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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
Study period:
2-9 November 2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
GLP study conducted in compliance with OECD Guideline No. 429 with deviations: tested only up to 50% (without rationale); no preliminary study conducted; no ear thickness measurements reported

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
tested only up to 50% (without rationale); no preliminary study conducted; no ear thickness measurements reported
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Appearance: Clear colorless liquid
- Storage condition of test material: Room temperature

In vivo test system

Test animals

Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Jackson Laboratories, Bar Harbor, ME 04609.
- Females - nulliparous and non-pregnant: Yes
- Microbiological status of animals, when known: Animals were healthy and free from infections.
- Age at study initiation: 9-11 weeks
- Weight at study initiation: 18-24 g
- Housing: Animals were group housed (5 per cage) upon receipt then 4 per cage when assigned to groups.
- Diet: Certified Rodent Chow 7012C, ad libitum.
- Water: Tap water, ad libitum
- Acclimation period: 7 days
- Indication of any skin lesions: None

ENVIRONMENTAL CONDITIONS
- Temperature: 22.2-26.7 °C
- Humidity: 22-44 %
- Photoperiod: 12 h light/12 h dark

- IN-LIFE DATES: 2-9 November 2004

Study design: in vivo (LLNA)

Vehicle:
other: Diethyl phthalate/ethanol (3:1)
Concentration:
2.5, 5, 10, 25 and 50% (w/v) in Diethyl phthalate/ethanol (3:1)
No. of animals per dose:
4
Details on study design:
DOSE PREPARATION
On each day of dosing, the test item was prepared 2.5, 5, 10, 25 and 50% (w/v) in volumetric flasks by dissolving the appropriate amount of test item in the vehicle. All preparations were vortexed to mix. The test item dosing solutions were clear colorless liquids.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: The criterion for a positive response is that one or more concentrations of a test item elicits a 3-fold or greater increase in isotope incorporation relative to the vehicle control.

TREATMENT PREPARATION AND ADMINISTRATION:
Mice were treated on the dorsal surface of both ears (25 µL/ear), once per day on Days 1, 2, and 3. Approximately 24 ± 2 h between applications of test item was maintained. On Day 6, the mice were injected i.v. with 20 µCi of 3H-thymidine in 250 µL of sterile saline. Five hours later the mice were euthanized by CO2 asphyxiation. At removal, the number of nodes collected per animal was recorded, and the nodes were examined for size/appearance and the data recorded. The lymph nodes from each group were pooled and a single cell suspension was prepared. Cells were washed twice with phosphate buffered saline (PBS) and precipitated with 5% trichloroacetic acid (TCA) overnight at 2-8 °C. The pellets were recovered by centrifugation and resuspended in 1 ml of TCA and transferred to a vial containing scintillation fluid. An additional 1 mL of TCA was used to rinse the tube, and it was also transferred to the scintillation fluid. Incorporation of 3H-thymidine was measured in a β-scintillation counter.
Increases in 3H-thymidine incorporation relative to the vehicle-treated control were derived for each group and recorded as stimulation indices (SI).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The evaluation of the equality of means for body weight data was made by a one-way analysis of variance using the F distribution to assess statistical significance. If statistically significant differences between the means are found, a Dunnett's test was used to determine the degree of significance from the control means.
No statistical analysis was done on the Disintegration per Minute (DPM).

Results and discussion

Positive control results:
Hexylcinnamaldehyde at 35 % induced skin sensitisation (SI = 3.45).

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1.19
Test group / Remarks:
2.5%
Key result
Parameter:
SI
Value:
1.07
Test group / Remarks:
5%
Key result
Parameter:
SI
Value:
0.97
Test group / Remarks:
10%
Key result
Parameter:
SI
Value:
1.28
Test group / Remarks:
25%
Key result
Parameter:
SI
Value:
2.16
Test group / Remarks:
50%
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
At termination, the lymph nodes from the mice treated with the test item at 50% were enlarged but appeared normal. The lymph nodes from the mice in the vehicle and the test item treated animals at all other levels were normal in size and appearance.

DISINTEGRATION PER MINUTE (DPM)
Exposure to test item at 0, 2.5, 5, 10, 25 and 50% (w/v) resulted in DPM of 286, 340, 307, 278, 366 and 618, respectively

STIMULATION INDEX
Exposure to test item at 2.5, 5, 10, 25 and 50% (w/v) resulted in stimulation indices of 1.19, 1.07, 0.97, 1.28, and 2.16, respectively.

CLINICAL OBSERVATIONS:
- There was no mortality and all animals appeared normal throughout the study.
- The application sites on the mice from the groups treated with the test item at 50% appeared wet on Days 3-5. There were no other findings. No edema or erythema was noted at the application sites on any of the mice.

BODY WEIGHTS
There were no statistically significant differences in body weights observed between any of the treatment groups.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The registered substance did not induce skin sensitisation in an in vivo test (LLNA) at concentrations up to 50% with a stimulation index (SI) of 2.16. This SI is considered to be high and it could be anticipated to be higher than 3 with higher doses. Moreover, the registered substance was predicted to be a skin sensitizer based on QSAR models (ToxTree, Toolbox and VEGA) and its metabolite eugenol is a known sensitser. Therefore, considering all the evidence, the registered substance is classified as a skin sensitiser Cat. 1B according to the Regulation (EC) No 1272/2008 (EC3 > 2%).
Executive summary:

A study was performed to assess the skin sensitisation potential of test item in the CBA/J strain mouse following topical application to the dorsal surface of the ear. The method was conducted according to the OECD test guideline No 429 and in compliance with GLP.

 

Six groups of 4 CBA/J female mice were treated on the dorsal surface of both ears once per day for 3 days with 2.5, 5, 10, 25 or 50% (w/v) of test item with the vehicle (Diethyl phthalate/ethanol (3: 1)). On Day 6, the mice were injected, i.v. with 20 µCi of 3H-thymidine in sterile saline. Five hours later, the mice were euthanized and the draining auricular lymph nodes were removed. The lymph node cells were precipitated with 5% trichloroacetic acid (TCA) and the pellets counted in a β-scintillation counter to determine incorporation of the 3H-thymidine.

 

There was no mortality and all animals appeared normal throughout the study. The application sites on the mice from the groups treated with the test item at 50% appeared wet on Days 3-5. There were no other findings. No edema or erythema was noted at the application sites on any of the mice. At termination, the lymph nodes from the mice treated with the test item at 50% were enlarged but appeared normal. The lymph nodes from the mice in the vehicle and the test item treated animals at all other levels were normal in size and appearance. There were no statistically significant differences in body weights observed between any of the treatment groups.

Exposure to test item at 2.5, 5, 10, 25 and 50% (w/v) resulted in stimulation indices of 1.19, 1.07, 0.97, 1.28, and 2.16, respectively.

 

The historical positive control, Hexylcinnamaldehyde, gave a SI of 3.45, when tested at 35 % w/v. The test system was therefore considered to be valid.

 

Under the test conditions, the registered substance did not induce skin sensitisation at concentrations up to 50% with a stimulation index (SI) of 2.16. This SI is considered to be high and it could be anticipated to be higher than 3 with higher doses. Moreover, the registered substance was predicted to be a skin sensitizer based on QSAR models (ToxTree, Toolbox and VEGA) and its metabolite eugenol is a known sensitser. Therefore, considering all the evidence, the registered substance is classified as a skin sensitiser Cat. 1B according to the Regulation (EC) No 1272/2008 (EC3 > 2%).

This study have been considered sufficient, together with in silico data to classify the registered substance without further testing using a weight of evidence approach.