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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An HPRT assay (OECD 476) is available for the substance itself. No Ames test is available for the substance itself, therefore data on other members of the subgroup is used. From this data no indications for genetic toxicity were found.

No in vitro cytogenicity study with the substance is available, but an in vitro micronucleus study is ordered for the substance. At the moment, only information on in vitro cytogenicity is available on one member of the subgroup (CAS 8013-07-8). Some differences in response between experiment 1 and 2 but no consistent indication of an effect on structure or frequency of chromosomal aberrations, with or without metabolic activation. However, no indications exist that ESBO induces CA when testetd to its limit of solublity.

 

 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8 May 1992 to 1 June 1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: There is no indication of the duration of the incubation period in Experiment 2. This is not expected to effect the validity of the results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

other: EEC Annex V Test B14

Qualifier:

according to guideline

Guideline:

other: UKEMS Guidelines

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine gene

Species / strain / cell type:

S. typhimurium, other: TA98; TA100; TA 1535; TA1537 and TA102

Additional strain / cell type characteristics:

other: biotin and histidine required for growth

Metabolic activation:

with and without

Metabolic activation system:

mammalian liver post-mitochondrial fraction (S9)

Test concentrations with justification for top dose:

Please see Table 1 and 2 below.
An initial toxicity range-finder was carried out in TA100 strain only, using final concentrations of Epoxidised Soybean Oil at 8, 40, 200, 1000 and 5000 µg/plate plus a solvent and a positive control. These treatments were non-toxic and the same dose range was used for experiment 1. For experiment 2 treatments, the dose range was narrowed to 312.5 - 5000 µg/plate in order to investigate those concentrations most likely to exhibit a mutagenic response.

Vehicle / solvent:

Test chemical solutions were prepared by dissolving Epoxidised Soybean Oil in analytical grade acetone, immediately prior to assay to give the required maximum concentration treatment solution. Further dilutions were then made using acetone. The test chemical solutions were protected from light and used within approximately 4 hours of the initial formulation of the test agent.

Untreated negative controls:

yes

Remarks:

solvent acetone

Positive controls:

yes

Remarks:

Please Table 2 below

Details on test system and experimental conditions:

An initial toxicity range-finder was carried out in TA100 strain only, using final concentrations of Epoxidised Soybean Oil at 8, 40, 200, 1000 and 5000 µg/plate plus a solvent and a positive control. These treatments were non-toxic and the same dose range was used for experiment 1.

Five strains of bacteria were used in this study. For all assays, bacteria were cultured for about 10 hours at 37 °C in nutrient broth (containing ampicillin for strains TA98 and TA100 and ampicillin and tetracycline for strain TA102). Bacteria were taken from vials of frozen cultures, which had been checked for strain characteristics (histidine dependence, rfa character and resistance to ampicillan (TA98 and TA100) or ampicillin plus tetracycline (TA102). Checks were carried out according to Maron and Ames and De Serres and Shelby. For all treatments, cultures were used within 2 hours of the end of the incubation period.

Epoxidised Soybean Oil was tested for mutation in 5 strains of Salmonella typhurium at the concentrations detailed in Table 1. Triplicate plates with and without S-9 mix were used. Negative (solvent) and positive controls were included in both assays, in quintuplicate without and with S-9 mix. In each experiment, bacterial strains were treated with diagnostic mutagens in triplicate in the absence of S-9. The activity of the S-9 mix used in each experiment was confirmed by AAN treatments (again in triplicate) of at least one strain in the presence of S-9.

Because the results of the first experiment were negative, treatments in the presence of S-9 in Experiment 2 included a pre-incubation step, where the quantities of test chemical or control solution, bacteria and S-9 mix detailed, were mixed together and incubated for 1 hour at 37 °C, before the addition of 2.5 mL molten agar at 46 °C. Plating of these treatments then proceeded as for the normal plate-incorporation procedure. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected in the assay.

Colony Counting:
Colonies were counted electronically using a Seescan Colony Counter or manually where minor agar damage might have interfered with automatic counting, and the background lawn inspected for signs of toxicity.

