Methyl methanesulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Mammalian cell gene mutation assay was performed for the test material Methyl methanesulfonate (MMS) using the Chinese Hamster V79 cells. For test SCE cells, V79 cells of the substationary phase were exposed to the test chemical dissoved in either distilled water, special grade DMSO or ethanol for 3 hrs. After treatment, the cells were washed with Hanks' balanced salt solution and cultured further for 27 hr in normal medium containing 5-bromodeoxyuridine. Metaphases were then arrested by replacing the medium by medium containing Colcemid but without 5-bromodeoxyuridine and incubating the cells for an additional 2 hr. Chromosome specimens were prepared by the usual air-drying method after hypotonie treatment with 0.075 M potassium chloride for 20 min and fixation with Carnoy's fixative. Before fixation, the cells were kept in the dark or under a safe light. Chromosomes were then differentially stained by the usual FPG (Fluorescence plus Giemsa) technique with some modifications. For each dose of agents, SCE were analyzed on 50 well-spread metaphases; the chromosome numbers ranged from 19 to 23, but the average chromosome number per cell was 21. SCE were expressed as average numbers per cell, and control values were subtracted to obtain induced SCE frequencies.

Methyl methanesulfonate (MMS) was found to be a potent inducer of SCE in the cell line used.

 

Gene toxicity in vitro result for the test compound Methyl methanesulfonate (MMS) is positive in the mammalian cell gene mutation assay performed using Chinese Hamster V79 cells.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting 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

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

In vitro gene toxicity study of methyl methanesulfonate by using Sister Chromatid Exchanges in Chinese Hamster V79 Cells

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

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

Target gene:

No data

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: Eagle's minimum essential medium plus 10% fetal bovine serum plus 10% fetal bovine serum, kanamycin amd neomycin
- 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:

1.6 µg per ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: The test chemical was either dissolved in distilled water, special-grade dimethyl sulfoxide or Ethanol
- Justification for choice of solvent/vehicle: The test chemical was either soluble in distilled water, special-grade dimethyl sulfoxide or Ethanol

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

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

DURATION
- Preincubation period: No data available
- Exposure duration: 3 hrs
- Expression time (cells in growth medium): 29 hrs
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): 29 hrs

SELECTION AGENT (mutation assays): No data available
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Fluorescence plus Geimsa stain

NUMBER OF REPLICATIONS: No data available

NUMBER OF CELLS EVALUATED: 50 well spread metaphases

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

Evaluation criteria:

Sister chromatid exchanges were analyzed

Statistics:

SCE-inducing potencies were expressed as increase in the number of mutants and SCE over the control values by plotting on a logarithmic scale

Species / strain:

Chinese hamster lung fibroblasts (V79)

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

Conclusions:

Gene toxicity in vitro result for the test compound Methyl methanesulfonate (MMS) is positive in the mammalian cell gene mutation assay performed using Chinese Hamster V79 cells.

Executive summary:

Mammalian cell gene mutation assay was performed for the test material Methyl methanesulfonate (MMS) using the Chinese Hamster V79 cells. For test SCE cells, V79 cells of the substationary phase were exposed to the test chemical dissoved in either distilled water, special grade DMSO or ethanol for 3 hrs. After treatment, the cells were washed with Hanks' balanced salt solution and cultured further for 27 hr in normal medium containing 5-bromodeoxyuridine. Metaphases were then arrested by replacing the medium by medium containing Colcemid but without 5-bromodeoxyuridine and incubating the cells for an additional 2 hr. Chromosome specimens were prepared by the usual air-drying method after hypotonie treatment with 0.075 M potassium chloride for 20 min and fixation with Carnoy's fixative. Before fixation, the cells were kept in the dark or under a safe light. Chromosomes were then differentially stained by the usual FPG (Fluorescence plus Giemsa) technique with some modifications. For each dose of agents, SCE were analyzed on 50 well-spread metaphases; the chromosome numbers ranged from 19 to 23, but the average chromosome number per cell was 21. SCE were expressed as average numbers per cell, and control values were subtracted to obtain induced SCE frequencies.

Methyl methanesulfonate (MMS) was found to be a potent inducer of SCE in the cell line used.

