Registration Dossier

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:
3 March 1987 - 21 April 1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP - Guideline study, tested with the source substance propane-1,2-diyl diacetate (CAS 623-84-7). In accordance to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989

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): Propylene glycol diacetate
- Analytical purity: > 99.5%
- Lot/batch No.: XZ 87265

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
Eighty-four virgin female New Zealand White rabbits were obtained from ENKI-konijnenfarm, Someren, The Netherlands. They arrived on February 17,
1987. The age of the animals at the start of the study was about 6 months, the body weight range was 3145-4321 g.

The hair of rabbits was shaved on the premises of the breeder, using electric clippers. It was removed from the back between the shoulder and the lumbar region, resulting in a bare area of about 150 cm2.

The animals were checked for signs of ill health and anomalies immediately upon arrival. Male rabbits of proven fertility and the female rabbits were
housed individually in suspended, galvanized cages, fitted with a wiremesh floor and front. The cages were placed in a room, controlled for light (a 12 hour light/dark cycle), temperature (18 +/- 3C), ventilation (ca. 3 air changes/hour ), and relative humidity (at least 40%) throughout the study.

A nutritionally adequate standard laboratory rabbit diet and unfluoridated tap water were provided ad libitum.

Administration / exposure

Route of administration:
dermal
Vehicle:
unchanged (no vehicle)
Details on exposure:
Considering the results of a preliminary dermal toxicity study with PGDA it was decided, after consultation with the sponsor, to apply undiluted
PGDA in amounts of 0.3 and 1.0 ml/kg body weight/day. The control group was treated with 1.0 ml distilled water/kg body weight/day.

The test and control substances were applied daily on the shaven dorsal skin, covering an area of approximately 25 cm2/ml. Prior to application, the treated skin was cleaned with lukewarm water-moistened tissues. To prevent the treated rabbits from ingesting the test compound orally, they were provided with neck collars. The collars were applied one week before administration of the test solution, which started at day 6 of pregnancy e and continued up to and including day 18.

Analytical verification of doses or concentrations:
no
Details on mating procedure:
A few days before the start of the experiment, on calendar dates February 26 and 27, 1987, the rabbits were shaved again.

Starting on March 3, 1987, small numbers of rabbits were inseminated artificially. Ovulation was induced by an intravenous injection of a luteinizing hormone (Pregnyl, Organon, Oss, The Netherlands), diluted to a final concentration of 50 IU/ml in 1 ml physiological saline. Immediately after injection of Pregnyl, the females were inseminated artificially with about 40 million spermatozoa, freshly obtained from dummy copulations of male New Zealand White breeders of proven fertility. The day of insemination was considered as day 0 of pregnancy. This procedure was continued on week days until a sufficient number of possibly pregnant animals was obtained for each group.

Inseminated females were assigned to each group in rotation. The selection of females to be inseminated was based on the body weight, measured on February 20, 1987. The animals with the highest body weight were inseminated on the first day. The sequence was continued from where it ended on the previous day until each of the three groups contained the desired number of 28 animals.
Duration of treatment / exposure:
Treatment with PGDA was started on day 6 of pregnancy, and continued up to and including day 18.
Frequency of treatment:
daily
Duration of test:
Days 6 - 18 of gestation
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0.3 and 1.0 mL/kg bw/day
Basis:
other: daily dermal dose
Remarks:
Doses / Concentrations:
317 and 1058 mg/kg bw/day
Basis:
other: calculated daily dermal dose based on a specific gravity of 1.058 g/cm³
No. of animals per sex per dose:
28
Control animals:
other: distilled water

Examinations

Maternal examinations:
Body weights
The animals were individually weighed on day 0, 6, 12, 19, 24 and 29 of pregnancy.

Food consumption
The quantity of food consumed by each animal of each group was determined during the following periods: day 0 to day 6, day 6 to day 19 and day 19 to day 29 of pregnancy.
Ovaries and uterine content:
On day 29 of pregnancy (the day before term) the rabbits were killed by intravenous injection of 1 ml/kg body weight Euthesate (Apharmo, Arnhem, The Netherlands).

Subsequently both ovaries and the uterus were exteriorised through a midline incision in the abdominal wall. The number of corpora lutea of pregnancy in each ovary was recorded and both ovaries and the gravid uterus were weighed.

Thereafter, the foetuses were removed from the uterus, dried of amniotic fluid, weighed and examined for gross abnormalities.
Fetal examinations:
The foetus length was measured from crown to tail. The placentas of the live foetuses were weighed and examined for macroscopic abnormalities. Early and late resorptions and dead foetuses were counted. Resorption was classified "early" when only placental tissue and "late' when placental as well as embryonic tissue was visible at termination. In addition, the number of implantation sites in both uterine horns was recorded and the empty uterus was weighed. All foetuses were killed by cooling of the body temperature down to 0°C.