Evaluation criteria:

Acceptance Criteria:
The assay was considered valid if the following criteria were met:
i) the mean negative control counts fell within the normal range as defined in Appendix 4
ii) the positive control chemicals induced clear increases in revertant numbers confirming discrimination between different strains, and an active S-9.
iii) no more than 5 % of the plates were lost through contamination or some other unforeseen event

Evaluation criteria:
A test compound was considered to be mutagenic if
i) the assay was valid
ii) Dunnett's test gave a significant response (p <= 0.01), and the data set showed a significant dose-correlation
iii) the positive responses described in (ii) were reproducible

Statistics:

The m-statistic was first calculated to check that the data were Poisson-distributed and then Dunnett's test was used to compare the counts of each dose with the control. The presence or otherwise of a dose response was then checked by linear regression analysis.

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Toxicity:
Only treatments of strain TA102 in Experiment 2 (+ S-9 only) showed signs of toxicity (as indicated by thinning of the background bacterial lawn) in this study. In this case, toxic effects were seen mostly at the 3 highest doses. It would appear that the use of a pre-incubation step particularly enhanced the toxicity of the test agent to this test strain.

Range-finder and Experiment 1 treatments were carried out using final concentrations of Epoxidised SOybean Oil at 8, 40, 200, 1000 and 5000 µg/plate, Precipitation, in the form of oil droplets, was observed at concentrations of 1000 and 5000 µg/plate. For experiment 2, testing was again carried out up to maximum concentration of 5000 µg/plate (despite the observation of precipitation), as it was possible that the compund formed an emulsion within the test system and it was felt important to maximise the exposure of the cells to this. A narrowed dose range was also used in this experiment (312.5 - 5000 µg/plate) in order to examine those doses most likely to exhibit a mutagenic response. Oil droplets were observed on test plates in this experiment, following treatments of 1250 µg/plate and above.

Mutation:
The individual plate counts were averaged to give mean values. From the data it can be seen that mean solvent control counts fell within the normal historical range, that the positive control chemicals all induced large increases in revertant numbers in the appropriate strains, and that < 5 % of plates were lost, leaving adequate numbers of plates at all treatments. The study was accepted as valid.

The mutation data were evaluated as follows:
No treatment with Epoxidised Soybean Oil of any of the tester strains, earlier in the absence or presence of S-9, resulted in a significant increase in revertant numbers. The data obtained therefore gave no indication of an ability of the test agent to induce mutation.

Remarks on result:

other: strain/cell type: S. typhimurium TA 102

Remarks:

Migrated from field 'Test system'.

No further information

Conclusions:

Interpretation of results (migrated information):
negative

It is concluded that Epoxidised Soybean Oil failed to induce mutation in 5 strains of Salmonella typhimurium, when treated up to a maximum concentration of 5000 µg/plate, in the absence and presence of a rat liver metabolic activation system.
According to Directive 67/548/EEC, no classification is warranted.
According to Regulation (EC) No. 1272/2008, no classification is warranted.

Executive summary:

Epoxidised Soyban Oil was assayed for mutation in 5 -histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and presence of metabolic activation by an Aroclor 1254 induced rat liver post-mitochondrial fraction (S-9), in two separate experiments.

It was concluded that Epoxidised Soybean Oil failed to induce mutation in 5 strains of Salmonella typhimurium, when treated up to a maximum concentration of 5000 µg/plate, in the absence and presence of a rat liver metabolic activation system. According to Directive 67/548/EEC, no classification is warranted. According to Regulation (EC) No. 1272/2008, no classification is warranted.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

GLP compliance:

yes (incl. QA statement)

Remarks:

Harlan Cytotest Cell Research GmbH, In den Leppsteinswiesen 19, 64380 Rossdorf, Germany

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

- Test-substance No.: 12/0029-1
- Lot/batch No.: CE80580015
- Expiration Date: February 27, 2013
- Name of test material (as cited in study report): Sovermol 1055

Target gene:

HPRT (hypoxanthine-guanine phosphoribosyl transferase)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Before freezing, the level of spontaneous mutants was depressed by treatment with HAT-medium. Each batch is screened for mycoplasm contamination and checked for karyotype stability and spontaneous mutant frequency.

