Branched and linear octadecanoic acid, esters with glycerol oligomers

Genetic toxicity: in vitro

Some information in this page has been claimed confidential.

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from similar mixture/product

Adequacy of study:

key study

Study period:

09 May 2012 - 12 Mar 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

29 July 2016

Deviations:

yes

Remarks:

200 metaphases scored; mitotic index used as parameter for cytotoxicity (no RPD or RICC values given), no C-charts or X-bar charts provided

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayrisches Landesamt für Gesundheit und Lebensmittelsicherheit, Erlangen, Germany

Type of assay:

other: in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: V79 cells (ATCC, CCL-93)
- Suitability of cells: yes
- Proliferation rate: 12 - 14 h:
- Modal number of chromosomes: diploid number, 2n = 22

MEDIA USED
- Type and identity of media: MEM medium supplemented with 10% fetale bovine serum, 100 U/100 µg/mL penicilline/streptomycin solution, 2 mM L-glutamine, 2.5 µg/mL amphotericin, 25 µM HEPES.

Metabolic activation:

with and without

Metabolic activation system:

Co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone

Test concentrations with justification for top dose:

Pre-experiment for Toxicity:
with and without metabolic activation: 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000, 2500 and 5000 µg/mL

Experiment I:
4 h treatment with and without metabolic activation: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/mL

Experiment II:
20 h treatment without metabolic activation: 10.0, 30.0, 100, 250, 500, 1000, 2500 and 5000 µg/mL
4 h treatment with metabolic activation: 100, 316, 1000, 3000, 4000 and 5000 µg/mL

The following concentrations were selected in the main experiments for the microscopic analyses:
Experiment I:
with and without metabolic activation: 1000, 2500 and 5000 µg/mL
Experiment II:
without metabolic activation: 500, 1000 and 5000 µg/mL
with metabolic activation: 1000, 3000 and 5000 µg/mL

Vehicle / solvent:

No vehicle was used.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

Remarks:

not applicable

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: 1E+04 - 5E+04 cells depending on preparation time

DURATION
- Exposure duration: 4 and 20 h
- Fixation time (start of exposure up to fixation or harvest of cells): 4 h treatment: 20 h; 20 h treatment: 20 h

SPINDLE INHIBITOR: 0.2 µg/mL Colcemid in medium

STAIN: Giemsa

NUMBER OF REPLICATIONS: duplicates each in 2 independent experiments

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Colcemid was added to the cultures 17.5 h after the start of each treatment. 2.5 h later, the cells were treated on the slides in the chambers with hypotonic solution (0.4% KCl) for 20 min at 37 °C. After incubation in the hypotonic solution the cells were fixed with methanol/glacial acetic acid (3:1 v/v). All the steps were carried out on precision hot plates at 37 °C. After fixation the slides were air dried and stained.
All slides were independently coded before microscopic analysis.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 200. Parallel cultures were treated at each concentration. 100 metaphases per culture were scored

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; relative cell density

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
If observed, structural chromosomal aberrations, including breaks, fragments, deletions, exchanges and chromosomal disintegration were recorded. Gaps were recorded but not included in the calculation of the aberration rates.

Evaluation criteria:

A test substance was considered positive (clastogenic) in the chromosome aberration test if:
- a clear and dose-related increase in the number of cells with aberrations,
- a biologically relevant response for at least one of the dose groups, which is higher than the laboratory negative control range (0.0% - 4.0% aberrant cells (with and without metabolic activation)) was observed.

A test item is considered to be negative if there is no biologically relevant increase in the percentage of aberrant cells above concurrent control levels, at any dose group. Although most experiments will give clearly positive or negative results, in some cases the data set will preclude making a definitive judgement about the activity of the test substance.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In experiment II without metabolic activation, a biologically relevant decrease of the relative mitotic index and the relative cell density was noted at 5000 µg/mL (54% relative mitotic index and 56% relative cell density)

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test item could be suspended in cell culture medium at a concentration of 5000 µg/mL.
- Precipitation: Precipitation of the test substance was observed in following experiments:
Experiment I without metabolic activation: starting at 1000 µg/mL
Experiment II without metabolic activation: starting at 500 µg/mL
Experiment II with metabolix activation: starting at 1000 µg/mL

RANGE-FINDING/SCREENING STUDIES: The highest concentration analysed in the pre-experiment for toxicity was 5000 µg/mL. The relative mitotic index was used as the parameter for evaluating toxicity. The concentrations evaluated in the main experiments were based on the results of the pre-experiment.

