β-Alanine, N-(2-carboxyethyl)-, N-coco alkyl derivs., disodium salts

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

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: Full GLP report to current guidelines, 2013

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

β-Alanine, N-(2-carboxyethyl)-, N-coco alkyl derivs., disodium salts

EC Number:

290-476-8

EC Name:

β-Alanine, N-(2-carboxyethyl)-, N-coco alkyl derivs., disodium salts

Cas Number:

90170-43-7

IUPAC Name:

β-Alanine, N-(2-carboxyethyl)-, N-coco alkyl derivs., disodium salts

Details on test material:

Sponsor's identification : β-Alanine, N-(2-carboxyethyl)-, N-coco alkyl derivs., disodium salts (EC 290-476-8)
Description : Pale yellow liquid, concentration 29.9% in water
Batch number : 89099
Formulations were adjusted so all concentrations reported relate to the actives
Date received : 06 December 2012
Expiry date : 1 year after receipt
Storage conditions : Room temperature in the dark

Method

Target gene:

Assessment of the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital/-naphthoflavone

Test concentrations with justification for top dose:

The dose range used in the preliminary toxicity test was 19.53 to 5000 μg/ml for all three of the exposure groups.
Experiment 1 treatments were performed in duplicate (A + B), both with and without metabolic activation (S9-mix) at eight dose levels of the test item (19.53 to 625 μg/ml in both the absence and presence of metabolic activation)
Experiment 2 treatments were performed in duplicate (A + B), both with and without metabolic activation (S9-mix) at eight dose levels of the test item (5 to 160 μg/ml in the absence of metabolic activation, and 100 to 450 μg/ml in the presence of metabolic activation),

Vehicle / solvent:

Water

Controls

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

The treatment vessels were incubated at 37°C for 4 hours with continuous shaking using an orbital shaker within an incubated hood.
At the end of the treatment period, for each experiment, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 105 cells/ml. The cultures were incubated at 37 °C with 5% CO2 in air and subcultured every 24 hours for the expression period of two days by counting and diluting to 2 x 105 cells/ml.
On Day 2 of the experiment, the cells were counted, diluted to 104 cells/ml and plated for mutant frequency (2000 cells/well) in selective medium containing 4 μg/ml 5-trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/ml and plated (2 cells/well) for viability (%V) in non-selective medium.

Evaluation criteria:

The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.

Statistics:

Small significant increases designated by the UKEMS statistical package will be reviewed using the above criteria, and may be disregarded at the Study Director's discretion.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In all three of the exposure groups there was evidence of marked dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls. The toxicity was very steep in all three exposure groups.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

The steep toxicity curve observed proved that it would be hard to achieve optimum toxicity. The excessive toxicity observed at and above 468.75 μg/ml in the absence of metabolic activation and at 625 μg/ml in the presence of metabolic activation resulted in these dose levels not being plated for viability or 5-TFT resistance. In the presence of metabolic activation a dose level (468.75 μg/ml) was later excluded from statistical analysis as the RTG value also fell below the acceptable level of toxicity.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.

Executive summary:

Valid study, but difficulties in finding optimum concentrations for test due to sharp cut-off in toxicity. This is considered common with surface active agents that will disrupt membranes at criticial concentrations.