Fatty acids, C16-18 and C18-unsatd., Me esters, epoxidized

Endpoint summary

Description of key information

The administration of the test substance by gavage to male and female Wistar rats for 28 days (OECD 407) did not cause test substance-related adverse signs of systemic toxicity. Therefore, under the conditions of the present study, the no observed adverse effect level (NOAEL) was 1000 mg/kg bw/day.

No subchronic toxicity study is available with the substance itself. Therefore, the results of chronic studies with another member of the subgroup (CAS 8013-07-8) were used for the assessment of chronic toxicity. Based on the observations of this study, the NOAEL was determined to be 1000 mg/kg bw/day in male and 1400 mg/kg bw/day female rat.

However, to further improve the availale data on the subgroup members, another subchronic toxicity is planned with a member of the subgroup (CAS 95370-96-0).

 

 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

chronic toxicity: oral

Remarks:

combined repeated dose and carcinogenicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1986

Reliability:

other: 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

Rationale for reliability incl. deficiencies:

other: 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.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

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 or test system 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

No further information

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 Wister 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. 

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Limit test:

no

Specific details on test material used for the study:

- Test-substance No.: 12/0029-1
- Lot/batch No.: CE80580015
- Homogeneity: Given (visually)
- Expiry date: 27 Feb 2013
- Physical state/appearance: liquid/colorless, clear
- Storage conditions: ambient (room temperature)
- Stability: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.
- Name of test material (as cited in study report): Sovermol 1055

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
- Age at study initiation: 42 ± 1 days
- Weight at study initiation: Male: about 154g, females: about 129g
- Housing: Five animals per cage in polysulfonate cages supplied by TECNIPLAST, Hohenpeißenberg, Germany (floor area about 2065 cm2). Dust-free wooden bedding was used. Wooden gnawing blocks (Typ NGM E-022) supplied by Abedd® Lab. and Vet. Service GmbH, Vienna, Austria, were added for environmental enrichment.
- Diet: Ground Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water: Ad libitum
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

The test item was applied as a suspension. To prepare this suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, corn oil was filled up to the desired volume, subsequently released with a magnetic stirrer. During administration of the test substance, preparations were kept homogeneous by stirring with a magnetic stirrer. The test substance preparations were produced at least once a week and were stored at room temperature.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- The stability of the test item in corn oil at room temperature for a period of 7 days was demonstrated before the start of the study.
- Homogeneity of the test item was verified in the highest and lowest concentration. Considering the low relative standard deviation in the homogeneity analysis, it was concluded that the test item was distributed homogeneously in corn oil.
- Concentration control was verified in all concentrations at the beginning of the study. The mean values of the test substance in corn oil were always found to be in the range between 90-110% of the nominal concentrations. These results demonstrated the correctness of the concentrations the test substance in corn oil.

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

MORTALITY
A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied.

CLINICAL OBSERVATIONS
All rats were checked daily for any abnormal clinically signs before the administration as well as within 2 hours and within 5 hours after the administration.

DETAILED CLINICAL OBSERVATIONS
Detailed clinical observations (DCO) were performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 × 37.5 cm with sides of 25 cm high). The following parameters were examined: abnormal behavior when handled, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmus, feces (appearance/consistency), urine, pupil size.

FOOD CONSUMPTION
Food consumption was determined weekly over a period of 1 day and calculated as mean food consumption in grams per animal and day.

WATER CONSUMPTION
Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume.

BODY WEIGHT
Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period the body weight was determined on day 0 (start of the administration period) and thereafter at weekly intervals. The difference between the body weight on the respective day of weighing and the body weight on day 0 was calculated as body weight change.

