Reaction mass of sodium hydrogen N,N-dibutyl-10-(sulphonatooxy)octadecanamidate and sodium sulphooctadecanoate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-10-11 to 2013-01-08

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 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

first experiment 4 hours treatment with and without metabolic activation second experiment 24 hours treatment without metabolic activation, 4 hours treatment with metabolic activation

GLP compliance:

yes (incl. QA statement)

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:

N,N-dibutyloleamide and oleic acid, sulphated, sodium salts

EC Number:

915-926-9

Molecular formula:

C18H34Na2O6S + C26H52NNaO5S

IUPAC Name:

N,N-dibutyloleamide and oleic acid, sulphated, sodium salts

Test material form:

liquid

Details on test material:

The water content of the actual test item was 57.6% (w/w); the water content of the REACH registration substance was analytically determined to be 3.8% (w/w).

Method

Target gene:

HPRT

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital / Beta-Naphtoflavone induced Rat liver S9

Test concentrations with justification for top dose:

Experiment I:
without metabolic activation: 0.7; 1.4; 2.8; 5.5; 11.0; 16.5; 22.0 µg/mL
with metabolic activation: 5.5; 11.0; 22.0; 44.0; 88.0; 132.0; 176.0(p) µg/mL
Experiment II:
without metabolic activation: 5.5; 11.0; 22.0; 44.0; 88.0; 132.0; 176.0(p) µg/mL
with metabolic activation. 5.5; 11.0; 22.0; 44.0; 88.0; 132.0; 176.0(p) µg/mL
p = precipitation
In experiment I the cultures at the two highest concentrations without metabolic activation and the highest concentration with metabolic activation were not continued due to exceedingly severe cytotoxicity. In experiment II the cultures at the highest concentration with and without metabolic activation were not continued for the same reason. The culture at the lowest concentration of experiment I with metabolic activation and of experiment II with and without metabolic activation were not continued as a minimum of only four analysable concentrations is required by the guidelines.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water (10 % v/v)
- Justification for choice of solvent/vehicle: suitability properties

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION:
- Exposure duration: Experiment I: 4 hours with and without metabolic activation, Experiment II: 24 hours without metabolic activation, 4 hours with metabolic activation
- Expression time (cells in growth medium): 72 hours
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: > 1.5 x 10 e6

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using. The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological relevance and statistical significance were considered together.

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not effected (measured in two pre-experiments: pH 7.38 / 7.31 in the solvent control versus pH 7.33 at 11800 µg test item/mL and pH 7.25 at 180 µg test item/mL)
- Effects of osmolality: No relevant increase (283 mOsm in the solvent control and 296 mOsm at 11800 µg test item/mL and 286 mOsm measured at 180 µg test item/mL)
- Evaporation from medium: Not examined
- Precipitation: Pre-experiment: The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal of the test item. Following 4 hours treatment precipitation occurred at 180 µg/mL and above without metabolic activation and at 184.4 µg/mL and above with metabolic activation. Following 24 hours treatment without metabolic activation precipitation was observed at the maximum concentration of 11800 µg/mL.
Main experiments: Precipitation at the end of treatment, visible to the unaided eye, was not observed at evaluated concentrations.
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES:
According to the current OECD Guideline for Cell Gene Mutation Tests at least four analysable concentrations should be used in two parallel cultures. For freely-soluble and non-cytotoxic test items the maximum concentration should be 5 mg/mL, 5 µL/mL or 10 mM, whichever is the lowest. For cytotoxic test items the maximum concentration should result in approximately 10 to 20 % relative survival or cell density at sub-cultivation and the analysed concentrations should cover a range from the maximum to little or no cytotoxicity. Relatively insoluble test items should be tested up to the highest concentration that can be formulated in an appropriate solvent as solution or homogenous suspension. These test items should be tested up or beyond their limit of solubility. Precipitation should be evaluated at the end of treatment by the unaided eye.
Relevant cytotoxic effects indicated by a relative suspension growth below 50 % were observed at 22.5 µg/mL and above without metabolic activation and 184.4 µg/mL and above with metabolic activation following 4 hours treatment. After 24 hours treatment without metabolic activation strong toxic effects were observed at 184.4 µg/mL and above.
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal of the test item. Following 4 hours treatment precipitation occurred at 180 µg/mL and above without metabolic activation and at 184.4 µg/mL and above with metabolic activation. Following 24 hours treatment without metabolic activation precipitation was observed at the maximum concentration of 11800 µg/mL.
There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.
Based on the results of the pre-experiments, the individual concentrations of the main experiments were selected. A series of concentrations spaced by a factor of 2 was placed into the lower range. Narrower spacing was used at high concentrations to cover the cytotoxic range more closely.

COMPARISON WITH HISTORICAL CONTROL DATA:
The range of the historical solvent control data (3.4 - 36.6 mutant colonies / 106 cells) was exceeded in the second culture of the first experiment with metabolic activation at 11.0 µg/mL (49.3 mutant colonies / 106 cells) and at 132.0 µg/mL (45.7 mutant colonies/106 cells). However, the induction factor did not reach the threshold of three times the corresponding solvent control and statistical analysis showed that there was no dose dependent increase. Therefore, the increases were judged as biologically irrelevant.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No relevant cytotoxic effects were noted in the first and the second experiment without metabolic activation up to the maximum analyzable concentration of 11.0 µg/mL and 132.0 µg/mL respectively. Based on the very steep gradient of cytotoxicity exceedingly severe toxic effects occurred at the next higher concentrations of 16.5 µg/mL or 176.0 µg/mL even though the concentration spacing was smaller than factor 2 requested by the current OECD guideline. In both main experiments with metabolic activation, relevant cytotoxic effects occurred at 132.0 µg/mL in one culture (experiment I) or both cultures (experiment II). The later onset of cytotoxicity in the experimental parts with metabolic activation and following 24 hour treatment without metabolic activation indicates possible protein binding of the test item as the protein concentration during treatment is higher based on the S9-mix or serum present during long term treatment.

