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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Under the experimental conditions according to OECD 476 the test item did not induce gene mutations at the HPRT locus in V79 cells and in a reverse mutation assay test according to OECD 471, the test item is considered to be non-mutagenic to Salmonella typhimurium and Escherichia coli. Therefore, the test item is considered to be non-mutagenic.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

Target gene:

HPRT

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

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.

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.

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

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-05-08 to 2012-05-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted July 21, 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

May 30, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Phenobarbital/β-naphthoflavone induced rat liver S9
- method of preparation of S9 mix: the S9 mix preparation was performed according to Ames et.al
- concentration or volume of S9 mix and S9 in the final culture medium: 10% v/v

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II
without S9 mix
TA 1535, TA 100: 1; 3; 10; 33; 100; 333; 1000; and 2500 µg/plate
TA 1537: 0.3, 1; 3; 10; 33; 100; 333; and 1000 µg/plate
TA 98, WP2 uvrA: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
with S9 mix all strains: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: better than others

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide; 4-nitro-o-phenylene-diamine; methyl methane sulfonate, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation, pre-incubation

DURATION
- Preincubation period: 1 hour
- Exposure duration: 72 hours

NUMBER OF REPLICATIONS: 3 plates

DETERMINATION OF CYTOTOXICITY
A reduction in the number of spontaneous revertants (below the induction factor of 0.5), or a clearing of the bacteria background lawn.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
The test item precipitated in the overlay agar in the test tubes from 2500 and 5000 µg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 2500 to 5000 µg/plate. The undissolved particles had no influence on the data recording
- Other confounding effects:
COMPARISON WITH HISTORICAL CONTROL DATA: performed

Table 1: Summary of Results: Experiment I

Metabolic Activation	Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ±SD)
Without Activation			TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
	Deionised water		17 ± 4	21 ± 0	31 ± 4	106 ± 5	66 ± 1
			12 ± 1	11 ± 3	34 ± 7	108 ± 10	63 ± 7
	Untreated Humectol C liq hc	3 µg	17 ± 4	15 ± 1	28 ± 6	109 ± 11	67 ± 8
		10 µg	16 ± 3	16 ± 3	28 ± 6	109 ± 8	68 ± 7
		33 µg	16 ± 1	15 ± 1	26 ± 4	88 ± 11	77 ± 12
		100 µg	13 ± 1	9 ± 8	26 ± 2	78 ± 6	62 ± 8
		333 µg	7 ± 3^R	5 ± 2	26 ± 9	73 ± 20^R	48 ± 1
		1000 µg	9 ± 1^R	5 ± 2^R	16 ± 4	44 ± 6^R	34 ± 8
		2500 µg	4 ± 2^P R	1 ± 1^P R	10 ± 2^P	11 ± 4^P R	31 ± 6^P
		5000 µg	4 ± 1^P R	1 ± 1^P M R	3 ± 3^P R	2 ± 1^P M R	27 ± 5^P
	NaN3 4-NOPD 4-NOPD MMS	10 µg	1709 ± 114			2287 ± 21
		10 µg			274 ± 2
		50 µg		81 ± 8
		3.0 µL					1364 ± 22
With Activation	Deionised water		15 ± 5	24 ± 5	51 ± 8	141 ± 24	59 ± 4
	Untreated		20 ± 4	20 ± 4	36 ± 2	133 ± 9	69 ± 10
	Humectol C liq hc	3 µg	18 ± 3	21 ± 2	49 ± 10	139 ± 10	67 ± 3
		10 µg	16 ± 3	20 ± 8	47 ± 11	144 ± 16	68 ± 6
		33 µg	16 ± 6	26 ± 9	48 ± 5	132 ± 7	70 ± 10
		100 µg	23 ± 7	23 ± 6	36 ± 6	138 ± 16	78 ± 6
		333 µg	16 ± 3	21 ± 1	44 ± 6	144 ± 12	70 ± 3
		1000 µg	12 ± 2	19 ± 6	33 ± 4	105 ± 6	69 ± 9
		2500 µg	5 ± 0^P	6 ± 3^P	29 ± 5^P	76 ± 8^P R	44 ± 5^P
		5000 µg	8 ± 4^P	5 ± 4^P	17 ± 2^P	64 ± 14^P R	24 ± 2^P
	2-AA	2.5 µg	396 ± 15	339 ± 6	2244 ± 227	2831 ± 201
	2-AA	10.0 µg					302 ± 66