FUNCTIONAL OBSERVATIONAL BATTERY
A functional observational battery (FOB) was performed in all animals at the end of the administration period starting at about 10:00 h. At least one hour before the start of the FOB the rats were transferred to single-animal polycarbonate cages. Drinking water was provided ad libitum, but no food was offered during the measurements. The FOB started with passive observations without disturbing the rats, followed by removal from the home cage, open field observations in a standard arena and sensory motor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random. Home cage observations: The rats were observed in their closed home cages; during this period any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the rats. Attention was paid to: Posture, Tremors, Convulsions, Abnormal movements, Gait abnormalities. Open field observations: The rats were transferred to a standard arena (50 × 50 cm with sides of 25 cm height) and observed for at least 2 minutes. The following parameters were examined: Behavior on removal from the cage, Fur, Skin, Salivation, Nasal discharge, Lacrimation, Eyes/ pupil size, Posture, Palpebral closure, Respiration, Tremors, Convulsions, Abnormal movements/ stereotypes, Gait abnormalities, Activity/ arousal level, Feces excreted within 2 minutes (number/ appearance/ consistency), Urine excreted within 2 minutes (amount/ color), Rearing within 2 minutes. Sensory motor tests/ reflexes: The rats were then removed from the open field and subjected to following sensory motor or reflex tests: Reaction to an object being moved towards the face (Approach response), Touch sensitivity (Touch response), Vision (Visual placing response), Pupillary reflex, Pinna reflex, Audition (Auditory startle response), Coordination of movements (Righting response), Behavior during handling, Vocalization, Pain perception (Tail pinch), Grip strength of forelimbs, Grip strength of hindlimbs, Landing foot-splay test, Other findings.

MOTOR ACTIVITY ASSESSMENT
Motor activity (MA) was also measured from 14:00 h onwards on the same day as the FOB was performed. The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. For this purpose, the rats were placed in new clean polycarbonate cages with a small amount of bedding for the duration of the measurement. Eighteen beams were allocated per cage. The number of beam interrupts was counted over 12 intervals for 5 minutes per interval. The sequence in which the rats were placed in the cages was selected at random. On account of the time needed to place the rats in the cages, the starting time was "staggered" for each animal. The measurement period began when the 1st beam was interrupted and finished exactly 1 hour later. No food or water was offered to the rats during these measurements and the measurement room was darkened after the transfer of the last rat.

ESTROUS CYCLE DETERMINATION
Vaginal smears for cycle determination were be prepared in the morning and evaluated according to the timetable for at least 3 weeks. The differentiation was conducted according to following stages: 1. Diestrous: leukocytes, few nucleated epithelial cells, 2 Proestrous: single leukocytes, many nucleated and few horny epithelial cells, 3 Estrous only horny epithelial cells, 4 Metestrous leukocytes, some horny epithelial cells and some nucleated epithelial cells.

CLINICAL PATHOLOGY
In the morning blood was taken from the retrobulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane (Isoba®, Essex GmbH, Munich, Germany). The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence. For urinalysis the individual animals were transferred to metabolism cages (withdrawal of food and water) and urine was collected overnight. Urine samples were evaluated in a randomized sequence The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results.

HAEMATOLOGY
The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany): Leukocyte count, Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Platelet count, Differential blood count, Reticulocytes. Furthermore, blood smears were prepared and stained according to WRIGHT without being evaluated, because of non-ambiguous results of the differential blood cell counts measured by the automated instrument. (reference: Hematology: Principles and Procedures, 6th Edition, Brown AB, Lea & Febiger, Philadelphia, 1993, page 101). Clotting tests (Prothrombin time) were carried out using a ball coagulometer (AMAX destiny plus model; Trinity biotech, Lemgo, Germany).

CLINICAL CHEMISTRY
An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, γ-Glutamyltransferase, Sodium, Potassium, Chloride, Inorganic phosphate, Calcium, Urea, Creatinine, Glucose, Total bilirubin, Total protein, Albumin, Globulins, Triglycerides, Cholesterol, Bile acids.

