Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

A screening study and a one-generation study do not identify any effects at the limit dose of 1000 mg/kg bw/d.

Link to relevant study records

Referenceopen allclose all

Endpoint:
one-generation reproductive toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
26 October 1992 to 2 February 1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Soybean Oil used as vehicle. No details in relation to ovaries or uterine content. 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, is justified based on similar toxicity profiles and structural and functional similarities.
Qualifier:
according to
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Principles of method if other than guideline:
Conducted according to OECD Guideline Method 415 and EEC Recommendation No 87/302/EEC
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River France
- Age at study initiation: Approx 8 weeks old
- Weight at study initiation: mean of 279 g for males and 222 g for females
- Housing: Individually in polycarbonate cages
- Diet (ad libitum): free access to A04C pelleted diet, batch Nos. 20721, 20922, 21019 and 21207. These were distributed weekly
- Water (ad libitum): free access to water
- Acclimation period: 7-day acclimatization period

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

Route of administration:
oral: gavage
Vehicle:
other: soybean oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was dissolved in the vehicle and homogenised using a magnetic stirrer. The test substance was administered by gavage using a glass syringe with a metal probe:

in the males - 71 days before mating and during the mating period (until sacrifice after the weaning of F1 litters)
in the females - 15 days before mating, during the mating period, during the pregnancy, during the lactation until sacrifice

VEHICLE
- Amount of vehicle (if gavage): A constant dose volume of 5 ml/kg bw/day was used.
- Lot/batch no. (if required): 08380327
Details on mating procedure:
Each male was paired with one female of the same treatment group for the night. The female was placed with the same male until mating occurred or 10 days had elapsed. If no evidence of mating was observed after 10 days, the female was placed after 3 days rest period with another male that had already successfully mated, until mating occurred or 11 days had elapsed. Each morning, a vaginal lavage was performed in order to detect the presence of spermatozoa. The day when spermatozoa was found was designated as day 0 of pregnancy.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance was measured before the beginning of the study. On weeks 1, 4, 8, 12 and 16 each preparation was checked for achieved concentration. The results revealed a good stability of low dose level preparation for 7 days at +4
Duration of treatment / exposure:
The test substance was administered by gavage using a glass syringe with a metal probe:

in the males - 71 days before mating and during the mating period (until sacrifice after the weaning of F1 litters)
in the females - 15 days before mating, during the mating period, during the pregnancy, during the lactation until sacrifice
Frequency of treatment:
Daily
Details on study schedule:
- Dose selection rationale: Dose levels were determined in agreement with ETTEC following a previously conducted study (8707 RSR) at the dose levels of 150, 450 and 1000 mg/kg bw/day. The results showed that neither toxic effect 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.
- Rationale for animal assignment (if not random): Rat is chosen as it is commonly requested by regulatory authorities.
- Other: The oral route was chosen as it is the most probable route of exposure in humans
Remarks:
Doses / Concentrations:
0 mg/kg bw/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
100 mg/kg bw/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
300 mg/kg bw/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
28 - see Table 1 for study design
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were determined in agreement with ETTEC following a previously conducted study (8707 RSR) at the dose levels of 150, 450 and 1000 mg/kg bw/day. The results showed that neither toxic effect 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.
- Rationale for animal assignment (if not random): Rat is chosen as it is commonly requested by regulatory authorities.
- Other: The oral route was chosen as it is the most probable route of exposure in humans

See Table 1 below
Positive control:
No data
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
All animals were checked at least twice a day for mortality and signs of morbidity. Any animal showing signs of poor clinical condiditon was asphyxiated by carbon dioxide. Any animal found dead or killed due to poor clinical condition was subjected to macroscopic examination and a full spectrum of tissues was preserved whenever possible.

BODY WEIGHT: Yes
Body weight was recorded for each male on the first day of treatment (day 1) and then once a week until sacrifice. Body weight was recorded for each female on the first day of treatment (day 1) once a week before mating and during mating periods on days 0, 7, 14 and 20 of pregnancy and on days 1, 7, 14 and 21 post-partum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
The quantity of food consumed by each male was recorded once a week over a 7 day period of treatment until sacrifice. The quantity of food consumed by each female was recorded as follows:
- during the premating period, once a week over a 7-day period
- during the pregnancy, at the intervals day 0-day7, day 7-day 14 and day 14-day 20
- during the lactation, at the intervals day 1 pp-day 7 pp, day 7-day 14 pp and day 14pp-day 21 pp.

For the males and females, food consumption was not measured during the mating period. Food intake per animal and per day was calculated using the amount of food given and left in each feeder.

OTHER:
Approximately 24 hours after treatment, blood samples were taken from the orbital sinus of the overnight fasted animals while they were under light under anaesthesia. The samples were collected in tubes containing the appropriate anticoagulant. At the necropsy, bone marrow samples were taken from the femoral bone of sampled animals.

