Fatty acids, C12-14 (even numbered), methyl ester

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

17 Jun - 01 August 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP guideline study, tested with the source substance CAS 544-35-4. According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl linoleate

EC Number:

208-868-4

EC Name:

Ethyl linoleate

Cas Number:

544-35-4

Molecular formula:

C20H36O2

IUPAC Name:

ethyl octadeca-9,12-dienoate

Details on test material:

- Name of test material (as cited in study report): ethyl linoleate
- Analytical purity: ca. 100%
- Composition of test material, percentage of components: 75% active substance, 11% octadecenoic acid ethylester (ethyl oleate), 6% hexadecanoic acid ethylester, 3% octadecanoic acid ethylester, 3% octadecadienoic acid glycerol monoester
- Physical state: hazy yellow liquid
- Expiry date: 31 March 2011

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

primary culture, other: cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium, supplemented with FCS, L-glutamine, pen/strep, heparin
- Properly maintained: yes
- Average Generation time of the cells: 14.3 - 14.5 h

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and ß-naphthoflavone.

Test concentrations with justification for top dose:

1st exp: up to 333 µg/mL (precipitation at 333 µg/mL)
2nd exp: up to 800 µg/mL (precipitation at 333 µg/mL)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: mitomycin C (0.5µg/mL) and cyclophosphamide (10 µg/mL)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
In the first cytogenetic assay, the test substance was tested up to 333 µg/mL for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction. Precipitation occurred in the culture medium at this dose level.

In the second cytogenetic assay, the test substance was tested up to 800 µg/mL for a 24 h continuous exposure time with a 24 h fixation time and up to 600 µg/mL for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at these dose levels. In the presence of S9-mix the test substance was tested up to 333 µg/mL for a 3 h exposure time with a 48 h fixation time. Precipitation occurred in the culture medium at this dose level.

SPINDLE INHIBITOR: colchicine (0.5 μg/mL medium)
STAIN: 5% (v/v) Giemsa after fixation

NUMBER OF REPLICATIONS: duplicates each in two independent experiments

NUMBER OF CELLS EVALUATED: 200

DETERMINATION OF CYTOTOXICITY
- Method: The mitotic index of each culture was determined by counting the number of metaphases per 1000 cells.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

At least three analysable concentrations were used for scoring of the cytogenetic assay. Chromosomes of metaphase spreads were analysed from those cultures with an inhibition of the mitotic index of about 50% or above whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control. Also cultures treated with an intermediate dose were examined for chromosome aberrations. The test substance was difficult to dissolve in aqueous solutions, the highest concentration analysed at the 3 h exposure time was determined by the solubility in the culture medium. If dose related cytotoxicity was observed, the highest concentration analysed at the 24 and 48 h continuous exposure times was based on toxicity irrespective of the solubility of test substance in the culture medium. However, the extent of precipitation may not interfere with the scoring of chromosome aberrations.

Evaluation criteria:

A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.

Statistics:

The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics.

Results and discussion

Test results

Species / strain:

primary culture, other: cultured peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

at 333 µg/mL and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: bad water solubility, soluble in dimethyl sulfoxide at concentrations of 33 mg/mL and below but formed a suspension at concentrations of 50 mg/mL and above
- Precipitation: at concentrations of 333 µg/mL and above

RANGE-FINDING/SCREENING STUDIES:
With S9 mix: up to 333 µg/mL for 3 hours. Without S9 mix: up to 1000 µg/mL for 24 and 48 hours (beyond limit of solubility).
No cytotoxicity was observed in the duplicate cultures of the 3 h exposure time and the test substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations

COMPARISON WITH HISTORICAL CONTROL DATA:
The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range.
Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Remarks on result:

other: strain/cell type: lymphocytes

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table1: Chromosome aberrations in human lymphocyte cultures treated with test substance in the absence of S9-mix in the first cytogenetic assay (3 h exposure time, 24 h fixation time)

Conc	DMSO (1.0% v/v)				33 µg/mL				100 µg/mL				333 µg/mL				MMC-C 0.5µg/mL
Culture	A            B     A+B				A        B     A+B				A        B     A+B				A            B     A+B				A            B     A+B
Mitotic Index (%)	100				104				98				93				76
No. of Cells scored	100   100      200				100   100     200				100   100     200				100   100     200				50     100     150
No. of Cells with aberrations (+ gaps)	0	1		1	0	0		0	2	0		2	1	1		2	26	22		***^) 48
No. of Cells with aberrations (- gaps)	0	1		1	0	0		0	2	0		2	1	1		2	26	22		***^) 48
misc.											endo		poly		endo

The numerical variations endoreduplication (endo) and polyploidy (poly) were not counted as an aberration.

