Methyl pyruvate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

modified version of the Ames test (OECD 471), designated Ames II Assay (microtiter version).

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Methyl pyruvate

EC Number:

209-987-4

EC Name:

Methyl pyruvate

Cas Number:

600-22-6

Molecular formula:

C4H6O3

IUPAC Name:

methyl 2-oxopropanoate

Method

Target gene:

His gene

Species / strain

Species / strain / cell type:

other: TA 98, TA Mix (TA 7001 - TA 7006)

Metabolic activation:

with and without

Metabolic activation system:

mix from induced male Wistar rats

Test concentrations with justification for top dose:

O; 4; 20; 100; 500; 2500 and 5000 μg/ml

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other:

Details on test system and experimental conditions:

Bacteria from overnight cultures were used. 5 ml of the overnight cultures were added to tubes containing 25 ml Ames II Exposure medium and were gently mixed. After thorough
pipetting the following components were added in 24-well plates:
Without S9 mix
240 μL Bacteria suspension (tester strain + exposure medium)
10 μL Test substance or vehicle or positive control

With S9 mix
200 μL Bacteria suspension
40 μL S9 mix (liver homogenate to buffer ratio: 3:7)
10 μL Test substance or vehicle or positive control

The 24-well plates were incubated at 37°C with shaking at 250 rpm for about 90 minutes. After this incubation period, 2.8 ml Ames II Reversion indicator medium (containing bromocresol purple) was pipetted to each well of the 24-well plate. The contents of each well of the 24-well plates were distributed in 50 μL aliquots over 48 wells of a 348-well Revertant Colony Selection plate (RCSP). The plates were sealed in plastic bags and incubated at 37°C in the dark. After 48 hours incubation each 48-well section of the RCSP were scored and the number of positive wells (yellow = high number of his+ revertants) were counted.

48 wells in triplicate plates per dose or per control were used

Rationale for test conditions:

This method shows a good accuracy concerning the prediction of the results in the regular Ames test.

Evaluation criteria:

An increase in the mean number of positive wells in dose groups was compared to the mean value of the concurrent negative control (Evaluation factor 1 F).
- An increase in the mean of revertant wells in dose groups was calculated on the basis of the baseline data of the actual experiment (Evaluation factor 2 F). The baseline was derived from the mean spontaneous revertant number plus the value of standard deviation (mean + SD) from the distribution of spontaneous data.
- An increase in the mean of revertant wells in dose groups was calculated on the basis of the baseline data of an experimental run (Evaluation factor 3 F). A run consists of a variable number of experiments generally testing different test substances together each using the same vehicle control. This leads to an accumulation of replicates for negative controls which was used to calculate the mean spontaneous reversion number for each run.
A test substance is considered mutagenic in this test system if more than a doubling of Evaluation factor 3 F is observed.

Results and discussion

Test results

Species / strain:

other: TA 98, TA Mix (TA 7001 - TA 7006)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

- TOXICITY
No bacteriotoxic effect (clearing of the background lawn, decrease in the number of yellow wells) was observed.

- MUTAGENICITY
An increase in the number of positive wells (his+ revertants) was not observed either without S9 mix or after the addition of a metabolizing system.

- Precipitation in plates: No precipitation occurred

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions of this study, the test substance Methyl pyruvat is not a mutagenic substance in the Ames II Assay (Salmonella typhimurium reverse mutation assay) in the absence and the presence of metabolic activation.

Executive summary:

No bacteriotoxic effect (clearing of the background lawn, decrease in the number of yellow wells) and no increase in the number of positive wells (his+ revertants) was observed either without S9 mix or after the addition of a metabolizing system.

Thus, Methyl pyruvat is not a mutagenic substance in the Ames II Assay (Salmonella typhimurium reverse mutation assay) in the absence and the presence of metabolic activation under the experimental conditions of this study.