Haematology:
The following parameters were determined in the first 5 males of each group and on the first 5 females of each group having surviving pups at weaning on the day of sacrifice (day 121 for males and 70 for females):
Leucocytes, Erythrocytes, Haemoglobin, Packed Cell Volume, Mean Cell Volume, Mean Cell Haemoglobin, Mean Cell Haemoglobin Concentration, Thrombocytes, Differential White Cell Count, Bone Marrow sample (Myelogram)

Blood Chemistry:
The following parameters were determined in the first 5 males of each group and on the first 5 females of each group having surviving pups at weaning on the day of sacrifice (day 121 for males and 70 for females):
Glucose, urea, creatinine, Total Bilirubin, Total Proteins, Albumin, Albumin/globulin ratio, Alkaline phosphatase, Aspartate aminotransferase, Alanine aminotransferase.

Sacrifice:
After the weaning of the last F1 litter, F0 males and females were asphyxiated using carbon dioxide and sacrificed by exsanguination. Throughout the study, moribund animals were sacrificed in the same way. F1 pups were not selected on day 4 post-partum and F1 pups whose mother died during lactation were sacrificed by asphyxiation using carbon dioxide. At weaning (between days 22 and 25 post-partum) F1 pups were sacrificed by asphyxiation using carbon dioxide.

Macroscopic examination:
A complete macroscopic examination was performed on all parental animals including those that died during the study or are sacrificed prematurely, and on the pups that died during lactation, or are sacrificed at weaning. In the parental females, the number and distribution of implantation sites were recorded, whenever possible. F1 pups not selected on day 4 post-partum were fixed in Bouin’s fluid. They were then submitted to a free-hand sectioning according to Wilson’s technique in order to examine soft tissue.

Preservation of tissues:
In all the F0 parental animals including those that died during the study or were sacrificed prematurely, all the macroscopic lesions and the following tissues were preserved in 10 % buffered formalin (except for the eyes and pituitary gland which have been fixed in formol-sublimate): coagulating gland, prostate, uterus, ovaries, seminal vesicles, horns and cervix, pituitary gland, testes, vagina and epididymides. Additionally, in the first 5 FO males and the first F0 females of each group having surviving pups at weaning, the following organs were preserved in 10 % buffered formalin: ileum, kidneys and liver.
In F1 pups including those that died during lactation, all the microscopic lesions were preserved in 10 % buffered formalin (except the eyes and pituitary gland which will be fixed in formol-sublimate).

Microscopic examination:
All tissues required for microscopic examination were embedded in paraffin wax, sectioned at approximately 4 microns in thickness and stained with hematoxylin-eosin. Microscopic examination was performed on:
- tissues listed above in animals of the high dose level and control groups sacrificed at the end of the study and in animals that died, or were sacrificed prematurely, or in females that aborted.
- tissues listed above in animals suspected of infertility of the low and intermediate dose level groups.
- liver was examined in one male given the 1000 mg/kg bw/day dose level where liver enlargement was noted at autopsy. Simultaneously the liver from one control male was also examined.
Oestrous cyclicity (parental animals):
No details provided
Sperm parameters (parental animals):
No details provided
Litter observations:
itter size:
The total litter size and number of pups of each sex was recorded after birth. Each litter was examined daily in order to note the number of live, dead or cannibalized pups. Any gross abnormalities in pups were noted.

Body weight:
The weight of each litter was measured on days 1, 4, 7 and 14 post-partum.
The body weight of each pup was measured on day 21 post-partum.

Selection on day 4 post-partum:
On day 4 post-partum, the size of each litter was adjusted by eliminating extra pups by random selection, as nearly as possible, at 4 males and 4 females per litter. Whenever, the number of male or female pups prevented having at least 4 of each sex per litter, partial adjustment (for example, 5 males and 3 females) was made. Adjustments were not made for litters of less than 8 pups.

Clinical Signs:
Litters were examined daily for possible clinical signs.

Pup Development:
The number of pups in each litter exhibiting the following characteristics was recorded:
Pinna unfolding (on day 5 post-partum), hair growth (on day 5 post-partum), incisor eruption (on day 13 post-partum), eye opening (on day 17 post-partum), auricular duct opening (on day 17 post-partum), surface righting reflex (on day 5 post-partum), cliff avoidance (on day 11 post-partum), air righting reflex (on day 17 post-partum).
Postmortem examinations (parental animals):
Sacrifice:
After the weaning of the last F1 litter, F0 males and females were asphyxiated using carbon dioxide and sacrificed by exsanguination. Throughout the study, moribund animals were sacrificed in the same way.

Macroscopic examination:
A complete macroscopic examination was performed on all parental animals including those that died during the study or are sacrificed prematurely, and on the pups that died during lactation, or are sacrificed at weaning. In the parental females, the number and distribution of implantation sites were recorded, whenever possible.