 

Gene toxicity in vitro result for the test compound Methyl methanesulfonate (MMS) is positive in the mammalian cell gene mutation assay performed using Chinese Hamster V79 cells.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In vivo repeated-dose liver (RDLMN) and bone marrow micronucleus assay was performed to determine the mutagenic nature of methyl methanesulphonate (MMS). 6 weeks old male Crl:CD (SD) rats were exposed, once daily, to MMS at dose levels of 0, 12.5, 25 or 50 mg/kg/day for 14 days.

In the RDLMN assay, 24 hours after the last administration of MMS in the 14 days study period, the rats were euthanized under anesthesia. 1 g of the left lateral lobe was sliced into several pieces that were 0.5–1-mm thick. The sliced tissues were rinsed with cold Hank’s balanced salt solution (HBSS) and then treated with a digestion solution containing 100 units/mL of collagenase to make a HEP suspension. Isolated HEPs were fixed with 10% neutral-buffered formalin, and the suspension was stored until microscopic observation. Before observation, 10µL of the HEP suspension was mixed with an equal volume of the staining solution containing acridine orange (AO: 500µg/mL) and 4,6-diamidino-2-phenylindole dihydrochloride (DAPI: 10µg/mL). The stained cell suspension was dropped onto a glass slide and covered with a cover slip. The specimens were observed under a fluorescent microscope at 400× magnification with U-excitation. 2000 parenchymal HEPs, including mono-, bi-, and multi-nucleated cells, number of micronucleated hepatocytes (MNHEPs)  were analyzed and the number of mitotic phase cells among the 2000HEPs was also recorded to calculate the mitotic index (MI). The liver weight was measured at necropsy, and the liver weight to body weight ratio was recorded before dissecting an aliquot for the MN assay. After the isolation of HEPs, the residual liver tissue of the left lateral lobe was fixed with 10% neutral-buffered formalin, embedded in paraffin, sectioned, and then stained with hematoxylin and eosin. Histopathological examination of liver was also performed.

For the Bone marrow MN assay, the rats were euthanized under anesthesia twenty four hours after the last administration of MMS in the 14 days study period. The BM cells were collected by washing the femur cavity with fetal bovine serum. After centrifugation, the supernatants were removed, and the remaining samples were re-suspended and smeared on a glass slide. The smears were dried and fixed with methanol and stored until analysis. Immediately prior to fluorescence microscopic observation, the smears were stained with AO solution (40µg/mL). The stained cell suspension was dropped onto a glass slide and covered with a cover slip. 2000 immature erythrocytes (IMEs), and the number of micronucleated immature erythrocytes (MNIMEs) were analyzed and as a parameter of the cytotoxicity, the ratio of IMEs to 1000 erythrocytes were observed.

Diethylnitrosamine (DEN) was used as a positive control for both the assays.

MMS induced micronucleated hepatocytes (MNHEPs) cell in the RDLMN assay and bone marrow immature erythrocytes (IMEs), and the micronucleated immature erythrocytes (MNIMEs) in the bone marrow micronucleus assay. No histopathological findings were observed but an increase in liver weight was noted at dose level of 12.5, 25 or 50 mg/Kg/day in the RDLMN assay.

Based on the observations made, Methyl methanesulfonate (MMS) was considered to be a positive gene mutant at a concentration of 0, 12.5, 25 or 50 mg/kg/day when male Crl:CD (SD) were treated with MMS for 14 days.

Link to relevant study records

Reference

Endpoint:

genetic toxicity in vivo

Remarks:

Type of genotoxicity: other: Liver and Bone marrow Micronucleus assays

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

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

In vivo repeated-dose liver (RDLMN) and bone marrow micronucleus assay was performed to determine the mutagenic nature of methyl methanesulphonate (MMS)

GLP compliance:

yes

Type of assay:

other: Liver and bone marrow micronucleus assay

Specific details on test material used for the study:

- Name of test material: Methyl methanesulfonate (MMS)
- Molecular formula: C2H6O3S
- Molecular weight: 110.1324 g/mol
- Substance type: Organic
- Physical state: Solid
- Purity: 99.9 %
- Impurities (identity and concentrations): 0.01 %