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

see any other information on materials and methods incl. tables

Vehicle / solvent:

On the day of the experiment (immediately before treatment), the test item was dissolved in ethanol. The final concentration of ethanol in culture medium was 0.5% v/v.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION:
In medium

DURATION
- Preincubation period: 24 hours
- After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 μL/mL S9 mix. Concurrent solvent and positive controls were treated in parallel. After 4 hours this medium was replaced with complete medium following two washing steps with "saline G". In the second experiment the cells were exposed to the test item for 24 hours in complete medium, supplemented with 10% FBS, in the absence of metabolic activation.
- Expression/fixation time: Three or four days after treatment 1.5E6 cells per experimental point were sub-cultivated in 175 cm² flasks containing 30 mL medium. Following the expression time of 7 days five 80 cm² cell culture flasks were seeded with about 3 - 5E5 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability. The cultures were incubated at 37 °C in a humidified atmosphere with 1.5% CO2 for about 8 days. The colonies were stained with 10% methylene blue in 0.01% KOH solution.

NUMBER OF REPLICATIONS:
- The study was performed in two independent experiments, using identical experimental procedures.

NUMBER OF CELLS EVALUATED:
- The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity of the test item is indicated by a reduction of the cloning efficiency (CE).

Evaluation criteria:

The gene mutation assay is considered acceptable if it meets the following criteria:
- The numbers of mutant colonies per 1E6 cells found in the solvent controls falls within the laboratory historical control data.
- The positive control substances should produce a significant increase in mutant colony frequencies.
- The cloning efficiency II (absolute value) of the solvent controls should exceed 50%.
- The data of this study comply with the above mentioned criteria.

Evaluation of Results:
- A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
- A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.

A positive response is described as follows:
- A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
- The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed. However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory's historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance was considered together.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
Phase separation was observed in experiment I at 120 μg/mL with and without metabolic activation. In experiment II phase separation occurred at 120.0 μg/mL with metabolic activation.

RESULTS GENOTOXICITY:
No relevant and reproducible increase in mutant colony numbers/1E6 cells was observed in the main experiments up to the maximum concentration. The mutation frequency exceeded the threshold of three times the mutation frequency of the solvent control in the second experiment without metabolic activation at 60.0 μg/mL in culture I and at 7.5 to 60.0 μg/mL in culture II. However, only the increase observed in culture I at 60.0 μg/mL exceeded the historical range of solvent controls (2.6 - 40.3 mutant colonies/10E6 cells). The apparent increase of the mutation frequency in culture II is based on the low solvent control of just 3.9 colonies per 1E6 cells. The absolute values of the mutation frequency remained within the historical range of solvent controls. Furthermore, the apparent increase in culture II was not dose dependent as indicated by the lacking statistical significance. Consequently, the isolated increase noted in the first culture at 60.0 μg/mL was judged as irreproducible fluctuation. A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of the mutation frequency. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in the second culture of experiment I without metabolic activation and in the first culture of experiment II without metabolic activation. However, the trends were judged as biologically irrelevant as they were not reproduced in the parallel cultures. Furthermore, the mutation frequency of the second culture of the first experiment without metabolic activation remained within the historical range of solvent controls. In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 3.9 up to 19.6 mutants per 1E6 cells; the range of the groups treated with the test item was from 6.1 up to 46.2 mutants per 1E6 cells. The cloning efficiency II of the solvent control of the first culture of the second experiment with metabolic activity reached but did not exceed the lower limit of 50%. The data are valid however, as the solvent control of the parallel culture exceeded this limit. EMS (150 μg/mL) and DMBA (1.1 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

RANGE-FINDING/SCREENING STUDIES:
The highest concentration used in the pre-test was 4000 μg/mL limited by the solubility of the test item in ethanol and aqueous medium. Test item concentrations between 31.3 μg/mL and 4000 μg/mL were used to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation. Relevant cytotoxic effects indicated by a relative suspension growth below 50 were noted at 62.5 μg/mL and above without metabolic activation and 500.0 μg/mL and above with metabolic activation following 4 hours treatment. Following 24 hours treatment without metabolic activation cytotoxic effects as described above occurred at 62.5 μg/mL and above. The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. Phase separation occurred at 62.5 μg/mL and above with and without metabolic activation following 4 and 24 hours treatment. There was no relevant shift of the osmolarity and pH value even at the maximum concentration of the test item.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% in both parallel cultures occurred in the first experiment at 120.0 μg/mL without metabolic activation and in the second experiment at 60.0 μg/mL without metabolic activation.