Preservation of tissues:
In all the F0 parental animals including those that died during the study or were sacrificed prematurely, all the macroscopic lesions and the following tissues were preserved in 10 % buffered formalin (except for the eyes and pituitary gland which have been fixed in formol-sublimate): coagulating gland, prostate, uterus, ovaries, seminal vesicles, horns and cervix, pituitary gland, testes, vagina and epididymides. Additionally, in the first 5 FO males and the first F0 females of each group having surviving pups at weaning, the following organs were preserved in 10 % buffered formalin: ileum, kidneys and liver.

Microscopic examination:
All tissues required for microscopic examination were embedded in paraffin wax, sectioned at approximately 4 microns in thickness and stained with hematoxylin-eosin. Microscopic examination was performed on:
- tissues listed above in animals of the high dose level and control groups sacrificed at the end of the study and in animals that died, or were sacrificed prematurely, or in females that aborted.
- tissues listed above in animals suspected of infertility of the low and intermediate dose level groups.
- liver was examined in one male given the 1000 mg/kg bw/day dose level where liver enlargement was noted at autopsy. Simultaneously the liver from one control male was also examined.

Postmortem examinations (offspring):
F1 pups were not selected on day 4 post-partum and F1 pups whose mother died during lactation were sacrificed by asphyxiation using carbon dioxide. At weaning (between days 22 and 25 post-partum) F1 pups were sacrificed by asphyxiation using carbon dioxide.
F1 pups not selected on day 4 post-partum were fixed in Bouin’s fluid. They were then submitted to a free-hand sectioning according to Wilson’s technique in order to examine soft tissue.
F1 pups including those that died during lactation, all the microscopic lesions were preserved in 10 % buffered formalin (except the eyes and pituitary gland which will be fixed in formol-sublimate).
Statistics:
The following statistical calculations were carried out:
Male mating index, female mating index, male fertility index, female fertility index, gestation index, live birth index, viability index on day 4 pp, viability index on day 21 pp.

Mean values were compared by one-way variance analysis and Dunnett's test. Percentage values were compared by Fisher's exact probability test. The following sequence was used for the statistical analysis of haematology and blood biochemistry data:
The normality of the distribution of the values in each group was checked by Komolgorov-Smirnov's test (1948). If the distribution was normal, the homogeneity of variances between the groups was assessed by Bartlett's test (1937) (more than 2 groups) or Fisher's test (1934) (2 groups). If no significant heterogeneity of the variances was established, the comparision between treated and control groups was perfomred by Dunnett's test (1955). If the variances were heterogenous, the comparision between treated and control groups was performed by Dunn's test (1964) (more than 2 groups) or by Mann Whitney's test (1947) (2 groups). If the distribution of values in the group was not normal, tha analysis was repeated after logarithmic transformation of the values. If this logarithmic transformation fails to normalise the distribution of the values, comparision of treated and control groups was performed by Dunn's test using original values.
Reproductive indices:
Male mating index, female mating index, male fertility index, female fertility index, gestation index, live birth index
Offspring viability indices:
Live birth index, viability index on day 4 pp, viability index on day 21 pp.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No clinical signs were observed in surviving males or females. No deaths occured in females given 0 or 300 mg/kg bw/day. In the 100 mg/kg bw/day group, one female (H21235) was sacrificed on day 1 post-partum due to a vaginal prolapsus and another female
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean food consumption was similar in males of the control and treated groups during all the study and in females of the control and treated groups during the premating, pregnancy and lactation periods. The mean body weight gain was similar in males
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The mean food consumption was similar in males of the control and treated groups during all the study and in females of the control and treated groups during the premating, pregnancy and lactation periods. The mean body weight gain was similar in males
Organ weight findings including organ / body weight ratios:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Regular oestrous cycle, as evidenced from the morphological changes in the ovaries, uterus and vagina, was noted in the control and treated female rats, except in 2 females given 100 mg/kg bw/day. In these two animals deciduomatosis was noted in one femal
Other effects:
not specified
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
see results below
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Only one female did not mate. This was not related to the treatment. Despite this all treated females responded the same as the control animals in respect of mating and fertility.
No clinical signs were observed in males or females.

The mean food consumption was similar in males of the control and treated groups during all the study and in females of the control and treated groups during the premating, pregnancy and lactation periods.

The mean body weight gain was similar in males of the control and treated groups during all the study and in females of the control and treated groups during the premating, pregnancy and lactation periods.

All treated males responded the same as the control animals in respect of mating and fertility.

Only one female did not mate. This was not related to the treatment. Despite this all treated females responded the same as the control animals in respect of mating and fertility.

Haematology:
The statistically significant differences noted between the control and treated animals in some haematological parameters, namely total white and red blood cell count in males, were considered to be of no toxicological importance as they were minor, not dose-related and the individual values were within the range of background data (white blood cells: 5.9 – 15.6 G/l; red blood cells: 8.05 – 9.80 T/l).

Blood biochemistry:
No treatment-related abnormalities were noted in the blood biochemical parameters investigated and the individual values were comparatively similar in both control and treated animals and were within the background data range.