Species:

rat

Strain:

other: Crl:CD (SD)

Details on species / strain selection:

No data

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc. (Atsugi, Hino or Tsukuba, Japan)
- Age at study initiation: 6 weeks
- Weight at study initiation: No data available
- Assigned to test groups randomly: [no/yes, under following basis: ] No data available
- Fasting period before study: No data available
- Housing: Animals were housed in air-conditioned room.
- Diet (e.g. ad libitum): Food, ad libitum
- Water (e.g. ad libitum): Drinking water, ad libitum
- Acclimation period: No data available

ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data available
- Humidity (%):No data available
- Air changes (per hr): No data available
- Photoperiod (hrs dark / hrs light): 12-h light/dark cycle

IN-LIFE DATES: From: To: No data available

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Saline
- Justification for choice of solvent/vehicle: No data available
- Concentration of test material in vehicle: 0, 12.5, 25 or 50 mg/kg/day
- Amount of vehicle (if gavage or dermal): No data available
- Type and concentration of dispersant aid (if powder): No data available
- Lot/batch no. (if required): No data available
- Purity: No data available

Details on exposure:

For oral route
PREPARATION OF DOSING SOLUTIONS: The test chemical was dissolved in saline to give dose levels of 0, 12.5, 25 or 50 mg/kg/day

DIET PREPARATION
- Rate of preparation of diet (frequency): No data available
- Mixing appropriate amounts with (Type of food): No data available
- Storage temperature of food: No data available

Duration of treatment / exposure:

14 days

Frequency of treatment:

Daily

Post exposure period:

No data available

Remarks:

0, 12.5, 25 or 50 mg/kg/day

No. of animals per sex per dose:

Total: 20
0 mg/kg/day: 5 male
12.5 mg/kg/day: 5 male
25 mg/kg/day: 5 male
50 mg/kg/day: 5 male

Control animals:

yes, concurrent vehicle

Positive control(s):

Diethylnitrosamine (DEN)
- Justification for choice of positive control(s): DEN was used as a model chemical
- Route of administration: Oral (Gavage)
- Doses / concentrations: 0, 3.13, 6.25 or 12.5 mg/kg/day

Tissues and cell types examined:

Liver micronucleated hepatocytes (MNHEPs) cell and bone marrow immature erythrocytes (IMEs), and the number of micronucleated immature erythrocytes (MNIMEs) were examined.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Optimal dose levels were used by using a dose-finding preliminary test, with the maximum tolerated dose (the dose inducing clinical signs without being lethal) as the top dose level.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): The animals were treated once a day for 14 days

DETAILS OF SLIDE PREPARATION:
For Liver MN assay: The animals were treated with MMS for 14 days and 24 hours after the last administration, the rats were euthanized under anesthesia. 1 g of the left lateral lobe was sliced into several pieces that were 0.5–1-mm thick. The sliced tissues were rinsed with cold Hank’s balanced salt solution (HBSS) and then
treated with a digestion solution containing 100 units/mL of collagenase to make a HEP suspension. Isolated HEPs were fixed with 10% neutral-buffered formalin, and the suspension was stored until microscopic observation. Before observation, 10µL of the HEP suspension was mixed with an equal volume of the staining solution containing acridine orange (AO: 500µg/mL) and 4,6-diamidino-2-phenylindole dihydrochloride (DAPI: 10µg/mL). The stained cell suspension was dropped onto a glass slide and covered with a cover slip. The specimens were observed under a fluorescent microscope at 400× magnification with U-excitation

For BM MN assay :The animals were treated with MMS for 14 days and 24 hours after the last administration, the rats were euthanized under anesthesia. The BM cells were collected by washing the femur cavity with fetal bovine serum. After centrifugation, the supernatants were removed, and the remaining samples were re-suspended and smeared on a glass slide. The smears were dried and fixed with methanol and stored until analysis. Immediately prior to fluorescence microscopic observation, the smears were stained with AO solution (40µg/mL). The stained cell suspension was dropped onto a glass slide and covered with a cover slip.