URINALYSIS
The dry chemical reactions on test strips (Combur-10-test M, Roche, Mannheim, Germany) used to determine urine constituents semiquantitatively were evaluated with a reflection photometer (Miditron M; Roche, Mannheim, Germany). Parameters: pH, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Blood, Specific gravity, Sediment, Color (turbidity), Volume.

SPERM PARAMETERS
Sperm motility, Sperm morphology, Sperm head count (cauda epididymis), Sperm head count (testis).

Sacrifice and pathology:

NECROPSY
The animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.

ORGAN WEIGHTS
The following weights were determined in all animals sacrificed on schedule: Anesthetized animals, Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Prostate, Seminal vesicles with coagulating glands, Spleen, Testes, Thymus, Thyroid glands, Uterus with cervix.

ORGAN/TISSUE FIXATION
The following organs or tissues were fixed in 4% buffered formaldehyde solution or in modified Davidson’s solution: All gross lesions, Adrenal glands, Aorta, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Epididymis (left), Esophagus, Extraorbital lacrimal glands, Eyes with optic nerve (modified Davidson’s solution), Femur with knee joint, Harderian glands, Heart, Ileum, Jejunum (with Peyer’s patches), Kidneys, Larynx, Liver, Lungs, Lymph nodes (mesenteric and axillary lymph nodes), Mammary gland (male and female), Nose (nasal cavity), Ovaries, Oviducts, Pancreas, Parathyroid glands, Pharynx, Pituitary gland, Prostate, Rectum, Salivary glands (mandibular and sublingual glands), Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Testis, left (modified Davidson’s solution), Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus, Vagina. From the livers, one additional slice of the Lobus dexter medialis and the Lobus sinister lateralis was fixed in Carnoy’s solution and embedded in paraplast. The left testis and left epididymis of all animals sacrificed at scheduled dates were fixed in modified Davidson’s solution, whereas the right testis and epididymis were used for sperm parameters. Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings. All the above mentioned organs/tissues of the control and high dose group were stained with hematoxylin and eosin (H&E) stain, except for the gross lesions (lesions in the low and mid dose groups were also stained with H&E stain) and the liver (livers of the low and mid dose groups were paraplast embed). The immunorelevant organs and tissues were evaluated according to the following parameters: Thymus: Increased/decreased grade of cortico-medullar ratio (related only to area), increase of starry sky cells, changes of cellular density in the cortex, and changes of cellular density in the medulla. Spleen: Changes of the cellularity of PALS, lymphoid follicles, marginal zone, red pulp, altered cellular composition of follicles, altered number of germinal centers. Lymph nodes (mesenteric and axillary lymph nodes): Changes in the cellularity of follicles, interfollicular area, paracortical area, medulla, altered cellular composition of paracortex, altered number of germinal centers, hyperplasia of high endothelial venules. Peyer's patches (of the jejunum): Changes of the cellularity of follicles (including mantle zone and germinal centers) and changes of the cellularity of interfollicular area. Bone marrow: Changes of the cellularity and changes of the myeloid/erythroid ratio. Whenever the histopathologic evaluation of the immunorelevant organs and tissues did not reveal a morphologic alteration of these items and/or whenever no other pathologic finding was noted, these organs were diagnosed as "no abnormalities detected”. Special attention was given for the synchrony of the morphology of the estrous cycle in ovaries, uterus, cervix, and vagina. A correlation between gross lesions and histopathological findings was attempted.

Statistics:

- Body weight, body weight change: A comparison of each group with the control group was performed using DUNNETT's test (two-sided) for the hypothesis of equal means
- Feces, rearing, grip strength forelimbs, grip strength hindlimbs, footsplay test, motor activity and weight parameters: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON test (two-sided) for the equal medians.
- Blood parameters: For parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians For parameters with unidirectional changes: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians.
- Urinalysis parameters (apart from pH, urine volume, specific gravity, color and turbidity): Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians.
- Urine pH, volume, specific gravity, color and turbidity: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal
medians. Urine color and turbidity are not evaluated statistically.
- Sperm analysis parameters: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) with Bonferroni-Holm adjustment for the hypothesis of equal medians For the percentage of abnormal sperms (ABNORMAL6_C) values <6% were set to 6% (cut off 6 %).