Macroscopic examination:
Liver enlargement, without any relevant histopathological findings, was noted in 1 out of 28 males given 1000 mg/kg bw/day. Liver pallor was found in 2 out of 28 males given 300 mg/kg bw/day. These findings were considered to be of spontaneous nature and irrelevant to the treatment with the test substance. The other macroscopic findings encountered were those which are commonly recorded, spontaneous in rats of this strain and age and were considered to be of no toxicological importance.

Microscopic examination:
Regular oestrous cycle, as evidenced from the morphological changes in the ovaries, uterus and vagina, was noted in the control and treated female rats, except in 2 females given 100 mg/kg bw/day. In these two animals deciduomatosis was noted in one female ; severe metritis and degenerated placenta were noted in the other female. These changes can be found in untreated rats, therefore this weak incidence is not considered of toxicological importance. No treatment-related abnormalities were found in the male genital organs of the animals examined.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: This was the highest dose tested. No effects were seen at 1000 mg/kg bw/day
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs attributed to the treatment were observed in pups of any group.
Mortality / viability:
no mortality observed
Description (incidence and severity):
The viability on day 4 post-partum was similar in the control (94.2 %) and the 100 (91.1 %), 300 (97.9 %) and 1000 (93.5 %) mg/kg bw/day groups. On day 21 post-partum, the lactation index was also similar in the control (97.8 %) and the 100 (98.9 %), 300
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean pup body weight was similar in the control and treated groups on days 1, 4, 7, 14 and 21 post-partum
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
See results detailed below
Histopathological findings:
not specified
Gestation:
The mean number of implantation sites was similar in the control and treated groups. The gestation index was 100 % in the control, 300 and 1000 mg/kg bw/day groups. In the 100 mg/kg bw/day group, it was 96.2 % as one pregnant female was sacrificed on day 9 of pregnancy due to misdosing. The mean duration of gestation was similar in all groups and within the normal range of 21-22 days.

Live Birth index:
The live birth index was 100 % in all groups.

Pup viability:
The viability on day 4 post-partum was similar in the control (94.2 %) and the 100 (91.1 %), 300 (97.9 %) and 1000 (93.5 %) mg/kg bw/day groups. On day 21 post-partum, the lactation index was also similar in the control (97.8 %) and the 100 (98.9 %), 300 (97.9 %) and 1000 (94.4 %) mg/kg bw/day groups.

Pup body weight:
The mean pup body weight was similar in the control and treated groups on days 1, 4, 7, 14 and 21 post-partum.

Pup development:
The physical development as assessed by the following criteria: pinna unfolding, hair growth, tooth eruption, eye opening and auditory canal opening was similar in the control and treated groups. The reflex development as assessed by the following criteria surface righting, cliff avoidance and air righting was similar in the control and treated groups.

Pup clinical examination:
No clinical signs attributed to the treatment were observed in pups of any group.

Pup macroscopic examination:
No treatment-related macroscopic changes were noted in pups sacrificed at the end of the study. Since almost all dead pups were autolytic no macroscopic changes could be noted. In those which were not autolytic, no macroscopic changes attributed to treatment were noted. In pups culled on day 4 post-partum, no macroscopic changes were noted and the 100 mg/kg bw/day groups. In the 300 and 1000 mg/kg bw/day groups 1 out of 208 (0.5 %) pups and 6 out of 203 (3.0 %; p < 0.05) pups had a dilated renal pelvis. In the 1000 mg/kg bw/day group, 5 out of the 6 affected pups provided from the same litter whose mother was found dead on day 7 post-partum. This litter had a mean body weight lower than that of the other litters of the same group. Consequently the dilatation of renal pelvis of these pups is related to their slight delayed development related itself to the clinical conditions of the mother. This dilatation of renal pelvis is not attributed to treatment with ESBO.
Key result
Dose descriptor:
NOAEL
Generation:
F1
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
Reproductive effects observed:
not specified

No further details

Conclusions:
The test substance ESBO was administered daily by oral gavage to male and female Sprague-Dawley rats at 100, 300 and 1000 mg/kg bw/day dose levels 71 days before mating in males and 15 days before mating in females until day 21 post-partum of F1 litters. ESBO did not induce any toxic effects in parental males and females, did not disturb their capacity for reproduction and did not impair the development of the F1 offspring.
Under the test conditions, the highest tested dose of 1000 mg/kg bw/day was found to be the NOEL for fertility, reproduction and developmental toxicity.
Executive summary:

The test substance Epoxidised Soybean Oil (ESBO) was given daily 7 times a week to three groups of 28 F0 male and 28 F0 female Sprague-Dawley rats by gavage at the dose levels of 100, 300 and 1000 mg/kg bw/day. The F0 control animals (28 males and 28 females) received the vehicle Soybean Oil (SBO).

After 71 days of treatment in males and 15 days of treatment in females; the F0 male and female rats were paired. Treatment was continued in females during the mating and pregnancy and lactation periods until day 21 post-partum and in males during the mating period until day 21 post-partum of F1 litters.

