Fatty acids, C16-18, iso-Bu 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:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

04 Nov - 02 Dec 2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP guideline study, tested with the source substance CAS 163961-32-8. 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:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the united Kingdom

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

163961-32-8

Cas Number:

163961-32-8

IUPAC Name:

163961-32-8

Details on test material:

- Physical state: amber colored liquid
- Analytical purity: 82%
- Storage condition of test material: RT in the dark
- Batch number: A0-282-26

Method

Target gene:

Duplicate cultures of human lymphocytes were evaluated for chromosome aberrations at up to three dose levels.

Species / strain

Species / strain / cell type:

primary culture, other: human lymphocytes

Details on mammalian cell type (if applicable):

For each experiment, whole blood was drawn from the peripheral circulation of a volunteer who had previously been screened for suitability. The cell cycle length of 17 h was determined by BrdU incorporation.

Metabolic activation:

with and without

Metabolic activation system:

PB/βNF induced S9-Mix from male Spraque-Dawley rats

Test concentrations with justification for top dose:

1. Experiment:
24 h continous exposure without S9: 312.5; 468.75; 625 µg/mL
4 h exposure to test material with S9 mix followe by 20 h culture in treatment free media: 625; 1250 and 2500 µg/mL

2. Experiment
4 h exposure to test material without S9 mix followe by 20 h culture in treatment free media: 625; 1250 and 2500 µg/mL
4 h exposure to test material with S9 mix followe by 20 h culture in treatment free media: 625; 1250 and 2500 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: arachis oil

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Mitomycin C (MMC, 0.2 + 0.4µg/mL; -S9); Cyclophosphamide (CP, 7,5µg/mL; +S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium (Eagle´s minimal essential medium with HEPES buffer supplemented with L-glutamine, Pen/Strep, amphotericin B and 15% FCS

DURATION
- Exposure duration: Experiment I: 4 hours with S9 mix, or 24 hours without S9 mix; Experiment II: 4 hours with and without S9 mix
- Expression time (cells in growth medium): 20 hours after 4 hour exposure in both experiments

NUMBER OF REPLICATIONS: 2

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (0.1 µg/mL)
STAIN (for cytogenetic assays): 5% Giemsa for 5 min

NUMBER OF CELLS EVALUATED: Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there were approximately 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or reÍurangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicrty testing.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index: A total of 2000 or 1000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

Evaluation criteria:

A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.

Results and discussion

Test results

Test results 1

Species / strain:

primary culture, other: human

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

max. 38% inhibition at 468,75 mg/mL and 24 h incubation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

primary culture, other: human

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

max. 11% (1st experiment) and 22% (2nd experiment) inhibition at 1250 mg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: Dosing was based on that utilised in a previous study on a different batch of the test material (produced by a different manufacturing method) and included a dose level that induced some mitotic inhibition.
2500 µg/mL was chosen as maximum dose due to limited solubility.

Remarks on result:

other: strain/cell type: lymphocytes

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Mean values of chromosome aberrations and polyploid cells

	Experiment 1				Experiment 2
	24h without S9		4h with S9		4h without S9		4h with S9
Dose level (µg/mL)	% cells with aberrations (-gaps)	% cells with polyploids	% cells with aberrations (-gaps)	% cells with polyploids	% cells with aberrations (-gaps)	% cells with polyploids	% cells with aberrations (-gaps)	% cells with polyploids
0	0	0	0	0	0	0	0.5	0.5
312.5	0.5	0	-	-	-	-	-	-
468.75	0.5	0	-	-	-	-	-	-
625	1.5	0	1.0	0	0	0	0.5	0
1250	-	-	1.0	0.5	0.5	0	0	0
2500	-	-	0	0	0	0	0	0
positive control	54.0	0	39.3	0	42.0	0	28.7	0

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative non-clastogenic to human lymphocytes in vitro

The numbers of chromosome aberrations and polyploid cells were not increased at any dose level in comparison to the control. The test substance is non-clastogenic in mammalian cells in vitro.