L-(+)-lactic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The genotoxicity of L(+) lactic acid was assessed in a weight of evidence approach. L(+) lactic acid was tested negative in a bacterial reverse gene mutation test conducted according to OECD 471, in an in vitro chromosome aberration assay conducted according to OECD 473 and in a mammalian cell gene mutation assay conducted according to OECD 476 (nowadays OECD 490). Based on results, the test item was considered to not induce genetic toxicity.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2014-01-23 to 2014-05-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted 21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

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
- Expiration date of the lot/batch: 2015-01-01
- Storage condition of test material: room temperature in the dark

Target gene:

n.a.

Species / strain / cell type:

lymphocytes: peripheral human lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells:
Blood was collected from healthy adult, non-smoking, male volunteers. Blood samples were collected by venipuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin (Vacuette, Greiner Bio-One, Alphen aan den Rijn, The Netherlands). Immediately after blood collection lymphocyte cultures were started.
- Suitability of cells:
Peripheral human lymphocytes are recommended in international guidelines (e.g. OECD and EC).

MEDIA USED
- Type and identity of media:
- Culture medium: Culture medium consisted of RPMI 1640 medium (Invitrogen Corporation), supplemented with 20% (v/v) heat-inactivated (56 °C; 30 min) foetal calf serum (Invitrogen Corporation), L-glutamine (2 mM) (Invitrogen Corporation), penicillin/streptomycin (50 U/ml and 50 μg/ml respectively) (Invitrogen Corporation) and 30 U/ml heparin (Sigma, Zwijndrecht, The Netherlands).
- Lymphocyte cultures: Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 mL (9 mg/mL) phytohaemagglutinin (Remel, Europe Ltd., United Kingdom) was added.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Dose range finding assay:
A concentration of 901 µg/mL showed no precipitation in the culture medium. Therefore, this concentration was used as the highest concentration in the dose range finding test.
With and without S9: 10, 33, 100, 333, 901 µg/mL (equal to concentrations of 0.1, 0.4, 1.1, 3.7 and 10 mM)

First cytogenetic assay:
Based on the results of the dose range finding test the following dose levels were selected for the cytogenetic assay:
With and without S9: 10, 100, 901 µg/mL (equal to concentrations of 0.1, 1.1 and 10 mM)

Second cytogenetic assay:
Without S9: 100, 333, 666, 901 µg/mL (equal to concentrations of 1.1, 3.7, 7.4 and 10 mM)
With S9: 10, 100, 901 µg/mL (equal to concentrations of 0.1, 1.1 and 10 mM)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: RPMI 1640 medium

Untreated negative controls:

yes

Remarks:

solvent

Negative solvent / vehicle controls:

yes

Remarks:

solvent: RPMI 1640

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

0.5 and 0.75 μg/mL for a 3 h exposure period, 0.2 and 0.3 μg/mL for a 24 h exposure period and 0.1 and 0.15 μg/mL for a 48 h exposure period. Solvent for positive controls: Hank´s Balanced Salt Solution (HBSS)

Untreated negative controls:

yes

Remarks:

solvent

Negative solvent / vehicle controls:

yes

Remarks:

solvent: RPMI 1640

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

10 μg/mL for a 3 h exposure period (24 h fixation time). Solvent for positive controls: Hank's Balanced Salt Solution (HBSS)

Details on test system and experimental 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.

Key result

Species / strain:

lymphocytes: peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

based on determination of the mitotic index.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

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/mL 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 aberrations found in the solvent control cultures was within the laboratory historical control data range.

For individual results see box 'Any other information on results incl. tables'.

Mitotic Indices:

Table 1: 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
48 h exposure time, 48 h 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 cytegenetic assay

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)	34-36	1009-1028	100
10	37-35	1002-1012	103
100	35-28	1008-1033	90
901	15-9	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)	33-48	1040-1028	100
10	37-27	1013-1007	79
100	47-35	1035-1001	101
901	43-32	1008-1007	93
CP; 10 µg/ml	21-14	1005-1025	43

a)     Duplicate cultures

b)     Culture medium

Table 3: Mitotic index of human lymphocyte cultures treated with L(+)-lactic acid in the second 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)
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.