METHOD OF ANALYSIS:
For Liver MN assay: 2000 parenchymal HEPs, including mono-, bi-, and multi-nucleated cells, number of micronucleated hepatocytes (MNHEPs) were analyzed and the number of mitotic phase cells among the 2000HEPs was also recorded to calculate the mitotic index (MI).

For BM MN assay: 2000 immature erythrocytes (IMEs), and the number of micronucleated immature erythrocytes (MNIMEs) were analyzed and as a parameter of the cytotoxicity, the ratio of IMEs to 1000 erythrocytes were observed.

OTHER: No data available

Evaluation criteria:

Micronucleus induction was examined

Statistics:

Incidences of MNHEPs and MNIMEs cells between the test and vehicle control groups were analyzed by the conditional binomial test reported by Kastenbaum and Bowman at upper-tailed significance levels of 5% and 1%.

The other quantitative data were analyzed for their statistical significance by the multiple comparison procedure. Namely, the homogeneity of variance was examined using Bartlett’s test. When a homogeneous variance was demonstrated, one-way analysis of variance was applied; otherwise, Kruskal–Wallis test was applied. When statistical nsignificance was demonstrated between the groups, the difference was assessed using Dunnett’s test or the Dunnett-type multiple comparison test.

Sex:

male

Genotoxicity:

positive

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

No data

Table: Histopathological findings and organ weights of the liver in the RDLMN assay.

Duration

Dose

Histopathological findings

Liver Weight

Mg/Kg/day

H

S

A

P

M

F

Pob

Inf

Death, the other pathological findings

Absolute

Relative

14 days

0, 12.5, 25 or 50

-

-

-

-

-

-

-

-

-

-

Gain (12.5, 25 or 50)

Conclusions:

Methyl methanesulfonate (MMS) was considered to be a positive gene mutant at a concentration of 0, 12.5, 25 or 50 mg/kg/day when male Crl:CD (SD) were treated with MMS for 14 days in the repeated dose liver micronucleus assay (RDLMN) and bone marrow micronucleus assay.

Executive summary:

In vivo repeated-dose liver (RDLMN) and bone marrow micronucleus assay was performed to determine the mutagenic nature of methyl methanesulphonate (MMS). 6 weeks old male Crl:CD (SD) rats were exposed, once daily, to MMS at dose levels of 0, 12.5, 25 or 50 mg/kg/day for 14 days.

In the RDLMN assay, 24 hours after the last administration of MMS in the 14 days study period, the rats were euthanized under anesthesia. 1 g of the left lateral lobe was sliced into several pieces that were 0.5–1-mm thick. The sliced tissues were rinsed with cold Hank’s balanced salt solution (HBSS) and then treated with a digestion solution containing 100 units/mL of collagenase to make a HEP suspension. Isolated HEPs were fixed with 10% neutral-buffered formalin, and the suspension was stored until microscopic observation. Before observation, 10µL of the HEP suspension was mixed with an equal volume of the staining solution containing acridine orange (AO: 500µg/mL) and 4,6-diamidino-2-phenylindole dihydrochloride (DAPI: 10µg/mL). The stained cell suspension was dropped onto a glass slide and covered with a cover slip. The specimens were observed under a fluorescent microscope at 400× magnification with U-excitation. 2000 parenchymal HEPs, including mono-, bi-, and multi-nucleated cells, number of micronucleated hepatocytes (MNHEPs)  were analyzed and the number of mitotic phase cells among the 2000HEPs was also recorded to calculate the mitotic index (MI). The liver weight was measured at necropsy, and the liver weight to body weight ratio was recorded before dissecting an aliquot for the MN assay. After the isolation of HEPs, the residual liver tissue of the left lateral lobe was fixed with 10% neutral-buffered formalin, embedded in paraffin, sectioned, and then stained with hematoxylin and eosin. Histopathological examination of liver was also performed.

