N,N-dimethyl-N-tetradecyltetradecan-1-aminium chloride

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides

EC Number:

269-924-1

EC Name:

Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides

Cas Number:

68391-05-9

Constituent 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

Constituent 3

Reference substance name:

Propan-2-ol

EC Number:

200-661-7

EC Name:

Propan-2-ol

Cas Number:

67-63-0

Molecular formula:

C3H8O

IUPAC Name:

propan-2-ol

Test material form:

liquid

Method

Target gene:

his (Salmonella typhimurium strains), trp (E. coli)

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

0.316, 1.0, 3.16, 10.0, 31.6 and 100 μg
Pronounced cytotoxicity was noted starting at a concentration of 100 μg/plate in the preliminary cytotoxicity tests.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: test item was not soluble in highly purified water or dimethylsulfoxide

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: 1st experiment: in agar (plate incorporation); 2nd experiment: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 - 72 h

NUMBER OF REPLICATIONS: 3


DETERMINATION OF CYTOTOXICITY
- Method: background lawn

Evaluation criteria:

A test item is considered to show a positive response if
- the number of revertants is significantly increased (p ≤ 0.05, U-test according to MANN and WHITNEY) compared to the solvent control to at least 2-fold of the solvent control for the Salmonella typhimurium test strains TA98, TA100, TA1535, TA1537 and Escherichia coli test strain WP2 uvrA in both independent experiments.
- in addition, a significant (p ≤ 0.05) concentration (log value)-related effect (Spearman’s rank correlation coefficient) is observed;
Biological relevance of the results should be considered first.
Positive results have to be reproducible and the histidine or tryptophan independence of the revertants has to be confirmed by streaking random samples on histidine or tryptophan-free agar plates.
A test item for which the results do not meet the above mentioned criteria is considered as non-mutagenic in the AMES test.

Results and discussion

Test results

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not reported
- Effects of osmolality: not reported
- Precipitation: not reported

HISTORICAL CONTROL DATA
- see attachment

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation, pronounced cytotoxicity (scarce background lawn and reduction of the number of revertants) were noted at the top concentration of 100 μg/plate, in all Salmonella typhimurium strains and in the Escherichia coli strain WP2 uvrA [pKM101].

Any other information on results incl. tables

Plate incorporation, without metabolic activation
		TA98	TA100	TA1535	TA1537	E. coli WP2
0.316	mean SD	27.3 1.2	117.0 2.6	15.3 1.5	6.3 1.2	30.0 0.0
1.0	mean SD	36.3 0.6	118.7 4.6	17.0 1.0	5.7 2.1	29.3 2.9
3.16	mean SD	36.3 1.5	127.0 13.1	17.3 1.5	7.7 0.6	37.7 8.7
10	mean SD	39.0 6.1	128.3 7.6	16.7 1.5	5.7 1.2	45.3 6.4
31.6	mean SD	29.0 15.5	106.3 2.1	18.0 1.7	9.0 1.0	53.7 6.8
100	mean SD	12.0 # 1.0	66.7 # 0.6	8.7 # 1.2	2.0 # 0.0	14.3 # 0.6
Negative control	mean SD	29.0 6.2	131.3 15.4	25.0 0.0	6.7 0.6	51.3 3.1
Positive control	mean SD	143.3 1.5	990.3 4.9	149.3 0.6	97.3 9.5	205.3 4.9
Plate incorporation, with metabolic activation
0.316	mean SD	33.3 1.2	143.3 17.0	17.3 2.1	5.3 2.3	42.7 2.1
1.0	mean SD	26.7 5.5	131.0 15.6	20.7 7.4	7.0 1.7	45.7 6.4
3.16	mean SD	33.0 4.6	128.0 22.3	17.7 0.6	7.0 1.7	47.0 4.6
10	mean SD	25.7 2.5	131.0 2.6	17.7 0.6	7.3 1.2	41.3 4.0
31.6	mean SD	31.0 7.8	129.3 3.2	16.3 0.6	7.0 0.0	39.0 14.4
100	mean SD	12.7 # 1.2	68.0 # 3.6	8.3 # 0.6	2.0 # 0.0	14.3 # 1.5
Negative control	mean SD	36.7 0.6	120.7 2.1	17.0 1.0	6.7 1.2	48.3 12.1
Positive control	mean SD	145.7 4.0	980.0 30.8	152.3 10.4	85.0 17.3	184.7 35.9
Preincubation, without metabolic activation
0.316	mean SD	30.7 4.7	152.7 7.6	19.0 1.0	6.3 0.6	45.0 1.7
1.0	mean SD	26.0 9.5	164.3 18.5	22.0 2.6	7.0 1.0	53.3 5.7
3.16	mean SD	30.7 9.3	160.7 22.9	21.7 1.5	7.3 1.2	52.7 2.1
10	mean SD	41.7 6.4	167.3 12.4	24.7 4.0	7.3 0.6	36.0 10.4
31.6	mean SD	31.0 1.7	108.7 5.9	24.3 3.1	6.3 0.6	43.0 1.0
100	mean SD	14.7 # 0.6	51.0 # 1.0	7.7 # 0.6	2.0 # 0.0	15.7 # 0.6
Negative control	mean SD	28.3 3.2	174.0 3.6	27.7 0.6	5.3 0.6	41.7 18.9
Positive control	mean SD	146.0 3.6	845.3 4.0	193.0 1.0	86.3 2.3	143.0 28.2
Preincubation, with metabolic activation
0.316	mean SD	30.7 3.5	139.3 24.6	25.0 4.6	6.3 2.1	50.3 1.5
1.0	mean SD	26.3 4.7	138.7 28.5	23.7 4.6	6.3 2.1	54.3 4.2
3.16	mean SD	29.7 5.5	111.7 1.2	20.0 0.0	6.3 1.2	57.7 1.2
10	mean SD	29.0 4.6	122.0 4.6	22.0 3.6	4.7 0.6	54.0 7.0
31.6	mean SD	23.3 3.1	109.7 2.1	21.0 2.0	8.0 1.7	44.7   0.6
100	mean SD	13.7 # 1.5	59.3 # 8.3	7.3 # 0.6	2.0 # 0.0	15.3 # 1.2
Negative control	mean SD	31.7 9.0	177.3 33.2	22.7 1.5	7.7 1.5	49.7 1.5
Positive control	mean SD	137.3 5.7	830.3 16.2	153.0 13.0	80.3 5.5	159.7 3.1

Applicant's summary and conclusion

Conclusions:

Di-C12-18 alkyldimethyl ammonium chloride was not mutagenic in this bacterial reverse mutation assay in the presence and absence of metabolic activation.

Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471 (1997) and EU method B.13/14 (2008), Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and E. coli WP2 uvr A were exposed to Di-C12-18 alkyldimethyl ammonium chloride in ethanol in concentrations of 0 (control), 0316, 1.0, 3.16, 10.0, 31.6 and 100 µg/plate in all strains in the absence and presence of mammalian metabolic activation (rat liver S9 mix). The assay was performed using the plate incorporation method (1st experiment) and pre-incubation method (2nd experiment; 20 min pre-incubation).

The test substance was tested up to cytotoxic concentrations. Pronounced cytotoxicity was noted at 100 μg/plate in in all strains.

The positive control items showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system. The results of the negative and positive control cultures were within the range of the historical data. Hence, all acceptance criteria are met.

No increase in revertant colony numbers as compared with control counts was observed for the test item in the Salmonella typhimurium and in the Escherichia coli test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation and preincubation test).

Under the conditions of the study, the test substance was negative for mutagenic potential.