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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Lactic acid is not genotoxic in the present, and reviewed in vitro tests. Most of them are conducted similar to the OECD Guidelines 471, 473 and 476.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Not all required strains were tested (see below)
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Target gene:
Default
Species / strain:
other: TA97, TA 98, TA100, TA104
Metabolic activation:
with and without
Metabolic activation system:
rat liver homogenate S9
Test concentrations with justification for top dose:
0.5, 1.0 and 2.0 microliter lactic acid/plate
Vehicle:
Medium/water
Negative controls:
yes
Remarks:
medium
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Details on test system and conditions:
Strains and culture medium:
Salmonella typhimurium strains TA97, T A98, TA100 and TA104 were provided by Professor B. N. Ames. Their genotypes were confirmed according to Maron and Ames (1983). The soltions and media were prepared according to Maron and Ames (1983).
Preparation of rat liver enzymes:
The method of Sezzano et al. (1982) was adopted for induction of cytochrome P-450 in male CD-COBS rats (200-250 g). 0, 40, 60, 80, 100 mg/kg bw of phenobarbital (PB) were administered intraperitoneally in 3 doses (1 ml/day). Liver homogenate S9 fraction was prepared as in Ames et aI. (1975). The activity of the prepared S9 was evaluated using 2 tester strains (TA100 and TA98) and the positive control mutagen 2-aminoanthracene (2-AA).
Mutagenesis assay:
Several concentrations of the acidity regulators anhydrous citric acid, phosphoric acid, malic acid and lactic acid, dissolved in distilled water, were tested for mutagenicity in the standard plate incorporation assay (Maron and Ames, 1983). Distilled water (solvent) was used as the negative control. All tests were done in triplicate, both with and without S9.
Protein assay:
The protein concentration of liver homogenate S9 fraction was determined according to Kalcker (1947).
Evaluation criteria:
Number of revertants as compared to number of convertants in the control
Statistics:
Not mentioned, most likely t-test, or possibly ANOVA.
Species / strain:
other: Strains TA 97, TA 98, TA 100, TA104
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: Strains TA 97, TA 98, TA 100, TA 104
Conclusions:
Lactic acid is not mutagenic.
Executive summary:

The activities of various concentrations of 4 acidity regulators (anhydrous citric acid, phosphoric acid, malic acid and lactic acid) used in food industries in Iraq was assayed using the Salmonella/microsome mutagenicity assay. None of the samples was mutagenic in the absence or in the presence of S9 to any of the tester strains of Salmonella typhimurium.

Endpoint:
genetic toxicity in vitro
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Reference:
Composition 0
Qualifier:
no guideline required
Principles of method if other than guideline:
Review on several mutagenicity studies with lactic acid and some lactates.
GLP compliance:
not specified
Type of assay:
other: Review on several mutagenicity studies with lactic acid and some lactates
Test material information:
Composition 1
Species / strain:
other: various
Metabolic activation:
with and without
Test concentrations with justification for top dose:
various
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks on result:
other:
Remarks:
Migrated from field 'Test system'.

The results of 11 studies are reviewed.

Conclusions:
Interpretation of results (migrated information): Negative
In a review on 11 mutagenicity studies with lactic acid and some lactates, lactic acid is considered not genotoxic.
Executive summary:

Interpretation of results (migrated information): Negative

In a review on 11 mutagenicity studies with lactic acid and some lactates, lactic acid is considered not genotoxic.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Target gene:
Sd-4
Species / strain:
other: Strains B/Sd-4/1,3,4,5 and B/Sd-4/3,4
Metabolic activation:
without
Test concentrations with justification for top dose:
0.01 and 0.02 %
Details on test system and conditions:
Strains B/Sd-4/1,3,4,5 and B/Sd-4/3,4 of E. coli were used throughout this work. For every experiment, bacteria were grown for 24 hours at 37 °C in an aerated broth culture containing 10 micrograms of streptomycin per milliliter. They reached a saturation titer of approximately 2 to 3.0E+09 cells per milliliter. Each culture was started from an inoculum, usually large, taken from streptomycin-agar slants kept in a refrigerator. Before treatment the bacteria were washed in saline and resuspended in distilled water. A sample of the new suspension was added to the desired solution of chemical in distilled water, and incubated at 37 °C for a certain period of time; no growth occurs under these conditions. Another sample of the same suspension was added to an equal amount of distilled water and incubated for the same period of time, as a control. At the end of the treatment period, both treated and control suspensions were assayed by plating suitable dilutions on streptomycin-agar plates. At the same time they were plated (0.1 ml per Petri dish), either undiluted or diluted not more than 1: 10 in plain broth, onto a number of streptomycin-free plates, using a glass spreader and a turntable. The assay plates were incubated for 48 hours, after which it was possible to count the colonies and calculate the titers of the two suspensions at the end of treatment, and the percentage of survivors. The streptomycin-free plates (mutant plates) were incubated for at least six days. After this time the colonies were scored, and the frequency of mutants calculated by dividing the number of colonies by the number of (viable) bacteria plated.
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity:
other: 0.02 %
Remarks on result:
other: strain/cell type: Streptomycine-dependent strains B/Sd-4/1,3,4,5 and B/Sd-4/3,4
Remarks:
Migrated from field 'Test system'.
Additional information on results:
Some of the experiments with lactic acid indicated a weak mutagenic effect; but this is rendered doubtful by the fact that experiments wIth sodium lactate consistently gave negative results.
Conclusions:
Interpretation of results (migrated information): Negative
Lactic acid is not mutagenic.
Executive summary:

Thirty-one chemicals, representing various organic and inorganic groups, were tested for ability to induce back-mutations from streptomycin dependence (Sd-4) to nondependence in E. coli. Nineteen were found to be mutagens. It was demonstrated that mutagenicity is not a specific property of any group of chemicals, but appears among widely different groups. Chemicals having such different properties as boric acid, ammonia, hydrogen peroxide, copper sulfite, acetic acid, formaldehyde, and phenol were found to be mutagenic, indicating that genetic changes may be induced by many agents that are able to enter the living cell and upset its metabolic functions. Lactic acid was not found to be mutagenic.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
no
Type of assay:
other: Salmonella/microsome test (Ames test) and chromosomal abberation test in vitro
Test material information:
Composition 1
Target gene:
Default
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
rat liver homogenate S9
Test concentrations with justification for top dose:
10 mg/plate, resp 1.0 mg/ml (Ames test)
Vehicle:
water: phosphate buffer (Ames test)
Negative controls:
yes
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
other: TA92, TA94, TA2637
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information): Negative
Lactic acid is not mutagenic.
Executive summary:

Salmonella/microsome tests (Ames tests) and chromosomal aberration tests in vitro using a Chinese hamster fibroblast cell line were carried out on 190 synthetic food additives and 52 food additives derived from natural sources, all of which are currently used in Japan. In an Ames test, with and without metabolic activation (S9 mix), conducted on theS. typhimuriumstrains TA92, T A1535, TA1OO, TA1537, TA94, TA98 and TA2637, were invariably negative.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
no
Type of assay:
in vitro mammalian chromosome aberration test
Test material information:
Composition 1
Target gene:
Default
Species / strain:
other: Chinese hamster fibroblast cell line, established from a newborn female at the Cancer Research Institute, Tokyo (Koyama, Utakoji & Ono, 1970), and maintained by 4 day passages in Minimum Essential Medium (MEM; GlBCO) supplemented by 10% calf serum.
Details on mammalian cell lines (if applicable):
Chinese hamster fibroblast cells (Ishidate & Odashima, 1977)
Metabolic activation:
with and without
Metabolic activation system:
rat liver homogenate S9
Test concentrations with justification for top dose:
10 mg/plate, resp 1.0 mg/ml (CHL cell test)
Vehicle:
water: saline (CHL test)
Negative controls:
yes
Species / strain:
other: Chinese hamster fibroblast cells (Ishidate & Odashima, 1977)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information): Negative
Lactic acid is not mutagenic.
Executive summary:

Salmonella/microsome tests (Ames tests) and chromosomal aberration tests in vitro using a Chinese hamster fibroblast cell line were carried out on 190 synthetic food additives and 52 food additives derived from natural sources, all of which are currently used in Japan. In a chromosome aberration test using Chinese hamster lung fibroblasts with and without activation (S9 mix) lactic acid was tested negative.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
no
Type of assay:
in vitro mammalian chromosome aberration test
Test material information:
Composition 1
Species / strain:
Chinese hamster Ovary (CHO)
Details on mammalian cell lines (if applicable):
The Chinese hamster ovary K1 cell line (CHO­Kl, Flow Laboratory, U.S.A.) was used. The cells were maintained in Ham's F12 medium (Flow) supplemented with 10% fetal calf serum, kanamycin (60 µg/ml) and 17 mM sodium bicarbonate (NaHC03). The cells were grown as monolayers at 37°C in a 5% CO2/95% air atmosphere. The cultures were regularly screened for mycoplasma contamination.
Metabolic activation:
with and without
Metabolic activation system:
rat liver homogenate S9
Test concentrations with justification for top dose:
8-35 mM
Details on test system and conditions:
Preparation of metabolic activation system
S9 (Oriental Yeast Co., Ltd., Japan) was derived from the livers of rats pretreated with pheno­barbital and 5,6-benzoflavone. The S9 activation system (S9 mix) was prepared just before use according to Matsuoka et al. (1979), and sterilized by filtration, except S9 fraction.
Chromosomal aberration test
Cells (2 × 104/dish) were cultured for 48 h before the test compounds were added. Each stock solution of formic acid, acetic acid or lactic acid was prepared in distilled water at 2 M and diluted appropriately with distilled water on the day of
use. An aliquot of 50-75 µl of diluted acid was added to the culture medium (5 ml). In the ab­sence of S9 mix, chromosome preparations were made by an air-drying method 24 h after the addition of each organic acid. Cytotoxicity of each acid was also examined by counting surviving cells. In the presence of S9 mix, the cells were washed with physiological saline after a 6-h treat­ment, and then incubated with fresh medium for 18 h before the chromosome preparations were made (Ishidate, 1987).
In order to study the effect of neutralization of the treatment medium, 2 kinds of treatment media were examined; one was adjusted to pH 5.8 or pH 6.0 with each of these acids and the other was so adjusted then immediately neutralized to pH 6.4 and pH 7.2 with 1 M NaOH. In order to study the effect of enhancement of the buffering ability, chromosomal aberration tests were carried out on these acids in the absence of S9 mix using F12 medium containing 34 mM NaHCO) (twice the concentration usually employed). Furthermore, to study the effect of alteration of the buffering system, the tests were performed on these acids in the absence of S9 mix using F12 medium contain­ing 30 mM HEPES instead of NaHC03 as a buffer. This medium was adjusted to pH 8.5 with NaOH, and the cells were incubated in closed culture vessels.
The pH of the medium was measured initially and at 6 hand 24 h after the treatment using a pH meter. 100 well-spread metaphases from 1 experi­ment were observed at each point to record the percentage (including gaps) of cells with chro­mosomal aberrations. At least 2 independent ex­periments were carried out in each case.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
other: 14-35 mM, when pH was <= 5.8

When the culture medium was first acidified by the lactic acid dose and neutralized to pH 6.4 or when medium is containing 30 mM HEPES as buffer, lactic acid was non-clastogenic. Pseudo-positive reactions are seen as a result of non-physiological low pH.

Conclusions:
Interpretation of results (migrated information): Negative results obtained need to be interpreted in the light of the medium pH.
Lactic acid is not genotoxic at phyisological pH.
Executive summary:

Using Chinese hamster ovary Kl cells, chromosomal aberration tests were carried out with formic acid, acetic acid and lactic acid, and the relationship between the pH of the medium and the clastogenic activity was examined. The medium used was Ham's F12 supplemented with 17 mM NaHCO3 and 10 % fetal calf serum. All of these acids induced chromosomal aberrations at the initial pH of ca. 6.0 or below (about 10–14 mM of each acid) both with and without S9 mix. Exposure of cells to about pH 5.7 or below (about 12–16 mM of each acid) was found to be toxic. When the culture medium was first acidified with each of these acids and then neutralized to pH 6.4 or pH 7.2 with NaOH, no clastogenic activity was observed. Using F12 medium supplemented with 34 mM NaHCO3 as a buffer, no clastogenic activity was observed at doses up to 25 mM of these acids (initial pH 5.8–6.0). However, it was found that about 10 % of the cells had aberrations at pH 5.7 or below (27.5–32.5 mM of each acid). Furthermore, when 30 mM HEPES was used as a buffer, chromosomal aberrations were not induced at doses up to 20 mM formic acid and acetic acid (initial pH 7.0–7.1), and at doses up to 30 mM lactic acid (initial pH 6.6). In the initial pH range of 6.4–6.7 (25–32.5 mM of each acid), chromosomal aberrations were observed. The above results show that these acids themselves are non-clastogenic, and the pseudo-positive reactions attributable to non-physio­logical pH could be eliminated by either neutralization of the treatment medium or enhancement of the buffering ability.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17-Feb-2014 to 03-Mar-2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study according to OECD 471.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes
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
Additional strain characteristics:
other: each strain contained the following additional mutations: rfa, gal, chl, bio, uvrB
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Species / strain:
E. coli WP2 uvr A
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
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 5000µg/plate
Vehicle:
- Vehicle(s)/solvent(s) used: water
Negative controls:
yes
Remarks:
water
Solvent controls:
yes
Remarks:
solvent for test substance was water
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191, 2-aminoanthracene
Remarks:
2-aminoanthracene was used for all strains, sodium azide used for TA1535, ICR-191 for TA1537, 2-nitrofluorene for TA98, mehtylmethanesulfonate for TA100 and 4-nitroquinoline N-oxide for WO2uvrA
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 ± 4 h
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 (2) 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 (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) 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.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility:
- 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.

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

L(+)-lactic acid is not genotoxic in the reverse mutation assay.
Executive summary:
In a reverse gene mutation assay in bacteria, S. typhimurium strains TA1535, TA1537, TA98, TA100 and E. coli strain WP2uvrA were exposed to L(+)-lactic acid at concentrations of 0, 100, 333, 1000, 3330 and 5000 µg/plate in the presence and absence of mammalian metabolic activation. The test with metabolic activation (10% S9) was a plate incorporation test.

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.

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

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23-Jan-2014 to 01-Apr-2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study in accordance with OECD guideline 473.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Test material information:
Composition 1
Species / strain:
lymphocytes: peripheral human lymphocytes
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
dose range finding assay: 0, 10, 33, 100, 333, 901 µg/ml (equal to concentrations of 0, 0.1, 0.4, 1.1, 3.7 and 10 mM)
1 experiment: 0, 10, 100, 901 µg/ml (equal to concentrations of 0, 0.1, 1.1 and 10 mM)
2 experiment: 0, 100, 333, 666, 901 µg/ml (equal to concentrations of 0, 1.1, 3.7, 7.4 and 10 mM)
Vehicle:
- Vehicle(s)/solvent(s) used: RPMI 1640 medium
Negative controls:
yes
Solvent controls:
yes
Remarks:
solvent: RPMI 1640
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Remarks:
solvent for positive controls: Hank´s Balanced Salt Solution (HBSS)
Details on test system and conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 ± 2 hours
- Exposure duration: experiment 1: 3 hours (with and without metabolic activation); experiment 2: 24 and 48 hours without metabolic activation and 3 hours with metabolic activation
- Expression time (cells in growth medium): experiment 1: 20 to 22 hours; experiment 2: 44 to 46 hours
- Fixation time (start of exposure up to fixation or harvest of cells): experiment 1: 24 hours; experiment 2: 48 hours

SPINDLE INHIBITOR (cytogenetic assays): colchicine 0.5 µg/ml medium during the last 2.5 to 3 hours of the culture period
STAIN (for cytogenetic assays): 5 % (v/v) Giemsa for 10 to 30 minutes

NUMBER OF CELLS EVALUATED: 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index from at least 1000 cells
Evaluation criteria:
A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if:
None of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.