Conclusions:

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.
Therefore, the test item is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The study was performed to investigate the potential of the test item to induce gene
mutations at the HPRT locus in V79 cells of the Chinese hamster.
The assay was performed in two independent experiments, using two parallel cultures
each. The first main experiment was performed with and without liver microsomal
activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.
The highest concentration used in the range finding pre-experiment was 4000 μg/mL of
the test item based on the solubility properties of the test item. The concentration range of the main experiments was limited by phase separation of the test item and cytotoxic effects. The test item was dissolved in ethanol.
No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation.
Appropriate reference mutagens (EMS and DMBA), used as positive controls, induced a
distinct increase in mutant colonies and thus, showed the sensitivity of the test system
and the activity of the metabolic activation system.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 3, 1992 - October 16, 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, according to guidelines

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Principles of method if other than guideline:

Not relevant

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

This test evaluated the effect of Epoxidised Soybean Oil on mitotic index/chromosomal aberrations.

Species / strain / cell type:

lymphocytes: Human Peripheral Blood Lymphocytes

Details on mammalian cell type (if applicable):

Two healthy, non-smoking volunteers (male in Experiment 1 and female in Experiment 2) were used in this study. Neither donor was suspected of any virus infection nor had been exposed to high levels of radiation or hazardous chemicals. For each experiment an appropriate volume of whole blood was drawn from the peripheral circulation on the day of culture. Whole blood cultures were established in sterile disposable centrifuge tubes by placing 0.4 mL heparinised blood into 9.0 mL Hepes-buffered RPMI medium containing 20 % (v/v) foetal calf serum and 50 ug/mL gentamycin. Photohaemagglutinin (PHA, reagent grade) was included at a concentration of 37.5 uL per ml of culture to stimulate the lymphocytes to divide. Cultures were rocked continuously during incubation. The blood cultures were incubated at 37 C for approx. 48 hours.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S-9 mammalian liver post-mitochondrial fraction

Test concentrations with justification for top dose:

Preliminary solubility data indicated that limited precipitation occurred when an acetone solution at 6 mg/mL was diluted 100-fold into culture medium. No precipitation was observed when a 5.3 mg/ml acetone solution was diluted similarly to give 53 ug/ml. A concentration of 55 ug/ml was therefore considered close to the solubility limit and an appropriate maximum concentration for the main study. Test chemical stock solutions were prepared by dissolving Epoxidised soybean oil in analytical grade acetone to give 5.5 mg/ml. Stock solutions were not membrane filter-sterilised (because of the nature of the solvent ) and further dilutions made in acetone. Solutions were then used within 2 hours of initial dissolution as seen in Table 1 and 2.

Vehicle / solvent:

Acetone

Negative solvent / vehicle controls:

yes

Remarks:

Acetone was added to cultures designated as negative controls

Positive controls:

yes

Remarks:

Positive control chemicals were dissolved in sterile anhydrous analytical grade dimethyl sulphoxide immediately prior to use

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Please see table 3 for concentration levels

Positive controls:

yes

Remarks:

Positive control chemicals were dissolved in sterile anhydrous analytical grade dimethyl sulphoxide immediately prior to use

Positive control substance:

cyclophosphamide

Remarks:

Please see table 3 for concentration levels

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Test chemical stock solutions were prepared by dissolving epoxidised soybean oil in analytical grade acetone to give 5.5 mg/ml. Solutions were made according to Tables 1 and 2.

Glucose-6-phosphate (180 mg/ml), NADP (25 mg/ml), 150 mM KCl and rat liver S-9 were mixed in the ratio 1:1:1:2. A 0.5 ml aliquot of the resulting S-9 mix was added to each cell culture 99.5 mL) containing the test chemical to achieve the required final concentration in a total of 10 mL. Cultures treated in the absence of S-9 received 0.5 ml 150 mM KCl.

