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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The results of the Ames Salmonella plate incorporation assay (OECD Guideline 471 (Bacterial Reverse Mutation Assay)) indicate, that the test compound 2-lauroyloxyethyltrimethylammonium chloride did not induce base pair or frame shift mutations with and without metabolic activation.

Furthermore, 2-lauroyloxyethyltrimethylammonium chloride does not induce structural chromosomal aberrations in CHO cells in the presence or absence of S9 metabolic activation (OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)).

Treatment of L5178Y mouse lymphoma cells with 2-lauroyloxyethyltrimethylammonium chloride did not induce gene mutations to trifluorothymidine (TFT) -resistance with and without metabolic activation (OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)).

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Remarks:
and TA 1538
Metabolic activation:
with and without
Metabolic activation system:
S9-mix, rat liver
Test concentrations with justification for top dose:
0.24, 1.20, 6.00, 30.0 and 150.0 µg/plate
Vehicle:
Solvent dimethylsulfoxide (DMSO)
Negative controls:
yes
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-aminoanthracene
Key result
Species / strain:
other: TA 1535, TA 1537, TA 1538, TA 98, TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Conclusions:
The results of the Ames Salmonella plate incorporation assay indicate, that the test compound lauroyl choline chloride did not induce base pair or frame shift mutations with and without metabolic activation under the test conditions reported.
Executive summary:

Lauroyl choline chloride was assayed in a bacterial gene mutation plate incorporation assay (Ames test) using the strains TA 100, TA 1535, TA 98, TA 1537 and TA 1538 according to OECD guideline no.471. Induced his( + )revertants were determined both in the absence and presence of metabolic activation by a rat liver post-mitochondrial fraction (S9) from Aroclor 1254 induced animals.

Depending on a preliminary toxicity experiment, in two independent mutation experiments, cells were exposed to concentrations of 0.24, 1.20, 6.00, 30.0 and 150.0 μg per plate in the absence and presence of S9 (1st and 2nd experiment). In order to demonstrate the sensitivity of the assay system, positive control agents were used and marked increases in his(+)-revertants were induced in all tester-strains.

In the first and second assay, lauroyl choline chloride did not induce statistically significant increases in histidine-prototroph revertants in any tester-strains with and without metabolic activation in the tested concentration range.

It is thus concluded that lauroyl choline chloride did not induce gene mutations in the bacterial mutagenicity assay with and without metabolic activation in vitro when tested under the experimental conditions reported.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Test material information:
Composition 1
Species / strain:
Chinese hamster Ovary (CHO)
Details on mammalian cell lines (if applicable):
The experiments were performed with the strain K1-BH(4) of Chinese hamster ovary cells. The modal chromosome number is 21. The cells were supplied by Dr.Kirkland, Microtest Research Ltd., Heslington, York, UK.
Metabolic activation:
with and without
Negative controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and conditions:
Activation System
S9 Homogenate
The 9000 g supernatant of rat liver homogenate (2) was purchased from CCR GmbH & Co.KG, 64380 Roßdorf, FRG, and used in tests with metabolic activation.

S9 Mix
The S9 mix contains per 5 ml:
NADP (Na salt) (25 mg/ml) - 1.0 ml
d-glucose-6-phosphate (180 mg/ml) - 1.0 ml
Rat liver homogenate - 2.0 ml
150 mM KCl - 1.0 ml
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Conclusions:
It is concluded that under the experimental conditions employed in this study, lauroyl choline chloride does not induce structural chromosomal aberrations in CHO cells in the presence or absence of S9 metabolic activation.
Executive summary:

Lauroyl choline chloride was assayed in an in vitro cytogenetic assay using cultures of Chinese hamster ovary (CHO) cells both in the absence and presence of metabolic activation by a rat liver post-mitochondrial fraction (S9) from Aroclor-1254 induced animals.

In an initial cytotoxicity and cell cycle experiment, dose levels and sampling times for the main study were determined on the basis of the mitotic indices and distribution of division metaphases in bromodeoxyuridine (BUdR) labelled cultures.