Half the number of the foetuses of each litter was then decapitated, the heads were fixed in Bouinfs fixative and subsequently free-hand sectioned
according to a modification of Wilson's technique, (van Julsingha and Bennett, 1977). The fresh bodies of these foetuses were examined for visceral anomalies by careful dissection. After these examinations the bodies were cleared and stained with Alizarine Red S for skeletal examination. The remainder of the pups of each litter was also examined for visceral anomalies by careful dissection. However, these foetuses were not decapitated. After visceral examination with exception of the heads, these intact foetuses were cleared and stained with Alizarin Red S for skeletal examination of head and body.

All examinations for foetal abnormalities were carried out under a stereo microscope at magnifications up to 40 times.
Statistics:
For the statistical analyses of differences in degree of ossification between the test and control groups the Student t-test was applied to transformed ossification values (DgOt) expressed in degrees and calculated by the formula:

DgOt = arcsine sqrt(DgO)

Statistical analysis of differences in body weight was carried out by applying analysis of co-variance, with body weight on day 0 as the co-variable, followed by Dunnett's multiple comparison test. Food consumption, organ weights, litter data, foetus weights and lengths and placenta weights were analysed by applying analysis of variance, followed by Dunnett's multiple comparison test, whereas skeletal and visceral anomalies were evaluated by the Chi-square test.
Indices:
Percentage of pre-implantation loss (PRIL) was calculated from each litter by the formula:

PRIL = (number of corpora lutea - number of implantation sites)/(number of corpora lutea) x 100%

Percentage post implantation loss (POIL) was similarly calculated for each litter by the formula:

POIL = (number of implantation sites - number of live young)/(number of implantation sites) x 100%

The degree of ossification of foetus skeletons (DgO) was calculated for each litter by the formula:

DgO = (number of bones without ossification (or with incomplete ossification))/(number of bones examined)
Historical control data:
Included as Annex 7 of report

Results and discussion

Results: maternal animals

Maternal developmental toxicity

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

Details on maternal toxic effects:
Clinical signs and dermal effects
During the study, no abnormalities in condition or behaviour were observed that could be related to the treatment with PGDA. No PGDA related mortality occurred.

Several rabbits, distributed over all groups showed inflammatory skin reactions or scaliness in the neck region or on the ears. These parts were
not treated with PGDA or water, and the effects were considered to be caused by chafing of the neck collars.

During the study no dermal reactions were observed at the application sites of the treated rabbits that could be related to the dermal application of PGDA.

Maternal performance
All 84 rabbits, injected with Pregnyl to stimulate ovulation, and subsequently artificially inseminated, showed evidence of ovulation. The number of pregnant animals per group was 24 to 26, and the fertility index ranged from 85.7 in the high-dose group to 92.9 in the control group. Animals A33, A23 and A7 of the control group aborted on nominal day 23, 24 and day 28 of pregnancy, respectively. Animal C33 of the high dose group aborted on nominal day 27 of pregnancy.

Animal B27 died day 28 of pregnancy; gross examination at autopsy revealed indications of inflammatory reactions in the lungs.

One dam in the high-dose group (C49) revealed only dead foetuses. All other animals showed litters with live foetuses.

The gestation index ranged from 91.7 in the high-dose group to 96.0 in the mid-dose group. All values were well within normal limits.

Growth and food intake
Mean maternal body weights at the start of the study varied only slightly between the groups. During the experiment the individual maternal weights and weight gain varied considerably within and between the groups. In all groups, a number of animals gained weight whereas others lost weight. Mean maternal body weights were always comparable in all groups. Mean weight gain during treatment was only somewhat lower in the low dose group, when compared with the controls. Mean food intake figures measured over the three periods did not differ significantly between the test groups and controls, nor was there a dose-related increase or decrease in food consumption over these three periods of time.

Gross observations of dams at sacrifice
At autopsy of does on day 29 of pregnancy, animal A23 of the control group as well as animal C49 of the high-dose group showed an uterus horn filled with purulent exsudate. Animal A55 showed an uterus horn filled with haemorrhagic fluid. Hypertrophic ovaria were seen in animal B43 of the mid-dose group. A swollen and red uterus horn was observed in animal C17 of the high-dose group. Animal A7 of the control group showed a pale liver and atelectasis of the lungs, the stomach and intestines of this animal were filled with a clear fluid.

No abnormalities were observed that could be related to the treatment with PGDA.

Autopsy findings and litter data
The mean number of corpora lutea, implantation sites, early and late resorptions and dead foetuses were comparable in all groups. Consequently, pre- and postimplantation loss did not reveal any treatment-related effect. No differences were observed between the groups in the mean number of live foetuses.

