[No public or meaningful name is available]

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

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

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1,2-Benzenedicarboxylic acid, di-C9-11-branched and linear alkyl esters

EC Number:

271-085-1

EC Name:

1,2-Benzenedicarboxylic acid, di-C9-11-branched and linear alkyl esters

Cas Number:

68515-43-5

IUPAC Name:

dioctyl phthalate

Details on test material:

- Name of test material (as cited in study report):DIPLAST L9 11P
- Physical state:yellow liquid
- Lot/batch No.:PLA/S20517/97
- Quantity: 590 g was used on this study
- Storage: ambient temperature

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:Charles River
- 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 acclimatisation 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/light on 6:00 GMT

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): Formulations were prepared on a weekly basis and divided into daily aliquots which were stored at ambie nt 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: for the following doses (0, 250, 500, 1000 mg/kg/day) the following concentrations are used: 0, 50, 100, 200 mg/ml
- Amount of vehicle (if gavage): 5 daily doses, each with 5 ml/kg of the relative concentrations.

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. Results of these analyses area presented in the Formulation Chemistry report.

Details on mating procedure:

The female are housed 1:1 with stock male from the same strain and source

Duration of treatment / exposure:

gavage

Frequency of treatment:

Daily dose in 5 application steps

Duration of test:

20 days (gestation)

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
50 mg/ml
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
100 mg/ml
Basis:
actual ingested

Doses / concentrations 4

Remarks:

Doses / Concentrations:
200 mg/ml
Basis:
actual ingested

No. of animals per sex per dose:

Three groups of 22 female + a control group (22 female)

Control animals:

yes, concurrent vehicle

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 are only reported for Days 0, 1, 3, 6, 9, 12, 15, 18 and 20 after mating.

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:

Foetuses were uniquely 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 by chilling on a cool plate (approximately 4°C).
Approximately half of each litter was allocated to fresh visceral examination at necropsy and were eviscerated and fixed in industrial methylated spirit (74° op.). These foetuses were processed and stained 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 using the programme (Startox) developed by Huntingdon Life Sciences. 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

Details on maternal toxic effects:
The incidence of general clinical signs did not indicate any adverse effect of treatment with D911P and all animals survived to termination.

Effect levels (maternal animals)

Dose descriptor:

NOAEC

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, 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.

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 necropsy 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 pregnant, however one female(no. 68) at 1000 rig/kg/day was found to only have three empty implantation sites (two an the 1ef and one in the right uterine horn) at necropsy examination on Day 20 of gestation. This litter has been 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 D911P during the extended treatment period adopted in this study. 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 D911P.

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 litters 32 and 33 at 250 mg/kg/day and 81 and 86 at 1000 mg/kg/day being particularly affected. In view of the litter clustering and the absence of any clear dosage relationship, these findings are considered to be coincidental, and are 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 be attributed to treatment with certainty.

Applicant's summary and conclusion

Conclusions:

All females survived to termination and, with the exception of one female at 1000 mg/kg/day which showed total litter resorption, all females had live offspring on Day 20 of gestation. Assessment of clinical signs, bodyweights and bodyweight gain, food intake and necropsy findings, including gravid uterine weight, did not indicate any adverse effects of treatment on the parent female.
Assessment of litter data, mean placental, litter and foetal weight and observations made at detailed visceral and skeletal examination of the foetuses did not reveal any clear adverse effect of treatment on enbryofoetal survival or development.
It was concluded that daily treatment with D911P from before implantation until just before the expected day of parturition, at dosages up to 1000 mg/kg/day, had no clear adverse effects on pregnancy of the female rat or on embryofoetal survival and development.

Executive summary:

A study of developmental toxicity revealed 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.