Two independent experiments for the induction of chromosomal aberrations were performed. In the absence of S9, cells were exposed to the test article for 15 hours at concentrations of 5.00, 10.0, 20.0, 30.0, 40.0, 50.0, 60.0 and 70.0 μg/ml (1st experiment) or for 15 hours at concentrations of 10.0, 20.0, 40.0, 60.0, 80.0 and 100.0 μg/ml (2nd experiment). With S9 added, cells were treated for two hours with concentrations of 25.0, 50.0, 100.0, 150.0, 200.0, 250.0, 300.0 and 350 μg/ml (1st experiment) or 25.0, 50.0, 75.0, 100.0, 125.0 and 150.0 μg/ml (2nd experiment). Following treatment, the cells were allowed to recover for 18h and 23 hours (1st experiment) or 18 hours (2nd experiment). This way metaphases were harvested at 20 and 25 hours (1st experiment) or at 20 hours (2nd experiment) after the start of treatment. Three parallel cultures were set up at each experimental point, and one of them was inoculated with BUdR for cell cycle analysis.

In order to demonstrate the sensitivity of the assay system, methylmethanesulphonate (10 μg/ml, without S9) and cyclophosphamide (6.25 μg/ml, S9 activated) were used as positive control agents. Both compounds induced statistically significant increases in cells bearing chromosomal aberrations.

With respect to cell cycle kinetics and cytotoxicity, the following lauroylcholine chloride treated cultures were chosen for chromosome analysis:

1st experiment

-S9 30.0, 50.0, 70.0 μg/ml (15 h sampling time)

+S9 25.0, 50.0, 100.0 μg/ml (20 h sampling time)

2nd experiment

-S9 10.0, 40.0, 80.0 μg/ml (15 h sampling time)

+S9 50.0, 100.0, 150.0 μg/ml (20 h sampling time)

From the chromosome analysis, no biologically relevant or statistically significant increase in structural chromosome aberrations was obtained at any concentration tested. This applied both to the absence and presence of S9 metabolic activation.

Conclusion: It is thus concluded that lauroyl choline chloride did not induce structural chromosome aberrations in Chinese hamster ovary cells with or without metabolic activation in vitro when tested up to concentrations which induced marked cytotoxicity.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell transformation assay
Test material information:
Composition 1
Target gene:
thymidine-kinase-locus
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
S9 liver homogenate prepared from Aroclor-1254 induced rats
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
methylmethanesulfonate
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Conclusions:
Treatment of L5178Y mouse lymphoma cells with Lauroyl choline chloride did not induce gene mutations to trifluorothymidine
(TFT) -resistance with and without metabolic activation under the test conditions.
Executive summary:

Lauroyl choline chloride was assayed in an in vitro gene mutation assay using cultures of mouse lymphoma L5178Y TK+/- cells according to OECD-guideline-no. 476. Induced mutants to trifluorothymidine (TFT)-resistance were determined both in the absence and presence of metabolic activation by a rat liver post-mitochondrial fraction (S-9) from Aroclor-1254 induced animals.

In the first experiment of the study, comprising two independent experiments, cells were exposed to concentrations of 3.12, 6.25, 12.5, 25.0, 50.0, 75.0 and 100.0 μg test substance per ml in the absence and in the presence of S9 for 2 hours. Two parallel cultures were set up at each experimental point. In order to demonstrate the sensitivity of the assay system, methylmethanesulphonate (50.0 μg/ml, without S-9) and benz-α-pyrene (3.0 μg/ml, with S9) were used as positive control agents and both compounds induced statistically significant increases in TK-/- -mutants.

After treatment without metabolic activation, the survival at a concentration of 100 μg/ml was reduced to 39.4% of the control value, with S9 survival of Lauroyl choline chloride treatments at the same concentration was reduced to 6.80% of the control cultures. Concentrations of 25.0, 50.0, 75.0 and 100.0 μg/ml without S9 and 12.5, 25.0, 50.0 and 75.0 μg/ml with S9 were plated out for viability and TFT resistance. With and without metabolic activation Lauroyl choline chloride did not induce statistically significant increases in TFT-mutant frequencies at all concentration levels tested.

In the second experiment, this result was confirmed. Neither with nor without S9 addition were any biologically relevant or statistically significant increases in mutation frequencies found.

It is thus concluded that lauroyl choline chloride did not induce gene mutations in mouse lymphoma L5178Y TK +/cells in the thymidine-kinase-locus with and without metabolic activation in vitro when tested under the experimental conditions reported.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

No positive results were found in mutagenicity and cytogenicity studies on bacteria and mammalian cells in which 2-lauroyloxyethyltrimethylammonium chloride itself were used. Therefore, the substance is regarded as not mutagenic and will accordingly not be classified.