Fatty acids, C16 and C18-20 (even numbered, unsaturated), 2-ethylhexyl esters

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28th October 1992 to 18th November 1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Current test guidelines indicates that the test material should be administered up to the day prior to the scheduled Caesarean section. In this study the rats were sacrificed on Day 20 and the fetuses were removed by Caesarean section. The test material was administered daily from day 6 to day 15 and therefore not up to the day prior to the Caesarean section. Read across to a study result from an investigation using a similar material is justified for members of the Epoxidised Oils and Derivatives group. Four epoxidised oils and esters (linseed, soybean,9-octadecanoate propylene glycol ester and 2-ethylhexyl tallate ester ETP). The C14-C22, 2-ethylhexylesters are listed as similar products on the market to ETP based on fatty acids from other naturally occurring fatty acids This group of epoxies are identified as sharing common structural and functional similarities, recognised in an OECD SIDS review as a single category, and therefore justifying read-across between data for different members of the group. Consequently data sharing between ESBO epoxidised soybean oil, ELO epoxidised Linseed oil and ETP epoxidised 2ethylhexyl tallate and fatty acids, C14-C22, 2-ethylhexylesters, epoxidised is commonly utilised in the preparation of this dossier. Read-across bridges are used for members of the EOD group where appropriate, is justified based on similar toxicity profiles and structural and functional similarities.

Justification for type of information:

Read across to a study result from an investigation using a similar material is justified for members of the Epoxidised Oils and Derivatives group. Four epoxidised oils and esters (linseed, soybean,9-octadecanoate propylene glycol ester and 2-ethylhexyl tallate ester ETP). The C14-C22, 2-ethylhexylesters are listed as similar products on the market to ETP based on fatty acids from other naturally occurring fatty acids. This group of epoxies are identified as sharing common structural and functional similarities, recognised in an OECD SIDS review as a single category, and therefore justifying read-across between data for different members of the group. Consequently data sharing between ESBO epoxidised soybean oil, ELO epoxidised Linseed oil and ETP epoxidised 2ethylhexyl tallate and fatty acids, C14-C22, 2-ethylhexylesters, epoxidised is commonly utilised in the preparation of this dossier. Read-across bridges are used for members of the EOD group, where appropriate, justified based on similar toxicity profiles and structural and functional similarities. Read across from the epoxidised oils to the unsaturated fatty acids is considered in document presented in point 7.1 Toxicokinetics, and this justifies, inter alia, the use of read -across at this point to avoid unnecessary use of laboratory animals.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

12th May 1981

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: E.E.C. Recommendation No. 87/302/E.E.C., 18th November 1987

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Epoxidised Soybean oil

IUPAC Name:

Epoxidised Soybean oil

Constituent 2

Reference substance name:

Soybean oil, epoxidized

EC Number:

232-391-0

EC Name:

Soybean oil, epoxidized

IUPAC Name:

232-391-0

Constituent 3

Reference substance name:

8013-07-8

Cas Number:

8013-07-8

IUPAC Name:

8013-07-8

Details on test material:

- Name of test material (as cited in study report): Epoxidised Soybean Oil (ESBO)
- Alias name: Reoplast 39
- Physical state: clear yellowish liquid
- Lot/batch No.: 08380306
- Expiration date of the lot/batch: July 1994
- Stability under test conditions: stable
- Storage condition of test material: Room temperature

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Sprague-Dawley Rats: CRL CD (SD) BR
- Source: Charles River, 76410 Saint-Aubain-lès-Elbeuf, France
- Age at study initiation: At the beginning of the treatment period, the females were 9 weeks
- Weight at study initiation: approximately 200 g
- Housing: The rats were housed individually in polycarbonate U.A.R. cages (48.0 x 27.0x 20.0 cm). Each cage contained autoclaved sawdust and was equipped with a water bottle. Bacteriological analyses of the sawdust and detection of possible contaminants were periodically made. Bottles and cages were changed at least once a week.
- Diet (e.g. ad libitum): U.A.R. A04 C pelleted diet, batch No. 20721
- Water (e.g. ad libitum): drinking water filtered using 0.22 micron filter
- Acclimation period: 5 days before the beginning of the treatment period

