1-Propanaminium, 2-hydroxy-N-(2-hydroxyethyl)-N-methyl-N-[2-[(1-oxo-9-octadecen-1-yl)amino]ethyl]-3-sulfo-, methyl sulfate, sodium salt (1:1:1)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item is considered non-mutagenic in a bacterial reverse mutation assay.

 

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 February 2018 - 20 April 2018

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:

21st July 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, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Sodium phenobarbitone and β-naphthoflavone induced rat liver S9 homogenate (S9 Mix)

Test concentrations with justification for top dose:

Based on the results of initial cytotoxicity test, concentrations of 0.005, 0.016, 0.05, 0.16 and 0.5 mg/plate are selected for testing in the mutation test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was found miscible in DMSO at 50 mg/mL.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

with S9 mix, TA98, TA100,TA102,TA1535,TA1537, 4 µg/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S9 mix, TA98, 2 µg/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9 mix, TA100,TA1535, 1 µg/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9 mix, TA102, 0.5 µg/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix, TA1537, 50 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 30 min

NUMBER OF REPLICATIONS: 3

Evaluation criteria:

There should be a dose related increase over the range tested and/or a reproducible increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing doses of the test item either in the presence or absence of the metabolic activation system.
The test will be judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 2 times the mean vehicle control value in Salmonella typhimurium strains TA98, TA100 and TA102 or equal to or greater than 3 times the mean vehicle control value in tester strains TA1535 and TA1537. An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose responsive increase that does not achieve the respective threshold cited above or a non dose responsive increase that is equal to or greater than the respective threshold cited. A response will be evaluated as negative, if it is neither positive nor equivocal.

Statistics:

None

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test item resulted in slight precipitation at 5 mg/plate and no precipitation at 1, 2, 3 and 4 mg/plate

RANGE-FINDING/SCREENING STUDIES:
Based on the results of precipitation test, the concentrations of 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 and 5 mg/plate were selected for the initial cytotoxicity test.
The test item resulted in cytotoxicity which was observed by a thinning of the bacterial background lawn. The tester strain exposed with test item in the presence and absence of metabolic activation system resulted in cytotoxicity and those concentrations are graded as 0 (Absent lawn) for 0.9, 1, 2, 3, 4 and 5 mg/plate, 1+ (extremely reduced lawn) for 0.7 and 0.8 mg/plate, 2+ (moderately reduced lawn) for 0.6 mg/plate and 3 + (Slightly reduced lawn) for 0.5 mg/plate when compared to vehicle control.
The test item was found to be cytotoxic up to the maximum concentration of 0.5 mg/plate tested when compared to the vehicle control. On the basis of cytotoxicity results 0.5 mg/plate was considered as the highest test concentration for the main mutagenicity assay.

TABLE 1.SUMMARY OF COLONY COUNTS OF REVERTANTS OF TRIAL-I

          Plate Incorporation Method

Treatment	Test Concentration  (mg/plate)		No. of Revertants (Mean of 3 Plates)
			With S9						Without S9
			Salmonella typhimurium						Salmonella typhimurium
			TA 98	TA 100	TA 102	TA 1535	TA 1537		TA 98	TA 100	TA 102	TA 1535	TA 1537
Vehicle Control	100 µL of Dimethyl sulphoxide	Mean	31.0	111.7	265.7	22.3	10.7		26.3	106.0	263.7	20.0	10.0
		±SD	5.6	6.4	4.5	1.2	1.5		1.5	5.3	12.1	3.0	2.0
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
Test item	0.005	Mean	26.3	107.3	264.7	19.7	8.7		24.7	104.7	257.7	18.3	7.0
		±SD	4.0	6.8	6.5	4.0	3.1		2.1	9.9	10.0	1.5	2.6
	Fold Increase		0.8	1.0	1.0	0.9	0.8		0.9	1.0	1.0	0.9	0.7
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.016	Mean	24.0	106.7	262.0	21.7	7.7		23.7	104.7	260.3	20.0	9.3
		±SD	2.6	6.8	8.2	3.2	2.1		4.0	8.3	7.1	3.5	1.5
	Fold Increase		0.8	1.0	1.0	1.0	0.7		0.9	1.0	1.0	1.0	0.9
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.05	Mean	24.7	107.3	260.0	21.3	8.0		23.7	103.0	267.3	18.0	7.7
		±SD	2.1	5.5	7.0	1.5	3.6		1.5	3.0	8.1	2.6	1.5
	Fold Increase		0.8	1.0	1.0	1.0	0.8		0.9	1.0	1.0	0.9	0.8
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.16	Mean	20.7	103.7	268.7	16.7	9.3		21.0	101.3	252.0	17.0	8.7
		±SD	2.5	3.1	7.8	2.1	1.5		1.0	4.0	2.6	1.0	2.3
	Fold Increase		0.7	0.9	1.0	0.7	0.9		0.8	1.0	1.0	0.9	0.9
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.5	Mean	16.0	56.7	234.3	13.0	4.0		15.7	53.7	233.3	11.3	3.3
		±SD	3.0	4.7	8.1	1.0	1.0		1.5	3.8	4.5	0.6	0.6
	Fold Increase		0.5	0.5	0.9	0.6	0.4		0.6	0.5	0.9	0.6	0.3
	Lawn Intensity		3+	3+	3+	3+	3+		3+	3+	3+	3+	3+
Positive Control	100 µL of respective Positive Control	Mean	373.0	408.3	603.3	139.0	121.7		358.7	399.0	597.7	137.0	108.3
		±SD	17.0	9.0	10.6	10.1	6.5		15.0	12.5	14.3	5.6	7.1
	Fold Increase		12.0	3.7	2.3	6.2	11.4		13.6	3.8	2.3	6.9	10.8
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+

