Methyl N-(2,6-dimethylphenyl)-DL-alaninate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

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 journal.

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Compilation of historical local lymph node data for evaluation of skin sensitization alternative methods.

Author:

Gerberick GF, Ryan CA, Kern PS, Schlatter H, Dearman RJ, Kimber I, Patlewicz GY, Basketter DA.

Year:

2005

Bibliographic source:

Dermatitis, 2005 Dec; 16(4):157-202

Reference 2

Reference Type:

publication

Title:

Fragrance material review on methyl salicylate

Author:

A. Lapczynski, L. Jones, D. McGinty, S.P. Bhatia, C.S. Letizia, A.M. Api

Year:

2007

Bibliographic source:

Food and Chemical Toxicology 45 (2007) S428–S452

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:

The skin sensitizing potential of the test chemical Methyl salicylate (CAS no: 119-36-8) was assessed by using the Mouse Local Lymphnode Assay.

GLP compliance:

not specified

Type of study:

mouse local lymph node assay (LLNA)

Justification for non-LLNA method:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

Methyl salicylate

EC Number:

204-317-7

EC Name:

Methyl salicylate

Cas Number:

119-36-8

Molecular formula:

C8H8O3

IUPAC Name:

Methyl 2-hydroxybenzoate

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): Methyl salicylate
- Molecular formula : C8H8O3
- Molecular weight: 152.15 g/mol
- Substance type: Organic
- Physical state: liquid
-Smiles: c1(c(cccc1)O)C(OC)=O
- InChI:1S/C8H8O3/c1-11-8(10)6-4-2-3-5-7(6)9/h2-5,9H,1H3

Specific details on test material used for the study:

- Name of test material (as cited in study report): Methyl salicylate
- Molecular formula : C8H8O3
- Molecular weight: 152.15 g/mol
- Substance type: Organic
- Physical state: liquid
-Smiles: c1(c(cccc1)O)C(OC)=O
- InChI:1S/C8H8O3/c1-11-8(10)6-4-2-3-5-7(6)9/h2-5,9H,1H3

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
Source: No data
Age at study initiation: 7-12 weeks

ENVIRONMENTAL CONDITIONS: No data

Study design: in vivo (non-LLNA)

Challenge controls:

Control animals were treated with the vehicle alone.

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

1%, 2.5%, 5%, 10% or 20% (25µl)

No. of animals per dose:

5 female mice/dose

Details on study design:

Treatment was performed daily for 3 consecutive days. Five days after initiation of exposure all mice were injected via the tail vein with 250µl of PBS containing 20µCi of tritiatied thymidine. The mice were sacrificed 5 hours later, and draining the auricular lymph nodes were excised and pooled for each experimental group or each individual animal. The incorporation of tritiated thymidine measured by β-scintillation counting and was reported in disintegrations /minute. An SI was calculated for each chemical group as the ratio of disintegrations/minute of the treated group to the disintegrations/minute of the concurrent vehicle control group. A substance was classified skin sensitizer , if at one or more than one concentrations, it induced a three-fold or greater increase in local lymph node proliferative activity when treated with the concurrent vehicle treated controls (SI ≥3)

Statistics:

The approach to estimation of the relative skin sensitization potential is based on the mathematical estimation of the concentration of chemical necessary to obtain a threshold positive response (SI = 3); this is termed as the EC3 value

Refer Table 1 and 2 for more information

Results and discussion

In vivo (LLNA)

Results

Parameter:

EC3

Value:

> 20

Remarks on result:

other: The calculated EC3 value for methyl salicylate was >20.0%

Cellular proliferation data / Observations:

The calculated EC3 value for methyl salicylate was >20.0%

Any other information on results incl. tables

Table 1:Classification of relative skin sensitization potency by local lymphnode assay (LLNA) EC3 values
EC3 values (%)	Potency classification
≥10 to ≤100	Weak
≥1 to <10	Moderate
≥0.1 to <1	Strong
<0.1	Extreme

CAS

Vehicle

LLNA%

LLNA%

LLNA%

LLNA%

LLNA%

LLNA SI

LLNA SI

LLNA SI

LLNA SI

LLNA SI

LLNA EC3

Relative Potency

122-40-7

AOO

1.0

2.5

5.0

10.0

20.0

1.0

1.1

1.6

1.4

0.9

NC

Nonsensitizer

Where AOO – Acetic acid in olive oil ; SI - Stimulation Index; NC-Not Calculated

Applicant's summary and conclusion

Interpretation of results:

other: not sensitizing

Conclusions:

The relative potency index of Methyl salicylate (CAS no: 119-36-8) was estimated to be >20.0%. Based on the relative potency index Methyl salicylate (CAS no: 119-36-8) was considered to be not sensitizing in the Mouse Local Lymphnode Assay.

Executive summary:

The skin sensitizing potential of the test chemical Methyl salicylate (CAS no: 119-36-8) was assessed by using the Mouse Local Lymphnode Assay.

 

The LLNA was conducted on groups of five female CBA mice (7-12 weeks of age) by mean of topical application of chemical on the dorsum of both ears at a dose of 25µl of 1%, 2.5%, 5%, 10% or 20% in acetone/olive oil (4:1). Treatment was performed daily for 3 consecutive days. Five days after initiation of exposure all mice were injected via the tail vein with 250µl of PBS containing 20µCi of tritiatied thymidine. The mice were sacrificed 5 hours later, and draining the auricular lymph nodes were excised and pooled for each experimental group or each individual animal. The incorporation of tritiated thymidine measured by β-scintillation counting and was reported in disintegrations /minute.

 

 An SI was calculated for each chemical group as the ratio of disintegrations/minute of the treated group to the disintegrations/minute of the concurrent vehicle control group.

 

A substance was classified skin sensitizer , if at one or more than one concentrations, it induced a three-fold or greater increase in local lymph node proliferative activity when treated with the concurrent vehicle treated controls (SI ≥3)

 

The approach to estimation of the relative skin sensitization potential is based on the mathematical estimation of the concentration of chemical necessary to obtain a threshold positive response (SI = 3); this is termed as the EC3 value.

 

For each concentration of methyl salicylate, a stimulation index (SI) relative to the concurrent vehicle-treated control was calculated. The calculated EC3 value for methyl salicylate was >20.0%. Thus based on the relative potency index Methyl salicylate (CAS no: 119-36-8) was considered to be not sensitizing in theMouse Local Lymphnode Assay.