Methyl (S)-(-)-lactate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1988-09-13 to 1988-11-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl lactate

EC Number:

202-598-0

EC Name:

Ethyl lactate

Cas Number:

97-64-3

IUPAC Name:

ethyl 2-hydroxypropanoate

Specific details on test material used for the study:

- Test material name: Ethyl lactate
- Test Article Description: clear colourless liquid
- Storage conditions: room temperature

Method

Target gene:

his-

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Aroclor 1254 induced male SD rat liver
- method of preparation of S9 mix:Liver microsomal enzymes were prepared from male SD rats that had been injected with Aroclor 1254 at 500 mg/kg. The Aroclor was diluted in corn oil to a concentration of 200 mg/mL. Five days after i.p. injection with the Aroclor, the rats were sacrificed by decapitation, and their livers were excised. The preparation of the microsomal enzyme fraction was carried out with sterile glassware and solutions at 0-4 °C. The liver from each rat was excised and placed in approximately 20 mL of 0.15 M KCl contained in a pre-weighed beaker. After the liver was weighed, it was transferred to another beaker containing 3 volumes of 0.15 M KCl (3 mL/g of wet liver) where it was minced with sterile scissors. The minced liver was homogenized and subseguently centrifuged at 9000 x g for 10 minutes. The supematant (referred to by Ames as the S-9 fraction) was removed, and small aliquots were distributed into freezing ampules which were stored at < -70 °C.
- concentration or volume of S9 mix and S9 in the final culture medium: 1 mL of the microsomal enzyme reaction mixture (S-9 mix) contained the following components:
H20 - 0.56 mL
1.00 M NaH2P04/K2HPO4, pH 7.4 - 0.10 mL
0.05 M Glucose-6-phosphate - 0.10 mL
0.04 M NADP - 0.10 mL
0.2 M MgCl2 / 0.825 M KCl - 0.04 mL
S-9 0.10 mL
- quality controls of S9: metabolic capability)

Test concentrations with justification for top dose:

Pre-test: 10, 33, 67, 100, 333, 667, 1000, 3333, 6667, 10000 µg/plate, both with and without activation
Main test: 667, 1000, 3333, 6667 and 10000 µg/plate, both with and without activation
See also Table 1 in box " Any other information on materials incl. tables".

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Deionized, distilled grade water.
- Justification for choice of solvent/vehicle: Ethyl lactate was serially diluted with deionized, distilled grade water to obtain the desired test concentrations.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

All strains with activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA98 and TA1538, without activation

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA100 and TA1535, without activation

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ICR-191

Remarks:

TA1537 without activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: In agar (plate incorporation).

DURATION
- Preincubation period: no
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 48 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 48 hours

NUMBER OF REPLICATIONS: Pre test: single, main test: triplicate

NUMBER OF CELLS EVALUATED: Revertant colonies for a given tester strain and activation condition were counted either entirely by automated colony counter or entirely by hand.

DETERMINATION OF CYTOTOXICITY
- Method: The condition of the background bacterial lawn was evaluated for evidence of test article toxicity by using a dissecting microscope.

Evaluation criteria:

For a test article to be considered positive, it must cause at least a doubling in the mean revertants per plate of at least one tester strain. This increase in the number of revertants per plate must be accompanied by a dose response to increasing concentrations of the test article. In those cases where the observed dose-responsive increase in TA1537 or TA1538 revertants per plate is less than three-fold, the response must be reproducable.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

see Table 2

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

Test results 2

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

see Table 2

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

Test results 3

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

see Table 2

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

Test results 4

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

see Table 2

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

Test results 5

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

See Table 2

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:

RANGE-FINDING/SCREENING STUDIES:
The results of the dose range-finding study conducted in the presence and absence of rat liver microsomes indicate that no appreciable toxicity was observed up to 10000 µg per plate.

Any other information on results incl. tables

Table 2: Results of Salmonella mutagenicity assay with ethyl lactate

Average Revertants Per Plate**± Standard Deviation
Liver Microsomes: None
Dose (µg/plate)	TA98	TA100	TA1535	TA1537	TA1538
0*	14 ± 5	96 ± 20	20 ± 3	5 ± 1	13 ± 3
667	19 ± 2	111 ± 6	20 ± 6	5 ± 1	13 ± 3
1000	9 ± 3	104 ± 14	22 ± 2	7 ± 3	14 ± 4
3333	14 ± 2	111 ± 12	19 ± 1	5 ± 1	19 ± 2
6667	17 ± 3	107 ± 11	17 ± 2	8 ± 2	12 ± 2
10000	20 ± 7	99 ± 6	18 ± 5	5 ± 2	11 ± 2
Positive control	519 ± 53	485 ± 12	281 ± 7	241 ± 23	599 ± 37
Liver Microsomes: Rat Liver S-9
Dose (µg/plate)	TA98	TA100	TA1535	TA1537	TA1538
0*	23 ± 3	105 ± 12	12 ± 4	9 ± 3	22 ± 2
667	34 ± 7	99 ± 14	15 ± 5	6 ± 2	20 ± 8
1000	21 ± 1	102 ± 9	13 ± 6	8 ± 3	22 ± 7
3333	27 ± 6	102 ± 5	13 ± 3	7 ± 3	21 ± 6
6667	28 ± 5	105 ± 13	13 ± 3	12 ± 5	16 ± 5
10000	28 ± 3	102 ± 4	12 ± 3	7 ± 2	21 ± 3
Positive control	950 ± 36	2358 ± 118	300 ± 4	167 ± 7	1387 ± 125

*Vehicle control with dionized, distilled grade water

** Mean of 3 plates

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions reported, Ethyl lactate did not cause gene mutations in an Ames Test (equivalent to OECD Guideline 471). Therefore, the test item is considered to be non-mutagenic in this bacterial reverse gene mutation assay.

Executive summary:

In a reverse gene mutation assay (equivalent to OECD Guideline 471) in bacteria, strains TA98, TA100, TA1535, TA1537 and TA1538 of S. typhimurium were exposed to Ethyl lactate at concentrations of 667, 1000, 3333, 6667 and 10000 µg/plate in the presence and absence of mammalian metabolic activation (plate co-incubation).

Ethyl lactate was tested up to the limit concentration of 10000 µg/plate. There was no dose-related increase in the number of revertants in any of the test strains with and without activation. 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. This study satisfies the requirement for OECD test guideline 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.