DURATION
- Preincubation period: approx 48 hours
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 20 or 44 hours. Please see Table 5.

SPINDLE INHIBITOR (cytogenetic assays): Colchicine
STAIN (for cytogenetic assays): the cells were stained for 5 minutes in 4 % (v/v) filtered Giemsa stain in pH 6.8 buffer.

NUMBER OF REPLICATIONS: Please see Table 4

DETERMINATION OF CYTOTOXICITY
Slides were examined, uncoded, for mitotic index or percentages of cells in mitosis

Evaluation criteria:

Slides were examined, uncoded, for mitotic index or percentages of cells in mitosis.
Scoring of aberrations - where possible 25 cells from each of the selected NQO and CPA positive control treatments were analysed to ensure that the system was operating satisfactorily. One hundred metaphases from each culture were analysed for chromosome aberrations. Only cells with 44-46 chromosomes were considered accepatble for analysis of structural aberrations. Any cell with more than 46 chromosomes, that is polyploid, endoreplicated and hyperdiploid cells, observed during this search was noted and recorded seperately.

Statistics:

None reported

Species / strain:

lymphocytes: Human Peripheral Blood Lymphocytes

Metabolic activation:

with and without

Genotoxicity:

other: not required

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The results of mitotic index determinations for the treatments in Experiment 1, without and with S-9 sampled at 20 hours are shown in Table 6. No mitotic inhibition was observed in the absence of S-9 and little (approx. 14 %) in its presence, although this was not clearly dose-related. The following doses were then chosen:

Mitotic Inhibition *
20 hours, -S-9: 26.95, 38.5, 55 ug/mL 0 %
20 hours, +S-9: 26.95, 38.5, 55 ug/mL 14 %

* = at highest analysed dose

The results of mitotic index determinations for the treatments in Experiment 2, without and with S-9 sampled at 20 and 44 hours are seen in Table 7.


In contrast to Experiment 1, mitotic inhibition was observed in the absence of S-9 at the 20 hour sampling in Experiment 2. Approximately 47 % mitotic inhibition was seen at the highest level in the absence of S-9 and 25 % in its presence. No mitotic inhibition was seen at the 44 hour sampling time. The following doses were selcted for analysis:

Mitotic Inhibition *
20 hours, -S-9: 30.94, 41.25, 55 ug/mL 47 %
20 hours, +S-9: 30.94, 41.25 55 ug/mL 25 %
44 hours, - and + S-9; 55 ug/mL 0%

* = at highest analysed dose

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 6: Mitotic Index Determinations for Experiment 1

Treatment (ug/ml)	Mitotic index (%)
	-S-9		+S-9
	A	B	A	B
Untreated	NS	NS	NS	NS
Solvent	3.2	2.3	4.3	3.5
1.554	NM	NM	NM	NM
2.219	NM	NM	NM	NM
3.171	NM	NM	NM	NM
4.530	NM	NM	NM	NM
6.471	NM	NM	NM	NM
9.244	NM	NM	NM	NM
13.21	NM	NM	NS	NS
18.87	NS	NS	NS	NS
26.95	3.1	2.2	3.6	2.7
38.5	2.1	2.3	2.9	4.0
55	2.8	2.5	3.0	3.7

NS = not scored

NM = slides not made

(Slides from solvent control cultures C and D not made)

Table 7: Mitotic index determinations for Experiment 2.