F0 animals were observed daily for clinical signs. Food consumption and body weight were measured at designated intervals. The females were allowed to deliver normally. The F1 litters were examined daily for clinical signs, viability, physical, and reflex development until day 21 post-partum. Pup body weights were recorded on days 1. 4, 7, 14 and 21 post-partum.

About 24 hours after the last administration, haematology and blood chemistry investigations were performed in 5 males and 5 females of each group. At the end of the study, macroscopic examination of all F0 males and females and F1 pups was performed. In all the parents, reproductive organs and macroscopic lesions were sampled and additionally in 5 males and 5 females of each group ileum, kidneys and liver were sampled. Microscopic examination of the reproductive organs was performed in all animals of the control and 1000 mg/kg bw/day groups and in animals suspected of infertility, died or sacrificed in the 100 and 300 mg/kg bw/day groups. In addition the liver of one male of the 1000 mg/kg bw/day group and of the control group were also examined microscopically.

Results:

Clinical parental data – no treatment-related mortalities or clinical signs were observed. The mean food consumption and body weight gain of males and females were similar in the control and treated groups. The variations of haematologic or blood chemistry parameters were not of toxicological importance. The macroscopic and microscopic examination of the animals did not reveal any changes attributed to the treatment.

Reproductive data - the mating and fertility indices of males or female was similar in the control and treated groups.

Litter data – the gestation index and the mean duration of gestation was similar in all groups. The live birth index was 100 % in all groups. The viability indices on day 4 and day 21 post-partum, the physical and reflex development of pups and the mean pup body weight were similar in the control and treated groups.

Conclusion – the test substance ESBO administered daily by oral route (gavage) to male and female Sprague-Dawley rats at the 100, 300 and 1000 mg/kg bw/day dose levels 71 days before mating in males and 15 days before mating in females until day 21 post-partum of F1 litters did not induce any toxic effects in parent males and females, did not disturb their capacity of reproduction and did not impair the development of the F1 offspring/

Under the conditions, the highest tested dose of 1000 mg/kg bw/day was found to be the NOEL.

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
November 24, 2004 - June 13, 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP compliant study completed in accordance with current guidelines - OECD 422. Only minor deviations occurred with no notable effect on the outcome of the investigations - see 'Overall comments' for details. The study was conducted with ETP rather than Fatty acids C14-22, 2ethylhexyl esters epoxidised, but read-across between the two esters is considered appropriate., based on similarity of structure and function. 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, is justified based on similar toxicity profiles and structural and functional similarities.
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
see 'Overall Remarks'
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: HanBrl:WIST (SPF)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd. Switzerland
- Age at study initiation: 10 weeks minimum
- Weight at study initiation: Males - 291-342 g Females - 180-223g
- Fasting period before study: Only fasted before collection of blood samples
- Housing: Animals were housed in Makrolon cages with wire mesh tops and standard granulated softwood bedding. During the prepairing period, males and females were housed individually. Cages of males were interspersed amongst those holding females to promote the development of regular estrous cycles. During the pairing period; rats were housed one male/one female in Makrolon pairing cages. After mating or at the end of the pairing period, the males and the females were housed individually again. During the lactation period (until day 4 of lactation), dams were housed together with their litters.
- Diet ( ad libitum): Pelleted standard Kliba-nafag 3433 rat/mouse maintenance diet (Batch no. 53/04)
- Water ( ad libitum): Fullinsdorf community potable tap water provided in cages bottles
- Acclimation period: Minimum of 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 5 °C
- Humidity (%): 30-70 %
- Air changes (per hr): 10-15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark and at least 8 hours light