Chromosome aberrations in human lymphocyte cultures treated with L(+)-lactic acid

1. Cytogenetic Assay:

Conc	Exposure Time [h]	Fixation Time [h]	Metabolic Activation	Culture	Mitotic Index [%]	Cells  scored	Cells  + gaps	Cells - gaps
Culture medium	3	24	without	A+B	100	200	0	0
10 µg/ml	3	24	without	A+B	103	200	5	5
100 µg/ml	3	24	without	A+B	90	200	0	0
901 µg/ml	3	24	without	A+B	34	200	4	4
MMC-C 0.5 µg/ml	3	24	without	A+B	16	150	76***	76***
Culture medium	3	24	with	A+B	100	200	2	2
10 µg/ml	3	24	with	A+B	79	200	3	3
100 µg/ml	3	24	with	A+B	101	200	2	2
901 µg/ml	3	24	with	A+B	93	200	2	2
CP 10 µg/ml	3	24	with	A+B	43	200	59***	59***

*) Significantly different from control group (Chi-square test), p < 0.001

2. Cytogenetic Assay:

Conc	ExposureTime [h]	FixationTime [h]	Metaboic Activation	Culture	Mitotic Index  [%]	Cells  scored	Cells  + gaps	Cells - gaps
Culture medium	3	48	with	A+B	100	200	2	1
10 µg/ml	3	48	with	A+B	81	200	1	1
100 µg/ml	3	48	with	A+B	72	200	0	0
10 µg/ml	3	48	with	A+B	77	200	3	3
CP 10 µg/ml	3	48	with	A+B	n.d.^b	100	53***	53***
Culture medium	24	24	without	A+B	100	200	1	1
100 µg/ml	24	24	without	A+B	90	200	1	1
666 µg/ml	24	24	without	A+B	79	200	0	0
901 µg/ml	24	24	without	A+B	46	200	3	3
MMC- 0.1 µg/ml	24	24	without	A+B	33	100	53***	53***
Culture medium	48	48	without	A+B	100	200	1	1
100 µg/ml	48	48	without	A+B	87	200	3	3
333 µg/ml	48	48	without	A+B	65	200	2	2
666 µg/ml	48	48	without	A+B	39	200	2	2
MMC- 0.1 µg/ml	48	48	without	A+B	21	100	53***	53***

^bCP was fixed after 24 hours. Therefore, the mitotic index could not be calculated as percentage of control.

*) Significantly different from control group (Chi-square test), p < 0.001

Conclusions:

L(+)-lactic acid is considered to be not clastogenic in the in vitro mammalian chromosomal aberration test using human lymphocytes, with and without metabolic activation.

Executive summary:

In an in vitro cytogenicity assay conducted according to OECD guideline 473, peripheral human lymphocyte cultures were exposed to L(+)-lactic acid (90% purity), solved in RPMI 1640 cell culture medium. In the first experiment, the doses were 0, 10, 100, 901 µg/mL with and without metabolic activation. In the second experiment doses were 0, 100, 333, 666, 901 µg/mL without metabolic activation and 0, 10, 100, 901 µg/mL with metabolic activation (rat liver S9-mix).

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 hours. The percentage of the mitotic index after 24 hours of 901 µg/mL was 55%, that after 48 hours of 901 µg/mL 49%. 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 responses. 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 the in vitro mammalian chromosomal aberration test according to OECD 473.

Reference 2

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

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

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.

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 / 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

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

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

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

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

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

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 applicable

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True 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.

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 conducted according to OECD guideline 471, Salmonella 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).

Reference 3

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

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 gene mutation tests using the thymidine kinase gene

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.

Target gene:

thymidine kinase (TK)

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

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.

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

True negative controls validity:

not examined

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'.

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 x 10^5)	Cell count after 24 hours of subculture (cells/mL x 10^5)	Cell count after 48 hours of subculture (cells/mL x 10^5)	SG(1) x 10^5 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 x 10^5)	SG(1) x 10^5 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 metabolic 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

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 conducted 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

L(+)-lactic acid was tested in an in vitro test battery, which were performed in accordance with the OECD guidelines 471 (Ames test), 473 (chromosome aberration) and 476 (MLA, nowadays OECD 490).