Values of Revertants are in Mean±SD

Positive controls: with S9 Salmonella typhimurium TA98, TA100, TA102, TA1535 and TA1537 = 4 µg/plate of 2-Aminoanthracene

without S9: TA98: 2 µg/plate of 2-Nitrofluorene, TA100 and TA1535: 1 µg/plate of Sodium azide, TA102: 0.5 µg/plate of Mitomycin C, TA1537: 50 µg/plate of 9-Aminoacridine

TABLE 2: SUMMARY OF COLONY COUNTS OF REVERTANTS OF TRIAL-II

Preincubation Method

Treatment	Test Concentration  (mg/plate)		No. of Revertants (Mean of 3 Plates)
			With S9						Without S9
			Salmonella typhimurium						Salmonella typhimurium
			TA 98	TA 100	TA 102	TA 1535	TA 1537		TA 98	TA 100	TA 102	TA 1535	TA 1537
Vehicle Control	100 µL of Dimethyl sulphoxide	Mean	28.0	103.0	271.0	21.0	9.0		25.7	97.0	266.0	21.0	10.7
		±SD	4.6	4.6	8.7	3.6	3.0		4.0	6.6	6.2	2.0	1.5
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
LYAF (Dried)	0.005	Mean	22.7	100.0	267.3	19.0	8.0		21.7	97.3	261.3	18.0	7.3
		±SD	5.1	3.0	6.0	2.6	2.6		2.5	5.7	9.6	3.6	2.1
	Fold Increase		0.8	1.0	1.0	0.9	0.9		0.8	1.0	1.0	0.9	0.7
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.016	Mean	26.3	94.7	257.3	18.3	8.7		21.7	98.0	264.0	21.3	9.3
		±SD	3.5	5.7	2.5	1.5	1.5		3.1	2.6	9.6	2.5	3.2
	Fold Increase		0.9	0.9	0.9	0.9	1.0		0.8	1.0	1.0	1.0	0.9
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.05	Mean	26.3	100.7	254.3	18.3	9.3		22.0	96.7	257.0	17.0	8.7
		±SD	4.5	7.6	3.5	3.2	3.2		3.0	5.7	6.2	2.6	3.1
	Fold Increase		0.9	1.0	0.9	0.9	1.0		0.9	1.0	1.0	0.8	0.8
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.16	Mean	22.0	84.7	246.0	19.0	6.7		20.3	86.3	242.7	17.3	6.7
		±SD	2.6	3.5	4.6	2.0	2.1		3.1	2.5	6.4	4.2	1.5
	Fold Increase		0.8	0.8	0.9	0.9	0.7		0.8	0.9	0.9	0.8	0.6
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.5	Mean	14.7	59.7	216.3	13.3	3.3		13.3	52.0	221.3	13.0	3.7
		±SD	1.2	3.5	4.5	1.5	0.6		1.5	2.6	6.1	1.0	1.5
	Fold Increase		0.5	0.6	0.8	0.6	0.4		0.5	0.5	0.8	0.6	0.3
	Lawn Intensity		3+	3+	3+	3+	3+		3+	3+	3+	3+	3+
Positive Control	100 µL of respective Positive Control	Mean	380.7	404.3	595.3	142.3	121.3		355.7	398.3	593.3	139.3	116.0
		±SD	10.7	8.5	17.6	11.7	11.1		14.3	10.5	9.6	8.6	6.0
	Fold Increase		13.6	3.9	2.2	6.8	13.5		13.9	4.1	2.2	6.6	10.9
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+

