Methyl ester of fatty acids, Cx-Cy, Hydroxymethylated

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted to the following guidelines - USEPA, OPPTS 870.2600 (2003), OECD, Guideline 429 (2002) and EC, Guideline B.42 (2004) and complied with the Principles of GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Concentration and stability of dosing solutions were not verified analytically

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

yes

Remarks:

Concentration and stability of dosing solutions were not verified analytically

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

yes

Remarks:

Concentration and stability of dosing solutions were not verified analytically

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): Methyl Polyhydroxymethyl Stearate
- Molecular formula (if other than submission substance): not specified in the report
- Molecular weight (if other than submission substance): not specified in the report
- Smiles notation (if other than submission substance): not specified in the report
- InChl (if other than submission substance): not specified in the report
- Structural formula attached as image file (if other than submission substance): not specified in the report
- Substance type: Mixture of functionalized fatty acid methyl esters (FAMEs), which may be derived from a variety of natural seed oils.
- Physical state: Waxy solid at room temperature
- Analytical purity: > 95.2 %
- Impurities (identity and concentrations): not specified in the report
- Composition of test material, percentage of components:
Methyl Palmitate: 9.68 %
Methyl Stearate: 17.8 %
Methyl Hydroxymethyl Stearate: 38.6 %
Methyl Dihydroxymethyl Stearate: 26.7 %
Methyl Trihydroxymethyl Stearate: 2.37 %
- Isomers composition: not specified in the report
- Purity test date: not specified in the report
- Lot/batch No.: (lot # 200500200-25-4)
- Expiration date of the lot/batch: not specified in the report
- Radiochemical purity (if radiolabelling): not applicable
- Specific activity (if radiolabelling): not applicable
- Locations of the label (if radiolabelling): not applicable
- Expiration date of radiochemical substance (if radiolabelling): not applicable
- Stability under test conditions: not specified in the report
- Storage condition of test material: not specified in the report

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan (Indianapolis, Indiana)
- Age at study initiation: 9-12 weeks
- Weight at study initiation: not specified in the report
- Housing: up to six per cage in filter tubs containing corncob bedding, during acclimation and experiment conduct
- Diet (e.g. ad libitum): ad libitum, LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St.Louis, Missouri) in pelleted form.
- Water (e.g. ad libitum): ad libitum, Municipal water source
- Acclimation period: at least one week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 1°C
- Humidity (%): 40-70 %
- Air changes (per hr): 12-15 times/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)


IN-LIFE DATES: not specified in the report

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Vehicle (4:1 AOO), 5%, 20% and 80% of natural oil monomer

No. of animals per dose:

Six female mice/group

Details on study design:

RANGE FINDING TESTS:
During the screening study, the mice were treated with three daily topical applications of 1%, 5%, 25%, 50%, 75% or 90% of NOM. Erythema was absent in the mice treated with 1% or 5%, while the mice dosed with 25%, 50% or 75% demonstrated slight erythema on day 3 which resolved by day 6. The mouse treated with 90% Methyl Polyhydroxymethyl Stearate, demonstrated well-defined erythema on day 3, which resolved by day 6. Body weights were unaffected in all dose groups


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: Based on Technical Report No.87, 2003 by an expert ECETOC panel - Test materials that elicit a stimulation index (SI) of > 3 (i.e., 3-fold greater proliferation than control animals) would be considered positive for dermal sensitization potential.


TREATMENT PREPARATION AND ADMINISTRATION:
Methyl Polyhydroxymethyl Stearate (5%, 20% and 80%) were dissolved in AOO (4:1 acetone:olive oil), preferred solvent in LLNA guidelines and also based upon maximum solubility. Solutions were prepared daily just prior to dosing. Preparation of the dosing materials was documented in the study file. The concentrations of the dosing solutions were not verified analytically.
The application of the test material (25 μl/ear) was made on the dorsal surface of both ears, using an adjustable pipette with a disposable tip. Ears were inspected prior to application of the test material solutions, and erythema was evaluated on days 2, 3, and 6. Body weights were recorded on days 1 and 6.

Positive control substance(s):

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

Statistics:

The Stimulation Index (SI) and EC3 calculation using appropriate methods were calculated for each mouse. Means and standard deviation (SD) were generated for body weight data (absolute and gain) and the LLNA response (dpm & SI values). These body weight and dpm data were analyzed by a one-way analysis of variance (Steele and Torrie, 1960). When differences were indicated by the ANOVA, a comparison of treated vs. control groups was done using a Dunnett’s t-test (Steele and Torrie, 1960). The alpha level at which all tests were conducted was 0.05.

Results and discussion

Positive control results:

30% HCA treated group elicited a stimulation index (SI) of 8.2 in comparison to vehicle-treated mice and this can be considered as a positive response from the positive control.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: Vehicle control 1.0 ± 0.2 5% NOM 2.0 ± 1.4 20% NOM 3.2 ± 1.3 80% NOM 7.4 ± 2.1

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Vehicle control 624.0 ± 215.59 5% NOM 1251.0 ± 856.01 20% NOM 2021.0 ± 792.50 80% NOM 4633.7 ± 1358.2

Applicant's summary and conclusion

Interpretation of results:

other: weak dermal sensitization potential

Conclusions:

Based on the results of the study, NOM has an estimated EC3 of 17.5% and can be classified as having a weak dermal sensitization potential.

Executive summary:

The Local Lymph Node Assay (LLNA) was conducted to assess the potential of natural oil monomer (NOM) to cause contact sensitization by measuring the lymphocyte proliferative responses from auricular lymph nodes following topical application of the test material to the mouse ear.

Screening Study: Three daily topical applications of 1%, 5%, 25%, 50%, 75%, or 90% of NOM were given to one animal at each dose level. Erythema was absent in the mice treated with 1% or 5%, while the mice dosed with 25%, 50%, or 75% demonstrated slight erythema on day 3 which resolved by day 6. The mouse treated with 90% NOM, demonstrated well-defined erythema on day 3 which resolved by day 6. Body weights were unaffected in all dose groups. Results from this stud y were used to determine the dosing concentrations for NOM in the LLNA.

LLNA: Six female mice/group received 5%, 20%, or 80% of NOM, or vehicle (4:1 AOO), on days 1-3. On day 6, uptake of 3H-thymidine into the auricular lymph nodes draining the site of chemical application was measured five hours post administration. Proper conduct of the LLNA was confirmed via a positive response using 30% a-hexylcinnamaldehyde (HCA), a moderate contact sensitizer, which elicited proliferation that was 8.2 in comparison to vehicle-treated mice.

Erythema was absent in the mice treated with 5%, while two of six mice treated with 20% NOM had slight erythema on day 3, which resolved by day 6. The mice treated with 80% demonstrated slight erythema on days 2 and 3, which persisted through day 6. Body weights were unaffected in all dose groups.

Mice treated with 5%, 20% and 80% NOM elicited proliferative responses with stimulation indices (SI) that were respectively 2.0, 3.2, and 7.4 in comparison to vehicle-treated mice. The concentration that would cause a 3- fold increase in proliferation (EC3) was calculated to be 17.5% which is consistent with weak dermal sensitization potential as described by an expert ECETOC panel (Technical Report No. 87, 2003).