Fatty acids, C14-22, 2-ethylhexyl esters, epoxidized

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 15, 1992 - June 25, 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP compliant. According to guidelines. Read across to a study result from an investigation using a similar material is justified for members of the Epoxidised Oils and Derivatives group. Four epoxidised oils and esters (linseed, soybean,9-octadecanoate propylene glycol ester and 2-ethylhexyl tallate ester ETP). The C14-C22, 2-ethylhexylesters are listed as similar products on the market to ETP based on fatty acids from other naturally occurring fatty acids This group of epoxies are identified as sharing common structural and functional similarities, recognised in an OECD SIDS review as a single category, and therefore justifying read-across between data for different members of the group. Consequently data sharing between ESBO epoxidised soybean oil, ELO epoxidised Linseed oil and ETP epoxidised 2ethylhexyl tallate and fatty acids, C14-C22, 2-ethylhexylesters, epoxidised is commonly utilised in the preparation of this dossier. Read-across bridges are used for members of the EOD group where appropriate, is justified based on similar toxicity profiles and structural and functional similarities.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

Not relevant

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

ESBO

IUPAC Name:

ESBO

Constituent 2

Reference substance name:

Epoxidised Soybean Oil

IUPAC Name:

Epoxidised Soybean Oil

Constituent 3

Reference substance name:

Soybean oil, epoxidized

EC Number:

232-391-0

EC Name:

Soybean oil, epoxidized

IUPAC Name:

232-391-0

Constituent 4

Reference substance name:

8013-07-8

Cas Number:

8013-07-8

IUPAC Name:

8013-07-8

Details on test material:

- Name of test material (as cited in study report): Epoxidised Soybean Oil
- Physical state: Yellow liquid
- Lot/batch No.: 08380306
- Storage condition of test material: Room temperature in the dark

Method

Target gene:

tk gene locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y TK +/- mouse lymphoma cells were obtained from the American Type Culture Collection. They were stored as frozen stocks in liquid nitrogen. Each batch of cells was purged of TK mutants, checked for spontaneous mutant frequency and that they were mycoplasma free. For each experiment the vials were thawed rapidly, the cells were diluted in RPMI 10 and incubated in a humidified atmosphere of 5 % v/v CO2 in air. When the cells were growing well, subcultures were established in an appropriate number of flasks.

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:

The concentrations were selected using a cytotoxicity range-finder. See Table 1 below.

Vehicle / solvent:

Acetone was used as a solvent. The test item was added and the culture was then vortexed for approx. 10 seconds to obtain a good emulsion. A top dose of 5000 ug/ml was achievable using an emulsion.

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

Comprised of solvent, acetone, diluted 100-fold in the treatment medium.

Controls 2

Positive controls:

yes

Remarks:

Please see Below

Positive control substance:

benzo(a)pyrene

Remarks:

The positive controls were also made to certain concentrations please see table 2 below.

Controls 3

Positive controls:

yes

Remarks:

Please see Below

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

The positive controls were also made to certain concentrations please see table 2 below.

Details on test system and experimental conditions:

METHOD OF APPLICATION:

Preparations of cofactor solutions with and without S-9

Quantity (ml)
With S-9 Without S-9
Glucose-6-phosphate (180 mg/ml) 1.0 -
NADP (25 mg/ml) 1.0 -
150 mM KCL 1.0 5.0
Rat Liver S-9 2.0 -

The above were used at the rate of 1.0 ml per 19 ml of cell culture containing the test chemical (to achieve the required final concentration in a total of 20 ml).


Three types of RPMI 1640 medium were prepared as follows:
Final Concentration in:
RPMI A RPMI 10 RPMI 20
Horse Serum (heat inactivated) 0 % v/v 10 % v/v 20 % v/v
Gentamycin 100 ug/ml 100 ug/ml 100 ug/ml
Fungizone 2.5 ug/ml 2.5 ug/ml 2.5 ug/ml
Pluronic 0.5 ug/ml 0.5 ug/ml -


DURATION
- Exposure duration: 9- 15 days
- Expression time (cells in growth medium): 2 days

NUMBER OF CELLS EVALUATED: 1 x E7 cells per flask

DETERMINATION OF CYTOTOXICITY
Following adjustment of the cultures to 2 x E5 cells/ml after treatment, samples from these were diluted to 8 cells/ml as seen in table 3. using a 8-channel pipette, 0.2 ml of concentration C of each culture was placed into each well of two 96-well microtitre plates (192 wells, at an average of 1.6 cells per wall). The plates were incubated at 37 C in a humidified incubator gassed with 5 % v/v CO2 in air until scorable (9-15 days). Wells containing viable clones were identified by eye using background illumination and counted.

Evaluation criteria:

At the end of the expression period the cell densities in the selected cultures were adjusted to 1 x E4/ml. TFT (300 ug/ml) was diluted 100-fold into these suspensions to give a final concentration of 3 ug/ml. Using a 8-channel pipette, 0.2 ml of each suspension was placed into each well of four 96-well microtitre plates (384 wells at 2 x E3 cells per well). Plates were incubated until scorable (13 days) and wells containing clones were identified as above and counted. In addition, the number of wells containing large colonies and the number containing small colonies were scored for the negative and positive controls and for doses of test chemical showing a significant increase in mutant frequency over the negative control.

Statistics:

All calculations were performed on a microcomputer.