Values of Revertants are in Mean±SD

Positive controls: with S9: Salmonella typhimurium TA98, TA100, TA102, TA1535 and TA1537 = 4 µg/plate of 2-Aminoanthracene

without S9 TA98: 2 µg/plate of 2-Nitrofluorene, TA100 and TA1535: 1 µg/plate of Sodium azide, TA102: 0.5 µg/plate of Mitomycin C, TA1537: 50 µg/plate of 9-Aminoacridine

Conclusions:

Under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, the test item is considered non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

The substance was evaluated for mutagenicity in a Bacterial Reverse Mutation Test according OECD TG 471. The test item formed suspension in dimethyl sulphoxide at a concentration of 50 mg/mL. Test item resulted in slight precipitation at 5 mg/plate. The highest concentration selected for initial cytotoxicity test was 5 mg/plate. Salmonella typhimurium TA100 strain was exposed to vehicle control, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 and 5 mg/plate. The test item resulted in cytotoxicity at 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 and 5 mg/plate tested concentration in initial cytotoxicity test. Based on the results of initial cytotoxicity test, concentrations of 0.005, 0.016, 0.05, 0.16 and 0.5 mg/plate were selected for testing in the mutation test by plate incorporation method.

The test item was assessed for its mutagenic effects using Salmonella typhimurium strains: TA98, TA100, TA 102, TA1535, and TA1537. The test item was tested at the concentrations of 0.005, 0.016, 0.05, 0.16 and 0.5 mg/plate for plate incorporation method and for preincubation method using dimethyl sulphoxide as vehicle based on the results of solubility, precipitation and initial cytotoxicity test. The study was conducted with and without metabolic activation (S9 fraction). The S9 fraction was prepared from sodium phenobarbitone and β-naphthoflavone induced rat liver. Vehicle control and appropriate positive controls (2-nitrofluorene, sodium azide, 9-Aminoacridine and Mitomycin C, for trials “without metabolic activation” and 2-Aminoanthracene for trials “with metabolic activation”) were tested simultaneously. Two trials were carried out for this study in triplicate. Trial I was carried out as plate incorporation method and trial II as pre incubation method.

Based on the experimental results obtained, the mean numbers of revertant colonies at the tested concentrations were comparable to those of the vehicle control in both the trials (in the presence and absence of metabolic activation). There was no appreciable increase in number of revertant colonies at any of the tested concentrations in both the trials. The number of revertant colonies in the positive controls resulted in 2.2 to 13.9 fold increase under identical conditions.

Under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, the test item is considered non-mutagenic in this bacterial reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro

The test item was evaluated for mutagenicity in a Bacterial Reverse Mutation Test according OECD TG 471.

The test item formed suspension in dimethyl sulphoxide at a concentration of 50 mg/mL. Test item resulted in slight precipitation at 5 mg/plate. The highest concentration selected for initial cytotoxicity test was 5 mg/plate. Salmonella typhimurium TA100 strain was exposed to vehicle control, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 and 5 mg/plate. The test item resulted in cytotoxicity at 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 and 5 mg/plate tested concentration in initial cytotoxicity test. Based on the results of initial cytotoxicity test, concentrations of 0.005, 0.016, 0.05, 0.16 and 0.5 mg/plate were selected for testing in the mutation test by plate incorporation method.

The test item was assessed for its mutagenic effects using Salmonella typhimurium strains: TA98, TA100, TA 102, TA1535, and TA1537. The test item was tested at the concentrations of 0.005, 0.016, 0.05, 0.16 and 0.5 mg/plate for plate incorporation method and for preincubation method using dimethyl sulphoxide as vehicle based on the results of solubility, precipitation and initial cytotoxicity test. The study was conducted with and without metabolic activation (S9 fraction). The S9 fraction was prepared from sodium phenobarbitone and β-naphthoflavone induced rat liver. Vehicle control and appropriate positive controls (2-nitrofluorene, sodium azide, 9-Aminoacridine and Mitomycin C, for trials “without metabolic activation” and 2-Aminoanthracene for trials “with metabolic activation”) were tested simultaneously. Two trials were carried out for this study in triplicate. Trial I was carried out as plate incorporation method and trial II as pre incubation method.

Based on the experimental results obtained, the mean numbers of revertant colonies at the tested concentrations were comparable to those of the vehicle control in both the trials (in the presence and absence of metabolic activation). There was no appreciable increase in number of revertant colonies at any of the tested concentrations in both the trials. The number of revertant colonies in the positive controls resulted in 2.2 to 13.9 fold increase under identical conditions.

Under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, the test item is considered non-mutagenic in this bacterial reverse mutation assay.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available information on the test item regarding genetic toxicity are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available experimental information, the test substance is not classified for genetic toxicity according to Regulation (EC) No 1272/2008 (CLP), as amended for the twelfth time in Regulation (EU) No 2019/521.