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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
chronic toxicity: oral
Remarks:
combined repeated dose and carcinogenicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
1986
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
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. However the reliability of the team preparing the review is considered to be 1 or 2. 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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1986
Report Date:
1986

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Epoxidised Soybean Oil (ESBO)
- Physical state: Pale yellow, viscous, oily liquid.
- Storage condition of test material: Room temperature

Test animals

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.


Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

open allclose all
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

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

No further information

Applicant's summary and conclusion

Conclusions:
It was concluded that ESBO was not carcinogenic when fed to rats at up to 2.5 % of the diet. Further it was concluded that 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. 
Executive summary:

Groups of 48 male and 48 female rats of a Wistar strain were fed diet containing 0.025, 0.25 or 2.5 % Epoxidised Soya Bean Oil (ESBO) for 2 years. Similar groups were given basal diet to serve as a control or 2.5 % Soya bean Oil (SBO).

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 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.

The incidence of most histological findings including tumours were similar in treated and control groups. There was a tendency for less severe glomerulonephrosis in the ESBO-treated rats. 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 ESBO was not carcinogenic when fed to rats at up to 2.5 % of the diet. Further it was concluded that 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.