Any other information on results incl. tables

Table 1: Summary of results

	conc. µg/mL	P	S9 mix	relative cloning efficiency I %	relative cell density %	relative cloning efficiency II %	mutant colonies/ 10^6cells	induction factor	relative cloning efficiency I %	relative cell density %	relative cloning efficiency II %	mutant colonies/ 10^6cells	induction factor
Column	1	2	3	4	5	6	7	8	9	10	11	12	13
Experiment I / 4 h treatment				culture I					culture II
Solvent control with water			-	100.0	100.0	100.0	19.2	1.0	100.0	100.0	100.0	17.8	1.0
Positive control (EMS)	150.0		-	81.7	106.4	91.2	136.4	7.1	78.1	109.7	96.1	110.9	6.2
Test item	0.7		-	89.4	95.6	99.9	21.6	1.1	86.6	87.0	74.6	15.0	0.8
Test item	1.4		-	89.2	105.8	100.4	29.7	1.5	92.6	126.9	120.7	14.6	0.8
Test item	2.8		-	89.9	99.9	111.8	24.8	1.3	96.2	102.3	96.5	22.4	1.3
Test item	5.5		-	88.6	121.5	90.4	24.9	1.3	95.6	98.3	84.4	35.5	2.0
Test item	11.0		-	87.4	105.6	77.5	18.1	0.9	91.5	85.6	66.8	27.5	1.5
Test item	16.5		-	0.0	culture was not continued^#				0.0	culture was not continued^#
Test item	22.0		-	0.0	culture was not continued^#				0.0	culture was not continued^#
Solvent control with water			+	100.0	100.0	100.0	11.2	1.0	100.0	100.0	100.0	27.2	1.0
Positive control (DMBA)	1.1		+	80.9	69.8	96.4	358.8	31.9	80.4	95.6	82.2	571.3	21.0
Test item	5.5		+	102.2	culture was not continued^##			107.9		culture was not continued^##
Test item	11.0		+	95.5	65.9	95.8	8.1	0.7	102.6	85.3	83.3	49.3	1.8
Test item	22.0		+	96.8	71.5	93.5	12.1	1.1	101.8	85.5	92.7	30.1	1.1
Test item	44.0		+	93.8	84.2	100.5	15.4	1.4	109.5	92.4	89.9	28.3	1.0
Test item	88.0		+	90.0	63.8	88.9	20.8	1.8	105.6	89.0	95.8	21.7	0.8
Test item	132.0		+	48.3	76.4	106.6	10.8	1.0	100.1	108.2	87.0	45.7	1.7
Test item	176.0	P	+	0.0	culture was not continued^#			0.0		culture was not continued^#
Experiment II / 24 h treatment				culture I				culture II
Solvent control with water			-	100.0	100.0	100.0	22.3	1.0	100.0	100.0	100.0	16.4	1.0
Positive control (EMS)	150.0		-	87.5	81.9	96.0	506.2	22.7	92.5	82.4	92.6	434.4	26.4
Test item	5.5		-	101.3	culture was not continued^##				102.6	culture was not continued^##
Test item	11.0		-	100.7	95.3	94.4	12.4	0.6	99.8	110.5	94.5	19.4	1.2
Test item	22.0		-	95.8	92.7	94.0	14.3	0.6	100.6	117.0	96.2	19.2	1.2
Test item	44.0		-	94.5	105.0	96.4	11.9	0.5	105.2	113.3	96.6	8.5	0.5
Test item	88.0		-	96.3	111.2	97.3	14.2	0.6	101.5	101.5	96.5	20.3	1.2
Test item	132.0		-	93.7	74.8	92.4	9.9	0.4	91.2	97.5	97.3	26.2	1.6
Test item	176.0	P	-	7.0	culture was not continued^#				3.4	culture was not continued^#
Experiment II / 4 h treatment
Solvent control with water			+	100.0	100.0	100.0	14.7	1.0	100.0	100.0	100.0	17.3	1.0
Positive control (DMBA)	1.1		+	92.2	77.3	98.0	314.7	21.5	87.6	112.1	97.8	331.0	19.2
Test item	5.5		+	97.9	culture was not continued^##				97.1	culture was not continued^##
Test item	11.0		+	99.3	106.7	102.4	26.8	1.8	98.0	142.9	97.6	20.4	1.2
Test item	22.0		+	94.2	88.1	97.7	17.9	1.2	94.6	126.9	100.2	17.2	1.0
Test item	44.0		+	94.7	88.5	98.4	13.7	0.9	93.2	131.9	104.6	14.1	0.8
Test item	88.0		+	78.4	95.6	98.9	7.9	0.5	78.1	123.7	103.0	9.7	0.6
Test item	132.0		+	37.5	81.3	102.5	17.8	1.2	39.9	133.1	103.3	16.4	1.0
Test item	176.0	P	+	0.0	culture was not continued^#				0.0	culture was not continued^#

P – Precipitation

^#culture was not continued due to exceedingly severe cytotoxic effects

^##culture was not continued as a minimum of only four analysable concentrations is required

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions according to OECD 476 the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test item is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The study was performed to investigate the potential of the test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster in a test according to OECD guideline 476. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The maximum test item concentration of 11800 µg/mL used in the pre-experiments was equal to approximately 5452 µg/mL of the REACH registration substance. The test item was dissolved in deionised water. The dose range of the main experiments was limited by cytotoxic effects. No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test item is considered to be non-mutagenic in this HPRT assay.