Fatty acids, C16-18, stearyl esters

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:

08 Mar - 29 Jun 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted April 1984

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

2016

Deviations:

yes

Remarks:

limited documentation, less then 2 million cells cultured during expression period

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Energie und Bundesangelegenheiten, Wiesbaden, Germany

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Test material information

Constituent 1

Reference substance name:

3687-45-4 (purity: 55-70%)

IUPAC Name:

3687-45-4 (purity: 55-70%)

Method

Target gene:

HPRT locus

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: minimal essential medium (MEM) supplemented with 10 % fetal calf serum (FCS)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes, by treatment with HAT-medium
- Doubling time 12 - 16 h in stock cultures

Metabolic activation:

with and without

Metabolic activation system:

Co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with 500 mg/kg bw Aroclor 1254 i.p. 5 days prior to sacrifice.

Test concentrations with justification for top dose:

10, 30, 60 and 100 µg/mL, with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol. The final concentration of ethanol in the culture medium did not exceed 1 % v/v.
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its non-toxicity to the cells.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ethylmethanesulphonate (600 µg/mL in MEM without FCS, -S9) and 7,12-dimethylbenz(a)anthracene (3.85 µg/mL in DMSO, +S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent):
Cloning efficiency: 7 days
Mutant selection: 9 and 12 days (experiments 2 and 1, respectively)
- Fixation time (start of exposure up to fixation or harvest of cells):
Cloning efficiency: 7 and 14 days
Mutant selection: 16 and 19 days (experiments 2 and 1, respectively)

Counting of colonies: the colonies were stained with 10 % methylene blue in 0.01 % KOH solution. Stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

SELECTION AGENT (mutation assays): thioguanine, 11 µg/mL medium (Sigma, Deisenhofen, Germany)

NUMBER OF REPLICATIONS: 1 replication in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: determination of concentration-related cloning efficiency on day 7 after start of exposure; determination of cell survival on day 14 after start of exposure (end of selection time)

Evaluation criteria:

The test substance is considered to be positive if it induces either a concentration-related increase of the mutant frequency or a reproducible positive response for one of the test points.
A test material producing neither a concentration-related increase in the mutant frequency nor a reproducible positive response in any of the test points is considered non-mutagenic in this system.
A significant response is:
1) test substance induces reproducibly with one of the concentrations a mutation frequency that is 3 times higher than the spontaneous mutation frequency in the experiment
2) test substance induces reproducible concentration-related increase of the mutation frequency. May also be considered in case a 3-fold increase of the mutant frequency is not observed.

In a case-by-case evaluation the decision depends on the level of the corresponding negative control data.

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:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

precipitation at limit of water solubility at 100 µg/mL observed.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Due to limited solubility of the test substance higher concentrations than 100 µg/mL for the cytogenetic evaluation were not feasible.
- Precipitation: observed at concentrations higher than 100 µg/mL

RANGE-FINDING/SCREENING STUDIES
Data on the pre-test were obtained from the chromosomal aberration study (IUCLID section 7.6.1: key, Emery, 1994, ChrAb, RL1)
A pre-test was performed to determine the toxicity of the test article. The test substance was dissolved in ethanol due to its solubility properties. The highest attainable concentration was 100 µg/mL, at which a slight precipitation was observed. No toxic effects were observed up to and including the highest dose level.

COMPARISON WITH HISTORICAL CONTROL DATA: yes (data not presented)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In parallel to the mutagenicity testing the cloning efficiency was tested. Without metabolic activation the relative cloning efficiencies were between 64.2 - 91.8 % in treated cells compared to control cells. With metabolic activation the relative cloning efficiencies were between 92.4 - 109.7 % in treated cells compared to control cells.

No statistically significant increase in gene mutations was observed (see Table 1 and 2). The positive controls were shown to be valid.

Any other information on results incl. tables

Table 1: mutagenicity data, experiment 1

	Concentration (µg/mL)	Metabolic activation	No. of mutant colonies after plating in TG medium* (mean of 5 flasks)	% cell survival	No. of mutant colonies per 10^6surviving cells	% cloning efficiency
Negative control	-	-	2.2	54	9.4	100.0
Vehicle control	-	-	1.0	66	3.9	100.0
EMS	600	-	89.6	50	416.4	73.8
Test substance	10	-	1.0	72	3.3	91.8
Test substance	30	-	2.0	72	6.8	72.0
Test substance	60	-	0.2	81	0.6	68.1
Test substance	100	-	2.8	65	10.2	64.2
Negative control	-	+	2.0	69	7.3	100.0
Vehicle control	-	+	3.2	68	11.4	100.0
DMBA	3.85	+	112.0	57	496.2	53.1
Test substance	10	+	5.4	78	16.1	92.4
Test substance	30	+	0.2	65	0.8	95.6
Test substance	60	+	1.4	67	5.4	97.3
Test substance	100	+	0.2	74	0.7	109.7

*only colonies with more than 50 cells 8 days after seeding in TG selection medium were scored

** ration of mean number of cells found after 7 days in normal growth medium / cell number seeded per flask

TG = Thioguanine

EMS = Ethylmethanesulphonate

DMBA = 7,12-dimethylbenz(a)anthracene

 

Table 2: mutagenicity data, experiment 2

	Concentration (µg/mL)	Metabolic activation	No. of mutant colonies after plating in TG medium* (mean of 5 flasks)	% cell survival	No. of mutant colonies per 10^6surviving cells	% cloning efficiency
Negative control	-	-	2.0	69	6.6	100.0
Vehicle control	-	-	1.8	64	6.4	100.0
EMS	600	-	145.8	60	496.9	61.5
Test substance	10	-	4.2	62	18.4	93.6
Test substance	30	-	1.0	63	4.1	107.8
Test substance	60	-	3.2	60	13.5	56.4
Test substance	100	-	0.8	59	3.5	61.4
Negative control	-	+	2.4	60	10.3	100.0
Vehicle control	-	+	2.2	61	9.0	100.0
DMBA	3.85	+	141.6	64	550.4	50.3
Test substance	10	+	1.4	61	5.7	104.8
Test substance	30	+	2.2	72	7.5	106.1
Test substance	60	+	0.6	69	2.1	102.8
Test substance	100	+	1.4	79	4.4	107.7

*only colonies with more than 50 cells 8 days after seeding in TG selection medium were scored

** ratio of mean number of cells found after 7 days in normal growth medium / cell number seeded per flask

TG = Thioguanine

EMS = Ethylmethanesulphonate

DMBA = 7,12-dimethylbenz(a)anthracene

Applicant's summary and conclusion

Conclusions:

CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.