Maternal organ weights
Mean absolute weights of the ovaries, gravid and empty uterus did not show significant differences between test groups and control, nor was a dose-related increase or decrease detected.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 058 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
1 058 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

Details on embryotoxic / teratogenic effects:
Foetus weights and lengths
Mean foetal and placental weight were slightly lower in the 1.0 ml/kg group when compared with the controls. However, no statistically significant differences were observed.

Macroscopic examination of foetuses
No macroscopically visible malformations were observed in foetuses that could be related to treatment.

Umbilical hernia was observed in one foetus of litter B1 of the 0.3 ml/kg group.

The number of foetuses with body weights of less than 60 percent of the mean body weight in the control group (= dysmature appearance) were comparable between the groups.

The dysmature foetus of litter B47 of the 0.3 ml/kg group also showed a flexure of both forelimbs. Torsio of both hindlimbs was observed in one
foetus of litter B45 of the 0.3 d/kg group.

Examination of foetal soft tissues
Visceral malformations were observed in 4 foetuses of the high-dose group. These malformations consisted of a missing aorta-arch (litter C19 foetus no. 9), unilateral agenesis of the right ovary and uterus horn accompanied with a hypertrophic left ovary (litter C1 foetus no. 3), a misshapen spleen (litter C25 foetus no. 3) and severe edema formation accompanied with a hypotrophic heart, lungs and pancreas in foetus no. 7 of litter C51.

The incidence of minor variations in arterial branching in the aortic arch area was slightly higher in the high dose group than in the controls. However, the incidence of litters in which these variations were observed was the same in both groups. Moreover, minor variations in arterial branching are frequently observed in control series and the observed differences were not statistically significant.

Examination of the placenta revealed a somewhat higher number of placental cysts in the high-dose group litters than in the controls.

Examination of foetal skeletons
Skeletal malformations were observed in both the control and the high-dose group.

A complexity of thoracic and spinal malformations was observed in foetus no. 6 of litter C7 of the 1.0 ml/kg group; foetus no. 11 of litter C31 showed a misshapen cervical vertebral body.

A malformed sternebra was observed in foetus no. 9 and foetus no. 3 of respectively litters A5 and A29 of the control group as well as in foetus
7 and foetus no. 4 of litters C51 and C55 of the high-dose group, respectively.

A malformed and fused sternebra was observed in foetus no. 9 of litter C19 of the high-dose group. The thoracic ribs of foetus no. 7 of litter A19
was misshapen; thoracic rib no. 11 of foetus no. 4 of litter C13 was bifurcated, and thoracic ribs nos. 6 and 7 of foetus no. 1 of litter C47 were fused. The pollex was missing bilaterally in foetus no. 1 of litter C9, and unilaterally in foetus no. 2, litter C51.

No statistically significant differences were observed in the incidence of skeletal malformations between the high dose group and the controls, either for each observation separately or for the total incidence of skeletal malformations.

Minor skeletal anomalies and variants occurred in a number of foetuses in all groups. There were no statistically significant differences between the control and high-dose group, which could be related to the treatment. The variation in ossification of foetal skeletons, expressed as the arcsine transformation of the different ossification in the high-dose group, was very well comparable with the control values. The only significant difference that was observed between high-dose group and controls (incomplete ossification of hindlimb phalanges) is considered a fortuitous finding due to an extremely low control value as compared with the species variation that is normally observed in control series.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Propylene glycol diacetate, applied dermally to rabbits in the sensitive period of pregnancy with a limit quantity of 1 ml/kg bw (1058 mg/kg bw) per day was not embryo/foetotoxic or teratogenic.
Executive summary:

Propylene glycol diacetate was examined for its potential embryotoxic and teratogenic properties in New Zealand white rabbits. Groups of at least 24 rabbits were dermally treated during days 6 through 18 with 0, 0.3 and 1.0 ml PGDA per kg bw (equal to 0, 0.317 or 1.058 g/kg bw). 1 g/kg bw is a limit quantity for substances with low toxicity, as specified by OECD.

The dose levels were chosen on the basis of a probe study. On day 29 of pregnancy the does were autopsied. The foetuses were weighed and examined microscopically. Half of the foetuses were examined by cross sections according to the Wilson technique. The remainder of the foetuses were also examined for visceral and skeletal abnormalities. No mortality or abnormality in condition or behaviour were observed in any of the groups that could be related to the treatment. Fertility and gestation were comparable in all groups and well above the average of the historical controls.

Body weights, food intake, autopsy findings and litter data did not reveal any treatment related effect.

No compound related visceral or skeletal malformations, anomalies or variants were observed in the foetuses.

Propylene glycol diacetate, applied dermally to rabbits in the sensitive period of pregnancy with a limit quantity of 1 ml/kg bw per day was not embryo/foetotoxic or teratogenic.