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was weighed into a glass beaker on a tared precision balance and approximately 80 % of the vehicle added (w/v). Using an appropriate homogenizer a homogenous mixture was prepared. The remaining vehicle was added until the required final volume was achieved. Separate formulations were prepared for each concentration at weekly intervals. During the daily administration period homogeneity of the test item in the vehicle was maintained using a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle (if other than water): ETP is stable in Corn Oil. Analytical evaluations were conducted by RCC.
Details on mating procedure:
No further details provided
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples for determination of concentration, homogeneity and stability (7 days) of the dose formulations were taken immediately after the first dose preparation. Determination of concentration and homogeneity of these samples was performed prior to the first dosing occasion. Additionally, samples for determination of concentration and homogeneity were taken once during gestation.
In addition to these two sets of samples,.samples were also retained from each set of weekly dose formulations (three samples from top, middle and bottom). These samples were not analysed since no discrepancy (outside the specified range) was noted in those samples scheduled for analysis.
For determination of concentration and homogeneity three samples of approximately 2 g were taken from the top, middle and bottom of each formulation and transferred into flat bottomed flasks. Stability samples (approximately 2 g) were taken from the middle only. The samples were frozen (-25 °C to -15 °C) pending analysis. Analysis was performed using GC.
Actual sample concentrations had to be within the accepted limit of 80 % - 120 % of the nominal concentration. Results of homogeneity must not deviate more than 15 % from the respective mean value. Stability results were accepted if the deviation from mean homogeneity results was not higher than 10 %. After analysis and evaluation of the results, the dose formulation samples were discarded at the discretion of the principle investigator for the analytical phase.
Duration of treatment / exposure:
After acclimatization, animals of both sexes received the test item for 14 days prior to pairing and during the pairing period. Daily dosing of the females was continued throughout pregnancy and up to Day 4 of lactation (4 or 5 days post-partum). Males were dosed for at least 28 days and until the day prior to scheduled necropsy. Males were terminated on day 29 and pups on day 4 post-partum. The dams were terminated on day 5 or 6 post-partum
Frequency of treatment:
ETP was administered once daily orally (by gavage). All animals received a dose volume of 2 mL/kg
Details on study schedule:
No details supplied
- Dose selection rationale: The purpose of this study was to generate preliminary information concerning the effects of Fatty acids, tall-oil, epoxidized,2-ethylhexylesters (ETP) on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. In addition, it
provided information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition. ETP was administered once daily orally (by gavage) to males for at least 28 days and to female rats throughout the prepairing and pairing periods until day 3 of lactation. The choice of 100, 300 and 1000 mg/kg bw/day is a standard selection for a dose range-finding investigation.

- Rationale for animal assignment (if not random): P generation allocated randomly
- Rationale for selecting satellite groups: Not applicable
- Post-exposure recovery period in satellite groups: Not applicable
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:

Remarks:
Doses / Concentrations:
300 mg/kg bw/day
Basis:

Remarks:
Doses / Concentrations:
100 mg/kg bw/day
Basis:

Remarks:
Doses / Concentrations:
0 mg/kg bw/day
Basis:
other: gavage control
No. of animals per sex per dose:
40 males, 10 per group
40 females, 10 per group
Please see Table 1 for more information
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The purpose of this study was to generate preliminary information concerning the effects of Fatty acids, tall-oil, epoxidized,2-ethylhexylesters (ETP) on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. In addition, it
provided information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition. ETP was administered once daily orally (by gavage) to males for at least 28 days and to female rats throughout the prepairing and pairing periods until day 3 of lactation. The choice of 100, 300 and 1000 mg/kg bw/day is a standard selection for a dose range-finding investigation.

- Rationale for animal assignment (if not random): P generation allocated randomly
- Rationale for selecting satellite groups: Not applicable
- Post-exposure recovery period in satellite groups: Not applicable
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
All animals were checked at least twice daily for any mortalities. All animals were observed at least twice daily for signs of reaction to treatment and/or symptoms of ill health . Additionally, the females were observed for signs of difficult or prolonged parturition.

DETAILED CLINICAL OBSERVATIONS: Yes
Prior to the first administration and then at weekly intervals thereafter, a detailed clinical observation was performed outside the home cage. Animals were observed for the following: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and automatic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern); changes in gait, posture and response to handling as well as the presence of clonic and tonic movements, stereotypies or bizarre behaviour.

At one time during the study (males: shortly before scheduled sacrifice; females: on day 3 or 4 post partum) relevant parameters from a modified Irwin screen test were performed for five P generation males and five P generation females randomly selected from each group. This FOB assessment was conducted following the daily dose administration. Animals were observed for the following: (a) Cage side observations: unusual body movements, abnormal behaviour, (b) Hand-held observations: palpebral closure, pinna reflex, lacrimation, pupil size, pupil reactivity, salivation (c) Open field observations: level of ambulatory activity including rearing, responsiveness to sharp noise (d) Categorical observations: hair coat, behaviour, respiration, muscle movements (e) Measurements/Counts: hindlimb/forelimb grip

BODY WEIGHT: Yes
The animals were weighed daily throughout the entire study.
Oestrous cyclicity (parental animals):
No details
Sperm parameters (parental animals):
No details
Litter observations:
LACTATION AND LITTER DATA:
Day 0 of lactation was the day on which a female had delivered all her pups. The litters were examined for litter size, live birth, stillbirth, and any gross anomalies. The sex ratio of the pups was recorded. The dams were caged together with their litters until day 4 of lactation. Pups were weighed individually (without identification) on days 0 (if possible), and with identification on days 1 and 4 post partum. The dams and pups were observed daily for survival and behavioural abnormalities in nesting and nursing.
Postmortem examinations (parental animals):
ROSS PATHOLOGY: Yes
Males were sacrificed on day 29 after treatment for 28 days. Pups were sacrificed on day 4 post partum. Females were sacrificed on day 5 or 6 post partum. The animals were examined macroscopically for any structural abnormalities or pathological changes, with special attention paid to the organs of the reproductive system. The number of implantation sites and corpora lutea was recorded for all dams with litters. Dead pups (except where excessively cannibalized) and pups killed at day 4 of lactation were examined macroscopically.