Determination of survival or viability: Poison distribution, plating efficiency, % relative survival
Determination of mutant frequency: Mutant frequency, plating efficiency,
Statistical significance of mutant frequencies.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Please see Tables 5 & 6 below

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

No marked toxicity was seen after the application of the highest dose. Please see Table 4

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: Please see table 4 below

Any other information on results incl. tables

Table 4: raw plate counts and % relative survival for Epoxidised Soybean Oil in the cytotoxicity range-finder.

Treatment ug/mL)	In the absence of S-9		In the presence of S-9
	Survival (1) at Day 0*	% Relative survival	Survival (1) at day 0*	% relative survival
0	18	100.0	23	100.0
78.125	21	129.2	21	84.2
156.25	16	83.8	17	59.7
312.5	18	100.0	19	71.0
625	15	76.5	15	49.9
1250	15	76.5	15	49.9
2500	16	83.8	18	65.2
5000	19	109.0	23	100.0

(1) 1.6 cells/well plated

* 32 wells scored

Table 5: Summary of Results Experiment 1

Treatment (ug/ml)	Absence of S-9		Treatment (ug/mL)	Presence of S-9
	% RS	Mutant Frequency		% RS	Mutant Frequency
0	100.0	312.57	0	100.0	401.83
312.5	95.8	389.82 NS	312.5	97.9	484.86 NS
625	86.6	326.20 NS	625	87.9	563.19 NS
1250	79.9	439.82 NS	1250	86.0	518.57 NS
2500	131.9	520.41 *	2500	152.9	426.40 NS
5000	143.9	467.96 *	5000	178.7	433.74 NS
Linear Trend		**	Linear Trend		NS
NQO			BP
0.05	83.5	1676.58	2	55.2	1794.55
0.1	45.3	2078.73	3	39.4	2122.84

 

Table 6: Summary of Results Experiment 2

Treatment (ug/ml)	Absence of S-9		Treatment (ug/mL)	Presence of S-9
	% RS	Mutant Frequency		% RS	Mutant Frequency
0	100.0	410.98	0	100.0	304.98
312.5	112.9	334.82 NS	312.5	91.7	331.46 NS
625	87.7	466.23 NS	625	82.6	331.03 NS
1250	71.4	404.74 NS	1250	87.7	313.73 NS
2500	84.5	461.15 NS	2500	84.5	283.11 NS
5000 $$	100.7		5000	83.2	311.46 NS
Linear Trend		NS	Linear Trend		NS
NQO			BP
0.05	67.5	1136.27	2	59.5	883.78
0.1	37.7	1143.11	3	32.1	1443.04

 

NS Not significant

$$ Treatment excluded due to excessive heterogeneity

*, **, *** Significant at 5 %, 1% and 0.1 % level respectively

Applicant's summary and conclusion

Conclusions:

Negative ESBO failed to demonstrate an ability to induce mutation at the tk locus of mouse lymphoma L5178Y cells.

It is concluded that, under the conditions employed in this study, ESBO failed to demonstrate the ability to induce mutation at the tk locus of L5178Y mouse lymphoma cells in the absence and presence of S-9. Therefore, ESBO is not considered to be mutagenic.
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 (ESBO) was assayed for its ability to induce mutation at the tk locus (5-trifluorothymidine resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity range-finder followed by 2 independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254 induced rat liver post-mitochondrial fraction (S-9).

Following a wide range of treatments in the range-finder experiment, separated by 2-fold intervals and ranging from 78.125 to 5000µg/ml, cells survived all doses of ESBO yielding 109.0 % relative survival in the absence and 100.0 % relative survival in the presence of S-9 at the top dose.

Accordingly, 5 doses were chosen for the first experiment, separated by 2-fold intervals and ranging from 312.5 to 5000µg/ml. All doses were plated for viability and 5-trifluorothymidine resistance 2 days after treatment. The top doses plated yielded 143.9 % and 178.7 % relative survival in the absence and presence of S-9. In the second experiment the same dose range was selected. The top dose plated in this experiment was again 5000µg/ml in the absence and presence of S-9, which yielded 100.7 % and 83.2 % relative survival respectively.

Negative (solvent) and positive control treatments were included in each experiment in the absence and presence of S-9. Mutant frequencies in negative control cultures fell within normal ranges, and statistically significant increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (without S-9) and benzo(a)pyrene (with S-9). Therefore the study was accepted as valid.

In the absence of S-9, reproducible statistically significant and dose-related increases in mutant frequency were not observed in the 2 experiments over the dose range 312.5 to 2500µg/ml. At 5000µg/ml, a positive point was obtained in Experiment 1 and due to heterogeneity in the data this dose was excluded from analysis in Experiment 2. However, if each of the replicate cultures at 5000µg/ml in Experiment 2 are considered in turn, neither yields a statistically significant increase in mutant frequency. This, combined with the fact that there were no absolute increases in mutant numbers in Experiment 1 at 5000µg/ml and that carry over of the test compound was a problem at this dose, suggests that the increased mutant frequency seen in experiment 1 was not the result of chemically induced mutation.

In the presence of S-9, no statistically significant increases in mutant frequency were observed at any dose level tested in Experiment 1 or 2.

It is concluded that, under the conditions employed in this study, ESBO failed to demonstrate the ability to induce mutation at the tk locus of L5178Y mouse lymphoma cells in the absence and presence of S-9. Therefore, ESBO is not considered to be mutagenic.

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

According to Regulation (EC) No. 1272/2008, no classiification is warranted.