For the Bone marrow MN assay, the rats were euthanized under anesthesia twenty four hours after the last administration of MMS in the 14 days study period. The BM cells were collected by washing the femur cavity with fetal bovine serum. After centrifugation, the supernatants were removed, and the remaining samples were re-suspended and smeared on a glass slide. The smears were dried and fixed with methanol and stored until analysis. Immediately prior to fluorescence microscopic observation, the smears were stained with AO solution (40µg/mL). The stained cell suspension was dropped onto a glass slide and covered with a cover slip. 2000 immature erythrocytes (IMEs), and the number of micronucleated immature erythrocytes (MNIMEs) were analyzed and as a parameter of the cytotoxicity, the ratio of IMEs to 1000 erythrocytes were observed.

Diethylnitrosamine (DEN) was used as a positive control for both the assays.

MMS induced micronucleated hepatocytes (MNHEPs) cell in the RDLMN assay and bone marrow immature erythrocytes (IMEs), and the micronucleated immature erythrocytes (MNIMEs) in the bone marrow micronucleus assay. No histopathological findings were observed but an increase in liver weight was noted at dose level of 12.5, 25 or 50 mg/Kg/day in the RDLMN assay.

Based on the observations made, Methyl methanesulfonate (MMS) was considered to be a positive gene mutant at a concentration of 0, 12.5, 25 or 50 mg/kg/day when male Crl:CD (SD) were treated with MMS for 14 days.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Additional information

Gene mutation in vitro:

Various peer reviewed publications were reviewed to determine the mutagenic nature of methyl methanesulphonate. The studies are as mentioned below:

Analysis for the induction of micronuclei in L5178Y mouse lymphoma cells by the methylating agent methyl methanesulfonate (MMS) was performed by Lutz et al (Toxicological sciences, 2005). 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.

In another study, Mammalian cell gene mutation assay was performed by Nishi et al (Cancer Research, 1984) for the test material Methyl methanesulfonate (MMS) using the Chinese Hamster V79 cells. For test SCE cells, V79 cells of the substationary phase were exposed to the test chemical dissoved in either distilled water, special grade DMSO or ethanol for 3 hrs. After treatment, the cells were washed with Hanks' balanced salt solution and cultured further for 27 hr in normal medium containing 5-bromodeoxyuridine. Metaphases were then arrested by replacing the medium by medium containing Colcemid but without 5-bromodeoxyuridine and incubating the cells for an additional 2 hr. Chromosome specimens were prepared by the usual air-drying method after hypotonie treatment with 0.075 M potassium chloride for 20 min and fixation with Carnoy's fixative. Before fixation, the cells were kept in the dark or under a safe light. Chromosomes were then differentially stained by the usual FPG (Fluorescence plus Giemsa) technique with some modifications. For each dose of agents, SCE were analyzed on 50 well-spread metaphases; the chromosome numbers ranged from 19 to 23, but the average chromosome number per cell was 21. SCE were expressed as average numbers per cell, and control values were subtracted to obtain induced SCE frequencies. Methyl methanesulfonate (MMS) was found to be a potent inducer of SCE in the cell line used. Gene toxicity in vitro result for the test compound Methyl methanesulfonate (MMS) is positive in the mammalian cell gene mutation assay performed using Chinese Hamster V79 cells. Methyl methanesulfonate (MMS) was found to be a potent inducer of mutation in the cell line used. Gene toxicity in vitro result for the test compound Methyl methanesulfonate (MMS)  is positive in the 6-thioguanine resistance cell gene mutation assay performed using Chinese Hamster V79 cells.

