Hexadecyl(2-hydroxyethyl)dimethylammonium dihydrogen phosphate

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Hexadecyl(2-hydroxyethyl)dimethylammonium dihydrogen phosphate

EC Number:

287-619-1

EC Name:

Hexadecyl(2-hydroxyethyl)dimethylammonium dihydrogen phosphate

Cas Number:

85563-48-0

Molecular formula:

C20H44NO.H2O4P

IUPAC Name:

dihydrogen hexadecyl(2-hydroxyethyl)dimethylazanium phosphate

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): Product name, 00/0259-4
- Physical state: liquid, yellowish, clear
- Expiration date of the lot/batch: 22 Aug 2016
- Stability under test conditions: The stability of the test substance under storage conditions is guaranteed until 22 Aug 2016
- Storage condition of test material: room temperature

Method

Target gene:

histidine gene

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 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

Controls

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

Results and discussion

Test results

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'.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.