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

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Qualifier:

according to guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Epoxidised Soybean oil

IUPAC Name:

Epoxidised Soybean oil

Constituent 2

Reference substance name:

ESBO

IUPAC Name:

ESBO

Constituent 3

Reference substance name:

Soybean oil, epoxidized

EC Number:

232-391-0

EC Name:

Soybean oil, epoxidized

IUPAC Name:

232-391-0

Constituent 4

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
- Physical state: Yellow liquid
- Lot/batch No.: 08380306
- Storage condition of test material: Room temperature in the dark

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system 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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0 mg/kg bw/day
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
100 mg/kg bw/day
Basis:
nominal conc.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
300 mg/kg bw/day
Basis:
nominal conc.

Doses / concentrations 4

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

Examinations

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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Details on results (P0)

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.

Effect levels (P0)

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

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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.

Effect levels (F1)

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

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

No further details

Applicant's summary and conclusion

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.