Methyl methanesulphonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

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:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed publication

Data source

Reference

Reference Type:

publication

Title:

Different Types of Combination Effects for the Induction of Micronuclei in Mouse Lymphoma Cells by Binary Mixtures of the Genotoxic Agents MMS, MNU, and Genistein

Author:

Werner K. Lutz, Oliver Tiedge, Roman W. Lutz, and Helga Stopper

Year:

2005

Bibliographic source:

TOXICOLOGICAL SCIENCES 86(2), 318–323 (2005)

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

In vitro induction of micronuclei was tested using L5178Y mouse lymphoma cells by Methyl methanesulfonate

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Methyl methanesulphonate

EC Number:

200-625-0

EC Name:

Methyl methanesulphonate

Cas Number:

66-27-3

Molecular formula:

C2H6O3S

IUPAC Name:

methyl methanesulfonate

Details on test material:

- Name of test material: Methyl methanesulfonate (MMS)
- Molecular formula: C2H6O3S
- Molecular weight: 110.1324 g/mol
- Substance type: Organic
- Physical state: Liquid
- Purity: No data available
- Impurities (identity and concentrations): No data available

Specific details on test material used for the study:

- Name of test material: Methyl methanesulfonate (MMS)
- Molecular formula: C2H6O3S
- Molecular weight: 110.1324 g/mole
- Substance type: Organic
- Physical state: Liquid
- Purity: No data available
- Impurities (identity and concentrations): No data available

Method

Target gene:

No data

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 cell culture medium supplemented with antibiotics,
0.25 mg L-glutamine/ml, 107 µg sodium pyruvate /ml, and 10% heat inactivated horse serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: No data available
- Periodically checked for karyotype stability: No data available
- Periodically "cleansed" against high spontaneous background: No data available

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

not specified

Metabolic activation system:

No data

Test concentrations with justification for top dose:

0, 100, 200, 300 µM

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The chemical is soluble in DMSO

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 200000 cells per mL

DURATION
- Preincubation period: No data available
- Exposure duration: 4 hrs
- Expression time (cells in growth medium): 20 hrs
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hrs

SELECTION AGENT (mutation assays): No data available
SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B
STAIN (for cytogenetic assays): Acridine orange

NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED: Two thousand cells (1000 per slide) were evaluated for each treatment. The score (MN) was the number of cells containing one or more micronuclei per 1000 binucleated cells (BN cells). The percentage of binucleated cells was evaluated as a marker of cell proliferation.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data available

OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other: No data available

OTHER: No data available

Rationale for test conditions:

No data available

Evaluation criteria:

The percentage of binucleated cells was evaluated as a marker of cell proliferation.

Statistics:

The hypothesis of response addition (addition of net effects of the components) was tested by the sign test. Under the null hypothesis of response addition and the assumption of symmetrical errors, the observed number of cells with micronuclei is with equal probability greater or less than the number of cells with micronuclei calculated for response addition.

The hypothesis of dose addition was tested with a linear model, using the logarithm of the score MN to linearize the sublinear dose responses for the components in a parallel manner. For the test of deviation from dose addition,
Testing for interaction according to
Log10 (MN) = a +(bXA)+(c X B) +(d X A X B)+Ɛ (error term)
Letters A and B stand for the concentrations of the two chemicals. AX B is the interaction term. The coefficients a, b, c, and d were estimated by linear regression. The error was proportional to the score MN. For the statistical handling of the replicates, random effects for the parameters were added if this resulted in a model improvement (mixed effects model).

Note: The absence of an interaction (d ¼ 0) is equivalent to dose addition, as can be shown by replacing B for A by normalization with c/b (last line):
MN= 10 (a+b X A+c X B+c) = 10a X 10(bXA) X 10(c X B) X 10c
=10a X 10(bXA) X 10(b X c/b X B) X 10c
= 10a X (10b)A X 10(c/b X B) X 10c
=10a X (10b)(A+c/bXB) X 10c

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

not specified

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

No data available

Applicant's summary and conclusion

Conclusions:

Methyl methanesulfonate (MMS) tested at a concentration of 0, 100, 200, 300 µM showed the induction of micronuclei in the mouse Lymphoma L5178Y cells and hence is positive for gene mutation in vitro.

Executive summary:

Analysis for the induction of micronuclei in L5178Y mouse lymphoma cells by the methylating agent methyl methanesulfonate (MMS) was performed. The test chemical was used at dose levels of 0, 100, 200 and 300 µM. Pilot experiment was performed to determine the tange for the test and the concentration range used was defined by similar effect magnitude, in order not to exceed the response range of the assay that may be limited by cytotoxicity. Mouse lymphoma L5178Y cells, clone 3.7.2c were cultured in suspension in RPMI 1640 cell culture medium supplemented with antibiotics, 0.25 mg L-glutamine/ml, 107 µg sodium pyruvate/ml, and 10% heat inactivated horse serum. Methyl methanesulfonate (MMS) was dissolved in DMSO and added to L5187Y mouse lymphoma cells at a density of 2 X 10⁵cells per ml. The cells were washed twice after 4 hrs chemical exposure and cytochalasin B was added. Cytochalasin B remained in the culture for the entire expression time of 20 h until the cells were harvested. Cytospin preparations on glass slides were prepared. After 2 h in methanol at -20C, the slides were incubated with acridine orange for 5 min, washed twice with Sorensen buffer for 5 min, and mounted for microscopy. Two thousand cells (1000 per slide) were evaluated for each treatment. The score (MN) was the number of cells containing one or more micronuclei per 1000 binucleated cells (BN cells). The percentage of binucleated cells was evaluated as a marker of cell proliferation. components. Two completely independent sets of experiments were run for each pairwise combination. In addition, controls were run in duplicate. A dose response relationship was observed for micronucleus induction in L5178Y mouse lymphoma cells. Methyl methanesulfonate (MMS) tested at a concentration of 0, 100, 200, 300 µM showed the induction of micronuclei in the mouse Lymphoma L5178Y cells and hence is positive for gene mutation in vitro.