of salivation after dosing were seen sporadically in all groups, including control. A greater number of animals were affected at 1000 mg/kg/day than in the Control and lower dosage groups; however, it was noted that post-dosing salivation is frequently observed in studies with oral gavage dosing and its occurrence is generally considered to reflect distaste of the dosing formulation rather than

indicating toxicological effect. Several animals in all groups (including control) showed staining of the coat on the head and body, and some incidences of hairloss: these observations were considered to be related to the use of oil as the dosing vehicle and to be of no toxicological significance.

Bodyweight

Bodyweights and bodyweight gain during gestation were unaffected by treatment. Adjustment of overall bodyweight gain for the contribution of the gravid uterus showed no adverse effect of treatment on bodyweight gain.

Food consumption

Food consumption during gestation was unaffected by treatment; group mean values for all treated groups were similar to the Control throughout

Macroscopic pathology

Macroscopic findings at neuropsy of females on Day 20 of gestation were unremarkable being generally restricted to confirmation of signs (hairloss staining) observed in-life. No findings considered to be of toxicological significance were observed.

LITTER RESPONSES:

Litter data

All females were pregnante , however one female at 1000 mg/kg/day was found to only have three empty implantation sites (two in the left and one in the right uterine horn). This litter was classified as total litter resorption (total litter loss in-utero). Given the isolated nature of this finding, and the absence of any adverse effect on the process of implantation (as judged by pre-implantation loss) or on foetal survival (as judged by post-implantation loss), an association with treatment was considered unlikely.

The number of implantations, resorptions and live foetuses were essentially similar in all groups and were unaffected by treatment. There was no evidence that the implantation process or in-utero survival had been adversely affected by administration of the test substance. Sex ratio, expressed as percentage males, was comparable in all groups.

Placental, litter and foetal weights

There were no obvious adverse effects of treatment on placental, litter or foetal weights.

It was noted that mean foetal weight was higher in both sexes at 1000 mg/kg/day, with differences from Control attaining statistical significance for females; this was not considered to be of any toxicological significance.

Foetal evaluation

There were 3, 1, 2 and 3 foetuses (3, 1, 2 and 2 litters affected) in Groups 1 to 4 respectively showing major abnormalities. Neither the type, incidence nor distribution of major abnormalities indicated any obvious adverse effect of treatment with the tested substance.

The incidence and distribution of minor visceral and skeletal abnormalities or skeletal variants did not indicate any adverse effect of treatment on foetal development.

At 1000 and 250 mg/kg/day, detailed visceral examination revealed a higher incidence of foetuses showing dilated ureter compared to the Control, although this incidence was within the recent background control data range. Accompanying this finding was a higher incidence of renal cavitation and hydroureter in animals allocated to skeletal processing, at these dosages compared to the Control.

Collectively these findings were noted to cluster, with two litters at 250 mg/kg/day and two litters at 1000 mg/kg/day being particularly affected. In view of the litter clustering and the absence of any clear dosage relationship, these findings were considered to be coincidental, and emphasised by a low incidence in the Control group.

At 1000 and 500 mg/kg/day there was also an increased incidence of foetuses with 13/14 or 14/14 ribs. Again there was no dose relationship and, as the total number of litters affected remained similar to that of the Control group, it was considered that this finding could not, with certainty, be attributed to treatment.

Applicant's summary and conclusion

Conclusions:
Potential developmental toxicity has been studied in the rat using methods in accordance with OECD TG414. Daily treatment from before implantation to just before the expected day of parturition resulted in no clear adverse effects on pregnancy of the female rat or on embryofoetal survival and development at dose levels up to 1000 mg/kg bw/day.




Executive summary:

Potential developmental toxicity has been studied in the rat using methods in accordance with OECD TG414. Daily treatment from before implantation to just before the expected day of parturition resulted in no clear adverse effects on pregnancy of the female rat or on embryofoetal survival and development at dose levels up to 1000 mg/kg bw/day.