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EC number: 252-036-3 | CAS number: 34451-19-9
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 2014-05-06 to 2014-08-26
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 014
- Report date:
- 2014
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- adopted July 21, 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian cell transformation assay
Test material
- 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
Constituent 1
- Specific details on test material used for the study:
- - Name of test material (as cited in study report): L(+)-lactic acid
- Analytical purity: 90%
- Lot/batch No.: 1208002033
- Physical state: clear, colourless liquid
- Expiration date of the lot/batch: 2015-01-01
- Stability under test conditions: valid expiry date
- Storage condition of test material: room temperature in the dark
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
The test substance was dissolved in RPMI 1640 (exposure medium, Hepes buffered medium (Dutch modification) (Invitrogen Corporation, Breda, The Netherlands). L(+)-lactic acid concentrations were used within 2 hours after preparation.
Method
- Target gene:
- thymidine kinase (TK)
Species / strain
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: American Type Culture Collection, (ATCC, Manassas, USA, 2001)
- Suitability of cells: L5178Y mouse lymphoma cells are used because they are sensitive indicators of mutagenic activity of a broad range of chemical classes.
CELL CULTURE
- Horse serum: Horse serum (Invitrogen Corporation) was inactivated by incubation at 56 °C for at least 30 minutes.
- Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) (Invitrogen Corporation) containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively) (Invitrogen), 1 mM sodium pyruvate (Sigma) and 2 mM L-glutamin (Invitrogen Corporation).
- Growth medium: Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
- Selective medium: Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20) and 5 μg/mL trifluorothymidine (TFT) (Sigma).
- Non-selective medium: Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20). - Additional strain / cell type 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). A concentration of 0.01 M (901 μg/ml) L(+)-lactic acid showed no precipitation in the culture medium. Therefore, a concentration of 0.01 M was used as the highest concentration of L(+)-lactic acid. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: RPMI 1640
Controlsopen allclose all
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- solvent: RPMI 1640
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- -S9, 15 and 5 µg/mL for 3 and 24 h treatment period
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- solvent: RPMI 1640
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- +S9, 7.5 µg/mL
- Details on test system and experimental 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: 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 su cultured once. After 24 hours of subculturing, the cells were counted. If less than 1.25 x 10^5 cells/mL were counted no subculture was performed. - 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 day 2) 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.
Results and discussion
Test results
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- 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 10^6 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.
Experiment 1 & 2: For individual results see Tables 3-5 in box 'Any other information on results incl. tables'
Any other information on results incl. tables
Table 1: Dose-range finding test: Cytotoxicity of L(+)-lactic acid (3 hours treatment)
Dose (µg/mL) |
Cell count after 3 hours of treatment (cells/mL x10^5) | Cell count after 24 hours of subculture (cells/mL x 10^5) | Cell count after 48 hours of subculture (cells/mL x10^5) | SG(1) x10^5 cells/mL) |
RSG (2) % |
without metabolic activation |
|||||
SC | 6.9 | 5.0 | 6.9 | 152 | 100 |
17 | 6.3 | 5.0 | 7.4 | 149 | 98 |
52 | 7.0 | 5.2 | 6.9 | 161 | 106 |
164 | 7.3 | 5.2 | 7.1 | 173 | 113 |
512 | 7.6 | 5.2 | 6.8 | 172 | 113 |
901 | 6.8 | 5.4 | 7.0 | 166 | 109 |
with metabolic activation |
|||||
SC | 5.3 | 4.9 | 7.8 | 130 | 100 |
17 | 5.2 | 5.2 | 7.5 | 130 | 100 |
52 | 4.2 | 5.2 | 7.6 | 106 | 82 |
164 | 4.1 | 5.3 | 7.2 | 100 | 77 |
512 | 5.0 | 5.1 | 7.5 | 122 | 94 |
901 | 4.3 | 5.1 | 7.4 | 104 | 80 |
Note: all calculations were made without rounding off
