Potassium (S)-lactate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2014-01-17 to 2014-05-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21 July 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

L-(+)-lactic acid

EC Number:

201-196-2

EC Name:

L-(+)-lactic acid

Cas Number:

79-33-4

Molecular formula:

C3H6O3

IUPAC Name:

(2S)-2-hydroxypropanoic acid

Specific details on test material used for the study:

- Name: L(+)-lactic acid
- CAS No.: 79-33-4
- Appearance: clear colourless liquid
- Batch No.: 1208002033
- Purity: 90%
- Storage: at room temperature in the dark
- Expiry date: 2015-01-01

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: L(+)-lactic acid was dissolved in Milli-Q water (Millipore Corp., Bedford, MA., USA). Preparation of test solutions started with solutions of 50 mg/mL applying vortexing resulting in a clear colourless solution.
The lower test concentrations were prepared by subsequent dilutions in Milli-Q water. The stock solution was filter (0.22 μm)-sterilized. Test substance concentrations were used within 2 hours after preparation.

Method

Target gene:

Salmonella typhimurium: Histidine
E. coli: Tryptophane

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Trinova Biochem GmbH, Germany (Master culture from Dr. Bruce N. Ames)

Additional strain / cell type characteristics:

other: each strain contained the following additional mutations: rfa, gal, chl, bio, uvrB

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

CELL SOURCES:
- Source of cells: Master culture from The National Collections of Industrial and Marine Bacteria, Aberdeen, UK

Additional strain / cell type characteristics:

other: The Escherichia coli WP2uvrA strain detects base-pair substitutions

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Selection of an adequate range of doses was based on a dose range finding test with the strains TA100 and WP2uvrA.

Strains TA100 and WP2uvrA: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
Strains TA1535, TA1537 and TA98: 100, 333, 1000, 3330 and 5000 µg/plate

The highest concentration of L(+)-lactic acid used in the subsequent mutation assay was 5000 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Milli-Q water

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Milli-Q water

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9; WP2uvrA; 15 μg/plate; dissolved in DMSO

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Milli-Q water

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9; TA100; 650 μg/plate; dissolved in DMSO

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Milli-Q water

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S9; TA98; 10 μg/plate; dissolved in DMSO

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Millli-Q water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ICR-191

Remarks:

without S9; TA1537; 2.5 μg/plate; dissolved in DMSO

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Milli-Q water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9; TA1535; 5 μg/plate; dissolved in saline

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Milli-Q water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9; 1 µg/plate (TA98: 5 and 10% S9, TA100: 5% S9), 2 µg/plate (TA100: 10% S9), 2.5 µg/plate (TA1535: 5 and 10% S9; TA1537: 5% S9); dissolved in DMSO

Details on test system and experimental conditions:

Dose Range Finding Test:
Selection of an adequate range of doses was based on a dose range finding test with the strains TA100 and WP2uvrA, both with and without 5% (v/v) S9-mix. Eight concentrations, 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate were tested in triplicate. The highest concentartion of lactic aqcid used in the subsequent mutation assay was 5000 µg/plate.

Mutation Assay:
At least five different doses (increasing with approximately half-log steps) of the test substance were tested in triplcate in each strain. In the first experiment the test item was tested both in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and TA98. In an independent repeat of the assay with additional parameters, the test substance was tested both on the absence and presence of 10% (v/v) S9-mix in all tester strains. The negative control (vehicle) and relevant postive controls were concurrently tested in each strain in the presence and absence of S9-mix.
Top agar in top agar tubes was melted by heating to 45 °C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (10^9 cells/mL) of one of the tester strains, 0.1 mL of a diltuin of the test substance in Milli-Q-water and either 0.5 mL S9-mix or 0.5 mL 0.1 M phosphate buffer. The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 °C +/- 1.0 °C for 48 +/- 4 hours. After this period revertant colonies were counted.

NUMBER OF REPLICATIONS: 3

Colony counting:
The revertant colonies were counted manually and evidenve of the test article precipitation on the plates was recorded.

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn was evaluated.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or
WP2uvrA is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three times the concurrent vehicle control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Statistics:

not applicaple

Results and discussion

Test results

Key result

Species / strain:

other: S. typhimurium TA1535, TA1537, TA98, TA100, E.coli WP2uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No

RANGE-FINDING/SCREENING STUDIES: Dose range finding test performed with TA100 and WP2uvrA. Doses tested: 0, 3, 10, 33, 100, 333, 1000, 3330, 5000 µg/plate. Based on the results, the following doses were selected for the main experiment with TA1535, TA1537 and TA98 in the presence and absence of S9-mix: 100, 333, 1000, 330 and 5000 µg/plate.

COMPARISON WITH HISTORICAL CONTROL DATA: The negative control values were within the laboratory historical control data ranges, except for TA100 in the absence of S9-mix, second experiment. Evaluation: The mean plate count (146) was just outside the limit of the range (144) and clear negative results are observed in all experiments. Therefore, this deviation in the mean plate count of the solvent control had no effect on the results of the study. The strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly, except the response for TA1535 in the absence of S9-mix, first experiment. Evaluation: The value (257) was just below the limit of the range (262). The purpose of the positive control is as a reference for the test system, where a positive response is required to check if the test system functions correctly. Since the value was more than 3 times greater than the concurrent solvent control values, this deviation in the mean plate count of the positive control had no effect on the results of the study.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.

MUTAGENICITY:
Experiment 1: No increase in the number of revertants was observed upon treatment with the test item under all conditions tested.
Experiment 2: Based on the results from the first experiment, the test item was tested up to 5000 µg/plate. No increase in the number of revertants was observed.

Applicant's summary and conclusion

Conclusions:

In conclusion, L(+)-lactic acid is not genotoxic in the bacterial reverse gene mutation assay (OECD 471) in the presence and absence of mammalian metabolic activation.

Executive summary:

In a reverse gene mutation assay in bacteria conducting according to OECD guideline 471, Salomella typhimurium strains TA1535, TA1537, TA98, TA100 and E. coli strain WP2uvrA were exposed to L(+)-lactic acid (90% purity) at concentrations of 0, 100, 333, 1000, 3330 and 5000 µg/plate in the presence and absence of mammalian metabolic activation.

L(+)-lactic acid was tested up to the limit concentration of 5000 µg/plate. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background. Based on the results, the test item can be considered to be non-mutagenic.

 

This study is classified as acceptable and satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation assay).