Soybean oil, epoxidized

Genetic toxicity: in vitro

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EEC Annex V Test B14

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: UKEMS Guidelines

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Epoxidised Soybean Oil

IUPAC Name:

Epoxidised Soybean Oil

Constituent 2

Reference substance name:

ESBO

IUPAC Name:

ESBO

Constituent 3

Reference substance name:

Soybean oil, epoxidized

EC Number:

232-391-0

EC Name:

Soybean oil, epoxidized

Cas Number:

8013-07-8

Molecular formula:

Not necessary, substance is a UVCB.

IUPAC Name:

Soybean oil, epoxidized

Details on test material:

- Name of test material (as cited in study report): Epoxidised Soybean Oil
- Abbreviation: ESBO
- Alias name: Reoplast 39
- Physical state: Clear yellow Liquid
- Lot/batch No.: 08380306
- Expiration date of the lot/batch: July 1994
- Stability under test conditions: Stable
- Storage condition of test material: Dark at room temperature
- Other:
- Colour value (Gardner): 1 - 2
- Refractive Index at 20 °C: 1.4731
- Iodine Value: 4.3 g J2/100g
- Acid Value: 0.27 mg KOH/g
- Oxirane Value: 6.4 %

Method

Target gene:

histidine gene

Species / strain

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.

Controls

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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'.

Any other information on results incl. tables

No further information

Applicant's summary and conclusion

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.