In the same study by Nishi et al (Cancer Research, 1984), 6-thioguanine resistance cell gene mutation assay was performed for the test material Methyl methanesulfonate (MMS) using the Chinese Hamster V79 cells. V79 cells of the substationary phase were exposed to the test chemical dissoved in either distilled water, special grade DMSO or ethanol for 3 hrs. After treatment, the cells were washed with Hanks' balanced salt solution and cultured further for 27 hr in normal medium containing 5-bromodeoxyuridine. Immediately after washing with Hanks' balanced salt solution, some cells were plated in dishes at 200 cells/6-cm dish to determine the toxicity of the agents (initial survival). The remaining cells were all replated in fresh flasks at about 0.5 to 1 x 10⁶cells per 75-sq cm flask, to allow the full expression time. Subculture was usually done twice or 3 times during the expression time at a split ratio of 1:2 to 1:8, depending on the recovery. The medium was changed every 2 days until the cells reached the stationary phase. Usually on Day 6, which is considered to be an adequate expression time under many conditions, some of the treated cells were plated at 200 cells/6-cm dish for survival adjustment (replated survival). The remaining cells were plated at 1 x 10⁵cells/6-cm dish in medium containing 10 µM 6-thioguanine. On Days 13 to 15, colonies were fixed, stained, and counted. Mutation frequencies were expressed as the numbers of mutants per 10" viable cells, and control values were subtracted to obtain induced mutation frequencies. Methyl methanesulfonate (MMS) was found to be a potent inducer of mutation in the cell line used. Gene toxicity in vitro result for the test compound Methyl methanesulfonate (MMS)  is positive in the 6-thioguanine resistance cell gene mutation assay performed using Chinese Hamster V79 cells .

Benz et al (Aerospace Medical Research Laboratory, 1979) performed in vitro micronucleus study to determine the mutagenic nature of Methyl methanesulfonate using canine perpheral lymphocytes. Peripheral blood samples were assessed for the induction of micronuclei in the lymphocte cell line used. Methyl methanesulphonate caused an an increase in number of chromosome breaks being caused by increasing chemical doses, followed by a decrease in number of micronuclei detected at even higher dose levels. For a cell to have a micronucleus, at least one cell division must occur. At the highest doses, MMS prevented cell division and therefore fewer micronuclei are seen even though, presumably, increase in chromosome breakage was observed. On the basis of observations made, Methyl methanesulfonate (MMS) is a considered to be a positive gene mutant to induce micronuclei in the canine peripheral lymphocytes exposed in vitro to the test chemical.

Based on the available data for the target chemical, Methyl methanesulphonate exhibits gene mutation toxicity in vitro. Hence, the chemical is likely to classify as a gene mutant in vitro.

Gene toxicity in vivo:

Various peer reviewed publications were reviewed to determine the mutagenic nature of methyl methanesulphonate. The studies are as mentioned below:

In vivo repeated-dose liver (RDLMN) and bone marrow micronucleus assay was performed by Hamada et al (Mutation Research, 2015) to determine the mutagenic nature of methyl methanesulphonate (MMS). 6 weeks old male Crl:CD (SD) rats were exposed, once daily, to MMS at dose levels of 0, 12.5, 25 or 50 mg/kg/day for 14 days and 0, 7.5, 15 or 30 mg/Kg/day for 28 days.

In the RDLMN assay, 24 hours after the last administration of MMS in the 14 days or 28 days study period, the rats were euthanized under anesthesia. 1 g of the left lateral lobe was sliced into several pieces that were 0.5–1-mm thick. The sliced tissues were rinsed with cold Hank’s balanced salt solution (HBSS) and then treated with a digestion solution containing 100 units/mL of collagenase to make a HEP suspension. Isolated HEPs were fixed with 10% neutral-buffered formalin, and the suspension was stored until microscopic observation. Before observation, 10µL of the HEP suspension was mixed with an equal volume of the staining solution containing acridine orange (AO: 500µg/mL) and 4,6-diamidino-2-phenylindole dihydrochloride (DAPI: 10µg/mL). The stained cell suspension was dropped onto a glass slide and covered with a cover slip. The specimens were observed under a fluorescent microscope at 400× magnification with U-excitation. 2000 parenchymal HEPs, including mono-, bi-, and multi-nucleated cells, number of micronucleated hepatocytes (MNHEPs)  were analyzed and the number of mitotic phase cells among the 2000HEPs was also recorded to calculate the mitotic index (MI). The liver weight was measured at necropsy, and the liver weight to body weight ratio was recorded before dissecting an aliquot for the MN assay. After the isolation of HEPs, the residual liver tissue of the left lateral lobe was fixed with 10% neutral-buffered formalin, embedded in paraffin, sectioned, and then stained with hematoxylin and eosin. Histopathological examination of liver was also performed.

