DL-alanine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

The recommended 5th bacterial strain (E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102) is not used in this study

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

his-

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Remarks:

S.typh. strain TA94 and TA92 also used

Metabolic activation:

with

Metabolic activation system:

S-9

Test concentrations with justification for top dose:

at least five different concentrations, up to 20.0 mg/plate

Vehicle / solvent:

Phosphate buffer

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Details on test system and experimental conditions:

Cells cultured overnight were pre-incubated with both the test sample and the S-9 mix for 20 min at 37 °C before plating. Duplicate plates were used for each of six different concentrations of the sample. The number of revertant (his+) colonies was scored after incubation at 37 °C for 2 days. The result was considered positive if the number of colonies found was twice the number in the control (exposed to the appropriate solvent or untreated). If no reasonable dose response was detected, additional experiments using different doses or induced mutation frequency assays (Yoshikawa, Nakadate, Watabe et al. 1980) were performed.

Evaluation criteria:

The result was considered positive if the number of his+ colonies found was twice the number in the control (exposed to the appropriate solvent or untreated). The maximum dose for positive results in the Ames test represents the dose at which the maximum effect was obtained, while the maximum dose for negative results represents the highest non-cytotoxic dose used in the experimet.
A negative result indicates that no significant increases in the numbers of revertant colonies were detected in any S.typhimurium strains at the maximum dose.

Statistics:

not reported

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

not examined

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not examined

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not examined

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not examined

Key result

Species / strain:

S. typhimurium, other: TA92 and TA94

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not examined

Conclusions:

DL-Alanine was negative with metabolic activation in all trains tested.

Executive summary:

In a reverse gene mutation test in bacteria (Ames test), strains TA 1535, TA 1537, TA 92, TA 98, TA94 and TA 100 of S. typhimurium were exposed to DL-Alanine in phosphate buffer in at least six different concentrations up to 20 mg/plate in the presence of mammalian metabolic activation (rat S9, preincubation). There was no evidence of induced mutant colonies over background.

The study was performed in accordance to OECD guideline 471 with minor deviations. Therefore, the results can be considered to be reliable.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1984

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

not specified

Type of assay:

other: in vitro mammalian chromosome aberration test (migrated information)

Test material

Test material information

Constituent 1

Reference substance name:

DL-alanine

EC Number:

206-126-4

EC Name:

DL-alanine

Cas Number:

302-72-7

Molecular formula:

C3H7NO2

IUPAC Name:

DL-alanine

Method

Species / strain

Species / strain / cell type:

other: Chinese hamster fibroblast CHL

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: The cell line was originally established from the lung of a newborn female at the Cancer Research Institute, Tokyo (Koyama, Utakoji & Ono, 1970)
- Methods for maintenance in cell culture if applicable: maintained by 4-day passages in Medium (see below)
- Modal number of chromosomes: 25
- Normal (negative control) cell cycle time: 15hr

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: MEM (GIBCO) supplemented with 10 % calf serum
- Properly maintained: yes

Metabolic activation:

without

Test concentrations with justification for top dose:

three different doses
max. 1.0 mg/ml

Vehicle / solvent:

physiological saline

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

no

Details on test system and experimental conditions:

METHOD OF APPLICATION: The cells were exposed to each sample at three different doses for 24 and 48 hr. In the present studies, no metabolic activation systems were applied. The maximum dose of each sample was selected by a preliminary test in which the dose needed for 50% cell-growth inhibition was estimated using a cell densitometer (Monocellater, Olympus Co., Ltd). Previous studies indicated that the osmotic pressure of the medium generally rose with sample concentrations of more than 10 mM, so that the maximum dose for some samples was limited to around this level, at which cytotoxic effects were not necessarily observed.

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (final concn 0.2 /µg/ml)

STAIN (for cytogenetic assays): After air-drying, the slides were stained with Giemsa solution (1.5%, at pH 6.8; E. Merck) for 12-15 min.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Chromosome preparations were made as follows. Colcemid (final concn 0.2 /µg/ml) was added to the culture 2 hr before cell harvesting. The cells were then trypsinized and suspended in a hypotonic KCl solution (0.075 M) for 13 min at room temperature. After centrifugation the cells were fixed with acetic acid-methanol (1:3, v/v) and spread on clean glass slides. After air-drying, the slides were stained with Giemsa solution (1.5%, at pH 6.8; E. Merck) for 12-15 min.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): A hundred well-spread metaphases were observed under the microscope (x 600 with a no-cover objective lens).

DETERMINATION OF CYTOTOXICITY
The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. Untreated cells and solvent-treated cells served as negative controls, in which the incidence of aberrations was usually less than 3.0%. The results were considered to be negative if the incidence was less than 4.9 %, equivocal if it was between 5.0 and 9.9 %, and positive if it was more than 10. 0%.

Evaluation criteria:

The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. Untreated cells and solvent-treated cells served as negative controls, in which the incidence of aberrations was usually less than 3.0%. The maximum dose for positive results in the Chromosome aberration test represents the dose at which the maximum effect was obtained, while the maximum dose for negative results represents the highest non-cytotoxic dose used in the experimet.
A result was considered positive if the total incidence of cells with aberrations (including gaps) was 10.0 % or more, equivocal if the incidence was between 5.0 and 9.9 % and negative if the incidence was 4.9 % or less.

Statistics:

not reported

Results and discussion

Test results

Key result

Species / strain:

other: Chinese hamster fibroblast cell line CHL

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not examined

Additional information on results:

The results of the chromosomal aberration tests:
The maximum dose of test substance used: 1.0 mg/ml
The solvent used: physiological saline
The incidence of polyploid cells at 48 hr: 1.0 %
The incidence of cells with structural aberrations at 48 hr after treatment: 1.0 %
The final result: D,L-Alanine does not induce chromosomal aberrations in CHO cells.

Applicant's summary and conclusion

Conclusions:

DL-Alanine was tested negative for chromosome aberration without metabolic activation.

Executive summary:

In a chromosomal aberration test Chinese hamster lung fibroblast CHO cells were exposed to DL-Alanine in physiological saline in at least three different concentrations up to 1 mg/plate for 24 and 48 hours without metabolic activation. The results were considered to be negative since the incidence of polyploid cells after 48 hr is 1.0% and the incidence of cells with structural aberrations at 24 or 48 hr after treatment is 1.0% as well.

The study was performed in accordance with OECD guideline 473 with minor deviations. Therefore, the results can be considered to be reliable.