SC = solvent control = exposure medium
(1) = suspension growth
(2) relative suspension growth
SG= (Cell count after 3 h treatment) x (Cell count after 24 h subculture)/(Cells subcultured (at t=3 h)(1.25x10^5 c/mL)) x (Cell count after 48 h subculture)/(Cells subcultured (at t=24 h) (1.25 x 10^5 c/mL))
RSG = [SG(test)/SG(control)] x 100
Table 2: Dose-range finding test: Cytotoxicity of L(+)-lactic acid (24 hours treatment)
Dose (µg/mL) |
Cell count after 24 hours of treatment (cells/mL x 10^5) | Cell count after 24 hours of subculture (cells/mL x10^5) | SG(1) x10^5 cells/mL) |
RSG (2) % |
without metabolic activation |
||||
SC |
9.5 | 5.9 | 45 | 100 |
17 | 8.9 | 5.9 | 42 | 93 |
52 | 9.3 | 5.7 | 42 | 93 |
164 | 9.1 | 5.2 | 39 | 85 |
512 | 8.8 | 5.5 | 39 | 87 |
901 | 7.2 | 4.6 | 26 | 58 |
Note: all calculations were made without rounding off
SC = solvent control = exposure medium
(1) = suspension growth
(2) relative suspension growth
SG = (Cell count after 24 h treatment) x (Cell count after 24 h subculture)/(Cells subcultured after treatment (1.25 x 10^5 c/mL)
RSG = [SG(test)/SG(control)] x 100
Cytotoxic and mutagenic response of L(+)-lactic acid in the mouse lymphoma L5178Y test system
Abbreviations:
RSG: Relative Suspension Growth
CE: Cloning Efficiency
RS: Relative Survival
RTG: Relative Total Growth
MF: Mutation Frequency per 10^6 Survivors
SC: Solvent Control (= Exposure Medium)
MMS: Methylmethanesulfonate
CP: Cyclophosphamide
Experiment 1
Table 3: 3 h treatment, without metablic activation
Dose [µg/mL] |
RSG [%] |
CEday2 [%] |
RSday2 [%] |
RTG [%] |
MF total |
MF small |
MS large |
SC1 | 100 | 97 | 100 | 100 | 89 | 70 | 16 |
SC2 | 100 | 80 | 100 | 100 | 86 | 66 | 18 |
0.54 | 107 | 86 | 98 | 105 | 98 | 71 | 25 |
1.7 | 118 | 79 | 89 | 106 | 98 | 72 | 23 |
5.4 | 126 | 83 | 93 | 117 | 94 | 62 | 28 |
17 | 129 | 77 | 87 | 112 | 122 | 91 | 26 |
52 | 108 | 75 | 84 | 91 | 124 | 97 | 23 |
164 | 112 | 78 | 88 | 99 | 104 | 66 | 34 |
512 | 106 | 72 | 82 | 87 | 147 | 99 | 41 |
901 | 101 | 88 | 99 | 100 | 116 | 89 | 23 |
MMS | 79 | 41 | 47 | 37 | 1149 | 870 | 191 |
Table 4: 3 h treatment, with metabolic activation
Dose [µg/mL] |
RSG [%] |
CEday2 [%] |
RSday2 [%] |
RTG [%] |
MF total |
MF small |
MS large |
SC1 | 100 | 68 | 100 | 100 | 51 | 26 | 23 |
SC2 | 100 | 64 | 100 | 100 | 55 | 30 | 25 |
0.54 | 92 | 78 | 118 | 108 | 53 | 31 | 20 |
1.7 | 78 | 111 | 168 | 132 | 26 | 17 | 8 |
5.4 | 56 | 93 | 140 | 79 | 38 | 28 | 10 |
17 | 60 | 97 | 146 | 88 | 31 | 22 | 9 |
52 | 65 | 66 | 100 | 65 | 62 | 47 | 14 |
164 | 92 | 74 | 111 | 102 | 53 | 45 | 7 |
512 | 64 | 77 | 116 | 74 | 45 | 12 | 32 |
901 | 93 | 81 | 123 | 114 | 45 | 25 | 19 |
CP | 37 | 29 | 43 | 16 | 849 | 647 | 167 |
Experiment 2
Table 5: 24 h treatment, without metabolic activation
Dose [µg/mL] |
RSG [%] |
CEday2 [%] |
RSday2 [%] |
RTG [%] |
MF total |
MF small |
MS large |
SC1 | 100 | 98 | 100 | 100 | 57 | 26 | 30 |
SC2 | 100 | 102 | 100 | 100 | 50 | 19 | 30 |
0.54 | 92 | 84 | 84 | 77 | 63 | 19 | 42 |
1.7 | 91 | 86 | 86 | 78 | 71 | 40 | 28 |
5.4 | 99 | 89 | 89 | 88 | 65 | 25 | 38 |
17 | 90 | 89 | 89 | 80 | 49 | 11 | 38 |
52 | 86 | 98 | 98 | 84 | 51 | 16 | 34 |
164 | 85 | 88 | 87 | 74 | 72 | 34 | 36 |
512 | 78 | 90 | 90 | 71 | 50 | 22 | 26 |
901 | 64 | 107 | 107 | 68 | 53 | 9 | 43 |
MMS | 80 | 61 | 61 | 49 | 621 | 198 | 368 |
Applicant's summary and conclusion
- Conclusions:
- In conclusion, L(+)-lactic acid is considered to be non-mutagenic in the in vitro mammalian cell gene mutation test (OECD 476, nowadays OECD 490) in the presence and absence of mammalian metabolic activation.
- Executive summary:
In a mammalian cell gene mutation assay in accordance to OECD guideline 476 (nowadays OECD 490), L5178Y mouse lymphoma cells cultured in vitro were exposed to L(+)-lactic acid (90% purity), solved in RPMI 1640 medium. In the first experiment, L(+)-lactic acid was tested up to concentrations of 901 µg/mL (0.01 M, the highest concentration recommended in the guidelines) in the absence and presence of S9-mix. The incubation time was 3 hours. In the second experiment, L(+)-lactic acid was again tested up to concentrations of 901 µg/mL in the absence S9-mix. The incubation time was 24 hours. No toxicity was observed at this dose level in the absence and presence of S9 mix. 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. Based on the results, it can be concluded, that L(+)-lactic acid is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described.
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