Registration Dossier

Administrative data

Description of key information

No repeated dose oral toxicity study is available for Soybean oil, epoxidized, reaction products with methanol. Therefore data from structural analogues, Epoxidized soybean oil (ESBO) and Fatty acids, tall-oil, epoxidized, 2-ethylhexylesters (ETP), is used. 
For ETP a combined repeated dose oral toxicity study with a reproduction/developmental screening study in rats, was performed according to OECD guideline 422. A NOAEL for general systemic toxicity of 1000 mg/kg bw/day was established for male and female.
For ESBO, a combined chronic toxicity/carcinogenicity study was performed in rats, according to OECD guideline 453. NOELs of 1000 mg/kg bw/day for males and 1400 mg/kg bw/day for females were established.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
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 ESBO but read-across between the two oils is considered appropriate.
Reason / purpose:
reference to other study
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
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 oral 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.

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
Remarks:
Doses / Concentrations:
0 mg/kg body weight/day
Basis:
other: gavage
Remarks:
Doses / Concentrations:
100 mg/kg body weight/day
Basis:
other: gavage
Remarks:
Doses / Concentrations:
300 mg/kg body weight/day
Basis:
other: gavage
Remarks:
Doses / Concentrations:
1000 mg/kg body weight/day
Basis:
other: gavage
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:
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
Positive control:
No positive control included in the study.
Observations and examinations performed and frequency:
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.

FOOD CONSUMPTION AND COMPOUND INTAKE :
Males: Food consumption was recorded weekly during the pre-pairing and post-pairing period.
Females: Food consumption was recorded for the following periods: days 1-8 and 8-14 of the pre-pairing period; days 0-7, 7-14 and 14-21 post coitum and days 0-4 post partum. Food consumption was not recorded during the pairing period since these would have been mixed values of males and females.

HAEMATOLOGY: Yes
Blood samples were obtained on the day of scheduled necropsy from all P generation males after they had been fasted overnight. Blood samples of P generation females were obtained on day 5 or 6 post partum after the females had been fasted overnight. Blood samples were collected from the retro orbital sinus with the animals under light isoflurane anaesthesia.
Blood samples were collected early in the working day to reduce biological variation caused by circadian rhythms. The following haematology parameters were determined: Erythrocyte count, Haemoglobin, Haematocrit, Mean corpuscular volume, red cell volume distribution width, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, haemoglobin concentration distribution width, platelet count, total leukocyte count, differential leukocyte count, coagulation, thromboplastin time, activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
The following clinical biochemistry parameters were determined: glucose, urea, creatinine, bilirubin (total), cholesterol (total), Aspartate aminotransferase, Alanine aminotransferase, bile acids, alkaline phosphatase, Gamma-glutamyl-transferase, Sodium, Potassium, Chloride, Calcium, Phosphorus inorganic, protein (total), albumin, globulin, albumin/globulin ratio.

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.
Sacrifice and pathology:
GROSS 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.
Other examinations:
Deailed above
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.
Mortality:
mortality observed, treatment-related
Description (incidence):
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
Description (incidence and severity):
No effects
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption was slightly higher in the male group 4. This was incidental and of no toxicological relevance.
Food efficiency:
not examined
Description (incidence and severity):
Test substance was administered by oral gavage
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
Test substance was administered by oral gavage
Ophthalmological findings:
not examined
Description (incidence and severity):
Not required in range-finding examination
Haematological findings:
no effects observed
Description (incidence and severity):
No effects
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No effects
Urinalysis findings:
not examined
Description (incidence and severity):
Not required
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Locomotor activity was statistically higher in group 3 and 4 males than in the respective control. This was attributable to normal biological variation.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
During necropsy of parent animals no treatment-related findings were noted. For males treated at 300 or 1000 mg/kg/day, mean absolute and relative liver weights were dose-dependently increased.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No effects
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Please see below
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
Not required
Details on results:
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
Critical 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

 

