Butyl (S)-2-hydroxypropionate

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:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

adopted July 21, 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 3

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

Test material information

Constituent 1

Reference substance name:

L-(+)-lactic acid

EC Number:

201-196-2

EC Name:

L-(+)-lactic acid

Cas Number:

79-33-4

Molecular formula:

C3H6O3

IUPAC Name:

(2S)-2-hydroxypropanoic acid

Specific details on test material used for the study:

- Name 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

Controls

Controls 1

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

Controls 2

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.