L-methionine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

publication

Title:

Assessment of the performance of the Ames II™ assay: a collaborative study with 19 coded compounds

Author:

S Flückiger-Isler, M Baumeister, K Braun, V Gervais, N Hasler-Nguyen,eR Reimann,J Van Gompel, H.-G Wunderlich,G Engelhardt

Year:

2004

Bibliographic source:

Mutation Research/Genetic Toxicology and Environmental Mutagenesis Volume 558, Issues 1–2, Pages 181–197

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Assessment of mutagenicity using the Ames II assay. The Ames II assay is a is a liquid microtiter modification of the Ames test and consists of the ‘strains’ TAMix and TA98. TAMix is a mixture of the Salmonella typhimurium strains TA7001, TA7002, TA7003, TA7004, TA7005 and TA7006.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

L-methionine

EC Number:

200-562-9

EC Name:

L-methionine

Cas Number:

63-68-3

Molecular formula:

C5H11NO2S

IUPAC Name:

L-methionine

Details on test material:

- Name of test material (as cited in study report): L-Methionine
- Analytical purity: 98%
- Other: Supplier: Sigma

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 2

Species / strain / cell type:

other: TAMix

Details on mammalian cell type (if applicable):

TAMix consists of the strains TA7001, TA7002, TA7003, TA7004, TA7005, and TA7006.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

Six concentrations, plus a negative (solvent) control and a positive control were tested each culture was treated and dispensed into microtiter plates in triplicate. Compounds were tested without any determination for viability or optimization for dose. The highest and the lowest dose level were 5000 and 4g/ml, respectively, and the intermediate doses were spaced at two- to five-fold intervals.
One group used its own internally validated setup for an automated system which differed from the prescribed protocol in that a 10
times lower top dose (500 g/ml) was used, the triplicate values derived from three different overnight cultures, there was no agitation during the 90 min of exposure, and the plate scoring was performed through spectrophotometry.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (final concentration of 4% in the assay)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

without S9 mix:
Into 1-well of a 24-well plate (one well/strain/dose/replicate), 0.215 ml of Exposure Medium and 0.025 ml of culture were aliquoted resulting in a total volume of 0.240 ml.
with S9 mix:
Into 1-well of a 24-well plate (one well/strain/dose/replicate), 0.1775 ml of Exposure Medium, 0.025 ml of culture and 0.0375 ml of 30% S9 mix were aliquoted resulting in a total volume of 0.240 ml.

To each of these cultures, 0.01 ml of test chemical, was added, to give a final volume of 0.250 ml. This mixture was incubated for 90 min at 37°C with agitation at 250 rpm. After 90-min incubation, each well of the 24-well plates containing the chemically treated cultures received 2.8 ml of Indicator Medium. The cultures were mixed gently with the histidine-deficient Indicator Medium that selects for prototrophic reversion before being distributed in 0.05 ml aliquots to 48 wells of a 384-well microtiter plate. One plate was used per strain and replicate. The plates were incubated at 37°C for 48 h. An essential constitution of the Indicator Medium (Bromocresol purple), turns yellow as the pH drops due to the catabolic activity of revertant cells which grow in the absence of histidine.

The number of positive (yellow) wells out of 48 wells per replicate and dose was compared with the number of spontaneous revertants obtained in the negative control section. The average number of wells containing revertants per culture and concentration was calculated from the triplicate sections, and the increases above the zero dose were determined at each concentration of the test chemicals.

Evaluation criteria:

Revertant yields greater than two times the baseline level obtained in the triplicate values of a given dose was classified as an increase in revertant yield of the assay. Multiple responses of greater than two-fold the baseline level led to the classification as a clear positive.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Test results 2

Species / strain:

other: TAMix

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

L-Methionine was tested in the Ames II assay in four laboratories. Non of the laboratories obtained a mutagenic effect with methionine.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

L-Methionine was tested in the Ames II assay in four laboratories. Non of the laboratories obtained a mutagenic effect with L-methionine.