Clinical signs:

no effects observed

Description (incidence and severity):

No test substance-related, adverse findings were observed in male and female animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d).

Mortality:

no mortality observed

Description (incidence):

No animal died prematurely in the present study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No significant differences with regard to the mean body weights and mean body weight change values were noted for male and female animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No test substance-related, adverse findings were observed in male and female animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d).

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among hematological parameters were observed. In females of test group 1 (100 mg/kg bw/d) hematokrit values were higher compared to controls and in females of test group 3 (1000 mg/kg bw/d) red blood cell (RBC) counts were lower compared to controls. The hematokrit was not changed dose-dependently. The RBC mean was marginally decreased (6% lower compared to controls), within the historical control range (RBC 6.71-8.41 Tera/L), and no other red blood cell parameter in these rats was altered. Therefore, both red blood cell alterations were regarded as incidental and not treatment-related

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No treatment-related changes among clinical chemistry parameters were observed.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No treatment-related changes among urinalysis parameters were observed.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

- Functional observational battery: Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test-substance treated groups and controls, were without a dose-response relationship or occurred in single rats only, these observations were considered to have been incidental. No test substance-related effects were observed for home cage observations, open field observations and sensorimotor tests/reflexes. Feces, rearing, grip strength of fore- and hindlimbs as well as foot splay test did not reveal significant changes in test groups 1-3 (100, 300 and 1000 mg/kg bw/d) when compared to the control animals.
- Motor activity measurement: There were no significant deviations concerning the overall motor activity (summation of all intervals) in male and female animals of all test groups in comparison to the concurrent control group. The single intervals Nos. 4 and 9 were significantly decreased in male animals of test group 3 (1000 mg/kg bw/d). The single interval No. 6 was significantly increased in male animals of test group 1 (100 mg/kg bw/d). The single interval No. 8 was significantly decreased in female animals of test groups 1 and 2 (100, 300 mg/kg bw/d). However, as there were no significant deviations with regard to the overall motor activity (summation of all intervals) in male animals of test groups 1 and 3 (100 and 1000 mg/kg bw/d) as well as in female animals of test groups 1 and 2 (100 and 300 mg/kg bw/d) in comparison to the concurrent control group these findings were assessed as being incidental and not related to treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The significant absolute and/or relative weight changes noted in the brain of males and in the uterus and liver of females were not considered to be treatment-related since they occurred without any dose-dependent relationship. The significant decrease of the absolute and relative mean weight of the thyroid gland in females of test group 3 (1000 mg/kg bw/d) was not consistent with any histopathological finding. Moreover, the absolute (13.0 mg) and relative (0.007%) mean weight of the thyroid gland was within the historical control range (absolute weight range: 11.8-21.2 mg; relative weight range: 0.007-0.012%). Therefore, the thyroid gland weight decrease was regarded as incidental and not related to treatment. The significant differences in absolute and relative uterus weights of female animals in test groups 1 and 2 (100 and 300 mg/kg bw/d) compared to the control animals were most likely related to the estrous cycle status. In addition, the mean uterus weight of the control group was influenced by the very high value animal No. 23. Thus, the changes were regarded to be incidental and not related to treatment (see 'any other information on results' for details).

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All findings occurred individually. They were considered to be incidental in nature and not related to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

All histopathological findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental in nature and not related to treatment.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

- No test substance-related effects on estrous cycle length and the number of cycles were obtained.
- Sperm parameters: Concerning the motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as the sperm head counts in the testis and in the cauda epididymidis no treatment-related effects were observed. Male animal No. 5 of the control group had a low motility (21%) and a high percentage of abnormal sperms in the cauda epididymidis (70%). But even without the values of this rat there was no dose-dependent trend of both parameters when regarding the medians as well as no statistical significant difference between the groups.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No toxic effects observed at the highest dose tested.