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2 °C
- Humidity (%): 50 ± 20 %
- Air changes (per hr): about 13 cycles/hour of filtered, non-recylced air
- Photoperiod (hrs dark / hrs light): 12 hours/12 hours

IN-LIFE DATES: From: 28/10/1992 To: 13/11/1992

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Soybean Oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was diluted in the vehicle and homogenised using a magnetic stirrer.
The preparations were made by the C.I.T. Pharmacy for a maximum of 7 days of use according to the stability already demonstrated at the concentrations used in the study.

DIET PREPARATION
The animals had free access to U.A.R. A04 C pelleted diet, batch No. 20721 (U.A.R., 91360, Villemoisson-sur-Orge, France) and water (drinking water filtered using 0.22 micron filter). Each batch of food was analysed by the supplier.

Bacteriological and chemical analyses of the water and detection of possible contaiminants are made periodically. There were no known contaminants in the diet, water or sawdust at levels likely to influence the outcome of the study.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Soybean Oil
- Lot/batch no. (if required): 08380327

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chemical Analysis of the Preparations:
On the first day of treatment of the first mated females and the last day of treatment of the last mated females, each preparation (control group included) was checked for achieved concentration of the test substance. Each preparation was sampled in duplicate for analysis.

Assay Method:

The solution was agitated with a magnetic stirrer. An intended volume of solution (15 mL for the 0 and 100 mg/kg bw/day groups, 5 mL for the 300 mg/kg bw/day group, 1.5 mL for the 1000 mg/kg bw/day group) was sampled in a flask using a calibrated glass pipette and diluted with 100 mL of CETAB (N-cetyl-N, N, N-trimethyl-ammonium bromide) before adding 4 drops of cristal violet indicator. The solution (agitated with a magnetic stirrer) was titrated by adding of perchloric acid solution (0.1 M perchloric acid in glacial acetic acid) with a volumetric distribution system (Dosimat 655, Methrom) until the colour of the indicator changed from violet to yellow.

Details on mating procedure:

The female rats were mated at the supplier's facilities. The day of mating was designated as day 0 of pregnancy.

Duration of treatment / exposure:

Dose levels were selected following the results of a previously conducted study (see other two studies presented in IUCLID Point 7.8.2). 1000 mg/kg bw/day was selected as the high dose level for this study; 100 and 300 mg/kg bw/day were selected as the low and intermediate dose levels respectively. Exposure was from day 6 to day 15 inclusive of pregnancy.

Frequency of treatment:

The test substance or the vehicle were given daily, at the same approximate daily time, 7 days a week from day 6 of pregnancy to day 15 inclusive.

Duration of test:

To day 20 of pregnancy

No. of animals per sex per dose:

There were 4 groups of rats, 1 group per dose concentration, and there 25 female rats per concentration tested

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected on basis of the results of a previously conducted study (CIT/Study No. 8707 RSR) performed at the dose levels of 150, 450 and 1000 mg/kg bw/day. The results showed that neither toxic effects, nor effects on reproduction and litters were noted at any of these dose levels. Therefore the 1000 mg/kg bw/day was selected as the high dose level for this study; 100 and 300 mg/kg bw/day were selected as the low and intermediate dose levels respectively. There was one group per dose concentration, with 25 animals per group, 0, 100, 300 and 1000 mg/kg bw/day.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes - animal were checked for mortaility or signs of morbidity
- Time schedule: twice a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: on days 2, 6, 9, 12, 15 and 20 of pregnancy

FOOD CONSUMPTION : Yes
- Food consumption for each animal was determined at the following intervals: day 2 -day 6, day 6 - day 9, day 9 - day 12, day 12 - day 15 and day 15 - day 20