Endpoint:
chronic toxicity: oral
Remarks:
combined repeated dose and carcinogenicity
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The toxicological profile review for long term dietary exposure of rats to epoxidised soybean oil has been prepared by a BIBRA who are very reliable but the content is not necessarily drawn from a single scorable study. Since the data prepared by BIBRA may have been drawn from a variety of sources to construct the profile, it is not possible to allocate a score for reliability.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Principles of method if other than guideline:
No specific guidelines are quoted. The study was completed to ensure compliance with a range of national and international requirements in relation to food contact. The study as completed was braodly in accordance with the requirements of OECD Guideline 453.
GLP compliance:
no
Remarks:
The study was undertaken before the effective date for GLP implementation in the USA
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Scientific Agribusiness Consultants International
- Weight at study initiation: Batch 1 - between 20-50g; Batch 2 30-50g
- Fasting period before study: rats were not fasted
- Housing: Four to a cage in grid bottom aluminium cages measuring approx. 36x36x13 cm. These were suspended in racks carrying 20 cages above sheets of waterproof paper for collection of excreta. The paper was renewed daily.
- Diet : Laboratory Animal Diet No. 1 ad libitum
- Water : ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23 °C
- Humidity (%): 50 - 70 %
- Air changes (per hr): Partial air conditioning maintained a positive air pressure, with no recirculation of filtered 0.5um air to give 12-15 changes of air per hour.


Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
Spratt's Lab Diet No.1 was ground to a powder. ESBO (0.025, 0.25, 2.5 %) or 2.5 % SBO were added by weight and thoroughly mixed in a commercial food mixer. The ground diet served as the control. Fresh batches were prepared weekly. Administration was by admixture with the diet which was given ad lib for 104 weeks. The dietary levels were 0 % (controls), 0.025, 0.25% and 2.5% for epoxidised soybean oil and 2.5% for soybean oil

Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
No analyses were carried out to verify the concentration of ESBO or SBO.
Duration of treatment / exposure:
Administration was by admixture with the diet which was given ad lib for up to 104 weeks.
Frequency of treatment:
Test item was provided via diet so the diet was accessible all day
Remarks:
Doses / Concentrations:
0 %
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0.025 %
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0.25 %
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
2.5 %
Basis:
nominal in diet
No. of animals per sex per dose:
The rats were distributed into five groups of 24 rats of each sex from each animal supply batch to give total of 48 animals of each sex in each group.
Control animals:
yes, plain diet
Details on study design:
No details supplied
Positive control:
No details supplied
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
Rats were observed at least once daily for abnormalities of appearance and behaviour or signs of ill-health. Those showing such signs were isolated and monitored more frequently. If the condition improved, the rat was returned to its original cage but if it deteriorated so that the welfare of the rat was impeded, or it was deemed unlikely to survive for another 24 hr the rat was killed for post-mortem examination and tissues kept for histological investigation. Any rat found dead in the cage was subjected to a post mortem examination and tissues preserved if not autolysed.

BODY WEIGHT: Yes
These were recorded on the first day of treatment (day 0), on days 2, 4, 7, 14, 21 and 28 and then at two-week intervals until week 104.

FOOD CONSUMPTION AND COMPOUND INTAKE : Yes
Both food and water were measured over the 24 hour period prior to bodyweight determinations.

HAEMATOLOGY: Yes
Blood was taken from a tail vein of selected rats from batch 1 from the groups given 0 (control), 0.025 and 0.25 % ESBO and from those given 2.5 % SBO. Ten males from each dose level were bled during week 13, 26, 53 and 79 and ten females were bled during week 15, 26, 53 and 79. The blood was examined for haemaglobin content, packed cell volume and erythrocyte and leucocyte counts. Appropriately stained microscope preparations were used to identify the different types of leucocytes and the proportion of erythrocytes present as reticulocytes. Examination of these slides was confined to those from the control group and the rats given 2.5 % ESBO or SBO. As part of the scheduled post-mortem examinations blood was taken from the aorta from each animal during the anaesthetic phase and examined as described above.

CLINICAL CHEMISTRY: Yes
Serum separated from the blood samples taken at the end of the study was used for biochemical analyses. This was analysed for the activities of glutamic-oxaloacetic and glutamic-pyruvic transaminases, lactic dehydrogenase and for content of glucose, urea, total protein and albumin.