The preceding criteria are not absolute and other modifying factors might enter into the final evaluation decision.
Statistics:
The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics:

(N-1) (ad-bc)^2
X^2 = ------------------------
(a+b) (c+d) (a+c) (b+d)

Where:
a = the total number of aberrant cells in treated cultures to be compared with the control.
b = the total number of aberrant cells in the control cultures.
c = the total number of non aberrant cells in treated cultures to be compared with the control.
d = the total number of non aberrant cells in the control cultures.
N = sum of n0 and n1.
n0 = the total number of cells scored in the control cultures.
n1 = the total number of cells scored in the treated cultures.

(N-1) (ad-bc)^2
If P X^2 > ----------------------- (one-tailed) is small (p< 0.05) the hypothesis that the
(a+b) (c+d) (a+c) (b+d)

incidence of cells with chromosome aberrations is the same for both the treated and the solvent control group is rejected and the number of aberrant cells in the test group is considered to be significantly different from the control group at the 95 % confidence level.
Species / strain:
lymphocytes: peripheral human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
based on determination of the mitotic index.
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: At the highest concentration of the test substance (10 mM equal to 901 µg/ml) the pH was 7.1 compared to a pH of 7.8 in the solvent control.
- Effects of osmolality: At the highest concentration of the test substance (10 mM equal to 901 µg/ml) the osmolarity was 275 mOsm/kg compared to an osmolarity of 269 mOsm/kg in the solvent control.
- Water solubility: miscible
- Precipitation: No

RANGE-FINDING/SCREENING STUDIES: In the dose range finding test blood cultures were treated with 10, 33, 100, 333, 901 µg L(+)-lactic acid/ml culture medium (equal to concentrations of 0.1, 0.4, 1.1, 3.7 and 10 mM) with and without S9-mix.

COMPARISON WITH HISTORICAL CONTROL DATA: The number of cells with chromosome abberations found in the solvent control cultures was within the laboratory historical control data range.

Mitotic Indices:

Table1:

Mitotic index of human lymphocyte cultures treated with L(+)-lactic acid in the dose range finding test

L(+)-lactic acid concentration

(µg/ml)                                     

Number of

metaphases:

 

Absolute

Number of

metaphases:

 

Number of cells scored

Number of

metaphases:

 

Percentage

of control

Without metabolic activation (-S9-mix)

 

 

 

3 h exposure time, 24 h fixation time

 

 

 

Control a)

96

1004

100

10

99

1007

103

33

80

1045

83

100

60

1008

63

333

66

1009

69

901

63

1003

66

24 h exposure time, 24 h fixation time

 

 

 

Control a)

65

1007

100

10

62

1042

95

33

71

1041

109

100

66

1016

102

333

68

1048

105

901

36

1028

55

48h exposure time,48h fixation time

 

 

 

Control a)

68

1017

100

10

64

1026

94

33

51

1013

75

100

65

1010

96

333

57

1017

84

901

33

1033

49

With metabolic activation (+S9-mix)

 

 

 

3 h exposure time, 24 h fixation time

 

 

 

Control a)

85

1044

100

10

70

1013

82

33

71

1008

84

100

68

1006

80

333

66

1020

78

901

71

1007

84

a.) culture medium

Table 2:

Mitotic index of human lymphocyte cultures treated with L(+)-lactic acid in the first cytogenetic assay

L(+)-lactic acid concentration (µg/ml)

Number of metaphases a)

 

Absolute 

Number of metaphases a)

 

Number of cells scored

Percentage of control

 

Without metabolic activation (-S9-mix)

 

 

 

3 h exposure time, 24 h fixation time

 

 

 

Control b)

3-36

1009-1028

100

10

3-35

1002-1012

103

100

3-28

1008-1033

90

901

1-9

1002-1040

34

MMC-C; 0.5 µg/ml

4-7

1026-1031

16

MMC-C; 0.75 µg/ml

7-5

1029-1004

17

With metabolic activation (+S9-mix)

 

 

 

3 h exposure time, 24 h fixation time

 

 

 

Control b)

3-48

1040-1028

100

10

3-27

1013-1007

79

100

4-35

1035-1001

101

901

4-32

1008-1007

93

CP; 10 µg/ml

21-14

1005-1025

43

a)     Duplicate cultures

b)     Culture medium

Table 5:

Mitotic index of human lymphocyte cultures treated with L(+)-lactic acid in the second cytogenetic assay 

L(+)-lactic acid concentration (µg/ml)

Number of

metaphasesa

 

Absolute

Number of

metaphasesa

 

Number

of cells scored

Percentage

of control

Without metabolic activation (-S9-mix)

 

 

 

24 h exposure time, 24 h fixation time

 

 

 

Control b)

90-85

1000-1000

100

100

75-83

1000-1003

90

333

67-65

1008-1000

75

666

73-66

1001-1000

79

901

39-42

1002-1000

46

MMC-C; 0.2 µg/ml

24-34

1000-1003

33

MMC-C; 0.3 µg/ml

21-33

1003-1000

31

48 h exposure time, 48 h fixation time

 

 

 

Control b)

93-88

1005-1000

100

100

71-87

1001-1000

87

333

66-51

1000-1000

65

666

34-37

1000-1002

39

901

22-24

1003-1000

25

MMC-C; 0.1 µg/ml

18-20

1002-1003

21

MMC-C; 0.15 µg/ml

17-19

1000-1004

20

With metabolic activation (+S9-mix)

 

 

 

3 h exposure time, 48 h fixation 

time

 

 

 

Control b)

88-87

1000-1000

100

10

66-75

1000-1045

81

100

62-64

1003-1005

72

901

71-63

1000-1000

77

CP; 10 µg/ml

22-18

1005-1000

-c.)

a) Duplicate cultures

b) Culture medium

c) CP was fixed after 24 hours. Therefore, the mitotic index could not be calculated as percentage of control.

Conclusions:
Interpretation of results (migrated information):
- negative with metabolic activation
- negative without metabolic activation
L(+)-lactic acid is considered not clastogenic in the in vitro mammalian chromosomal aberration test.
Executive summary:

In a mammalian cell cytogenetics assay peripheral human lymphocyte cultures were exposed to L(+)-lactic acid, solved in RPMI 1640 cell culture medium. In the first and second experiment the doses were 0, 10, 100, 901 µg/ml for 3 hours with and without metabolic activation. In the second experiment additional treatment to doses of 0, 100, 333, 666 and 901 µg/ml was carried out for 24 and 48 hours exposure time. S9 was derived from phenobarbital plus ß-naphtoflavone treated rats and supplemented with cofactors.

L(+)-lactic acid was tested up to 901 µg/ml which was cytotoxic based on determination of the mitotic index after an exposure time of 24 and 48 hour. The percentage of the mitotic index after 24 hours of 901 µg/ml was 55 %, that after 48 hours of 901 µg/ml 59 %. Concentrations lower than 901 µg/ml did not cause a dose-dependent decrease in the percentage of the mitotic index after 24 and 48 hours of exposure. The mitotic index after 3 hours of exposure was lower compared to control (66 % in experiment 1, 84 % in experiment 2) but did not reach the threshold value of 45 ± 5 % according to OECD guideline 473 for cytotoxicity. Positive controls induced the appropriate response. There was no evidence for a concentration related positive response of chromosome aberration induced over background.

This study is classified as acceptable and satisfies the requirement for OECD test guideline 473.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06-May-2014 to 04-Aug-2014
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)
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Test material information:
Composition 1
Target gene:
thymidine kinase (TK)
Species / strain:
mouse lymphoma L5178Y cells
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
0.54, 1.7, 5.4, 17, 52, 164, 512, 901 µg/ml which is equal to concentrations of 0.006, 0.02, 0.06, 0.6, 1.8, 5.7 and 10 mM.
The highest dose of 901 µg/ml is a limit test concentration of 10 mM (= 0.01 M).
Vehicle:
- Vehicle(s)/solvent(s) used: RPMI 1640
Negative controls:
yes
Solvent controls:
yes
Remarks:
solvent: RPMI 1640
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours in the first experiment (with and without metabolic activation), 24 hours in the second experiment (without metabolic activation)
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 or 12 days