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: heart, lung, liver, kidneys, stomach, intestines

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea
- Number and distribution of live and dead foetuses
- Number and distribution of early and late resorptions
- Number of implantation sites

Fetal examinations:

Examination of Fetuses:
- Body weight - each live foetus was weighed
- External Examination: each foetus was submitted to an external examination (including palate) to check for the presence of malformations
- Soft Tissue Examination: the soft tissue findings were classified into malformations and anomalies.
- Skeletal Examination: The skeletal findings were classified into skeletal variations, anomalies and malformations
- Sex of Fetuses: The sex of foetuses was determined at the time of the evisceration after fixation in alcohol or at the time of Wilson's sections.

Statistics:

The mean values were compared by one-way analysis of variances and Dunnett's test. Percentage values were compared by Fisher's exact probability test.

Indices:

No data supplied

Historical control data:

C.I.T historical control data:
Fetal Soft Tissue Anomalies: Dilated renal pelvis, mean: 0.9 %, range of means: 0. - 2.8 %; ureteral dilatation: mean: 1.4 %, range of means: 0 to 6.1 % .

Fetal Skeletal Variations:
Reduced Ossification of the 6th Sternebra
Fetal incidenc mean: 30.3 % - range of means: 16.0 % to 39.1 %, litter incidence: mean: 75.7 % - range of means: 57.1 % to 100.0 %.

Unossification of the 5th Sternebra
Fetal incidence: mean: 15.1 % - range of means: 1.7 % to 31.8 %; litter incidence: mean: 47.3 % - range of means: 9.5 % to 100 %.

Fetal Skeletal Anomalies:
mean: 0.7 % - range of means: 0.0 % to 3.2 %

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
No treatment-related macroscopic changes were observed at necropsy of the females. No deaths occurred in the females of any group. No abortions were noted. The mean food consumption for the females with completed pregnancy was similar in the control and treated groups. The mean body weight gain of the females with completed pregnancy was similar in the control and treated groups.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: other:

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
LITTER DATA
Corpora lutea, implantation sites and pre-implantation loss
The mean number of corpora lutea and implantation sites were similar in the control and treated groups.
The pre-implantation loss was higher in each treated group when compared to the control group. (23 %; p < 0.05 in the 100 mg/kgday group - 20.4 % N.S. in the 300 mg/kg/day - 24.4 %; p < 0.01 in the 1000 mg/kg/day group vs. 16.6 % in the control group). As the treatment of the females began after the implantation of the ova, the increase of the pre-implantation loss is considered not to have a toxicological significance.

Total Post-Implantation Loss:
- Resorptions: The rate of resorptions was similar in the control (2.6 % ) and the 100 (2.7 %), 300 (1.6 %) and 1000 (1.1%) mg/kg bw/day groups.
- Dead Fetuses: No dead foetuses were noted in the control, 100 and 300 mg/kg bw/day groups. In the 1000 mg/kg bw/day group, 1 out of 269 foetuses died. This very low incidence of dead foetuses (0.4 %) was considered to be of no toxicological significance.
- Post-implantation Loss: The post-implantation loss was similar in the control and treated groups.

Live Fetuses:
- Mean Number: The mean number of live foetuses was similar in the control and treated groups.
- Body Weight: The mean fetal body weight was similar in the control and treated groups.
- Sex-ratio: No treatment-related effects were noted on the sex-ratio.

FETAL OBSERVATIONS
Fetal External Observation: No external malformations were observed in the fetuses of the control and treated groups.
Fetal Soft Tissue Observations:
- Anomalies: No soft tissue anomlies were noted in the fetuses of the control and 100 mg/kg bw/day groups.
In the 300 mg/kg bw/day group, 3 out of 122 fetuses (2.5 % ) had dilated renal pelvis associated for two of them (1.6 % ) with ureteral dilatation.
In the 1000 mg/kg bw/day group, 3 out of 130 fetuses (2.3 % ) had dilated renal pelvis associated for one of them (0.8 % ) with ureteral dilatation.
These two findings which are within the range of C.I.T. Historical control data are considered to be incidental.