URINALYSIS: Yes
During week 26, 52, 78-80 and 104 urine samples were collected from 10 rats of each sex from batch 1 of each dose level except those given 0.025 % ESBO. Collections made at week 13 were confined to the controls and 2.5 % ESBO group. The samples were obtained by placing the animals individually in metabolism cages without food or water. three samples were collected from each rat at each interval. The conditions of the collection and examinations were:
- Collected over a 6 hr period following the normal overnight feeding and drinking and examined for volume, concentration (by refractive index), pH and the presence of glucose, blood, bile salts, ketones and proteins
- Collected over a 2 hour period immediately following an oral water load of 25 ml/kg and examined for volume, concentration and content of cells.
- Collected over a 4 hour period commencing 16 hour after an oral water load of 25 ml/kg. Food but not water was available to the animals during the 16 hour period. These samples were examined for volume and concentration.

Sacrifice and pathology:
After 104 weeks all surviving rats were killed, subject to post-mortem examination with recording of organ weights and smaples of tissues preserved. Feeding with the appropriate diet continued until all rats had been killed. The tissues from all rats were processed for microscopic examination.

GROSS PATHOLOGY: Yes
Rats surviving to the end of the study were weighed and the weights of brain, heart, liver, spleen, kidneys, stomach, small intestine, caecum (with and without contents), gonads, pituitary and thyroid recorded. When possible the following tissues, together with any other abnormal tissue, were preserved in 10 % buffered formalin: adrenal glands, aorta, bladder (urinary), brain, caecum, colon, eye, gonads, harderian gland, heart, kidneys, liver, lungs, lymph nodes, mammary gland, muscle (skeletal), nerve (sciatic), oesophagus, pancreas, pituitary gland, prostate, salivary glands, seminal besicles, skin, small intestine, spinal cord, spleen, stomach, thymus gland, thyroid gland, trachea, uterus, vagina.

HISTOPATHOLOGY: Yes
All tissues collected were preocessed for embedding in paraffin-wax, sectioned at approx. 5 um and stained with haematoxylin and eosin. Deviations from normal seen by the light microscope were noted.
Other examinations:
No details supplied
Statistics:
Analyses of variance and least significance difference test for Body weights, Haematology, Urine volume, Refractive index, pH and cell count, Food intake, Water intake, Serum analyses, Organ weights and organ weights relative to bodyweight. Chi2 for heterogeneity using data from all groups and Fisher's exact test for comparing individual groups: Semi-quantitative urine analyses, histological findings, tumour incidence. In all cases treated groups were compared with the controls and a probability of less than 0.05 taken to indicate statistical significance.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There were a few deaths at Week 52 but these were not dose related. Please see below for more details.
Mortality:
mortality observed, treatment-related
Description (incidence):
There were a few deaths at Week 52 but these were not dose related. Please see below for more details.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Bodyweight changes at the high dose level were not indicative of treatment related effects
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Slightly lower food consumption at higher doses, suggesting a compensatory nutritive value for the soybean oil, since bodyweights increased as diet intake went down.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
No treatment related effects
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
No treatment related effects
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
No adverse effects and possible beneficial effects on long term renal function from soybean oil administration
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
No treatment related effects.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
see results section below
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see results section below
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see results section below
Details on results:
CLINICAL SIGNS AND MORTALITY
No observations of condition or behaviour that could be related to treatment were reported. An eye infection at 11 and 12 weeks affected from 10-25 % of rats of each group indiscriminately and the animals recovered uneventfully. It is likely that this was as a result of dacryoadentis. In the first 52 weeks there were few deaths and they were not dose related. By week 78, 17-19 % of the controls had died. This increased to 38-42 % by week 96 and 48-52% at week 102. With the exception of the females given SBO and the females given the lowest dose (0.025 %) of ESBO there were less deaths in the treated groups than the controls. Please see table 1. Since this result did not demonstrate an adverse effect of treatment the data were not subject to detailed statistical analysis. However, in both sexes the number of deaths by 102 weeks in the high dose ESBO group was significantly (p<0.05 in males and p<0.01 in females by Fisher test) less than the control.