SELECTION AGENT (mutation assays): 5 µg/ml trifluorothymidine
STAIN (for cytogenetic assays): 0.5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth; other: relative survival, suspension growth, relative suspension growth, growth rate
Evaluation criteria:
A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.
Statistics:
The mutation frequency was expressed as the number of mutants per 10^6 viable cells. The plating efficiencies of both mutant and viable cells (CE day2) in the same culture were determined and the mutation frequency (MF) was calculated as follows:
MF = {-ln P(0)/number of cells plated per well}/ CE day2 x 10^6
Small and large colony mutation frequencies were calculated in an identical manner.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: At the highest concentration of test substance (0.01 M equal to 901 µg/ml) the pH was 6.84 compared to a pH of 7.31 in the solvent control.
- Effects of osmolality: At the highest concentration of test substance (0.01 M equal to 901 µg/ml) the osmolarity was 0.319 Osm/kg compared to an osmolarity of 0.299 Osm/kg in the solvent control
- Water solubility: miscible
- Precipitation: No

RANGE-FINDING/SCREENING STUDIES: Cytotoxicity data were obtained by treating 8 x 10^6 cells (10^6 cells/ml for 3 hours treatment) or 5 x 106 cells (1.25 x 10^5 cells/ml for 24 hours treatment) with 0, 17, 52, 164, 512 and 901 µg of test substance for 3 hours in the presence of S9-mix and for 3 and 24 hours in the absence of S9-mix.
After exposure, the cells were separated from treatment solutions centrifugation steps and re-suspended in RPM 1640 medium supplemented with 10% (v/v) inactivated horse serum (R10 medium). Cells were counted with the coulter particle counter.
For determination of the cytotoxicity, the surviving cells of the 3 hours treatment were subcultured twice. After 24 hours of subculturing, the cells were counted (day 1) and subcultured again for another
24 hours, after which the cells were counted (day 2). The surviving cells of the 24 hours treatment were subcultured once. After 24 hours of subculturing, the cells were counted. If less than 1.25 x 105 cells/ml were counted no subculture was performed.
The suspension growth expressed as the reduction in cell growth after approximately 24 and
48 hours or only 24 hours cell growth, compared to the cell growth of the solvent control, was used to determine an appropriate dose range for the mutagenicity tests.

COMPARISON WITH HISTORICAL CONTROL DATA: Spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control range.
Conclusions:
Interpretation of results (migrated information):
- negative without metabolic activation
- negative with metabolic activation
L(+)-lactic acid is considered not genotoxic in the in vitro mammalian cell gene mutation test.
Executive summary:

In a mammalian cell gene mutation assay L5178Y TK+/--3.7.2C mouse lymphoma cells cultured in vitro were exposed to L(+)-lactic acid, solved in RPMI 1640 medium at concentrations of 0.54, 1.7, 5.4, 17, 52, 164, 512 and 901 µg/mL in the presence and absence of mammalian metabolic activation. The S9 -mix was a S9 fraction derived from phenobarbital and ß-naphtoflavone treated male SD rats supplemented with cofactor mix. Metabolic activation was only performed in experiment 1 with a treatment duration of 3 hours, but not in experiment 2 with a treatment duration of 24 hours.

L(+)-lactic acid was tested to the maximum concentration of 0.01 M, equivalent to 901 µg/ml. The induced mutation frequency with and without metabolic activation was not increased compared to control in all tested concentrations. The positive controls did induce the appropriate response. The spontaneous mutation frequencies in the solvent-treated controls were within the historical control data ranges.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.

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

Genetic toxicity in vivo

Description of key information

Lactic acid is not genotoxic in the complete battery of in-vitro assays.

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Lactic acid is a ubiquitous and essential molecule of life in all higher animals and many micro-organisms. It is found in a multitude of food components. For such a molecule the determination of any genotoxic effect is irrelevant, since the internal exposure, and to a lesser extent the external exposure to this molecule is fixed and unavoidable.

Justification for classification or non-classification

Genotoxicity of lactic acid was assessed in a standard in vitro testing battery under GLP. The substance was not genotoxic in any of the test systems and thus does need not be classified.

This information is used in a read-across approach partly using data on L(+)-lactic acid, which is contained in lactic of unspecified stereochemistry as a major constituent together with its toxicologically equivalent D(–)-lactic acid. For details and justification of read-across please refer to the read-across report attached to IUCLID section 13.