Treatment (ug/mL)	Mitotic index (%)
	20 hours				44 hours
	A	B	A	B	A	B	A	B
Untreated	NS	NS	NS	NS	NS	NS	NS	NS
Solvent	4.7	3.8	5.0	6.9	3.8	3.5	5.9	6.7
5.506	NT	NT	NT	NT	NM	NM	NT	NT
7.342	NT	NT	NT	NT	NM	NM	NT	NT
9.789	NT	NT	NT	NT	NM	NM	NT	NT
13.05	NT	NT	NT	NT	NM	NM	NM	NM
17.4	NT	NT	NT	NT	NM	NM	NM	NM
23.2	NS	NS	NS	NS	NS	NS	NM	NM
30.94	3.2	3.7	5.5	6.8	NS	NS	NS	NS
41.25	4.2	3.8	5.4	6.0	NS	NS	NS	NS
55	1.5	3.0	3.6	5.3	3.8	4.1	6.0	6.7

NS = not scored

NM = slides not made

NT = not tested

Conclusions:

Interpretation of results (migrated information):
ambiguous Some differences in response between experiment 1 and 2 but no consistent indication of an effect on structure or frequency of chromosomal aberrations, with or without metabolic activation

It is concluded that Epoxidised soybean oil was unable to induce chromosome aberrations in cultured human peripheral blood lymphocytes when tested to its limit of solubility in both the absence and presence of S-9.
According to Directive 67/548/EEC, no classification is warranted.
According to Regulation (EC) No. 1272/2008, no classification is warranted.

Executive summary:

Epoxidised soybean oil was tested in an in vitro cytogenetics assay using duplicate human lymphocyte cultures from a male and female donor in 2 independent experiments. The highest dose level used, 55 ug/ml, was close to the solubility limit of Epoxidised soybean oil in culture medium. Treatments covering a broad range of doses, separated by narrow intervals, were performed both in the absence and presence of metabolic activation by a rat liver post-mitochondrial fraction (S-9) from Aroclor 1254 induced animals. In Experiment 1, treatment in the absence of S-9 was continuous for 20 hours. Treatment in the presence of S-9 was for 3 hours only followed by a 17 hour recovery period prior to harvest. The test compound dose levels for chromosome analysis were selected by evaluating the effect of Epoxidised soybean oil on mitotic index. Chromosome aberrations were analysed at 3 consecutive dose levels. The highest concentration chosen for analysis at this time, 55 ug/ml, induced no mitotic inhibition in the absence of S-9 and approximately 14 % in its presence, although this was not clearly dose-related. Experiment 2 included a delayed sampling time. Treatment in the absence of S-9 was continuous for 20 or 44 hours. Treatment in the presence of S-9 was for 3 hours followed by 17 or 41 hour recovery period. The highest concentration chosen for analysis at 20 hours, was again 55 ug/ml which on this occasion induced approximately 47 % and 25 % mitotic inhibition in the absence and presence of S-9 respectively. The effect of this single concentration only was investigated at the delayed harvest at which time no mitotic inhibition was induced.

Appropriate negative (solvent and untreated) control cultures were included in the test system in both experiments at both sampling times. Acceptable numbers of cells with structural aberrations were observed in solvent control cultures, slides from untreated cultures were not analysed. 4-Nitroquinoline 1-oxide (NQO) and cyclophosphamide (CPA) were employed as positive control chemicals in the absence and presence of liver S-9 respectively. Cells receiving these sampled in each experiment 20 hours after the start of treatment; both compounds induced statistically significant increases in the proportion of cells with structural aberrations.

In most cases, treatment of cultures with Epoxidised soybean oil in either the absence or presence of S-9 resulted in frequencies of cells with aberrations which were similar to and not significantly different from those seen in concurrent negative controls. Small increases in cells with aberrations were seen at the 20 hour sampling time following treatment with 26.95 ug Epoxidised soybean oil/ml in the presence of S-9 in Experiment 1 and 41.25 ug Epoxidised soybean oil/ml in the absence of S-9 in Experiment 2. In neither case, however, was the increase characterised by both statistical significance and frequencies of aberrant cells outside negative historical control ranges and could not therefore be considered biologically important.

It is concluded that Epoxidised soybean oil was unable to induce chromosome aberrations in cultured human peripheral blood lymphocytes when tested to its limit of solubility in both the absence and presence of S-9.

According to Directive 67/548/EEC, no classification is warranted.

According to Rgulation (EC) No. 1272/2008, no classification is warranted.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No in vivo genotoxicity data on the substance are available. Information is therefore derived from two in vivo micronucleus assays (OECD 474) with structurally related substances. Both substances are negative in this assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

An HPRT assay (OECD 476) is available for the substance itself. No Ames test is available for the substance itself, therefore data on other members of the subgroup is used. From this data no indications for genetic toxicity were found.