BODY WEIGHT AND WEIGHT GAIN
Neither 0.025 nor 0.25 % ESBO in the diet affected the body weights of males; no statistically significant difference from control weights were seen. Mean body weights of the males given 2.5 % ESBO were greater than the control and the differences were statistically significant compared with the controls, from week 58 to 92. For the remainder of the study the weights were greater than control but not always to a statistically significant degree. The mean bodyweights of the SBO-treated male rats were higher than those of the ESBO-treated animals and were statistically significantly different from the controls on most occasions from week 8 to the end of the experiment. The individual statistical comparisons were supported by an overall significant value in the analysis of variance from week 28. The pattern of bodyweights in the females was different. The weights of those given diet containing SBO were similar to the control up to week 74. After that time they tended to be heavier than the controls although the differences were seldom statistically significant. The difference appears to be mainly due to an irregularity in the growth curve of the controls at week 76 and 78. Although there was an early period (week 16-34) when the weights of the females given 2.5 % ESBO were higher than control by week 46-48 they were lower the difference first being statistically significant at week 54. Most of the values were significantly less than control up to week 90 although the differences were small, never being more than 8 % of the control weight. After week 90 the treated animals maintained their weight compared with a loss in the controls so that any differences were reduced. Statistically significantly lower body weights were seen in females given 0.25 % ESBO at some intervals between 64 and 74 weeks but, again the differences from control were small (not more than 6 % of the control).

FOOD CONSUMPTION AND COMPOUND INTAKE
The treated animals tended to eat slightly less than the controls although most of the values were within 10 % of the concurrent control. The analysis
of variance of the data from all groups of one sex was statistically significant on very few occasions. The comparison of individual groups of one sex
was statistically significant on very few occasions. The comparison of individual groups with the control revealed relatively few statistically significant differences. Many of these did not appear to be related to treatment as they were present only in the low dose groups or were similar at all dose levels. The intake by the male high dose ESBO group over the whole study was reduced by approximately 0.5 g/day and the females by 1 g/day. Similar reductions of food intake were apparent in the groups given SBO. The average intakes during the study by the males were approx. 10, 100 and 1000 g/kg for the rats given 0.025, 0.25 and 2.5 % ESBO respectively. The corresponding values for the females were approximately 14, 140 and 1400 mg/kg/day.

FOOD EFFICIENCY
The calculated intakes of ESBO and SBO in relation to bodyweight found that young rats consumed a higher dose level than the older rats. The intake by the females was greater than those of the males.

HAEMATOLOGY
The haematological measurements made on a proportion of the rats during the course of the study which related to the erythrocytes (red cell count, haemoglobin concentration, packed cell volume and reticulocyte count) showed little variation between the treated and control groups. There were isolated statistically significant differences, mainly increases, but these were not repeated at the later examinations. Similarly the total and differential white counts showed variations, sometimes statistically significant but these tended to be small and not to persist with prolonged treatment.

CLINICAL CHEMISTRY
There was a marked variation of results of the serum analyses in the old animals surviving to the end of the study. However, there were no apparent dose-related trends in the mean values. The only statistically significant differences from control were lower total protein concentrations in the male rats given SBO or the intermediate dose of ESBO and lower lactic dehydrogenase activity in those females given the highest dietary concentration of ESBO.

URINALYSIS
The majority of results of the renal concentration and dilution tests and of the urinary pH and cell excretion counts were similar in treated and control rats with no apparent dose - or time-related differences. In the males the only statistically significant difference was more concentrated urine collected in the 18-24 hour period when the rats were examined at 13 weeks. The females from the highest dose group of ESBO sampled at 78 and 104 weeks produced a lower volume of more concentrated urine than the controls in the 6-hour collection. In the same group the urinary cell excretion was higher than control from week 52 although, due to a marked variation, the differences were significant only at 1 year. The pH of the same urines was low although only by 10 % or less of the control. The only urine sample to give a reaction for bilirubin was from a control female at the end of the study. The number of samples with positive reactions for blood and the various concentrations of protein were similar in treated and control groups throughout the study. At the examination at 78-80 weeks, compared with controls, there were more males from the group given 2.5 % ESBO with glucose in the urine. After a further six months treatment this finding was not repeated even though the control incidence was still low. At the end of the study there were more positive reactions for ketones in the females given 2.5 % ESBO or 2.5 % SBO than in the controls.

ORGAN WEIGHTS
The organ weights and relative organ weights of both sexes given 0.025 or 0.25 % ESBO did not differ significantly from those of the controls. At the highest dose of ESBO (2.5 %) in males the liver, small intestine and thyroids were statistically significantly heavier than those of the control but these differences were not apparent when the values were expressed relative to body weight. The corresponding females had a significantly higher value for liver weight again not apparent when expressed relative to body weight. In the females the weights of the adrenal glands were less than the control and this persisted when the values were related to body weight. The male rats given SBO, with their higher body weights, had significant increases of most organ weights although again in relation to bodyweight they were comparable with the controls. In these rats the brain was one of the few organs not heavier than control and when expressed relative to the higher body weight the mean was significantly lowered. Both the organ weights and relative organ weights of the females given SBO were similar to the control values.

