Ethanol, 2,2'-iminobis-, N-tallow alkyl derivs., N-oxides

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In an in vitro bacterial reverse mutation assay (Ames) according to OECD guideline 471, the test item did not show a mutagenic potential.

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:

09 April - 08 June, 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:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

his

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:

0.001, 0.003, 0.01, 0.03 and 0.1 mg/plate
Based on the results of initial cytotoxicity test, the concentrations for testing in the mutation test were selected.

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent: 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, TA1535, 50 µg/plate

Details on test system and experimental conditions:

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

DURATION
- Preincubation period: 30 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth, viable count

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:

cytotoxicity

Remarks:

0.1 mg/plate

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:

cytotoxicity

Remarks:

0.1 mg/plate

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:

cytotoxicity

Remarks:

0.1 mg/plate

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:

cytotoxicity

Remarks:

0.1 mg/plate

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:

cytotoxicity

Remarks:

0.1 mg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

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 Salmonella typhimurium TA100 tester strain was exposed to vehicle control and the above indicated concentrations of the test item in the presence and absence of metabolic activation system. Incubation with the test item resulted in cytotoxicity, indicated by a thinning and disappearance of the bacterial background lawn.
Based on the results of initial cytotoxicity test, the tester strain Salmonella typhimurium TA100 was exposed with test item in the presence and absence of metabolic activation system at concentrations of 0.4, 0.2, 0.1, 0.05, 0.025, 0.0125, 0.00625 and 0.003125 mg/plate. The test item resulted in cytotoxicity at 0.1, 0.2 and 0.4 mg/plate.
Based on the results of initial cytotoxicity test, concentrations of 0.001, 0.003, 0.01, 0.03 and 0.1 mg/plate was selected for testing in the mutation test.

HISTORICAL CONTROL DATA
see "Any other information on materials and methods"

Table 1: Historical Data

Plate Incorporation Method

Metabolic Activation		With Metabolic Activation						Without Metabolic Activation
Tester strain		Salmonella typhimurium						Salmonella typhimurium
		TA 98	TA 100	TA 1535	TA 1537	TA 102		TA 98	TA 100	TA 1535	TA 1537	TA 102
Vehicle Control (DMSO)	Mean	21.3	103.4	21.2	9.2	260.3		21.2	102.8	21.2	9.4	261.8
	±SD	3.2	5.5	2.2	1.8	5.2		2.7	4.7	2.3	1.9	5.2
	Min	16	96	13	6	251		15	91	15	7	254
	Max	40	130	26	14	272		32	126	27	15	274
Positive Control	Mean	401.7	383.6	140.1	119.2	606.2		396.6	379.7	141.1	120.1	613.4
	±SD	23.4	16.9	8.0	6.6	27.6		27.4	18.3	9.9	7.3	22.6
	Min	256	246	118	100	510		290	270	110	98	589
	Max	440	419	182	149	670		430	446	190	158	694
Preincubation Method
Metabolic Activation		With Metabolic Activation						Without Metabolic Activation
Tester strain		Salmonella typhimurium						Salmonella typhimurium
		TA 98	TA 100	TA 1535	TA 1537	TA 102		TA 98	TA 100	TA 1535	TA 1537	TA 102
Vehicle Control (DMSO)	Mean	21.2	102.9	20.9	9.4	262.3		20.9	102.1	21.1	9.6	263.8
	±SD	2.8	4.6	2.2	2.0	4.6		2.8	4.3	2.3	2.6	5.5
	Min	16	89	14	4	256		15	88	16	6	251
	Max	32	128	28	17	275		32	129	28	23	275
Positive Control	Mean	402.5	383.9	140.6	118.6	606.5		398.3	381.3	141.3	119.5	616.0
	±SD	18.5	16.1	9.3	6.3	26.7		24.5	16.2	11.0	6.4	25.9
	Min	290	293	103	99	508		281	312	110	97	590
	Max	429	425	187	151	670		428	450	200	173	699

    Min: Minimum no. of colonies, Max: Maximum no. of colonies, SD: Standard Deviation

