Methyl methanesulphonate

Genetic toxicity: in vivo

Administrative data

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

Data source

Reference

Reference Type:

publication

Title:

Evaluation of the repeated-dose liver and gastrointestinal tract micronucleus assays with 22 chemicals using young adult rats.

Author:

Shuichi Hamada, Wakako Ohyama, Rie Takashima, Keisuke Shimada, Kazumi Matsumotod, Satoru Kawakami, Fuyumi Uno, Hajime Sui, Yasushi Shimada, Tadashi Imamura, Shoji Matsumura, Hisakazu Sanada, Kenji Inoue, Shigeharu Muto, Izumi Ogawa, Aya Hayashi, Tomomi Takayanagi, Yosuke Ogiwara, Akihisa Maeda, Emiko Okada, Yukari Terashima, Hironao Takasawa, Kazunori Narumi, Yumi Wako, Kazufumi Kawasako, Masaki Sano, Nobuyuki Ohashi, Takeshi Morita, Hajime Kojima, Masamitsu Honma, Makoto Hayashi

Year:

2015

Bibliographic source:

Mutation Research 780-781 (2015) 2–17

Materials and methods

Test guideline

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

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

Test material form:

other: Liquid

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/mol
- Substance type: Organic
- Physical state: Solid
- Purity: 99.9 %
- Impurities (identity and concentrations): 0.01 %

Test animals

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

Administration / exposure

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

Doses / concentrations

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

Examinations

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.

Results and discussion

Test results

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

Any other information on results incl. tables

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)

Applicant's summary and conclusion

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.