Any other information on results incl. tables

Table 1: Results of the pre-experiment

Concentration in µg/mL	Mitotic Index in %		Cell Density in %
		relative		relative
without S9 mix
Control	72	100	67	100
7.8	72	100	90	135
15.6	68	94	73	109
31.3	68	92	62	93
62.5	67	93	39	59
125	76	106	77	115
250	55	76	70	105
500	53	74	69	100
1000	52	72	49	73
2500	64	89	72	108
5000	52	72	59	88
with S9 mix
Control	94	100	68	100
7.8	74	79	65	95
15.6	75	80	76	112
31.3	96	102	87	129
62.5	84	89	76	113
125	99	105	73	109
250	82	87	66	96
500	83	88	77	114
1000	80	85	59	87
2500	84	89	69	102
5000	88	94	63	93

The mitotic index was determined in 1000 cells per culture of each test group.

The relative values of the mitotic index are related to the negative control.

The cell density was determined in 20 cell counts for each test group.

Table 2. Test results of experiment I and II

Test item	Concentration	Mitotic Index		Aberrant cells in %
	in µg/mL	in %		with gaps	without gaps
Exp. I, Exposure period 4 h, fixation time 20 h, without S9 mix
Control	medium	100		3.5	2.0
EMS	900	92		12.5	8.5
Test substance	1000 P	101		4.0	2.0
	2500 P	81		3.5	2.0
	5000 P	77		1.0	0
Exp. I, Exposure period 4 h, fixation time 20 h, with S9 mix
Control	medium	100		2.5	2.0
CPA	0.83	115		11.5	9.5
Test substance	1000	107		2.0	0.5
	2500	115		5.0	2.5
	5000	94		4.0	2.0
Exp. II, Exposure period 20 h, fixation time 20 h, without S9 mix
Control	medium	100		3.0	1.5
EMS	400	96		11.0	9.5
Test substance	500 P	70		2.5	1.0
	1000 P	54		4.0	2.0
	5000 P	64		1.5	0.5
Exp. II, Exposure period 20 h, fixation time 20 h, withS9 mix
Control	medium	100		6.0	3.5
CPA	0.83	112		15.0	14.0
Test substance	1000 P	94		5.0	4.0
	3000 P*	104		3.5	2.5
	5000 P	65		4.5	2.5

EMS: Ethylmethanesulfonate; CP: Cyclophosphamide (positive controls)

The mitotic index was determined in 1000 cells per culture of each test group.

For evaluation of aberrant cells, 100 cells per culture of each test group were scored (in total 200 cells)

* For evaluation of aberrant cells, 400 cells were scored.

Table 3: Historical Laboratory Control Data

	Number of aberrant cells
	with S9 mix		without S9 mix
Negative control	+ gaps	- gaps	+ gaps	- gaps
mean in %	4.3	2.0	3.6	1.5
SD	1.43	0.81	1.55	0.82
RSD in %	33.5	41.4	42.4	56.6
min in %	1.0	0.0	0.5	0.0
max in %	8.5	4.0	9.0	4.0
n	244	244	244	244
Positive control
mean in %	13.1	9.7	13.1	9.7
SD	2.29	1.66	2.70	1.96
RSD in %	17.4	17.1	20.7	20.3
min in %	8.5	8.0	8.5	8.0
max in %	23.0	16.0	26.5	20.5
n	235	235	235	235

mean = mean number of aberrant cells

SD = Standard Deviation

RSD = relative Standard Deviation

min = minimum number of aberrant cells

max = maximum number of aberrant cells

n = Number of assays

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