*)  Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.

 

Table2           : Chromosome aberrations in human lymphocyte cultures treated with test substance in the presence of S9-mix in the first cytogenetic assay (3 h exposure time, 24 h fixation time)

Conc                                            Conc	DMSO (1.0% v/v)			33 µg/mL			100 µg/mL			333 µg/mL			CP 10µg/mL
Culture	A           B A+B			A           B A+B			A           B A+B			A           B A+B			A           B    A+B
Mitotic Index (%)	100			106			101			103			42
No. of Cells scored	100  100 200			100  100 200			100  100 200			100  100 200			50    100    150
No. of Cells with aberrations (+ gaps)	1	0	1	0	1	1	0	1	1	1	1	2	27	16	***^) 43
No. of Cells with aberrations (- gaps)	1	0	1	0	1	1	0	1	1	1	1	2	27	16	***^) 43

 

Table3: Chromosome aberrations in human lymphocyte cultures treated with test substance in the absence of S9-mix in the second cytogenetic assay (24 h exposure time, 24 h fixation time)

Conc	DMSO (1.0% v/v)			100 µg/mL			600 µg/mL			800 µg/mL			MMC-C 0.2µg/mL
Culture	A           B    A+B			A           B    A+B			A           B    A+B			A           B    A+B			A           B    A+B
Mitotic Index (%)	100			102			85			35			53
No. of Cells scored	100  100     200			100   100     200			100   100     200			100   100     200			100   100     200
No. of Cells with aberrations (+ gaps)	1	1	2	0	1	1	0	2	2	1	0	1	29	30	***^) 59
No. of Cells with aberrations (- gaps)	1	1	2	0	0	0	0	1	1	1	0	1	29	29	***^) 58

 

Table4           : Chromosome aberrations in human lymphocyte cultures treated with test substance in the absence of S9-mix in the second cytogenetic assay (48 h exposure time, 48 h fixation time)

Conc	DMSO (1.0% v/v)			100 µg/mL			500 µg/mL			600 µg/mL			MMC-C 0.1µg/mL
Culture	A           B    A+B			A           B    A+B			A           B    A+B			A           B    A+B			A           B    A+B
Mitotic Index (%)	100			99			60			49			77
No. of Cells scored	100  100    200			100  100    200			100  100    200			100  100    200			50    100    150
No. of Cells with aberrations (+ gaps)	1	1	2	1	1	2	4	1	5	1	2	3	25	31	***^) 56
No. of Cells with aberrations (- gaps)	1	0	1	1	1	2	3	1	4	1	1	2	25	28	***^) 53

 

Table5: Chromosome aberrations in human lymphocyte cultures treated with test substance in the presence of S9-mix in the second cytogenetic assay (3 h exposure time, 48 h fixation time)

Conc	DMSO (1.0% v/v)			33 µg/mL			100 µg/mL			333 µg/mL			CP 10µg/mL
Culture	A           B    A+B			A           B    A+B			A           B    A+B			A           B    A+B			A           B    A+B
Mitotic Index (%)	100			101			107			103			-^a
No. of Cells scored	100  100    200			100  100    200			100  100    200			100  100    200			100  100    200
No. of Cells with aberrations (+ gaps)	0	0	0	0	0	0	0	1	1	1	0	1	33	29	***^) 62
No. of Cells with aberrations (- gaps)	0	0	0	0	0	0	0	0	0	1	0	1	33	29	***^) 62

^aCP was fixed after 24 hours. Therefore, the mitotic index could not be calculated as percentage of control.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative