Methyl methanesulphonate

Endpoint summary

Description of key information

Repeated Dose Toxicity: Oral

In Repeated dose subacute oral toxicity study, Male Sprague – Dawley rats were treated with Methyl Methansulfonate (MMS) at a concentration of 0, 20 or 40 mg/kg body weight/day orally by gavage for 2 weeks. The animals were observed for clinical signs, mortality, body weight changes, food consumption, gross pathology and histopathology. No effects were observed on mortality, clinical sign, body weight and food consumption at 20 mg/kg body weight/day treated rat as compared to control. Observation made suggest the No observed adverse effect level (NOAEL) was considered to be 20 mg/kg body weight /day when Sprague – Dawley male rats were exposed to Methyl Methansulfonate (MMS) for 2 weeks.

Repeated dose toxicity: Inhalation

Repeated dose inhalation toxicity study was performed by Sellkumar et al (Jounal of National Cancer Institute, 1987) to determine the toxic nature of Methylmethane sulfonate (MMS) in rat upon repeated exposure. 11 -12 week old Male Sprague Dawley rat were exposed to MMS at a dose level of 0 or 5 mg/L for 30 days. All animals were observed daily, weighed monthly, and allowed to die spontaneously or sacrificed when moribund. A complete necropsy was performed on each animal. On the basis of observations made, the low-observed-adverse-effect concentration (LOAEC)  was considered to be 5 mg/L (50ppm) when Sprague-Dawley male rat were exposed to Methylmethane sulfonate (MMS) upon repeated exposure for 30 days.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

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:

14 days repeated-dose toxicity tudy of Methyl methanesulfonate (MMS) was performed by oral exposure in male rats

GLP compliance:

not specified

Limit test:

no

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

Details on route of administration:

No data

Vehicle:

physiological saline

Details on oral exposure:

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
- Mixing appropriate amounts with (Type of food): No data
- Storage temperature of food: No data

VEHICLE
- Justification for use and choice of vehicle (if other than water): Saline
- Concentration in vehicle: 0, 12.5, 25 or 50 mg/kg/day
- Amount of vehicle (if gavage): No data
- Lot/batch no. (if required): No data
- Purity: No data

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No data available

Duration of treatment / exposure:

14 days

Frequency of treatment:

Daily

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

Details on study design:

- Dose selection rationale: 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.
- Rationale for animal assignment (if not random): No data available
- Rationale for selecting satellite groups: No data available
- Post-exposure recovery period in satellite groups: No data available
- Section schedule rationale (if not random): No data available

Positive control:

Diethylnitrosamine (DEN)

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: No data available
- Cage side observations checked in table [No.?] were included. The animals were observed for mortality

DETAILED CLINICAL OBSERVATIONS: No data available
- Time schedule: No data available

BODY WEIGHT: Yes
- Time schedule for examinations: Before dissecting the animals

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No data available
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data available
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data available

FOOD EFFICIENCY: No data available
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data available


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data available
- Time schedule for examinations: No data available

OPHTHALMOSCOPIC EXAMINATION: No data available
- Time schedule for examinations: No data available
- Dose groups that were examined: No data available

HAEMATOLOGY: No data available
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: No data available
- Animals fasted: No data available
- How many animals: No data available
- Parameters checked in table [No.?] were examined. No data available

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Twenty-four hours after the last administration of dose.
- Animals fasted: No data available
- How many animals: No data available
- Parameters checked in table [No.?] were examined: micronucleated hepatocytes (MNHEPs) of the liver and Immature erythrocytes (IMEs) and number of micronucleated immature erythrocytes (MNIMEs) of bone marrow were examined.

URINALYSIS: No data available
- Time schedule for collection of urine: No data available
- Metabolism cages used for collection of urine: No data available
- Animals fasted: No data available
- Parameters checked in table [No.?] were examined. No data available

NEUROBEHAVIOURAL EXAMINATION: No data available
- Time schedule for examinations: No data available
- Dose groups that were examined: No data available
- Battery of functions tested: sensory activity / grip strength / motor activity / other: No data available

OTHER:
Organ weight: Yes, Liver weight were recorded

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, Liver weight was examined at necropsy

HISTOPATHOLOGY: Yes
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 according to the standard method. A histopathological examination was performed under a light microscope to observe for findings including hepatocyte hypertrophy, single cell necrosis, anisokaryosis, prominent nucleolus, mitotic figure, focus of altered hepatocyte, proliferation oval cell or bile duct and cell infiltration.

Other examinations:

Gene toxicity of liver and bone marrow were 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.

Clinical signs:

not specified

Mortality:

no mortality observed

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Details on results:

Clinical signs and mortality: No mortality was observed in the treated dosed animals

Body weight and weight gain: No data available

Food consumption and compound intake: No data available

Food efficiency: No data available

Water consumption and compound intake: No data available

Opthalmoscopic examination: No data available

Haematology: No data available

Clinical chemistry: No data available

Urinanalysis No data available

Neurobehaviour: No data available

Organ weights: When treated with 12.5, 25 and 50 mg/kg/day, relative liver weight was increased as compare to control.

Gross pathology: No data available

Histopathology: No histopathological findings including hepatocyte hypertrophy, single cell necrosis, anisokaryosis, prominent nucleolus, mitotic figure, focus of altered hepatocyte, proliferation oval cell or bile duct and cell infiltration was noted. No increased frequency of mitotic cells in the liver was observed regardless of hepatocarcinogenicity.

Dose descriptor:

LOAEL

Effect level:

12.5 other: mg/kg/day

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Effect observed on liver weight

Critical effects observed:

not specified

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)

H: hepatocyte hypertrophy, S: single cell necrosis, A: anisokaryosis, P: prominent nucleolus, M: mitotic figure, F: focus of altered hepatocyte, Pob: proliferation oval cell or bile duct, Inf: cell infiltration.

Conclusions:

The Low observed adverse effect level (LOAEL) was considered to be 12.5 mg/kg/day when Crl:CD (SD) male rats were were exposed to Methyl methanesulfonate (MMS) for 14 days

Executive summary:

14 days repeated-dose oral toxicity study was performed on male Crl: CD (SD) rats to determine the oral toxic nature of Methyl methanesulfonate (MMS). MMS was dosed at a concentration of 0, 12.5, 25 and 50 mg/kg/day orally by gavage. The animals were observed for mortality, liver weight and histopathological parameters like hepatocyte hypertrophy, single cell necrosis, anisokaryosis, prominent nucleolus, mitotic figure, focus of altered hepatocyte, proliferation oval cell or bile duct and cell infiltration. Relative liver weight was increased in 12.5, 25 and 50 mg/kg/day dose group. No other histopathological findings were observed. On the basis of observations made, the Low observed adverse effect level (LOAEL) was considered to be 12.5 mg/kg/day when Crl:CD (SD) male rats were were exposed to Methyl methanesulfonate (MMS) for 14 days.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

20 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Data is from peer reviewed publication

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

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:

Repeated dose inhalation toxicity study was performed to determine the toxic nature of Methylmethane sulfonate in rat upon repeated exposure

GLP compliance:

not specified

Limit test:

yes

Specific details on test material used for the study:

- Name of test material : Methylmethane sulfonate (MMS)
- Molecular formula: C2H6O3S
- Molecular weight: 110.1324 g/mol
- Substance type: Organic
- Physical state: Liquid
- Purity: > 95%
- Impurities (identity and concentrations): < 5 %

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

No data

Sex:

male

Details on test animals or test system and environmental conditions:

Details on test animal
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, MA
- Age at study initiation: 11 to 12-week-old
- Weight at study initiation: 325 ± 16.8 g,
- Fasting period before study: No data available
- Housing: No data available
- Diet (e.g. ad libitum): Purina Lab Chow, ad libitum, except during exposure periods.
- Water (e.g. ad libitum): Water, ad libitum, except during exposure periods.
- Acclimation period: 2 weeks

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

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

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: No data available

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Dynamic exposure Chambers
- Method of holding animals in test chamber: No data available
- Source and rate of air: Airstream
- Method of conditioning air: Airstream over the liquids in a generating flask and then feeding the effluent vapor into the chamber air supply.
- System of generating particulates/aerosols: Test atmospheres of the chemicals were generated by passing an airstream over the liquids in a generating flask and then feeding the effluent vapor into the chamber air supply.
- Temperature, humidity, pressure in air chamber: No data available
- Air flow rate: 1.0 m3 or 1.3 m3
- Air change rate: No data available
- Method of particle size determination: Wilks Miran infrared gas analyzer were use to determine the particle size.
- Treatment of exhaust air: No data available

TEST ATMOSPHERE
- Brief description of analytical method used: Chamber concentrations of the chemicals were analyzed at half-hour intervals during the daily exposures by means of a Wilks Miran infrared gas analyzer with the use of appropriate wavelength.
- Samples taken from breathing zone: No data available

VEHICLE (if applicable)
- Justification for use and choice of vehicle: Air
- Composition of vehicle: No data available
- Type and concentration of dispersant aid (if powder): No data available
- Concentration of test material in vehicle: 0 or 50 ppm (0 or 5 mg/L)
- Lot/batch no. of vehicle (if required): No data available
- Purity of vehicle: No data available

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No data

Duration of treatment / exposure:

30 days

Frequency of treatment:

6 hr/day X 5 days/wk

Remarks:

0 or 5 mg/L

No. of animals per sex per dose:

Total : 178
0 ppm (0 mg/L): 98
50 ppm (5 mg/L): 80

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: 50 ppm (5 mg/L) dose was used for MMS because of the cost of compound.
- Rationale for animal assignment (if not random): Animal was assigned to test group randomly.
- Rationale for selecting satellite groups: No data available
- Post-exposure recovery period in satellite groups: No data available
- Section schedule rationale (if not random): No data available

Positive control:

No data

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations checked in table [No.?] were included: Mortality

DETAILED CLINICAL OBSERVATIONS: No data available
- Time schedule: No data available

BODY WEIGHT: Yes
- Time schedule for examinations: Monthly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No data available
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data available
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data available

FOOD EFFICIENCY: No data available
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data available

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data available
- Time schedule for examinations: No data available

OPHTHALMOSCOPIC EXAMINATION: No data available
- Time schedule for examinations: No data available
- Dose groups that were examined: No data available

HAEMATOLOGY: No data available
- Time schedule for collection of blood: No data available
- Anaesthetic used for blood collection: No data available
- Animals fasted: No data available
- How many animals: No data available
- Parameters checked in table [No.?] were examined: No data available

CLINICAL CHEMISTRY: No data available
- Time schedule for collection of blood: No data available
- Animals fasted: No data available
- How many animals: No data available
- Parameters checked in table [No.?] were examined: No data available

URINALYSIS: No data available
- Time schedule for collection of urine: No data available
- Metabolism cages used for collection of urine: No data available
- Animals fasted: No data available
- Parameters checked in table [No.?] were examined: No data available

NEUROBEHAVIOURAL EXAMINATION: No data available
- Time schedule for examinations: No data available
- Dose groups that were examined: No data available
- Battery of functions tested: sensory activity / grip strength / motor activity / other: No data available

OTHER: No data available

Sacrifice and pathology:

Sacrifice and pathology
GROSS PATHOLOGY: Yes

Organs examined :
Trachea, larynx, liver, kidneys, testes, and any other organs

HISTOPATHOLOGY: Yes
A complete necropsy was performed on each animal. The nasal passages were flushed with 10% neutral buffered Formalin; then the entire head and other organs were fixed in the same fixative. The head was then decalcified; and stepwise cross-sections were taken in the dorsoventral plane perpendicular to the long axis of the skull, beginning just posterior to the nostrils and extending caudal as far as the orbit. Histologic sections were taken from each lobe of the lung and from the trachea, larynx, liver, kidneys, testes, and any other organs exhibiting gross pathology. Paraffin sections, 4-5 /µm thick, were prepared in the usual fashion and stained with hematoxylin-eosin and with special stains, if necessary.

Organs examined :
Nasal passages, entire head, lung, Trachea, larynx, liver, kidneys, testes, and any other organs were examined

Other examinations:

No data available

Statistics:

No data available

Clinical signs:

not specified

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Other effects:

not specified

Details on results:

Clinical signs and mortality
Mortality: Cumulative mortality was observed in treated rat as compare to control.

Clinical signs: No data available

Body weight and weight gain: No data available

Food consumption and compound intake: No data available

Food efficiency: No data available

Water consumption and compound intake: No data available

Opthalmoscopic examination: No data available

Haematology: No data available

Clinical chemistry: No data available

Urinanalysis: No data available

Neurobehaviour: No data available

Organ weights: No data available
Gross pathology: Metastatic lesions in the lungs were noted in 2 animals dying of nasal squamous cell carcinomas, Squamous metaplasia, Polyp or papillomas lesions were observed in treated rat as compare to control.

Histopathology:
High incidences of nasal tumor, potent carcinogenic effect on the nasal epithelium, adenomatous polyps in larynx, adenomatous polyps in trachea, malignant lymphoma, adenoma in adrenal, adenocarcinoma in adrenal, papilloma in skin, adenomas in thyroid, islet cell adenoma in pancreas, fibro adenomas in mammary gland, adenocarcinoma in salivary gland, adenoma in salivary gland, lipomas in subcutaneous, fibromas in subcutaneous and adenoma in parathyroid.

47 (59%) developed tumors of the nasal mucosa with the first carcinoma being observed in animal dying at 256 days.

Dose descriptor:

LOAEC

Effect level:

5 mg/L air (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Effect on Mortality, gross pathology and histopathology.

Critical effects observed:

not specified

Table: Median life-span and time of tumor appearance following exposure to alkylating agents

Treatment	No. of animals	Median life days	Animals with tumors, No (%)	Time of tumor appearance, days
				Median	Range
5 mg/L X 30 exp	80	495	47 (59)	513	256-775

 

Table: Specific types of tumors and other lesions of the nasal mucosa in rats exposed to various alkylating agents

	Treatment	MMS, 5 mg/L X 30 exp
	No. of animals examined	80
No. of tumors and other lesions of nasal mucosa	Tumors bearing animals, No (%)	47 (59)
	Rhinitis	49
	Squamous metaplasia	5
	Polyps or papillomas	11
	Squamous cell carcinoma	33
	Adenocarcinoma	2
	Mixec carcinoma	2
	Osteogenicsarcome	-

Conclusions:

The low-observed-adverse-effect concentration (LOAEC) was considered to be 5 mg/L air (50ppm) when Sprague-Dawley male rat were exposed to Methylmethane sulfonate (MMS) upon repeated exposure for 30 days.

Executive summary:

Repeated dose inhalation toxicity study was performed to determine the toxic nature of Methylmethane sulfonate (MMS) in rat upon repeated exposure. 11 -12 week old Male Sprague Dawley rat were exposed to MMS at a dose level of 0 or 5 mg/L for 30 days. All animals were observed daily, weighed monthly, and allowed to die spontaneously or sacrificed when moribund. A complete necropsy was performed on each animal. The nasal passages were flushed with 10% neutral buffered Formalin and the entire head and other organs were fixed in the same fixative. The head was then decalcified; and stepwise cross-sections were taken in the dorsoventral plane perpendicular to the long axis of the skull, beginning just posterior to the nostrils and extending caudal as far as the orbit. Histologic sections were taken feom each lobe of the lung and from the trachea, larynx, liver, kidneys, testes, and any other organs exhibiting gross pathology. Paraffin sections, 4-5 µm thick, were prepared in the usual fashion and stained with hematoxylin-eosin and with special stains for histologic examination. Cumulative mortality was noted in the test animals. Gross pathological examinations revealed the presence of metastatic lesions in the lungs in 2 animals dying of nasal squamous cell carcinomas, squamous metaplasia and polyp or papillomas lesions were observed in treated rat as compare to control. In addition, High incidences of nasal tumor, potent carcinogenic effect on the nasal epithelium, adenomatous polyps in larynx, adenomatous polyps in trachea, malignant lymphoma, adenoma in adrenal, adenocarcinoma in adrenal, papilloma in skin, adenomas in thyroid, islet cell adenoma in pancreas, fibro adenomas in mammary gland, adenocarcinoma in salivary gland, adenoma in salivary gland, lipomas in subcutaneous, fibromas in subcutaneous and adenoma in parathyroid were observed. 47 (59%) developed tumors of the nasal mucosa with the first carcinoma being observed in animal dying at 256 days. On the basis of observations made, the low-observed-adverse-effect concentration (LOAEC)  was considered to be 5 mg/L air (50ppm) when Sprague-Dawley male rat were exposed to Methylmethane sulfonate (MMS) upon repeated exposure for 30 days.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

5 000 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

Data is from peer reviewed publication

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose toxicity: Oral

Various peer reviewed publications were reviewed to determine the toxic nature of methyl methansulphonate upon repeated exposure by oral route. The studies are as mentioned below:

In Repeated dose subacute oral toxicity study by Ozawa et al (Journal of toxicological sciences, 2000), Male Sprague – Dawley rats were treated with Methyl Methansulfonate (MMS) at a concentration of 0, 20 or 40 mg/kg body weight/day orally by gavage for 2 weeks. The animals were observed for clinical signs, mortality, body weight changes, food consumption, gross pathology and histopathology. No effects were observed on mortality, clinical sign, body weight and food consumption at 20 mg/kg body weight/day treated rat as compared to control. However, when treated with 40 mg/kg body weight/day, significant decrease in body weight was observed from day 8 of treatment in treatedmale rat as compared to control and there was a tendency of decrease on day 2 of treatment in the 40 mg/Kg group and significant differences between 40 mg/Kg bw/day and control group were observed from day 9 of treatment. When treated with 40 mg/kg body weight/day, significant decrease in absolute testes and epididymides weight was observed in treated male rat as compared to control. However, there were no significant differences in relative organ weight. Slight atrophy of testes was observed in 40 mg/kg body weight/day as compared to control and spermatic granuloma was observed in the right epididymis in one animal of control group. No other gross abnormalities were observed. Slight atrophy of seminiferous tubules (1/10), decrease (8/10) and exfoliation (3/10) of germ cells, vacuolar degeneration of sertoli cells of testes in and moderate cell debris in caput, slight caudal epididymal ducts, decrease of permatids (elongated) and spermatic granuloma of epididymis was observed in 40 mg/kg body weight/day dose group. Atrophy of seminiferous tubules (1/10) and dilatation of the seminiferous tubules (1/10), Slight vacuolar degeneration of sertoli cells of testes and cell debris in caput and caudal epididymal ducts of epididymis (4/10) was observed in 20 mg/kg body weight/day as compared to control. Significant decreased in spermatogonia was observed in stage XII seminiferous tubules of 40 mg/kg body weight/day dose group. Significant decreased in pachytene spermatocytes at stage XII were observed in 20 mg/kg body weight/day as compared to control. Observation made suggest the No observed adverse effect level (NOAEL) was considered to be 20 mg/kg body weight /day when Sprague – Dawley male rats were exposed to Methyl Methansulfonate (MMS) for 2 weeks.

Kuriyama et al (The journal of toxicological sciences, 2005) performed repeated dose oral toxicity study to determine the toxic nature of Methyl methanesulphonate (MMS) in rats upon repeated oral exposure. Crj: CD (SD), SPF male and female were treated with Methyl methanesulphonate (MMS) dissolved in ditilled water in the concentration of 0, 20, 30 and 40 mg/kg/body weight /day for 5 days followed by a 5 weeks recovery period. On the after final administration, 5 out of 10 males from each dose group were sacrificed for sperm examination and the other male animals were used for mating in the fifth week. All male animals were sacrificed by inhalation of carbon dioxide, examined for gross abnormalities, testis and epididymides were weighed and right cauda epididymis from the male unused for mating was employed for sperm examination such as motility, sperm morphological examination and caudal epodidymal sperm count. The right caput epididymis was used for sperm morphological examination. The animals were also observed for clinical signs, mortality, gross and histopathology. No MMS related clinical signs and mortality was noted. A slight but statistically significant decrease in body weight was observed on 5 day of treatment which is at the end of recovery period almost comparable to control. A significant decrease was observed in absolute epididymis weight in the 30 and 40 mg/Kg dose group animals. When treated with 30 mg/kg, significantly increased frequencies of morphologically abnormal sperm in cauda epididymis were observed from week 3 to 5 and when treated with 40 mg/kg, Slightly decreased sperm counts were observed throughout the study period. Chronological change such as tailless sperm followed by no-hook head sperm in caput epididymis was also observed. Significantly increased frequencies of tailless sperm in 4 week and no-hook sperm in 4 and 5 week were observed as compared to control in recovery at 30 mg/Kg dose level. During the recovery period at 40 mg/Kg, number of cauda epidiymis was significantly decreased in 1 week, significantly increased frequencies of morphologically abnormal sperm in cauda epididymis were observed from week 3 to 5. The frequency of morphologically abnormal sperm in caput epididymis increased in 2 and 3 week which recovered in 4 week. Based on the observations made, the no-observed-adverse-effect-level (NOAEL) was considered to be 20 mg/kg body weight /day when Crj: CD (SD), SPF male and female were treated with Methyl methanesulphonate (MMS) orally for 5 days.

In the study by Hamada et al (Mutation research, 2015), 28 days repeated-dose oral toxicity study was also performed on male Crl: CD (SD) rats to determine the oral toxic nature of Methyl methanesulfonate (MMS). MMS was dosed at a concentration of 0, 7.5, 15 and 30 mg/kg/day orally by gavage. The animals were observed for mortality, liver weight and histopathological parameters like hepatocyte hypertrophy, single cell necrosis, anisokaryosis, prominent nucleolus, mitotic figure, focus of altered hepatocyte, proliferation oval cell or bile duct and cell infiltration. Relative liver weight was increased in 30 mg/kg/day dose group. No other histopathological findings were observed. On the basis of observations made, the No Observed Adverse Effect Level (NOAEL) was considered to be 15 mg/kg/day when Crl:CD (SD) male rats were exposed to Methyl methanesulfonate (MMS) for 28 days.

14 days repeated-dose oral toxicity study was performed by Hamada et al (Mutation research, 2015) on male Crl: CD (SD) rats to determine the oral toxic nature of Methyl methanesulfonate (MMS). MMS was dosed at a concentration of 0, 12.5, 25 and 50 mg/kg/day orally by gavage. The animals were observed for mortality, liver weight and histopathological parameters like hepatocyte hypertrophy, single cell necrosis, anisokaryosis, prominent nucleolus, mitotic figure, focus of altered hepatocyte, proliferation oval cell or bile duct and cell infiltration. Relative liver weight was increased in 12.5, 25 and 50 mg/kg/day dose group. No other histopathological findings were observed. On the basis of observations made, the Low observed adverse effect level (LOAEL) was considered to be 12.5 mg/kg/day when Crl:CD (SD) male rats were exposed to Methyl methanesulfonate (MMS) for 14 days.

Another repeated dose subacute oral toxicity study was given by Ozawa et al (Journal of toxicological sciences, 2000). Male Sprague – Dawley rats were treated with Methyl Methansulfonate (MMS) at a concentration of 0, 20 or 40 mg/kg body weight/day orally by gavage for 4 weeks. The animals were observed for clinical signs, mortality, body weight changes, food consumption, gross pathology and histopathology. No effects were observed on mortality, clinical signs and body weight at 20 mg/kg body weight/day treated rat as compared to control. However, significant changes in food consumption were observed on day 2 in 20 mg/Kg bw/day dose group as compared to controls. When treated with 20 mg/kg body weight/day, significant decrease in epididymides weight was observed in treated male rats. However, there was no significant difference in relative organ weight. Slight atrophy of testes was observed in one animals of 20 mg/kg body weight/day and spermatic granuloma was observed in the right epididymis in one animal of control group. No other gross abnormalities were observed. In the testis, a decrease in germ cells (2/10) and exfoliation of germ cells (3/10) was noted in the treated group. Cell debris was observed in the epididymal ducts (6/10) of 20mg/Kg bw/day dosed group. Moderate vacuolar degeneration of sertoli cells were observed in control and treated group animals. Spermatic granulomas were also seen in some animals. Significant difference from the control group was observed in the pachytene spermatocytes/sertoli cells of stage II-III. No other significant alterations were noted. These observations suggest that the Low observed adverse effect level (LOAEL) was considered to be 20 mg/kg body weight /day when Sprague – Dawley male rats were exposed to Methyl Methansulfonate (MMS) for 4 weeks.

Kuriyama et al (Journal of Reproduction and Development, 2005) performed subacute testicular toxicity and sperm morphology study, 30 Crj: CD (SD), in which SPF male rats were treated with Methyl Methanesulphonate (MMS) in the concentration of 0 or 40 mg/kg body weight/day orally for 5 days. This was followed by a recovery period of 5 weeks. The animals were observed for clinical signs, mortality, morbidity, body weight changes, gross and histopathology and sperm parameters. No deaths, morbidity and clinical signs were observed during the study period. Slight but statistically significant decreased in body weight were observed on day 1 in treated rat as compared to control. Similarly, statistically significant increase in the morphologically abnormal sperm in cauda epididymis between week 3 and week 5 of recovery was observed, an increased frequencies of tailless sperm and no hook head sperm during recovery was noted and high frequencies of retention of step 19 spermatids at stage IX were observed from day 1 through week 3. Necrosis of germ cells in week 1 and vacuolar degeneration of sertoli cells, atrophy of semniferous tubules and spermatic granuloma in epididymis were observed at very low frequency. In addition, significantly decreased number of round spermatids in stage V, spermatogones in stage X, zygotene spermatocytes in stage XII and pachytene spermatocytes in stage XII was observed. Substantially lowered numbers of round spermatids were observed in stage II-II on day 1 and significantly increased frequency of TUNEL positive seminiferous tubules was observed in stage XIV-1 in week 1 of recovery in 40 mg/kg body weight/day for F0 treated rat as compared to control. On the basis of observations made, the Low Observed Adverse Effect Level (LOAEL) was considered to be 40 mg/kg body weight/day when Crj: CD (SD), SPF male rats were treated with Methyl Methanesulphonate (MMS).

Yet another study was performed by Yokoi et al (The journal of toxicological sciences, 2008). Combined repeated dose repro-developmental toxicity study was performed for Methyl methanesulphonate in rat exposed orally by repeated exposure. Female Crj: CD (SD) IGS rats were treated with Methyl methanesulphonate (MMS) at a concentration of 200 mg/kg body weight/day for 20 days. The animals were observed for clinical signs, mortality, body weight changes and food consumption, immunohistochemical findings were performed, gross and histopathology. No clinical signs and mortality was observed at the 200 mg/Kg bw/day dose level. Significant decrease in body weight was observed in treated rat as compare to control from GD7 to GD20. However, the gain in body weight from GD7 to term was similar for treated and control animals. On GD7 significantly decreased food consumption was observed in treated rat as compared to control. Clinical chemistry findings revealed significant decrease in total amount of glycogen per placenta and amount per gram of tissue on GD14. On GD20, presence of GLUT1 –positive cells in the JZ were observed in rat as compared to control. Plasma glucose levels did not differ from GD7 to GD20 significantly between treated and control animals. On GD12, significantly decreased RBC, HGB and HCT were observed as compare to control. Blood erythrocyte parameters revealed a slight anemia on GD 12. Histopathological examinations indicated marked hypoplasia of placenta on GD12. On GD 20, thickness of Labyrinth Zone (LZ) of placenta was significantly thinner in treated rat. Histology of the uterus including embryos showed no difference between MMS treated and control groups on GD7. The thickness of the LZ of the placenta was significantly smaller in the MMS group on GD12, 14 and 17 but not on GD20. The JZ of the placenta was significantly thinner n the MMS treated group than in the control group. No marked differences were observed in the decidua basalia between the two groups. The number of glycogen trophoblast cells in the juctional zone (JZ) of MMS treated rats was smaller than the control group on GD14 and had no marked decrease by GD20. The glycogen content of the placenta was lower on GD14 in the treated group. The amount of glycogen/g/tissue, but not the total amount/placenta was significantly greater in the MMS group than in the control group at GD20. On the basis of observations made, the low-observed-adverse-effect level (LOAEL) was considered to be 200 mg/kg body weight/day when female Crj: CD (SD) IGS rats were treated with Methyl methanesulphonate upon repeated oral exposure.  

Based on the available data summarized, it can be concluded that methyl methane sulphonate is not toxic upto a dose level of 20 mg/Kg bw/day. Although studied indicating LOAEL are also represented for MMS but the studies are not range dose studies. These studies are being conducted using single dose for repeated exposure. Also, the details on the overall parameters to clearly judge the repeated dose LOAEL nature are also not available for some studies. Hence, it is considered that MMS may be toxic above the NOAEL concentration of 20 mg/Kg bw/day.

Repeated dose toxicity: Inhalation

Various peer reviewed publications were reviewed to determine the toxic nature of methyl methansulphonate upon repeated exposure by inhalation route. The studies are as mentioned below:

Repeated dose inhalation toxicity study was performed by Sellkumar et al (Jounal of National Cancer Institute, 1987) to determine the toxic nature of Methylmethane sulfonate (MMS) in rat upon repeated exposure. 11 -12 week old Male Sprague Dawley rat were exposed to MMS at a dose level of 0 or 5 mg/L for 30 days. All animals were observed daily, weighed monthly, and allowed to die spontaneously or sacrificed when moribund. A complete necropsy was performed on each animal. The nasal passages were flushed with 10% neutral buffered Formalin and the entire head and other organs were fixed in the same fixative. The head was then decalcified; and stepwise cross-sections were taken in the dorsoventral plane perpendicular to the long axis of the skull, beginning just posterior to the nostrils and extending caudal as far as the orbit. Histologic sections were taken feom each lobe of the lung and from the trachea, larynx, liver, kidneys, testes, and any other organs exhibiting gross pathology. Paraffin sections, 4-5 µm thick, were prepared in the usual fashion and stained with hematoxylin-eosin and with special stains for histologic examination. Cumulative mortality was noted in the test animals. Gross pathological examinations revealed the presence of metastatic lesions in the lungs in 2 animals dying of nasal squamous cell carcinomas, squamous metaplasia and polyp or papillomas lesions were observed in treated rat as compare to control. In addition, High incidences of nasal tumor, potent carcinogenic effect on the nasal epithelium, adenomatous polyps in larynx, adenomatous polyps in trachea, malignant lymphoma, adenoma in adrenal, adenocarcinoma in adrenal, papilloma in skin, adenomas in thyroid, islet cell adenoma in pancreas, fibro adenomas in mammary gland, adenocarcinoma in salivary gland, adenoma in salivary gland, lipomas in subcutaneous, fibromas in subcutaneous and adenoma in parathyroid were observed. 47 (59%) developed tumors of the nasal mucosa with the first carcinoma being observed in animal dying at 256 days. On the basis of observations made, the low-observed-adverse-effect concentration (LOAEC)  was considered to be 5 mg/L (50ppm) when Sprague-Dawley male rat were exposed to Methylmethane sulfonate (MMS) upon repeated exposure for 30 days.

Snyder et al (Cancer letters, 1986) performed subacute repeated dose inhalation toxicity study to determine the toxic nature of Methylmethane sulfonate (MMS) in rat upon repeated exposure. Male Sprague Dawley rats were exposed to MMS at a concentration of 0 or 5 mg/L. Exposures were conducted in 1 .0 m3or 1.3 m3 stainless steel and plexiglass dynamic exposure chambers. The compounds were vaporized by passing a measured stream of over the surface of the liquid (for low chamber concentrations), or by nebulizing the liquid and vaporizing the resultant droplets (for high chamber concentrations) . Methyl methanesulfonate required heating in addition nebulization . Micrometering valves were used to control flow rates, and flow of air was monitored by rotometers. All exposures were conducted for 6 h/day, 5 days per week for 6 weeks (30 exposures). At least 50 rats were used in treatment group. In addition, two air-control groups totaling 150 rats were exposed to filtered air in duplicate chambers, using the same exposure protocol . A complete autopsy was performed on each animal for tissues that appeared abnormal and grossly evident masses . The examination of the skull, brain, and nasal cavity was performed by fixation of the entire skull with the brain in situ . After fixation in 10% neutral, buffered formalin, the heads were decalcified . Four stepwise cross sections, equally spaced, were taken in the dorsal-ventral plane perpendicular to thc long axis of the skull, from the anterior to the posterior of the nose . Sections were stained with hematoxylin and eosin and resultant slides were read. Cumulative mortality was observed for test chemical. In addition, carcinogenic effect were observed on the nasal mucosal macromolecules and abilities to bind to nasal mucosal DNA was also observed in treated rat as compare to control. However, MMS showed 33-39 times as much as activity in nasal cavity macromolecules compared to nasal cavity DNA. Hence, the lowest-observed-adverse-effect concentration (LOAEC)  was considered to be 5 mg/L (50ppm) for carcinogenic effect when Sprague-Dawley male rat were exposed with Methylmethane sulfonate (MMS).

Based on the available data for the target chemical, Methyl methanesulphonate exibits toxic nature uon repeated exposure by inhalation route and hence is likely to classify as a

toxicant upon inhalation exposure.

Repeated dermal study

The acute toxicity value for Methyl methanesulfonate (66-27-3 )(as provided in section 7.2.3) is 6926 mg/kg body weight. Also, given the use of the chemical; repeated exposure by the dermal route is unlikely since the use of gloves is common practice in industries. Thus, it is expected that Methyl methanesulfonate shall not exhibit 28 day repeated dose toxicity by the dermal route. In addition, there is no data available that suggests that Methyl methanesulfonate shall exhibit repeated dose toxicity by the dermal route. Hence this end point was considered for waiver.

Justification for classification or non-classification

Based on the above annotation for the target chemical Methyl methanesulfonate (66-27-3 ) does not exhibit toxic nature upon repeated exposure by  oral route. Hence it is not likely to classify as per the criteria mentioned in CLP regulation for oral route.

But the data available for the target chemical Methyl methanesulfonate (66-27-3 ) exhibit toxic nature upon repeated exposure by  inhalation .Hence it is likely to classify as per the criteria mentioned in CLP regulation for oral and inhalation route.

While dermal route of exposure has not classified value and least exposure of chemical. Hence is not likely to classify as per the criteria mentioned in CLP regulation for dermal route.