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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From June 06 to 12, 2001
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
GLP study performed equivalent or similar to OECD test guideline No. 429 with deviations: Individual weights of animals at start of dosing and at scheduled kill not reported; animal room humidity level was outside the target range; no ear thickness measurement, no range-finding test performed.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report date:
2001

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
(individual weights of animals at start of dosing and at scheduled kill not reported; animal room humidity level was outside the target range; no ear thickness measurement, no range-finding test performed)
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Reference substance name:
Isoeugenol
EC Number:
202-590-7
EC Name:
Isoeugenol
Cas Number:
97-54-1
IUPAC Name:
2-methoxy-4-prop-1-en-1-ylphenol
Test material form:
liquid
Details on test material:
- Physical state: Liquid
- Storage condition of test material: Stored at room temperature.

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/J Hsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague-Dawley, Inc., Indianapolis, IN.
- Age at study initiation: 6-8 weeks
- Weight at study initiation: 17.2-22.0 g (mean body weight: 20.0 g)
- Housing: Animals were housed individually in plastic shoebox-style cages.
- Diet: Purina Rodent Chow 5002, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature: 70-76 °F
- Humidity: 56-84 %
- Photoperiod: 12 h dark/12 h light

IN-LIFE DATES: From: June 06, 2001 To: June 12, 2001.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0.25, 0.5, 1.0, 2.5, and 5.0 % in 4:1 acetone/olive oil
No. of animals per dose:
6
Details on study design:
MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: A substance is considered a sensitizer if at least one concentration of the test material results in a statistically significant 3-fold or greater stimulation index (SI).

TREATMENT PREPARATION AND ADMINISTRATION:
- The vehicle and dilutions of the control and test materials were prepared on the bench top daily, prior to dosing. All suspensions were mixed by vortexing. 25 µL of control or test material was applied to the dorsum of each ear using a calibrated Finnpipet daily for three consecutive days. Animals were not treated on Days 4 and 5. On Day 6, animals were injected i.v. in the lateral tail vein with 0.25 mL containing 2 µCi of 125I-labelled Iododeoxyuridine and 10^-5 M FuDR in phosphate buffered saline (PBS). Approximately 5 h later, animals were euthanized by CO2 asphyxiation and the auricular lymph nodes were excised. The nodes were dissociated using the frosted ends of glass slides. The cell suspension was washed with Hanks' Balanced Salt Solution (HBSS) and then with PBS, prior to being resuspended in 5 % tricholoroacetic acid (TCA) and refrigerated at approximately 4°C. Approximately 18 h later the cells were centrifuged and resuspended in fresh 5 % TCA. The radioactivity was measured using a gamma counter (Packard Instruments).
Disintegrations per minute (DPM) and stimulation index (Sl) were calculated for each dose group.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
- To test if the compound was a sensitizer, a one-sample t test was performed for testing if the individual untransformed SI values of each dose level of each compound were different than 3.0.
- The natural log transformed DPM values for each test material concentration were compared against vehicle by first performing a Bartlett's Chi-Square test for variance homogeneity. If this was found to be non-significant, a one-way analysis of variance was used using dose (concentration). If this was found to be significant, then a Dunnett's t test was performed using an alpha of 0.05.
- If the Bartlett's Chi-Square was found to be significant, non-parametric analyses (specifically a Kruskal-Wallis test) were performed. If this was found to be significant, then a Jonckheere's-Terpera test was performed for dose-dependent trends.
- A confirmatory analysis was performed against the known standard hexylcinnamic aldehyde at two concentrations, 1 % and 25 % using the above methods.
- A fitted quadratic equation (a linear term and a square term of the concentration) was used to fit the data from the concentrations tested and to determine the concentration of test material required to elicit a stimulation index of 3 (EC3). The fitted quadratic equation was selected based on Loveless et al. (1996) and as stated the study protocol.
- A fitted linear equation was used to determine the concentration of hexylcinnamic aldehyde required to elicit a stimulation index of 3 (EC3) as only 3 doses [0 (vehicle), 1 and 25 %] of hexylcinnamic aldehyde were tested and the quadratic term was not significant.
- All calculations were performed using Microsoft Excel and SAS, version 6.12. PROCs GLM, FREQ, NPAR1WAY and MEANS were utilized.

Results and discussion

Positive control results:
Stimulation index for positive control group treated with 25 % of hexylcinnamic aldehyde in acetone:olive oil (4:1) was found to be 6.3; classified as skin sensitizer.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
EC3
Value:
< 2
Parameter:
SI
Value:
2.5
Variability:
± 0.5
Test group / Remarks:
0.25%
Parameter:
SI
Value:
1.2
Variability:
± 0.2
Test group / Remarks:
0.5%
Parameter:
SI
Value:
1.5
Variability:
± 0.3
Test group / Remarks:
1.0%
Parameter:
SI
Value:
4
Variability:
± 0.9
Test group / Remarks:
2.5%
Parameter:
SI
Value:
12.2
Variability:
± 1.9
Test group / Remarks:
5.0%
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
DPM (as mean ± SE) for 0 (vehicle), 0.25, 0.5, 1.0, 2.5 and 5.0 % were 5.1 ± 1.7, 12.7 ± 2.5, 6.1 ± 1.1, 7.9 ± 1.7, 20.3 ± 4.6 and 62.1 ± 9.8, respectively.

DETAILS ON STIMULATION INDEX CALCULATION
Stimulation index (as mean ± SE) for test material at 0.25, 0.5, 1.0, 2.5 and 5.0 % were 2.5 ± 0.5, 1.2 ± 0.2, 1.5 ± 0.3, 4.0 ± 0.9 and 12.2 ± 1.9, respectively.

EC3 CALCULATION
Calculated EC3 value for the test material was found to be 1.54 % and EC-3 potency value was 385 µg/cm2.

CLINICAL OBSERVATIONS
No mortality, irritation or other adverse toxic effects were noted in any of the animals.

BODY WEIGHTS
Not reported

Applicant's summary and conclusion

Interpretation of results:
Category 1A (indication of significant skin sensitising potential) based on GHS criteria
Conclusions:
Under the test conditions, test material is classified as “Category 1A” skin sensitizer according to the annex VI of the Regulation EC No. 1272/2008 (CLP) and to the GHS since EC3 < 2%.
Executive summary:

In a Local Lymph Node Assay (LLNA), groups of female CBA/J Hsd mice (6 females/group) were topically applied with test material at the dose concentrations of 0.25, 0.5, 1.0, 2.5 and 5.0 % final concentration in 1:4 acetone: olive oil to the dorsum of both ears (25 µL/ear) daily for three consecutive days. A vehicle control group was treated with 1:4 acetone: olive oil alone and a positive control group was treated with hexylcinnamic aldehyde at the dose concentration of 1 and 25 % in acetone:olive oil (4:1) in same manner to confirm the sensitivity and reliability of the test method. Three days after the final auricular application (on Day 6), animals were injected intravenously with 125I- labelled luDR to label proliferating cells. 125I-incorporation was quantified using a gamma counter and disintegrations per minute (DPM) and stimulation index (Sl) were calculated for each dose group.

No mortality, irritation or other adverse toxic effects were noted in any of the animals. Mean DPM for 0 (vehicle), 0.25, 0.5, 1.0, 2.5 and 5.0 % were 5.1, 12.7, 6.1, 7.9, 20.3 and 62.1, respectively. Stimulation Index (SI Value) calculated for test material treated groups was found to be 2.5, 1.2, 1.5, 4.0 and 12.2 for the dose concentrations of 0.25, 0.5, 1.0, 2.5 and 5.0 %, respectively. Calculated EC3 value for the test material was found to be 1.54 % and EC-3 potency value was 385 µg/cm2. Stimulation index for positive control group treated with 25 % of hexylcinnamic aldehyde in acetone: olive oil (4:1) was found to be 6.3; classified as skin sensitizer and confirming the validity of the study.

Under the test conditions, test material is classified as “Category 1A” skin sensitizer according to the annex VI of the Regulation EC No. 1272/2008 (CLP) and to the GHS since EC3 < 2%.

This study is considered as acceptable and satisfies the requirement for sensitisation endpoint.

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