Key result

Critical effects observed:

no

Absolute organ weights

When compared to the control group 0 (set to 100%), the following mean absolute weights were significantly increased or decreased (statistically significant changes printed in bold):

	Male animals			Female animals
Test group (mg/kg bw/d)	1 (100)	2 (300)	3 (1000)	1 (100)	2 (300)	3 (1000)
Brain	106%*	99%	101%
Thyroid gland				117%	103%	74%*
Uterus				55%*	53%*	88%

*p ≤ 0.05

All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

 

Relative organ weights

When compared to the control group 0 (set to 100%), the mean relative weights of the following organs were significantly decreased (statistically significant changes printed in bold):

	Male animals			Female animals
Test group (mg/kg bw/d)	1 (100)	2 (300)	3 (1000)	1 (100)	2 (300)	3 (1000)
Liver				90%	96%	103%
Thyroid gland				112%	104%	73%**
Uterus				53%*	53%**	87%

*p ≤ 0.05; **p ≤ 0.01

All other mean relative weight parameters did not show significant differences when compared to the control group 0.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

The quality of the whole database will be improved with the planned OECD 408 with a member of the subgroup.

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 adverse effect observed

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

For the substance itself a 28-day repeated dose toxicity study is available in which no adverse effects are observed up to 1000 mg/kg bw/day.

No subchronic toxicity study is available with the substance itself. Therefore, the results of chronic studies with another member of the subgroup (CAS 8013-07-8) were used for the assessment of chronic toxicity. Based on the observations of this study, the NOAEL was determined to be 1000 mg/kg bw/day in male and 1400 mg/kg bw/day female rat.

However, to further improve the availale data on the subgroup members, another subchronic toxicity is planned with a member of the subgroup (CAS 95370-96-0).

28-day repeated dose toxicity:

In a GLP compliant repeated-dose 28-day toxicity study performed according to OECD Guideline 407, the test item was administered orally by gavage to groups of 5 male and 5 female Wistar rats at dose levels of 0 mg/kg bw/d (test group 0), 100 mg/kg bw/d (test group 1), 300 mg/kg bw/d (test group 2) and 1000 mg/kg bw/d (test group 3) over a period of 4 weeks. Clinical examinations (mortality, clinical observations, food consumption, body weight, functional observational battery, and motor activity measurement) did not reveal treatment-related, adverse effects up to a concentration of 1000 mg/kg bw/d. In addition, no test substance-related effects on estrous cycle length and the number of cycles were obtained. Concerning clinical pathology (hematology, clinical chemistry, and urinalysis) no treatment-related, adverse effects were observed up to a dose of the test substance of 1000 mg/kg bw/d. Furthermore, no test substance-related effects on sperm parameters were obtained. Regarding pathology (organ weights, gross lesions, and histopathology) no treatment-related organ weight changes, gross lesions or histopathological findings were observed in the treated male and female animals including the reproductive organs. In conclusion, the administration of the test item by gavage to male and female Wistar rats for 4 weeks did not cause test substance-related adverse signs of systemic toxicity. Therefore, under the conditions of the present study the no observed adverse effect level (NOAEL) was 1000 mg/kg bw/d in male and in female Wistar rats (BASF 2013).

 

Chronic toxicity (CAS 8013-07-8)

Groups of 48 male and 48 female rats of a Wister 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. 

 

In another study two epoxidised soybean oils, Paraplex G-60 and Paraplex G-62, were added to the diets of rats for a two-year period and of dogs for one year in concentrations up to 5 %. No effects were observed 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.

 

Justification for classification or non-classification

Based on the available data, classification for repeated dose toxicity is not warranted in accordance with EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.