Table 2: Summary of Results: Experiment II

Metabolic Activation	Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ±SD)
			TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	Deionised water		12 ± 3	18 ± 7	31 ± 3	181 ± 5	57 ± 6
	Untreated		18 ± 5	23 ± 1	30 ± 9	172 ± 18	59 ± 6
	Humectol C liq hc	0.3 µg		21 ± 6
		1 µg	13 ± 4	19 ± 5		167 ± 14
		3 µg	12 ± 3	16 ± 5	29 ± 2	181 ± 14	53 ± 2
		10 µg	14 ± 5	21 ± 5	31 ± 5	174 ± 10	50 ± 15
		33 µg	20 ± 4	21 ± 6	29 ± 2	110 ± 4	51 ± 2
		100 µg	11 ± 4^R	9 ± 1^R	29 ± 2	84 ± 2^R	59 ± 4
		333 µg	6 ± 2^R	6 ± 2^R	28 ± 5	63 ± 5^R	35 ± 7
		1000 µg	8 ± 5^R	3 ± 1^M R	21 ± 1	30 ± 6^R	33 ± 11
		2500 µg	4 ± 3^P R		10 ± 8^P R	6 ± 1^P R M	22 ± 1^P
		5000 µg			2 ± 2^P M R		18 ± 3^P R
	NaN3	10 µg	1838 ± 55			1918 ± 53
	4-NOPD	10 µg			509 ± 8
	4-NOPD	50 µg		80 ± 15
	MMS	3.0 µL					716 ± 31
With Activation	Deionised water		20 ± 4	22 ± 11	43 ± 6	200 ± 5	68 ± 5
	Untreated		18 ± 1	25 ± 4	39 ± 9	210 ± 9	58 ± 9
	Humectol C liq hc	3 µg	22 ± 1	28 ± 3	39 ± 2	205 ± 15	63 ± 7
		10 µg	21 ± 7	27 ± 1	40 ± 10	207 ± 12	73 ± 6
		33 µg	16 ± 1	28 ± 2	44 ± 8	197 ± 16	79 ± 5
		100 µg	17 ± 6	27 ± 6	37 ± 5	200 ± 8	70 ± 10
		333 µg	16 ± 3	32 ± 3	45 ± 3	236 ± 23	76 ± 16
		1000 µg	12 ± 4	24 ± 4	40 ± 8	194 ± 37	66 ± 5
		2500 µg	14 ± 2^P R	10 ± 5^P R	27 ± 3^P R M	110 ± 12^P R	62 ± 12^P
		5000 µg	6 ± 2^P M R	1 ± 2^P M R	15 ± 3^P M R	53 ± 29^P M R	57 ± 6^P M
	2-AA	2.5 µg	300 ± 57	226 ± 10	1911 ± 178	2342 ± 80
	2-AA	10.0 µg					318 ± 12

NaN3: sodium azide                                                               R: Reduced background growth

2-AA: 2-aminoanthracene                                                        P: Precipitate

4-NOPD: 4-nitro-o-phenylene-diamine                                      M: Manual count

MMS: methyl methane sulfonate

Conclusions:

In a reverse mutation assay test according to OECD 471, the test item is considered to be non-mutagenic to Salmonella typhimurium and Escherichia coli

Executive summary:

The study according OECD 471 was performed to investigate the potential of the test item to induce gene mutations in the plate incorporation test and the pre-incubation test using the Salmonella typhimurium strains TA1535, TA1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The plates incubated with the test item showed reduced background growth in all strains. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation. There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

It can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used. Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Neither bacterial nor mammalian cells exhibited an increase mutation rate after in vitro exposure to the test item. An in vivo micronucleus assay in mice confirmed that there was no genetic hazard posed by the test item.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1999-03-29 to 1999-06-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