No in vitro cytogenicity study with the substance is available, but an in vitro micronucleus study is ordered for the substance. At the moment, only information on in vitro cytogenicity is available on one member of the subgroup (CAS 8013-07-8). Some differences in response between experiment 1 and 2 but no consistent indication of an effect on structure or frequency of chromosomal aberrations, with or without metabolic activation. However, no indications exist that ESBO induces CA when testetd to its limit of solublity.

Gene mutation assay in mammalian cells (HPRT) with test substance itself:

A GLP-compliant gene mutation assay, tested according to OECD guideline 476, was performed to investigate the potential of the test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The test item was dissolved in ethanol. The concentration range of the main experiments was limited by phase separation of the test item and cytotoxic effects. Phase separation was observed in experiment I at 120 μg/mL with and without metabolic activation and in experiment II at 120.0 μg/mL with metabolic activation. Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% in both parallel cultures occurred in the first experiment at 120.0 μg/mL without metabolic activation and in the second experiment at 60.0μg/mL without metabolic activation. No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. Therefore, the test item is considered to be non-mutagenic in this HPRT assay (Harlan 2013).

 

HPRT test CAS 8013-07-8:

Epoxidised Soyban Oil was assayed for mutation in 5 -histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and presence of metabolic activation by an Aroclor 1254 induced rat liver post-mitochondrial fraction (S-9), in two separate experiments. It was concluded that Epoxidised Soybean Oil failed to induce mutation in 5 strains of Salmonella typhimurium, when treated up to a maximum concentration of 5000 µg/plate, in the absence and presence of a rat liver metabolic activation system. According to Directive 67/548/EEC, no classification is warranted. According to Regulation (EC) No. 1272/2008, no classification is warranted.The potential of the test item to induce gene mutations was examined by means of two independent S. typhimurium reverse mutation assays (Ames test) according to OECD 471, adopted in 1997. The bacterial strains of TA 1535, TA 1537, TA 98, TA 100, and TA 102 were used. Experiment I was performed as a plate incorporation assay. Since a negative result was obtained in this experiment, experiment Il was performed as a pre-incubation assay. The test item was incubated in nominal concentrations of 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/ plate in experiment I and 33; 100; 333; 1000; 2500; and 5000 µg/ plate in experiment II. The test was performed in triplicates. After an incubation period of 48 hours, revertant colonies were counted. Negative and positive controls were included. The plates incubated with the test item showed normal background growth up to 5000 µg/ plate, with and without S9 mix in all strains used. No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups, with and without S9 mix in experiment I. Minor toxic effects were observed in experiment Il in strain TA 100 without S9 mix and in strains TA 1537 and TA 100 with S9 mix. No increase in revertant colony numbers of any of the five tested strains was observed following treatment with the test item at any concentration and neither with or without S9 mix. In experiment Il without S9 mix, the data in the negative control of strain TA 100 were slightly above our historical control range. This slight effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies. Based on the results the test item is considered to be not mutagenic in bacteria (RCC 2006). 

In-vitro MN (CAS 8013-07-8):

