Hexadecyl(2-hydroxyethyl)dimethylammonium dihydrogen phosphate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: - Key: OECD 471 (2015): negative with and without metabolic activation - Supporting: OECD 471 (1983): negative with and without metabolic activation in-vitro micronucleus test (OECD 487): in-vitro mammalian cell gene mutation test (HPRT, OECD 476): negative with and without activation

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well performed guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine gene

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 fraction

Test concentrations with justification for top dose:

SPT: 0, 33, 100, 333, 1000, 2500, 5000 µg/L
PIT: 0, 1, 3.3, 10, 33, 100, 333 µg/L

Vehicle / solvent:

water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: 2-aminoanthracene, N-methyl-N'-nitro-N-nitrosoguanidine, 4-nitro-o-phenylenediamine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

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

Evaluation criteria:

Generally, the experiment was considered valid if the following criteria were met:
• The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
• The sterility controls revealed no indication of bacterial contamination.
• The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
• Fresh bacterial culture containing approximately 109 cells per mL were used.
The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control data range under all experimental conditions in at least two experiments carried out independently of each other.

Statistics:

Arithmetic mean and standard deviation of back-mutant colonies per plate

Species / strain:

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

Remarks:

A strong bacteriotoxic effect was observed depending on the strain and test conditions from about 100 μg/plate onward

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Precipitation of the test substance was found depending on the
test conditions from about 100 μg/plate onward.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

SPT without metabolic activation (1st Run):

	Mean revertants per plate
	TA 1535	TA 100	TA 1537	TA 98	E. coli
Control	8.3	97.7	9.0	14.0	28.0
33 µg/plate	11.7	101.0	6.7	17.7	21.3
100 µg/plate	10.0	64.0	6.7	21.7	19.7
333 µg/plate	0.0	0.0	0.0	0.0	0.0
1000 µg/palte	0.0	0.0	0.0	0.0	0.0
2500 µg/plate	0.0	0.0	0.0	0.0	0.0
5000 µg/plate	0.0	0.0	0.0	0.0	0.0
Positive control	4472.7	3568.3	645.7	461.7	1265.3

SPT with metabolic activation:

	Mean revertants per plate
	TA 1535	TA 100	TA 1537	TA 98	E. coli
Control	7.7	94.0	8.0	22.0	24.3
33 µg/plate	8.3	102.7	8.7	24.3	21.3
100 µg/plate	11.0	8.3	8.3	13.3	22.0
333 µg/plate	8.7	7.3	2.7	4.0	13.0
1000 µg/palte	0.0	0.0	0.0	0.0	0.0
2500 µg/plate	0.0	0.0	0.0	0.0	0.0
5000 µg/plate	0.0	0.0	0.0	0.0	0.0
Positive control	284.0	2360.3	213.7	2427.3	149.0

SPT without metabolic activation (2nd Run):

	Mean revertants per plate
	TA 1535	TA 100	TA 1537	TA 98	E. coli
Control	12.3	97.7	11.0	20.0	31.3
1.0 µg/plate	13.7	101.0	11.3	20.0	30.0
3.3 µg/plate	9.3	92.7	9.7	18.3	29.0
10 µg/plate	13.0	109.0	12.0	21.0	29.0
33 µg/palte	12.7	97.0	8.0	21.0	22.3
100 µg/plate	13.7	68.3	8.7	9.0	15.3
333 µg/plate	3.0	0.0	0.0	0.0	0.0
Positive control	4570.3	3632.7	897.7	440.3	1281.3

SPT with metabolic activation (2nd Run):

	Mean revertants per plate
	TA 1535	TA 100	TA 1537	TA 98	E. coli
Control	9.7	97.7	14.3	23.3	28.0
1.0 µg/plate	10.0	97.7	10.7	25.7	34.4
3.3 µg/plate	12.0	84.0	10.7	24.7	21.0
10 µg/plate	11.0	95.0	11.0	24.3	30.3
33 µg/palte	11.3	109.0	12.7	24.7	29.3
100 µg/plate	12.0	99.0	12.7	23.7	24.0
333 µg/plate	6.3	7.7	5.0	9.0	7.7
Positive control	256.3	1840.0	192.7	1980.7	166.7

PIT without metabolic activation (1st Run):

	Mean revertants per plate
	TA 1535	TA 100	TA 1537	TA 98	E. coli
Control	9.0	100.7	6.3	18.0	22.3
1.0 µg/plate	8.0	84.3	7.0	13.3	27.0
3.3 µg/plate	8.3	98.3	5.3	18.0	19.3
10 µg/plate	10.7	101.0	8.3	18.7	17.7
33 µg/palte	9.3	86.7	7.0	13.0	24.0
100 µg/plate	3.7	33.7	3.7	6.0	26.0
333 µg/plate	0.0	0.0	0.0	0.0	0.0
Positive control	2553.0	1885.0	498.7	473.3	297.3

PIT with metabolic activation (1st Run):

	Mean revertants per plate
	TA 1535	TA 100	TA 1537	TA 98	E. coli
Control	10.7	98.0	6.0	23.7	21.3
1.0 µg/plate	10.3	127.3	8.0	26.0	23.7
3.3 µg/plate	10.0	121.7	9.3	24.3	25.0
10 µg/plate	10.3	105.7	9.7	29.0	25.3
33 µg/palte	8.3	113.7	6.0	20.0	22.7
100 µg/plate	10.3	110:7	5.7	30.0	20.7
333 µg/plate	6.0	9.7	0.0	0.0	0.0
Positive control	234.0	1880.7	139.0	1391.0	90.0

Conclusions:

Interpretation of results (migrated information):
negative

Under the experimental conditions of this study, the test substance was not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

Executive summary:

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains (TA 1535, TA 100, TA 1537, TA 98, E.coli WP2 uvrA) in a reverse mutation assay. The mutagenic potential was tested in a dose range of 1.0 o 5000 µg/plate in the SPT and in a range of 1.0 to 333 µg/plate in the PIT. The PIT and the SPT were both performed with and without metabolic activation (liver S9 mix from induced rats). Precipitation of the test substance was found depending on the strain and test conditions from about 100 µg/plate onward. A strong bacteriotoxic effect was observed depending on the strain and test conditions from about 100 µg/plate onward. A relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Bacterial reverse mutation test

Key study:

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains (TA 1535, TA 100, TA 1537, TA 98, E.coli WP2 uvrA) in a reverse mutation assay. The mutagenic potential was tested in a dose range of 1.0 o 5000 µg/plate in the SPT and in a range of 1.0 to 333 µg/plate in the PIT. The PIT and the SPT were both performed with and without metabolic activation (liver S9 mix from induced rats). Precipitation of the test substance was found depending on the strain and test conditions from about 100 µg/plate onward. A strong bacteriotoxic effect was observed depending on the strain and test conditions from about 100 µg/plate onward. A relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system.

Under the experimental conditions of this study, the test substance was not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

Supporting study:

The test item was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium. The investigations were performed on strains TA 98, TA 100, TA 1535 and TA 1537 with the following concentrations of the trial substance without and with microsomal activation: 1, 3, 9, 27 and 81 µg/plate. In order to confirm the results, the experiments were repeated with the concentrations of 6, 18, 54, 162 nad 486 µg/plate.

These tests permit the detection of point mutations in bacteria induced by chemical substances. Any mutagenic effects of the substances are demonstrable on comparison of the number of bacteria in the treated and control culures that have undergone back-mutation to histidine-phototrophism. To ensure that mutagenic effects of metabolites of the test substances formed in mammals would also be detected, experiments were performed in which the cultures were additionally treated with an activation mixture (rat liver microsomes and co-factors).

In the experiments performed without and with microsomal activation, comparison of the number of back-mutant colonies in the controls and the cultures treated with the various concentrations of the test item revealed a reduction in the colony count due to a growth-inhibiting effect of the compound at the upper concentrations.

No evidence of the induction of point mutations by the test item or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium used in these experiments.

Mammalian cell gene mutation assay

The substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Two independent experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation). According to an initial range-finding cytotoxicity test for the determination of the experimental doses and taking into account the cytotoxicity actually found in the main experiments, the following concentrations were tested. Test groups printed in bold type were evaluated for gene mutations:

1st Experiment:

- without S9 mix: 0; 2.3; 4.7; 9.4; 18.8; 37.5; 75.0; 150.0 μg/mL

- with S9 mix: 0; 2.3; 4.7; 9.4; 18.8; 37.5; 75.0; 150.0 μg/mL

2nd Experiment:

- without S9 mix: 0; 3.1; 6.3; 12.5; 25.0; 50.0; 100.0 μg/mL

- with S9 mix: 0; 3.1; 6.3; 12.5; 25.0; 50.0; 100.0; 150.0 μg/mL

Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in 6-thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted. The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, ethyl methanesulfonate (EMS) and 7,12-dimethylbenz[a]- anthracene (DMBA), led to the expected increase in the frequencies of forward mutations. In this study, in the 1st and 2nd Experiment, the highest concentrations evaluated for gene mutations were clearly cytotoxic in the absence and the presence of metabolic activation. Based on the results of the present study, the test substance did not cause any relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other.

Under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

In vitro Micronucleus Assay in V79 cells

The test substance was assessed for its potential to induce micronuclei in V79 cells in vitro (clastogenic or aneugenic activity). Two independent experiments were carried out, both with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation). According to an initial range-finding cytotoxicity test for the determination of the experimental doses, the following concentrations were tested. The test groups printed in bold type were evaluated.

1st Experiment

4 hours exposure, 24 hours harvest time, without S9 mix

0; 3.13; 6.25; 12.50; 25.00; 50.00 and 100.00 μg/mL

4 hours exposure, 24 hours harvest time, with S9 mix

0; 6.25; 12.50; 25.00; 50.00; 100.0 and 200.00 μg/mL

2nd Experiment

24 hours exposure, 24 hours harvest time, without S9 mix

0; 1.56; 3.13; 6.25; 12.50; 25.00 and 50.00 μg/mL

4 hours exposure, 44 hours harvest time, with S9 mix

0; 3.13; 6.25; 12.50; 25.00; 50.00 and 100.00 μg/mL μg/mL

A sample of at least 1000 cells for each culture were analyzed for micronuclei, i.e. 2000 cells for each test group. The negative controls gave frequencies of micronucleated cells within our historical negative control data range for V79 cells. Both positive control substances, ethyl methanesulfonate (EMS) and cyclophosphamide (CPP), led to the expected increase in the number of cells containing micronuclei. Cytotoxicity indicated by clearly reduced cell count (given as relative population doubling [RPD]) or proliferation index (CBPI) or low slide quality was observed at least at the highest applied test substance concentrations in all experimental parts of this study. On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system.

Justification for selection of genetic toxicity endpoint
Reliable and well documented study which is the basis for classification.

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available studies are considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance is not considered to be classified for genetic toxicity under Directive 67/548/EEC, as amended for the 31st time in Directive2009/2/EC.

 Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the fifth time in Directive EC 944/2013.