Table 2: Summary of the Colony Counts of Revertants, 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	27.7	112.0	267.0	22.0	10.0		28.0	106.7	264.0	21.7	9.0
		±SD	3.1	4.0	5.6	2.6	2.0		2.0	9.1	4.6	2.5	2.0
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
Humectol LYS Mod	0.001	Mean	22.3	113.0	263.7	22.0	11.0		22.7	111.3	262.3	20.3	10.0
		±SD	4.9	7.2	7.1	2.6	2.0		1.5	4.2	4.2	2.1	2.0
	Fold Increase		0.8	1.0	1.0	1.0	1.1		0.8	1.0	1.0	0.9	1.1
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.003	Mean	23.0	107.0	261.0	20.7	9.3		21.0	101.7	256.0	20.3	9.7
		±SD	3.6	5.6	9.2	1.5	2.1		1.0	7.0	5.3	2.5	2.1
	Fold Increase		0.8	1.0	1.0	0.9	0.9		0.8	1.0	1.0	0.9	1.1
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.01	Mean	22.0	107.0	264.0	20.7	9.7		21.3	105.0	258.3	22.0	9.3
		±SD	3.6	7.0	4.6	3.1	2.1		2.5	6.0	10.4	3.0	2.1
	Fold Increase		0.8	1.0	1.0	0.9	1.0		0.8	1.0	1.0	1.0	1.0
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.03	Mean	19.7	108.0	254.0	18.7	7.0		19.3	101.3	251.3	18.3	7.0
		±SD	1.5	9.5	5.0	1.5	1.0		2.5	8.0	5.5	2.5	1.7
	Fold Increase		0.7	1.0	1.0	0.8	0.7		0.7	1.0	1.0	0.8	0.8
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.1	Mean	13.7	60.3	223.3	13.0	4.0		13.0	60.0	219.3	12.0	3.7
		±SD	1.5	4.5	7.8	1.0	1.0		2.0	2.6	7.0	1.0	0.6
	Fold Increase		0.5	0.5	0.8	0.6	0.4		0.5	0.6	0.8	0.6	0.4
	Lawn Intensity		3+	3+	3+	3+	3+		3+	3+	3+	3+	3+
Positive Control	100 µL of respective Positive Control	Mean	357.3	400.0	607.3	139.3	108.3		351.0	401.0	606.7	128.3	102.0
		±SD	10.7	5.6	11.9	8.5	11.7		9.5	7.5	5.9	12.0	11.0
	Fold Increase		12.9	3.6	2.3	6.3	10.8		12.5	3.8	2.3	5.9	11.3
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
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 3: Summary of the Colony Counts of Revertants, 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	27.3	112.7	262.3	23.0	10.0		24.7	108.0	263.3	22.0	9.3
		±SD	3.1	5.7	7.1	1.0	2.6		4.2	8.2	4.5	2.6	2.1
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
Humectol LYS Mod	0.001	Mean	21.7	112.0	263.3	21.7	9.3		22.0	106.7	258.7	20.3	9.3
		±SD	3.5	3.6	7.1	3.1	3.1		2.6	6.1	9.0	2.5	1.5
	Fold Increase		0.8	1.0	1.0	0.9	0.9		0.9	1.0	1.0	0.9	1.0
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.003	Mean	23.3	106.0	260.3	20.7	9.3		20.3	104.3	262.3	20.3	9.7
		±SD	2.5	4.6	9.3	2.5	1.5		1.5	5.0	9.3	1.5	2.5
	Fold Increase		0.9	0.9	1.0	0.9	0.9		0.8	1.0	1.0	0.9	1.0
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.01	Mean	23.3	102.3	263.3	20.3	9.7		20.7	107.0	263.7	21.7	9.3
		±SD	4.0	7.8	9.1	4.9	2.5		3.1	5.6	7.0	2.1	1.5
	Fold Increase		0.9	0.9	1.0	0.9	1.0		0.8	1.0	1.0	1.0	1.0
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.03	Mean	21.0	99.0	260.3	18.3	7.0		18.7	88.7	266.0	18.0	6.7
		±SD	2.0	3.6	8.6	2.5	1.0		1.5	2.5	7.5	1.0	1.5
	Fold Increase		0.8	0.9	1.0	0.8	0.7		0.8	0.8	1.0	0.8	0.7
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.1	Mean	13.7	59.3	210.0	13.0	4.0		14.0	58.3	210.3	13.0	3.7
		±SD	2.1	4.2	8.2	2.0	1.0		1.0	3.1	5.5	1.0	0.6
	Fold Increase		0.5	0.5	0.8	0.6	0.4		0.6	0.5	0.8	0.6	0.4
	Lawn Intensity		3+	3+	3+	3+	3+		3+	3+	3+	3+	3+
Positive Control	100 µL of respective Positive Control	Mean	356.7	405.0	595.0	128.3	110.3		347.7	402.0	596.7	113.0	109.3
		±SD	4.9	5.6	16.7	7.5	18.2		7.5	5.6	12.7	9.6	9.5
	Fold Increase		13.0	3.6	2.3	5.6	11.0		14.1	3.7	2.3	5.1	11.7
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
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:

In an in vitro bacterial reverse mutation assay (Ames) according to OECD guideline 471, the test item did not show a mutagenic potential.

Executive summary:

The test item was evaluated for mutagenicity in an in vitro bacterial reverse mutation assay according to OECD 471. The test item was found soluble in dimethyl sulphoxide at a concentration of 50 mg/mL. In an preliminary cytotoxicity test, the tester strain Salmonella typhimurium TA100 was exposed to vehicle control, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 and 5 mg/plate. Precipitation of the test item was observed at 3, 4 and 5 mg/plate. Cytotoxicity was observed at all the tested concentration in an initial cytotoxicity test. To further assess the cytotoxicity and to select the appropriate test concentration for the mutation assay a follow up cytotoxicity test was performed at 0.4, 0.2, 0.1, 0.05, 0.025, 0.0125, 0.00625 and 0.003125 mg/plate. Cytotoxicity was observed, indicated by a thinning of the bacterial background lawn at 0.1, 0.2 and 0.4 mg/plate. Based on these results, concentrations of 0.001, 0.003, 0.01, 0.03 and 0.1 mg/plate were selected for the main mutation test.

The following Salmonella typhimurium tester strains were tested: TA98, TA100, TA 102, TA1535, and TA1537. The test item was tested at the concentrations of 0.001, 0.003, 0.01, 0.03 and 0.1 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 test and trial II as pre incubation test.

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 increase in number of revertant colonies at any of the tested concentrations in both the trials. The mean numbers of revertant colonies of vehicle control values were comparable to negative control values. The number of revertant colonies in the positive controls resulted in 2.3 to 14.1 fold increase under identical conditions. The positive controls and solvent controls were valid.

Based on the results of the study, it is concluded that the test item is “non-mutagenic” in the Bacterial Reverse Mutation Test up to the highest tested concentration of 0.1 mg/plate under the described test conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The test item was evaluated for mutagenicity in an in vitro bacterial reverse mutation assay according to OECD 471. The test item was found soluble in dimethyl sulphoxide at a concentration of 50 mg/mL. In an preliminary cytotoxicity test, the tester strain Salmonella typhimurium TA100 was exposed to vehicle control, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 and 5 mg/plate. Precipitation of the test item was observed at 3, 4 and 5 mg/plate. Cytotoxicity was observed at all the tested concentration in an initial cytotoxicity test. To further assess the cytotoxicity and to select the appropriate test concentration for the mutation assay a follow up cytotoxicity test was performed at 0.4, 0.2, 0.1, 0.05, 0.025, 0.0125, 0.00625 and 0.003125 mg/plate. Cytotoxicity was observed, indicated by a thinning of the bacterial background lawn at 0.1, 0.2 and 0.4 mg/plate. Based on these results, concentrations of 0.001, 0.003, 0.01, 0.03 and 0.1 mg/plate were selected for the main mutation test.

The following Salmonella typhimurium tester strains were tested: TA98, TA100, TA 102, TA1535, and TA1537. The test item was tested at the concentrations of 0.001, 0.003, 0.01, 0.03 and 0.1 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 test and trial II as pre incubation test.

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 increase in number of revertant colonies at any of the tested concentrations in both the trials. The mean numbers of revertant colonies of vehicle control values were comparable to negative control values. The number of revertant colonies in the positive controls resulted in 2.3 to 14.1 fold increase under identical conditions. The positive controls and solvent controls were valid.

Based on the results of the study, it is concluded that the test item is “non-mutagenic” in the Bacterial Reverse Mutation Test up to the highest tested concentration of 0.1 mg/plate under the described test conditions.

Justification for classification or non-classification

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