Of all parental animals the following tissues were preserved in neutral phosphate buffered 4 % formaldehyde solution: gross lesions, prostate, testes (in Bouin’s fixative), seminal vesicles with coagulation gland, ovaries, epididymides (in Bouin’s fixative), brain, spinal cord, small and large intestines (incl. Peyer’s patches), stomach, liver, kidneys, adrenals, spleen, heart, uterus with vagina, thymus, trachea and lungs (preserved by inflation with fixative and then immersion), thyroid, lymph nodes (mesenteric and mandibular), urinary bladder, peripheral nerve, and bone marrow.

HISTOPATHOLOGY: Yes
Full histopathology was carried out on the preserved organs and tissues of the animals in the vehicle control and high dose group (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure). For liver and thyroid examinations were extended to the animals of the other dosage groups, since treatment-related changes were seen in the highest dose group. All gross lesions were examined.

ORGAN WEIGHTS: Yes
For all adult female and male animals in each group the following organs were trimmed from any adherent tissue, as appropriate, and their wet weight taken: liver, brain, adrenals, thymus, kidneys, spleen, heart, testes and epididymides.
Statistics:
Dunnett t-test, Steel (rank) test, Fischer's Exact test
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
One female in group 4 died during the blood sampling procedure. This was considered an accident. No further deaths occured.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see below
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
CLINICAL SIGNS AND MORTALITY
No mortalities were observed. No treatment-related effects were seen. Neither food consumption nor body weight development were affected at any dose level.

BODY WEIGHT AND WEIGHT GAIN
No effects seen in males or females at any dose level

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food consumption was slightly higher in the male group 4. This was incidental and of no toxicological relevance.

HAEMATOLOGY
No effects seen

CLINICAL CHEMISTRY
No effects seen

NEUROBEHAVIOUR
Locomotor activity was statistically higher in group 3 and 4 males than in the respective control. This was attributable to normal biological variation.

ORGAN WEIGHTS
During necropsy of parent animals no test item-treated findings were noted. For males treated at 300 or 1000 mg/kg/day, mean absolute and relative liver weights were dose-dependently increased. See table 3 for further information.

GROSS PATHOLOGY
The intergroup incidence of various spontaneous changes gave no indication of an effect of treatment. No treatment related macroscopic abnormalities were noted.

HISTOPATHOLOGY:
Liver- Minimal hepatocellular hypertrophy in animals treated at 1000 mg/kg bw/day. This change was considered to represent an adaptive reaction most likely induced by an increased biotransformation of the test item. Therefore, it was not assessed as an adverse effect.
Thyroid- Increased incidence of minimal follicular cell hypertrophy in animals treated at 1000 mg/kg bw/day. No test item- related histopathological findings were noted in the reproductive organs of either sex. In particular, the assessment of the integrity of the spermatogenetic cycle did not reveal any evidence of impaired spermatogenesis.

REPRODUCTION DATA
Fertility rate was high resulting in at least 9 litters per group for evaluation of reproduction data. There were no treatment-related effects on precoital time, fertility indices, mean duration of gestation, number of implantations, post-implantation loss, pup survival or litter size from birth through to scheduled pup sacrifice on Day 4 post partum at any dosage.
Litter Data:
No test item-related abnormal findings were noted for pups at first litter check or during the first 4 days post partum. Sex ratios at first litter check and on day 4 post partum were unaffected by treatment with the test item.
Mean pup weights on day 0 and day 1 post partum were unaffected by treatment with the test item. Mean pup weight development during the first 4 days post partum was unaffected by treatment with the test item.
F1 Pups Necropsy findings:
No test item-related macroscopic findings were noted during necropsy of F1 pups.

Please see table 2 for F0 animal breeding statistics.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The only effect noted at this concentration was an increased liver weight
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Higher doses resulted in increased liver weight
Reproductive effects observed:
not specified

Table 2: F0 animals breeding for F1 litters

Group (mg/kg/day)

1

(0)

2

(100)

3

(300)

4

(1000)

# of females paired

10

10

10

10

# of females mated

10

10

10

10

# of pregnant females

10

10

10

10

# of females delivering pups

10

10

10

10

# of females with live pups on day 4 post partum

10

9 (A)

10

10

A = For female 52 only two dead pups were noted at first litter check

Table 3: Organ Weights (Gram)

 

Group 1

0 mg/kg

Group 2

100 mg/kg

Group 3

300 mg/kg

Group 4

1000 mg/kg

Liver

Males

Mean

8.33

8.73

9.38*

9.71*

St. Dev.

0.78

1.00

0.81

0.56

N

10

10

10

10

 

Liver

Females

Mean

8.02

9.03

8.40

9.43*

St. Dev.

