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

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:

experimental study

Adequacy of study:

key study

Study period:

From 2000-03-23 To 2000-10-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed according to OECD guideline 473 and GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

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:

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

EC Number:

267-015-4

EC Name:

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

Cas Number:

67762-38-3

Molecular formula:

UVCB substance, not univocal molecular formula available

IUPAC Name:

UVCB substance, no IUPAC name avalilable Chemical name: C16-C18 and C18 unsaturated alkyl carboxylic acids methyl esters

Details on test material:

- Name of test material (as cited in study report): Esterol C
- Physical state: light yellow liquid
- Analytical purity: 75.75 %
- Composition of test material, percentage of components: Mixture of methylesters of saturated and unsatured C16 to C20 fatty acids, no data.
- Lot/batch No.: 0006503
- Expiration date of the lot/batch: February 2001
- Storage condition of test material: at room temperature and protected from light

Method

Species / strain

Species / strain / cell type:

lymphocytes: primary cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 containing 20% fetal calf serum, L-glutamine (2mM), penicillin (100 U/ml), streptomycin (100µg/ml) and phytohaemagglutinin.

Metabolic activation:

with and without

Metabolic activation system:

S9 from induced Aroclor 1254 rat liver

Test concentrations with justification for top dose:

- 18.96, 37.93, 75.85, 151.70, 303.41, 606.82, 1213.64 and 2427.27 µg/ml (in the first experiment)
- 75.85, 151.70, 303.41, 606.82, 1213.64 and 2427.27 µg/ml (in the second experiment)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: no justification in the study report

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: mitomycin C without S9 mix (3µg/ml for 3 hours of treatment, 0.2 µg/ml for continuous treatment), cyclophosphamide with S9 mix (50 µg/ml)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION (first experiment)
- Exposure duration: 3h
- Expression time (cells in growth medium): 17h
- Fixation time (start of exposure up to fixation or harvest of cells): 20h

DURATION (second experiment)
- Exposure duration: 3h (without S9); 20h and 44h (with S9)
- Expression time (cells in growth medium): 17h and 41h (without S9); 20h and 44h (with S9)
- Fixation time (start of exposure up to fixation or harvest of cells): 20h and 44h (with and without S9)

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: approximately 1.5 normal cell cycles or approximately 1.5 normal cell cycles and 24 hours later.

NUMBER OF CELLS EVALUATED: 100 metaphase/culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Other: gaps, chromatid and chromosome breaks and exchanges, multipleaberrations and pulverization

OTHER: blind scoring

Evaluation criteria:

A reproducible and statistically significant increase in the frequency of cells with structural aberrations for at least one of the dose-levels and one of
the two harvest times was considered as a positive result. Reference to historical data or other considerations of biological relevance, was also taken into account in the evaluation of the findings.

Statistics:

The comparison was performed using the Chi-square test (p = 0.05)

Results and discussion

Test results

Species / strain:

lymphocytes: primary culture

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Cytotoxicity:
Without S9:
After 3-hour treatment, a slight decrease in the mitotic index was noted at dose-levels of 1213.64 and 2427.27 µg/ml (up to 34%).
After 20- hour treatment, a 35% decrease in the mitotic index was induced at 2427.27 µg/ml.
After 44-hour treatment, a 33-69% decrease in the mitotic index was noted at dose-levels of 1213.64 and 2427.27 µg/ml.

With S9 mix:
No noteworthy decrease in the mitotic index was noted at the 20 hour harvest time

Chromosal aberration analyses:
The test substance did not induce any significant increase in the frequency of cells with chromosome aberrations in both experiments and at both harvest times, with and without S9 mix.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Under the experimental conditions, the test substance Esterol C does not induce chromosome aberrations in cultured human lymphocytes.

Executive summary:

In a GLP mammalian cell cytogenetic assay (chromosome aberration) (Haddouk, 2000), primary lymphocyte cultures were exposed to Esterol C (batch no. 0006503) at concentration of 18.96, 37.93, 75.85, 151.70, 303.41, 606.82, 1213.64 and 2427.27 µg/ml with and without metabolic activation. Positive controls induced the appropriate response. There was no evidence of chromosome aberration induced over background.