In the bacterial reverse mutation test four strains of Salmonella typhimurium, TA1535, TA1537, TA100 and TA98 plus the Escherichia coli strain WP2uvrA were treated with L(+)-lactic acid up to the limit concentration of 5000 µg/plate. No precipitation occurred and the bacterial growth was undisturbed, indicative of the non-cytotoxicity of L(+)-lactic acid. No increase in the rate of revertant colonies was observed in all of the triplicates tested for each concentration (0, 100, 333, 1000, 3330 and 5000 µg/plate) with and without addition of S9-mix. The positive controls induced the appropriate response; the negative controls laid within the historical background. Thus, L(+)-lactic acid was not mutagenic in the bacterial reverse mutation assay.

Two further in vitro test were performed in mammalian cells. A gene mutation assay used L5178Y mouse lymphoma cells was conducted to study the impact of L(+)-lactic acid on the rate of forward mutation at the thymidine-kinase locus. The test was conducted in the presence and absence of metabolic activation with exposure times of 3 hours (with and without) and 24 hours (without). L(+)-lactic acid failed to be cytotoxic in a dose-dependent manner, reducing the cloning efficiency, given as percent, to roughly 84 percent. The highest test concentration was chosen in accordance to the proposed limit test concentration of OECD guideline 476, which is 0.01 M and corresponded to a dose of 901 µg/mL L(+)-lactic acid. 8 doses between 0.54 and 901 µg/mL L(+)-lactic acid were tested, not resulting in an increase of mutation frequency per 10^6 survivors in the presence or absence of metabolic activation with 3 or 24 hours exposure time. The positive controls induced the appropriate response and negative controls were in the historical control range. Thus, L(+)-lactic acid is considered as not mutagenic.

The third in vitro test was a chromosome aberration test in cultured peripheral human lymphocytes. Five doses of L(+)-lactic acid were tested ranging from 10 to 901 µg/mL. For cytotoxicity assessment, the mitotic index was determined. 2 experiments were conducted; the first one used 3 hours exposure time, followed by a fixation time of 24 hours in the presence and absence of metabolic activation. The second experiment exposed peripheral human lymphocytes for 24 and 48 hours to L(+)-lactic acid followed by equal fixation time (24 and 48 hours, respectively) and in a third conditions with metabolic activation exposed the cells for 3 hours, followed by a 48 hours fixation time. Upon 3 hours of exposure the mitotic index, given as percent, was reduced to 34 percent in the absence of metabolic activation, but only 93 and 77 percent with metabolic activation in the highest concentration of 901 µg/ml. Upon 24 and 48 hours exposure time, the mitotic indices were 46 percent (24 h, 901 µg/mL) and 39 percent (48 hours, 666 µg/mL). Thus, the reduction of the mitotic indices were significant at a dose of 901 (or 666) µg/mL. It has to be mentioned, that the mitotic index gives rather an estimate about the rate of the cells undergoing proliferation then the actual percentage of dead cells. With this, the difference in assessment of cytotoxicity between the gene mutation test (where no cytotoxicity has been observed) and the lowered rate of mitotic indices in the chromosome aberration test is not conflicting.

After the fixation time, cells with chromosome aberrations were counted, resulting in no significant increase over control levels for all exposure and fixation times tested in the presence and absence of metabolic activation. The positive controls induced the appropriate response and the negative controls were within the historical background range.

In addition to these three guideline studies with the target substance, four publications and one review article report further results on the genotoxic potential of lactic acid. No positive results were reported, with exception of Morita (1990), where some chromosome aberrations had been found under non-physiological conditions, i.e. low pH. The same study reports negative findings under physiological conditions, whereas the positive finding is of no toxicological relevance. The review article by Andersen (1998) displays the findings of 11 studies, which were all negative. Therefore, in view of all genotoxicity tests, L(+)-lactic acid is considered as not genotoxic.

Justification for classification or non-classification

Based on the available results, the target substance L(+) lactic acid is not considered to be genotoxic and no classification is warranted in accordance with CLP Regulation 1272/2008.