- Malformations: No soft tissue malformations were noted in the fetuses of the control, 100 and 1000 mg/kg bw/day groups.
In the 300 mg/kg bw/day group, 1 out of 122 fetuses had a cerebral venticular dilatation. This malformation which was noted only in one fetus and which was not observed at a higher dose level is considered as incidental.

Fetal Skeletal Observations:
Fetal Skeletal Variations:
- The fetal and litter incidences of reduced ossification of the 6th sternebra were significantly different in the 1000 mg/kg bw/day group from those of the control group. As these incidences are lower than those of the control group and within the range of C.I.T. control historical data, they are considered to have no toxicological significance.
In the same way, the fetal incidence and the litter incidence of unossification of the 5th sternebra were significantly different from those of the control group in the 1000 mg/kg/day group but lower and within the range of C.I.T. historical data. Therefore they are considered not to have a toxicological significance. In the 300 mg/kg/day group, the fetal incidence was not significantly different from that of the control group, but the litter incidence was lower and within the range of the CIT historical data. The fetal incidence of unossification of the 4th metacarpal was significantly higher in the 1000 mg/kg/daya group., when compared to the control group. This incidence is very low and below the range of CIT control historical data and therefore is not considered related to treatment.
No other dose-related effects were noted on the incidence of the skeletal variations.

Fetal Skeletal Anomalies:
The fetal incidence of reduced ossification of thoracic vertebrae was higher in the 300 and 1000 mg/kg/day groups. As the differences from the control groups are very slight and within the range of CIT control historical data, a treatment-related effect is ruled out. The incidence of the few other skeletal anolmaies was similar in the control and treated groups.

Fetal Skeletal Malformations:
No skeletal malformations were observed in fetuses of any group.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Throughout the study, a satisfactory concordance between obtained and nominal concentrations was found for the administered preparation.

Table 2       The pregnancy status is summarised in the table below:

Dose (mg/kg bw/day)	0	100	300	1000
Mated Females	25	25	25	25
Non-Pregnant Females	2	1	7	5
Pregnant Females	23	24	18	20
dead/sacrificed	0	0	0	0
aborted	0	0	0	0
total resorption	0	0	0	0
completed pregnancy	23	24	18	20

Applicant's summary and conclusion

Conclusions:

The test substance Epoxidised Soybean Oil (ESBO) when administered daily by oral gavage to pregnant female Sprague-Dawley rats during organogenesis at the dose levels of 100, 300 and 1000 mg/kg bw/day was well tolerated by the dams at all the dose levels and was neither embryotoxic or teratogenic.

The No Observable Adverse Effect Level (NOAEL) is defined as 1000 mg/kg bw/day in terms of maternotoxic effects and embryofetal development.

The No Observable Effect Level (NOEL) is also defined as 1000 mg/kg bw/day in terms of maternotoxic effects and embryofetal development.

Executive summary:

The objective of the study was to evaluate the potential toxic effects of the test substance Epoxidised Soybean Oil (ESBO) on embryonic and fetal development when administered by oral route (gavage) to pregnant female Sprague-Dawley rats during organogenesis (day 6 to day 15 of pregnancy inclusive).

The test substance Epoxidised Soybean Oil (ESBO) when administered daily by oral gavage to pregnant female Sprague-Dawley rats during organogenesis at the dose levels of 100, 300 and 1000 mg/kg bw/day was well tolerated by the dams at all the dose levels and was neither embryotoxic or teratogenic.

The No Observable Adverse Effect Level (NOAEL) is defined as 1000 mg/kg bw/day in terms of maternotoxic effects and embryofetal development.

The No Observable Effect Level (NOEL) is also defined as 1000 mg/kg bw/day in terms of maternotoxic effects and embryofetal development.