GROSS PATHOLOGY
Most of the findings in the treated male groups were of lower or similar incidence to the controls. None of the findings in the ESBO-treated males were present with a statistically significantly greater incidence than in the controls. All the male rats had some degree of glomerulonephrosis and within the control and ESBO-treated animals there was a trend for the higher dietary levels to have the less severe grades. An analysis for linear trend by Chi2 was significant (p<0.05) although there was no overall heterogeneity in the data. The distribution of the different grades of glomerulonephrosis was similar between the control and SBO-treated rats. The males given SBO had significantly higher incidences of Harderian glands showing secretion, congested lymph nodes and cystic seminal vesicles. In the females again most of the findings in the treated and the control groups were similar. As with the males there was a suggestion of a trend to less severe glomerulonephrosis but the differences or trend were not statistically significant. There were isolated non dose-related findings. The number of rats from the lowest dose with cystic spaces in the adrenal, with cardiac interstitial fibrosis and with congestion of the lymph nodes were higher than control. A similar incidence of lymph node congestion was seen in the rats given SBO. The SBO group also showed an increased incidence of pituitary haemorrhage compared with the control. There were a number of endometrial changes at the highest dose of ESBO. The incidence of cystic endometrium and of hyperplastic endometrium were significantly greater tan the control. Some animals had more than one of the three findings and the total numbers with one or more of the changes were 2, 4, 1, 10 and 4 for the control, three dose levels of ESBO and for SBO respectively. analysed by Chi2 these data showed significant heterogeneity and the incidence in the high dose ESBO group was significantly (p < 0.05) greater than the control. The difference between the 2.5 % ESBO and the SBO groups was not significant.

HISTOPATHOLOGY: NEOPLASTIC
in the males there were a total of 69 tumours, the most common being basophilic adenoma of the pituitary representing 35 % of the total with an even distribution through groups. The remainder were small incidences and only one, pancreatic-cell adenoma showed heterogeneity of distribution. This was due to the presence of four of the five tumours in the SBO group. Many of the tumours in males occurred in the controls alone with, the same incidence in treated and control groups or in the SBO group. Single cases of hepatocellular carcinoma, subcutaneous carcinoma, lymphoid leukaemia and thyroid adenoma were found in high dose (2.5 %) ESBO group with no corresponding control finding. A thyroid adenoma was found also in a male given 0.05 % ESBO. Some tumours; a Harderian-gland adenoma, a lymphoblastoma, two lymphosarcoma, a pancreatic-cell adenoma, a pituitary basophilic carcinoma, two subcutaneous fibrosarcoma, a parathyroid adenoma, and a fibrosarcoma in the abdomen were found in one of the lower-dose ESBO groups without a similar findings in the control or high dose group. There were more tumours in females with a total of 120 but of these 78 (65%) were pituitary adenoma with the highest incidence in the control but no significant heterogeneity between the groups. The remaining tumours were of low incidence in the ESBO-treated animals than in the controls. None of the individual tumours or the total incidences of rats affected showed significant heterogeneity among the groups or any significant increases in treated groups compared with the controls. Those present in the high-dose ESBO females without a corresponding control finding were single incidences of adrenal cortical carcinoma, pancreatic-cell adenoma and thymic lymphoblastomas as well as two thymic lymphosarcoma and uterine fibroleiomyoma. In the case of the uterine tumour there was also a single affected animal amoung the 0.25 % ESBO-treated rats. There were, in addition, single incidences of a number of tumours in the females given 0.025 or 0.25 % ESBO with no parallel finding in the controls or high dose. The lesions involved were a squamous carcinoma of the eye, a hepatic haemangioma, a mesenteric haemangioma, an oesophageal squamous carcinoma, an ovarian adenocarcinoma, a splenic reticulum-cell sarcoma, a splenic lymphosarcoma, a parathyroid adenoma and an uterine fibrolipoma.
Dose descriptor:
NOEL
Effect level:
1 other: g/kg
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Please see results
Dose descriptor:
NOEL
Effect level:
1.4 other: g/kg
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Please see results
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
chronic
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

No repeated dose oral toxicity test is available with Soybean oil, epoxidized, reaction products with methanol. Based on the category approach, studies performed with Epoxidized soybean oil (ESBO) and Fatty acids, tall-oil, epoxidized,2-ethylhexylesters (ETP) are applied. Please refer to the category justification document supplied along with the Chemical Safety report.