0.81

1.42

1.46

0.98

N

10

10

10

9

 

Conclusions:
In order to generate preliminary information on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time, Fatty acids, tall-oil, epoxidized,2-ethylhexylesters (ETP) was administered orally by gavage at dosages of 0 (corn oil), 100, 300 or 1000 mg/kg body weight/day to male rats for 28 days and to female rats throughout the pre-pairing, pairing and gestation periods until the individual day 4 post partum.
Administration of 1000 mg/kg/day resulted in increased liver weights (males and females) and minimal hepatocellular hypertrophy (males and females), that were considered to represent an adaptive change most likely induced by an increased biotransformation of the test item. Therefore, it was considered not to be adverse.
Administration of 300 mg/kgbw/day resulted in increased liver weights (males and females). In the absence of any histopathological correlate, these increased weights were considered not to be adverse.

Based in these data, the NOAEL was considered to be 1000 mg/kg bw/day.

In the absence of any adverse effects on reproductive parameters, the NOAEL and NOEL for reproduction/developmental toxicity were considered to be
1000 mg/kg bw/day.
Executive summary:

The purpose of this study was to generate preliminary information concerning the effects of Fatty acids, tall-oil, epoxidized,2-ethylhexylesters (ETP) on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. In addition, it provided information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition.

ETP was administered once daily orally (by gavage) to males for at least 28 days and to female rats throughout the prepairing and pairing periods until day 3 of lactation.

The following dose levels were applied:

Group 1:             0 mg/kg body weight/day (vehicle control)

Group 2:            100 mg/kg body weight/day

Group 3:             300 mg/kg body weight/day

Group 4:             1000 mg/kg body weight/day

A standard dose volume of 2 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (corn oil).

The following results were obtained:

Parent animals:

General Tolerability

No mortalities were observed. No test-item related effects were seen. Neither food consumption nor body weight development were affected at any dosage.

Reproduction Data:

Fertility rate was high resulting in at least 9 litters per group for evaluation of reproduction data. There were no treatment-related effects on precoital time, fertility indices, mean duration of gestation, number of implantations, post-implantation loss, pup survival or litter size from birth through to scheduled pup sacrifice on Day 4 post partum at any dosage.

Clinical Laboratory Investigations:

The assessment of clinical chemistry and haematology parameters indicated no differences between animals treated with the test item and vehicle controls.

Terminal Examinations:

During necropsy of parent animals no test item-treated findings were noted. For males treated at 300 and 1000 mg/kg/day, mean absolute and relative liver weights were dose-dependently increased.

Histopathology:

The following findings distinguished test item-treated animals from vehicle controls:

Liver- Minimal hepatocellular hypertrophy in animals treated at 1000 mg/kg/day. This change was considered to represent an adaptive reaction most likely induced by an increased biotransformation of the test item. Therefore, it was not assessed as an adverse effect.

Thyroid- Increased incidence of minimal follicular cell hypertrophy in animals treated at 1000 mg/kg/day. No test item- related histopathological findings were noted in the reproductive organs of either sex. In particular, the assessment of the integrity of the spermatogenetic cycle did not reveal any evidence of impaired spermatogenesis.

Litter Data:

No test item-related abnormal findings were noted for pups at first litter check or during the first 4 days post partum. Sex ratios at first litter check and on day 4 post partum were unaffected by treatment with the test item.

Mean pup weights on day 0 and day 1 post partum were unaffected by treatment with the test item. Mean pup weight development during the first 4 days post partum was unaffected by treatment with the test item.

F1 Pups Necropsy findings:

No test item-related macroscopic findings were noted during necropsy of F1 pups.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The test substance did not induce any toxic effects in parent males and females, did not disturb their capacity of reproduction and did not impair the development of the F1 offspring. Under the conditions, the highest tested dose of 1000 mg/kg bw/day was found to be the NOEL.

Effects on developmental toxicity

Description of key information

A rat PNDT study is available and reports no effects at the limit dose.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
read-across based on grouping of substances (category approach)
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, is justified based on similar toxicity profiles and structural and functional similarities.
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
12th May 1981
Qualifier:
according to
Guideline:
other: E.E.C. Recommendation No. 87/302/E.E.C., 18th November 1987
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals 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
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.
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 %
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.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: other:
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.
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
Key result
Abnormalities:
no effects observed
Developmental effects observed:
not specified

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) 

100 

300 

1000 

Mated Females  

25 

25 

25 

25 

Non-Pregnant Females  

1

Pregnant Females 

23 

24 

18 

20 

dead/sacrificed

0

0

aborted

0

total resorption

completed pregnancy

23

24

18

20

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.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

The test substance was well tolerated by the dams at all the dose levels and was neither embryonic or teratogenic. The No Observable Adverse Effect Level is defined as 1000 mg/kg bw/day in terms of maternotoxic effects and embryofetal development.The No Observable Effect Level is defined as 1000 mg/kg bw/day in terms of maternotoxic effects and embryofetal development.

Justification for classification or non-classification

According to Regulation (EC) No. 1272/2008, no classification is warranted in the absence of any indication of reproductive toxicity.