Epoxidised soybean oil was tested in an in vitro cytogenetics assay using duplicate human lymphocyte cultures from a male and female donor in 2 independent experiments. The highest dose level used, 55 ug/ml, was close to the solubility limit of Epoxidised soybean oil in culture medium. Treatments covering a broad range of doses, separated by narrow intervals, were performed both in the absence and presence of metabolic activation by a rat liver post-mitochondrial fraction (S-9) from Aroclor 1254 induced animals. In Experiment 1, treatment in the absence of S-9 was continuous for 20 hours. Treatment in the presence of S-9 was for 3 hours only followed by a 17 hour recovery period prior to harvest. The test compound dose levels for chromosome analysis were selected by evaluating the effect of Epoxidised soybean oil on mitotic index. Chromosome aberrations were analysed at 3 consecutive dose levels. The highest concentration chosen for analysis at this time, 55 ug/ml, induced no mitotic inhibition in the absence of S-9 and approximately 14 % in its presence, although this was not clearly dose-related. Experiment 2 included a delayed sampling time. Treatment in the absence of S-9 was continuous for 20 or 44 hours. Treatment in the presence of S-9 was for 3 hours followed by 17 or 41 hour recovery period. The highest concentration chosen for analysis at 20 hours, was again 55 ug/ml which on this occasion induced approximately 47 % and 25 % mitotic inhibition in the absence and presence of S-9 respectively. The effect of this single concentration only was investigated at the delayed harvest at which time no mitotic inhibition was induced. Appropriate negative (solvent and untreated) control cultures were included in the test system in both experiments at both sampling times. Acceptable numbers of cells with structural aberrations were observed in solvent control cultures, slides from untreated cultures were not analysed. 4-Nitroquinoline 1-oxide (NQO) and cyclophosphamide (CPA) were employed as positive control chemicals in the absence and presence of liver S-9 respectively. Cells receiving these sampled in each experiment 20 hours after the start of treatment; both compounds induced statistically significant increases in the proportion of cells with structural aberrations. In most cases, treatment of cultures with Epoxidised soybean oil in either the absence or presence of S-9 resulted in frequencies of cells with aberrations which were similar to and not significantly different from those seen in concurrent negative controls. Small increases in cells with aberrations were seen at the 20 hour sampling time following treatment with 26.95 ug Epoxidised soybean oil/ml in the presence of S-9 in Experiment 1 and 41.25 ug Epoxidised soybean oil/ml in the absence of S-9 in Experiment 2. In neither case, however, was the increase characterised by both statistical significance and frequencies of aberrant cells outside negative historical control ranges and could not therefore be considered biologically important.

It is concluded that Epoxidised soybean oil was unable to induce chromosome aberrations in cultured human peripheral blood lymphocytes when tested to its limit of solubility in both the absence and presence of S-9.

 

Micronucleus test in vivo (CAS# 151661-88-0):

The substance was tested in vivo for its DNA damaging potential in a micronucleus test. The study was performed in albino mice of the strain CFW1 following the draft OECD 474 (initial assessment). Six male and six female mice were used per experimental group. The test item was administered by oral gavage, using arachis oil as vehicle. Based on the results of the dose range finding study a dosage of 5000 mg/kg bw was applied in the main trial. The test item was administered once, and six male and six female animals per group were sacrificed at time intervals of 24, 48 and 72 hours after the administration. Bone marrow smears from both femurs of each animal were prepared and the bone marrow preparations from the first five animals of each group were examined for micronuclei in 1000 polychromatic erythrocytes of each animal. A vehicle control and MMS as positive control were included in this study. No statistically significant enhanced mean values of micro-nucleated cells in polychromatic erythrocytes were observed at all examined time intervals compared to negative control values. Toxic effects as indicated by an enhanced mortality rate or by a reduction in the ratio of polychromatic to normochromatic erythrocytes (PCE/ NCE) were not noticed. Under the experimental conditions selected the test item did not induce chromosomal mutations in the bone marrow of mice. The test item is considered to have no DNA damaging potential in mice in vivo (Henkel 1990).

 

Micronucleus test in vivo (CAS# 135800-37-2):

The potential to induce micronuclei in vivo was examined in an OECD 474 Guideline study. Single doses of 0, 1075, 2150 and 4300 µL/kg bw (calculated based on a density of 860 mg/mL) were applied intraperitoneally to each male and female CD-1 mice. After post exposure periods of 24, 48 or 72h, at least 1000 polychromatic erythrocytes were counted for each category. The test item did not induce additional micronuclei compared to the vehicle control. In the concurrent positive control (cyclophosphamide), the mean number of micronucleated cells was significantly elevated. No toxic effects were reported. Under the conditions of the study, the test item exhibited no mutagenic potential in mice (Toxicon 1991).

Justification for classification or non-classification

The substance was not mutagenic in the HPRT assay in V79 cells, and it was, based on information on other subgroup members, not mutagenic in the Ames test nor clastogenic in vivo in the mouse micronucleus test. Therefore, based on the available data, classification for genetic toxicity is not warranted in accordance with EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.