Methyl methanesulphonate

Endpoint summary

Description of key information

Skin sensitization

The skin sensitization potential of target chemical Methyl methanesulfonate (CAS No: 66-27-3)  was assessedin various experimental studies which were conducted on mouse (The Mouse local lymphnode assay (LLNA)) and humans for target chemicalMethyl methanesulfonate (CAS No: 66-27-3)  andits structurally similar read across substances3 Methanesulphonyl chloride (CAS no: 124-63-0)andSodium Lauryl Sulfate(CAS no:151-21-3).Based on the available data for the target and read across substances, it can be concluded that chemical Methyl methanesulfonate (CAS No: 66-27-3)  is able to cause skin sensitization and thus can be considered as sensitizing. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Skin Sensitizer”.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

Data is from publication.

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Principles of method if other than guideline:

In Local Lymph Node Assay, the Methyl methanesulphonate is administered on three consecutive days, followed by a 2-day rest period before analysis. On day 6 (5 days after initiation of treatment), the mice are injected intravenously, by the tail vein, with 250 μL of sterile phosphate-buffered saline (PBS) containing 20 μCi of [3H]methyl thymidine and the lymph nodes are excised five hours later. A lymph node cell suspension is then prepared and tritium thymidine or iododeoxyuridine 125 incorporation is determined by scintillation counting.

GLP compliance:

not specified

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

- Name of test material (as cited in study report): Methyl methanesulphonate
- Molecular formula (if other than submission substance): C2H6O3S
- Molecular weight (if other than submission substance): 110.132 g/mol
- Substance type: organic
- Physical state: solid

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

Details on test animal
TEST ANIMALS
- Source:
- Age at study initiation: 6 to 16 wk-old
- Weight at study initiation: no data
- Housing: no data
- Diet (e.g. ad libitum): no data
- Water (e.g. ad libitum): no data
- Acclimation period: no data

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

IN-LIFE DATES: From: To: no data

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

25 μL

No. of animals per dose:

4-5 animals

Details on study design:

Details on study design

PRE-SCREEN TESTS:
- Compound solubility: no data
- Irritation: no data
- Systemic toxicity: no data
- Ear thickness measurements: no data
- Erythema scores: >3

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph node asssay
- Criteria used to consider a positive response: Si > 3

•TREATMENT PREPARATION AND ADMINISTRATION:
Groups of mice (n 4 or 5) are treated by topical application, on the dorsum of both ears, with 25 μL of 1 of several concentrations (100%,50%,25%, 10%, 5%, 2.5%, 1%,0.5%, 0.25%, and 0.1 %) of test material, or with an equal volume of the relevant vehicle alone.
They are treated daily for 3 consecutive days, followed by a 2 day rest period before analysis. On the sixth day (5 days after initiation of treatment), the mice are injected intravenously, by the tail vein, with 250 μL of sterile phosphate-buffered saline (PBS) containing 20 μCi of [3H] methyl thymidine. Five hours later, the mice are killed, and the draining auricular lymph nodes are excised and pooled for each experimental group or for each individual animal. Single cell suspensions of lymph node cells are prepared. Lymph node cells are washed twice with an excess of PBS and precipitated with 5% trichloroacetic acid (TCA) at 4°C. The samples, pelleted by centrifugation, are resuspended 12 to 18 hours later in 1mL of 5% TCA and transferred to 10 mL of scintillation cocktail. Incorporation of tritiumlabeled thymidine [3H-TdR] is measured by beta-scintillation counting and expressed as disintegrations per minute (dpm).

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

The data are expressed as mean dpm for each experimental group, and the Stimulation Index (SI) is derived for each experimental group by dividing the mean dpm of that group by the mean dpm of the vehicle-control group. Experimental groups are compared with vehicle-treated controls.
SI of 3 (EC3) can be calculated EC3 determination is that data from the entire dose response curve are used to produce a single value of intrinsic potency.14 The EC3 value can be used to rank the relative skin-sensitizing potential of chemicals.

Key result

Parameter:

SI

Value:

> 3

Test group / Remarks:

Yes

Remarks on result:

other: Positive indication of skin sensitization .

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA/observation

CELLULAR PROLIFERATION DATA
The stimulation indices (SIs) for each experimental group are determined as the increase in 3H-TdR incorporation relative to concurrent vehicle-treated controls. A Methyl methanesulphonate that, at 1 or more concentrations, causes an SI of 3 or greater is considered to have skin-sensitizing activity. Thus, whether the draining auricular lymph nodes are excised and pooled for each experimental group or for each individual animal, the three-fold or greater increase in proliferative activity compared with concurrent vehicle-treated control animals is the sole criterion for a classification of skin-sensitizing activity.

DETAILS ON STIMULATION INDEX CALCULATION
Stimulation indices (SIs) for each experimental group are determined as the increase in 3H-TdR incorporation relative to concurrent vehicle-treated controls. A Methyl methanesulphonate that, at 1or more concentrations, causes an SI of 3 or greater is considered to have skin-sensitizing activity.

EC3 CALCULATION:
The EC3 values (3-fold increase in stimulation index)

CLINICAL OBSERVATIONS: no data

BODY WEIGHTS
No data

Interpretation of results:

other: sensitizing

Conclusions:

Methyl methanesulphonate(66-27-3) was observed for its skin sensitization potential in mouse by LLNA mode .It is concluded from the Mouse local lymphnode assay (LLNA) in vivo test of skin sensitization potential for Methyl methanesulphonate is skin sensitizing to the female CBA/Ca mouse.

Executive summary:

The Mouse local lymphnode assay (LLNA) in vivo test was performed on 6-16 weeks old, 4-5 female CBA/Ca mice to study the skin senisitization potential of Methyl methanesulphonate(66-27-3).Groups of mice (n 4 or 5) are treated by topical application, on the dorsum of both ears, with 25μLof 1of several concentrations of test material, or with an equal volume of the relevant vehicle alone. They are treated daily for 3 consecutive days, followed by a 2 day rest period before analysis. On the sixth day (5 days after initiation of treatment), the mice are injected intravenously, by the tail vein, with 250μLof sterile phosphate-buffered saline (PBS) containing 20μCi of [3H] methyl thymidine. Five hours later, the mice are killed, and the draining auricular lymph nodes are excised and pooled for each experimental group or for each individual animal. Single cell suspensions of lymph node cells are prepared. Lymph node cells are washed twice with an excess of PBS and precipitated with 5% trichloroacetic acid (TCA) at 4°C. The samples, pelleted by centrifugation, are resuspended 12 to 18 hours later in 1mL of 5% TCA and transferred to 10 mL of scintillation cocktail. Incorporation of tritiumlabeled thymidine [3H-TdR] is measured by {beta-scintillation counting and expressed as disintegrations per minute (dpm). The drainined auricular lymph nodes are excised and pooled for each experimental group or for each individual animal, the three-fold or greater increase in proliferative activity compared with concurrent vehicle-treated control animals is the sole criterion for a classification of skin-sensitizing activity.

Thus from the result obtained from the The Mouse local lymphnode assay (LLNA) in vivo test on mice for Methyl methanesulphonate,at 1or more concentrations, causes an SI of 3 or greater is considered to have skin-sensitizing activity. ThereforeMethyl methanesulphonate(66-27-3) was considered to be sensitizing to the female CBA/Ca mouse.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Skin sensitization:

Various studieshas been investigated for the test chemicalMethyl methanesulfonate (CAS No: 66-27-3)  to observe the potential for skin sensitization to a greater or lesser extent. The studies are based on in vivo experiments in mouse (The Mouse local lymphnode assay (LLNA)) and humans for target chemicalMethyl methanesulfonate (CAS No: 66-27-3) and its structurally similar read across substancesMethanesulphonyl chloride (CAS no: 124-63-0)andSodium Lauryl Sulfate(CAS no:151-21-3)which have beensummarized as below;

 

 

The key study was conducted by G. Frank Gerberick,et al.(American Journal of Contact Dermatitis 2000) to evaluate the skin sensitizing potential of target substance Methyl methanesulphonate (CAS No:66-27-3)in mouse by LLNA mode. The Mouse local lymphnode assay (LLNA) in vivo test was performed on 6-16 weeks old, 4-5 female CBA/Ca mice to study the skin senisitization potential of Methyl methanesulphonate (CAS No: 66-27-3).Groups of mice (n 4 or 5) are treated by topical application, on the dorsum of both ears, with 25μLof 1of several concentrations of test material, or with an equal volume of the relevant vehicle alone. They are treated daily for 3 consecutive days, followed by a 2 day rest period before analysis. On the sixth day (5 days after initiation of treatment), the mice are injected intravenously, by the tail vein, with 250μLof sterile phosphate-buffered saline (PBS) containing 20μCi of [3H] methyl thymidine. Five hours later, the mice are killed, and the draining auricular lymph nodes are excised and pooled for each experimental group or for each individual animal. Single cell suspensions of lymph node cells are prepared. Lymph node cells are washed twice with an excess of PBS and precipitated with 5% trichloroacetic acid (TCA) at 4°C. The samples, pelleted by centrifugation, are resuspended 12 to 18 hours later in 1mL of 5% TCA and transferred to 10 mL of scintillation cocktail. Incorporation of tritiumlabeled thymidine [3H-TdR] is measured by {beta-scintillation counting and expressed as disintegrations per minute (dpm). The drained auricular lymph nodes are excised and pooled for each experimental group or for each individual animal, the three-fold or greater increase in proliferative activity compared with concurrent vehicle-treated control animals is the sole criterion for a classification of skin-sensitizing activity.Thus from the result obtained from the Mouse local lymphnode assay (LLNA) in vivo test on mice for Methyl methanesulphonate ,at 1or more concentrations, causes an SI of 3 or greater is considered to have skin-sensitizing activity. Therefore Methyl methanesulphonate(66-27-3) was considered to be sensitizing to the female CBA/Ca mouse .

 

 The another Mouse local lymphnode assay (LLNA) for target chemical Methyl methanesulphonate (CAS No:66-27-3) was conducted by Gerberick GF et al.(Dermatitis. 2005 ) on 7-12 weeks old, 4-5 female CBA mice to study the skin sensitization potential of Methyl methanesulphonate. Groups of mice (n 4 or 5) were exposed topically, on the dorsum of both ears, with 25μLof 1of several concentrations of Methyl methanesulphonate, or with an equal volume of the relevant vehicle alone.They are treated daily for 3 consecutive days, followed by a 2 day rest period before analysis. On the sixth day (5 days after initiation of treatment), the mice are injected intravenously, by the tail vein, with 250μLof sterile phosphate-buffered saline (PBS) containing 20μCi of [3H] methyl thymidine. Five hours later, the mice are killed, and the draining auricular lymph nodes are excised and pooled for each experimental group or for each individual animal.The incorporation of tritium thymidine is measured by {beta-scintillation counting and expressed as disintegrations per minute (dpm).The SI was calculated for each chemical-treated group as the ratio of the disintegrations per minute in the treated group (or mean disintegrations per minute when individual animal were assessed) to the disintegrations per minute or mean disintegrations per minute of the concurrent vehicle control group. On the basis of value obtained of stimulation Index i.e 3.6 and EC3 Value i.e 8.1% from skin sensitization : local lymph node assay, it is concluded that the Methyl methanesulphonate is skin sensitizing to the female CBA mouse.

 

The above results were supported by a case report study conducted by van Joost T et.al, (Contact Dermatitis, Vol. 10, No. 3, pages 187-188, 1984) on a 40 years old man who worked for 2 years in a chemical factory for the read across chemical Methanesulphonyl chloride (CAS no: 124-63-0) in accordance with ICDRG batteries by performing patch test.During the patch test, a patient was tested at a dose of 1% in petrolatum which resulted into strong positive (+++) reaction after 48 and 72 hours. After 6 weeks the patient was retested at lower concentrations of0.5%, 0.2%, 0.1% and 0.005% in petrolatum and skin was examined after 48 and 72 hours.Out of 10 control persons, no positive reactions were observed at 0.2% and a negative reaction was observed at 0.1% in test patient.From the tested concentrations, a strong positive(++)reaction was observed at 0.5% and 0.2%. Thus the chemical Methanesulphonyl chloride (CAS no: 124-63-0) was appeared to acquire allergic contact dermatitis at these concentrations and hence considered as skin sensitizer.

 

The above mentioned results were further supported by the Local Lymph Node Assay (LLNA) conducted by D. A. BASKETTR et.al, (Food and Chemical Toxicology Vol. 32. No. 6. pp. 543-547. 1994) on Female CBA/Ca mice for read across chemical Sodium Lauryl Sulfate(CAS no:151-21-3).The LLNA was conducted on groups of CBA/Ca mice (8-12 weeks of age) by mean of topical application of chemical on the dorsum of both ears at a dose of 25µl of one of three concentrations (5%, 10% and 25%)of the test chemical. 5 days after the first topical application, all mice were injected iv with 250µl phosphate buffered saline (PBS) containing 20µCi of [³H]methyl thymidine (³HTdR) .The mice were killed 5 hr later and the draining auricular lymph nodes excised and pooled for each experimental group. A single cell suspension of lymph node cells (LNC) was prepared by gentle disaggregation through 200 mesh stainless steel gauze. Pooled LNC were pelleted by centrifugation at 190g for 10 min, washed twice with 10 ml PBS and resuspended in 3 ml 5% trichloroacetic acid (TCA). After incubation overnight at 4°C, the precipitate was recovered by centrifugation, resuspended in I ml 5% TCA and transferred to 10 ml scintillation fluid. Incorporation of³HTdR was measured by p-scintillation counting.The proliferative response of LNC was expressed as mean radioactive disintegrations per minute per lymph node (dpm/node for each experimental group and as the ratio of³HTdR incorporation into LNC of test nodes relative to control nodes [test: control (T:C) ratio]. A chemical was regarded as a sensitizer in the LLNA if at least one concentration resulted in a T:C ratio of 3 or greater and the data were not incompatible with a biological dose response. The test: control (T:C) ratios were 3.2, 4.0 and 4.2 at concentration of 5%, 10% and 25% respectively. Since the resulted test: control (T:C) ratio was greater than 3 at each concentration, the chemical Sodium Lauryl Sulfate(CAS no:151-21-3)was considered to be sensitizing in Local Lymph Node Assay (LLNA).

 

Thus Based on the above key and supporting studies for Methyl methanesulfonate (CAS No: 66-27-3)  and its structurally similar read across substancesMethanesulphonyl chloride (CAS no: 124-63-0)andSodium Lauryl Sulfate(CAS no:151-21-3),it can be concluded thatchemical Methyl methanesulfonate (CAS No: 66-27-3)  is able to cause skin sensitization. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Skin Sensitizer”.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The skin sensitization potential of test substance Methyl methanesulfonate (CAS No: 66-27-3)  and its structurally similar read across substancesMethanesulphonyl chloride (CAS no: 124-63-0)andSodium Lauryl Sulfate(CAS no:151-21-3)were observed in various studies. From the results obtained from these studies it is concluded that the chemical Methyl methanesulfonate (CAS No: 66-27-3)  is likely to cause skin sensitization and hence can be classified as “Skin Sensitizer”.