For the Bone marrow MN assay, the rats were euthanized under anesthesia twenty four hours after the last administration of MMS in the 14 days or 28 days study period. The BM cells were collected by washing the femur cavity with fetal bovine serum. After centrifugation, the supernatants were removed, and the remaining samples were re-suspended and smeared on a glass slide. The smears were dried and fixed with methanol and stored until analysis. Immediately prior to fluorescence microscopic observation, the smears were stained with AO solution (40µg/mL). The stained cell suspension was dropped onto a glass slide and covered with a cover slip. 2000 immature erythrocytes (IMEs), and the number of micronucleated immature erythrocytes (MNIMEs) were analyzed and as a parameter of the cytotoxicity, the ratio of IMEs to 1000 erythrocytes were observed.

Diethylnitrosamine (DEN) was used as a positive control for both the assays.

MMS induced micronucleated hepatocytes (MNHEPs) cell in the RDLMN assay in the 14 days study and bone marrow immature erythrocytes (IMEs), and the micronucleated immature erythrocytes (MNIMEs) in the bone marrow micronucleus assay in the 14 days and 28 days study both. No histopathological findings were observed but an increase in liver weight was noted at dose level of 12.5, 25 or 50 mg/Kg/day in the RDLMN assay. However, MMS did not induce micronucleated hepatocytes (MNHEPs) cell in the RDLMN assay in the 28 days study. No histopathological findings were observed in the 28 days study but an increase in liver weight was noted at 30 mg/Kg/day dose level.

Based on the observations made, Methyl methanesulfonate (MMS) was considered to be a positive gene mutant when male Crl:CD (SD) were treated with MMS for 14 days in both the RDLMN and 14 days and 28 days in the bone marrow miconucleus assay and was considered negative in the 28 days RDLMN assay .

In another study performed by Madrigal-Bujaidar ( Basic & Clinical Pharmacology & Toxicology, 2008), the assay was used to determine the effects of methyl methanesulfonate as an inducer of micronucleated polychromatic erythrocytes (MNPE) and micronucleated normochromatic erythrocytes (MNNE) in mice. The study was performed using six 2 months old male CD-1 mice. Methyl methane sulphonate was dissolved in ditilled water at a dose level of 0 or 30 mg/Kg/day and the animals were allowed to drink this for 4 weeks. The control group drank water without the tested chemical. The weight of the animals and the amount of water consumed were registered daily. The frequency of micronuclei was determined before the experimental treatment and at the end of each study week. Two blood smears were made from the tail of each animal, fixed in methanol for 5 min. and stained for 20 min. with a 4% Giemsa solution made in phosphate-buffered saline. One thousand erythrocytes per animal were scored to determine the rate of MNPE and MNNE. Besides, in order to evaluate bone marrow cytotoxicity, 1000 erythrocytes per animal were scored and the rate of polychromatic erythrocytes (PE) to the number of normochromatic erythrocytes (PE/NE index) was established. In the control animals, a significant increase in weight gain was observed from the first week of treatment, with a gain of 7.3 g at the fourth week. Animals treated with MMS increased only 3.8 g at the fourth week. When the administration of chemical was suspended, the animals showed a weight increase. The control group animals showed a homogeneous low amount of damaged cells with respect to the frequency of MNPE. The administration of MMS gave rise to a significant increase in the number of micronuclei in the 4 weeks, reaching to more than three times the value determined before the treatment. In the recovery period (weeks 5–8), the micronuclei values presented a decrease approaching the control level. The bone marrow-cytotoxic effect induced by the control group animals indicated a PE/NE index between 0.046 and 0.053. MMS was considered to be cytotoxic from the first week, reaching about a 50% index decrease at the fourth week with a partial recovery in the following 4 weeks. Thus on the basis of observations made, gene toxicity in vivo result for methyl methanesulfonate is positive when the CD-1 male mice were exposed to the test compound during the 4 week period.

Based on the available data for the target chemical, Methyl methanesulphonate exhibits gene mutation toxicity in vivo. Hence, the chemical is likely to classify as a gene mutant in vivo.

Justification for classification or non-classification

Based on the available data for the target chemical, Methyl methanesulphonate exhibits gene mutation toxicity in vitro and in vivo. Hence, the chemical is likely to classify as a gene mutant in vitro and in vivo.