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Diss Factsheets

Toxicological information

Skin sensitisation

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Administrative data

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.

Data source

Reference
Reference Type:
publication
Title:
Local Lymph Node Assay: Validation Assessment for Regulatory Purposes.
Author:
G. Frank Gerberick, Cintfy A. Ryan, Ian Kimber, Rebecca]. Dearman, Linda]. Lea, and DavidA. Basketter
Year:
2000
Bibliographic source:
American Journal of Contact Dermatitis 2000 Mar; 11(1):3-18.

Materials and methods

Test guideline
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)

Test material

Constituent 1
Chemical structure
Reference substance name:
Methyl methanesulphonate
EC Number:
200-625-0
EC Name:
Methyl methanesulphonate
Cas Number:
66-27-3
Molecular formula:
C2H6O3S
IUPAC Name:
methyl methanesulfonate
Details on test material:
- Name of test material (as cited in study report): Methyl methanesulfonate (MMS)
- Molecular formula (if other than submission substance): C2H6O3S
- Molecular weight (if other than submission substance): 110.1324 g/mole
- Substance type: Organic
- Physical state: Liquid
Purity: >95 %
- Impurities (identity and concentrations): , 5%
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

In vivo test system

Test animals

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

Study design: in vivo (LLNA)

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.

Results and discussion

In vivo (LLNA)

Results
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

Applicant's summary and conclusion

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.