In a combined chronic toxicity/carcinogenetic study according to OECD 453 but not according to GLP male and female Wistar rats were exposed to Epoxidized soybean oil (ESBO) in their diet at concentrations of 0.0, 0.025, 0.25 and 2.5% for 104 weeks (BIBRA & Hazelton 1986). In addition one group was treated with Soybean oil at a concentration of 2.5%. Each treatment group contained 48 rats of each sex. There was no adverse effect on survival. The males given 2.5% ESBO gained more weight than the controls whilst the females were slightly lighter. The same rats consumed slightly less food than controls, the difference being greater in females (approximately 1 g/rat/day) than males (approximately 0.5 g/rat/day). The water intake of the females given 2.5% ESBO was lower than the control especially in the second year of the study. Haematological examination and investigations of urine at 3, 6, 12, 18 and 24 months did not reveal any adverse effects. A lower volume of more concentrated urine was excreted by the females given 2.5% ESBO compared with the controls with occasional increases in urinary cell excretion. The organ weights in females were similar to controls, with the exeption of the adrenal weights in the highest ESBO dose group which were significantly lower compared to controls, whilst in males given 2.5% ESBO and more noticeably in those given 2.5 % SBO several organs were heavier than control. This was related to the growth changes since when expressed relative to bodyweight the values were normal. There was a marginally increased incidence of uterine changes in the females given 2.5% ESBO. Since there were similar changes in the females given SBO these changes could not be clearly related to ESBO. It was concluded that for ESBO the NOEL was 2.5% providing an average daily intake of approximately 1.0 g/kg in the males and 1.4 g/kg in the females. 

In a combined repeated dose toxicity study with reproduction and developmental screening conducted according to GLP and OECD guideline 422 (Marburger et al., 2005) 40 male and 40 female Wistar rats were exposed to Fatty acids, tall-oil, epoxidized,2-ethylhexylesters (ETP). The rats were exposed by oral gavage at doses of 0, 100, 300 or 1000 mg/kg bw/day. Males were exposed for at least 28 days while female were dosed until day 3 of lactation. No mortalities were observed. No test-item related effects were seen. Neither food consumption nor body weight development were affected at any dosage. The assessment of clinical chemistry and haematology parameters indicated no differences between animals treated with the test item and vehicle controls. 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. 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. Based on this study a NOEL of 100 mg/kg bw/day was derived as an increase in absolute and relative liver weight was observed at the higher dose groups. Based on these effects a NOAEL of 1000 mg/kg bw/day was determined.

In a supporting non-GLP, non-guideline study (Larson et al., 1960) albino rats and mongrel dogs were exposed to ESBO through their diet. The mongrel dogs (three dogs in total) were exposed one year and the rats were divided into subgroups. A subgroup of 5 rats per treatment group were exposed for one year the other rats for two years. In total 15 male and 15 female rats were used in this study. The rats were daily exposed to concentrations of 0, 0.1, 0.5, 1.0, 2.5 and 5.0%, the dogs to 0.1, 1.0 and 5.0%. Addition of either epoxidised soybean oils, Paraplex G-60 or Paraplex G-62, to the diet of rats and mongrel dogs did not result in effects on survival or on the blood picture and histologic examination of tissues revealed no lesions attributable to treatment. Early depression in growth was observed in rats from both materials (Paraplex G-62 being the most potent), but this effect disappeared on continued feeding. Dogs receiving the 5% concentrations lost weight, an effect which persisted throughout the feeding period. Elevated liver to body weight ratios resulted at the 5% concentration in male rats receiving Paraplex G-60 and at lower concentrations in both male and female rats receiving Paraplex G-62; kidney to body weight ratios also elevated with the latter material in female rats. Based on this study a NOAEL of 5% in diet was determined for male and female rats as well as for mongrel dogs.

Justification for classification or non-classification

Based on the findings of the repeated dose toxicity studies with ESBO and ETP, Soybean oil, epoxidized, reaction products with methanoldoes not need to be classified according to the Directive 67/548/EEC and the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.