Adopted 21st, July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

August 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH Gartenstrasse 27, 33178 Borchen
- Age at study initiation: approximately 9 weeks
- Weight at study initiation: males 37.6 g (33 – 42); females 28.2 g (25 – 31)
- Assigned to test groups randomly: yes
- Housing: macrolon cage type 3, 5 per cage, separatd by sex
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Photoperiod: 12 hours daily

Route of administration:

oral: gavage

Vehicle:

- Vehicle: water
- Concentration of test material in vehicle: 44.4 % (v/v)
- Amount of vehicle: 10 mL/kg

Frequency of treatment:

The test item was administered twice at an intervals of 24 hours.

Post exposure period:

24 hours

Dose / conc.:

4 504.5 mg/kg bw/day

Remarks:

Dose of test substance received. Corresponds to ca. 2082 mg/kg bw of the REACH registration substance.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide;
- Justification for choice of positive control: Standard positive control substance
- Route of administration: gavage
- Doses / concentrations: 50 mg/kg bw
- frequency: once

Tissues and cell types examined:

bone marrow from femora

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: limited mortality in (2/6 animals) in a preliminary test at 4504.5 mg/kg bw of test substance (2000 mg/kg bw calculated based on water free content) combined with no systemic toxicity at lower dose

DETAILS OF SLIDE PREPARATION:
Staining was performed as follows:
- 5 minutes in methanol
- 5 minutes in May-Grünwald's solution
- brief rinsing twice in distilled water
- 10 minutes staining in 1 part Giemsa solution to 6 parts buffer solution, pH 7.2 (Weise)
- rinsing in distilled water
- drying
- coating with Enteltan®

METHOD OF ANALYSIS:
2000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded, not the number of individual micronuclei. In addition, the ratio of polychromatic erythrocytes to 200 total erythrocytes was determined. Main parameter for the statistical analysis, i.e. validity assessment of the study and mutagenicity of the test substance, was the proportion of polychromatic erythrocytes with micronuclei out of the 2000 counted erythrocytes. All bone marrow smears for evaluation were coded to ensure that the group from which they were taken remained unknown to the investigator.

Evaluation criteria:

Both biological and statistical significances were considered together for evaluation purposes. A substance is considered positive if there is a significant increase in the number of micronucleated polychromatic erythrocytes compared with the concurrent negative control group. A test substance producing no significant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.

Statistics:

A one-sided Wilcoxon-Test was evaluated to check the validity of the study. The study was considered as valid in case the proportion of polychromatic erythrocytes with micronuclei in the positive control was significantly higher than in the negative control (p=0.05).

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 3378.4 - 4504.5 mg/kg bw test substance (1500 - 2000 mg/kg bw calculated based on water free content)
- Solubility: aqueous solution
- Clinical signs of toxicity in test animals: motor activity decreased, squatting posture, coat bristling, palpebral fissure closed, respiratory sounds

Conclusions:

The test item did not lead to a substantial increase of micronucleated polychromatic erythrocytes and is not mutagenic in the micronucleus test under the conditions described according to OECD 474.

Executive summary:

The micronucleus test was carried out with the test item according to OECD 474. The test item was dissolved in deionized water and was given twice at an interval of 24 hours as an orally dose of 4504.5 mg/kg bw test substance (corresponding to 2082 mg/kg body weight of the REACH registration substance) to male and female mice, based on the results of a previous dose range finding assay. According to the test procedure the animals were killed 24 hours after administration. Endoxan® was used as positive control substance and was administered once orally at a dose of 50 mg/kg body weight. The number of polychromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic erythrocytes to total erythrocytes in both male and female animals remained unaffected by the treatment with the test item and was not less than 20 % of the control value. Endoxan® induced a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the test system. The ratio of polychromatic erythrocytes to total erythrocytes was not changed to a significant extent. Under the conditions of the present study the results indicate that the test item is not mutagenic in the micronucleus test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

All three test systems confirmed that there was neither a gene mutation hazard nor a potential for chromosomal damage posed by the test item.

Justification for classification or non-classification

Not classified:

All available in vitro and in vivo assays showed no genetic hazard.