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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:
From December 08 to 14, 2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
GLP study performed according to OECD test guideline No. 429 with some deviations: no ear thickness measurement. The study was not fully reliable but was considered sufficient for the purpose of risk assessment.

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:
no ear thickness measurement
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
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:
solid: particulate/powder
Details on test material:
- Physical state: White powder
- Storage condition of test material: Stored at 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
- Age at study initiation: 10 weeks
- Weight at study initiation: 18-24 g
- Housing: Animals were housed in group of 4 per cage.
- Diet: Certified Rodent Chow 7012C, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 23.3-23.9 °C
- Humidity: 16-36 %
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: From: December 08, 2004 To: December 14, 2004.

Study design: in vivo (LLNA)

Vehicle:
other: Diethyl phthalate/ethanol (3:1)
Concentration:
1, 2.5, 5, 10 and 25 %
No. of animals per dose:
4
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: up to 25% (w/v) (the test item was not soluble at 50% w/w)
- Irritation: in general, the doses were selected so that the highest concentration maximizes exposure while avoiding excessive local irritation.
- Systemic toxicity: in general, the doses were selected so that the highest concentration maximizes exposure while avoiding systemic toxicity.
- Ear thickness measurements: not measured
- Erythema scores: not reported

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 as a sensitiser if one or more concentrations of test material results in a 3-fold or greater increase in isotope incorporation relative to the vehicle control.
TREATMENT PREPARATION AND ADMINISTRATION:
- Dosing solutions were prepared on each day of dosing. All preparations were vortexed to mix.
- 25 µL of control or test material was applied topically on the dorsal surface of both ears using a micropipette daily for three consecutive days (Days 1-3). Approximately 24 ± 2 h between applications of test material was maintained. On Day 6, animals were injected i.v. with 20 µCi of 3H-thymidine in 250 µL of sterile saline. 5 h later animals were euthanized by CO2 asphyxiation. 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.
- 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:
- 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.

Results and discussion

Positive control results:
SI for the positive control substance hexyl cinnamic aldehyde was 5.80 which demonstrates the validity of this study.

In vivo (LLNA)

Results
Key result
Parameter:
SI
Value:
< 3
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
At termination, the lymph nodes from the mice in the vehicle and test material treated animals at all levels were normal in size and appearance.
DPM (as mean ± sem) for 0 (vehicle), 1, 2.5, 5, 10 and 25 % were 285, 304, 197, 184, 286 and 280, respectively.

DETAILS ON STIMULATION INDEX CALCULATION
Stimulation index for test material at 1, 2.5, 5, 10 and 25 % were 1.07, 0.69, 0.65, 1.00 and 0.98, respectively.

EC3 CALCULATION
As a SI of 3 or more was not recorded for all of the concentrations tested, test item was not considered to have the potential to cause skin sensitization. Thus no EC3 was calculated.

CLINICAL OBSERVATIONS:
No mortality or clinical signs were observed. No oedema or erythema was noted at the application sites on any of the animals.

BODY WEIGHTS
No statistically significant differences in mean body weight and body weight changes observed between any of the treatment groups.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the test conditions, test material is not considered as skin sensitiser according to the annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.
Executive summary:

In a Local Lymph Node Assay (LLNA) performed according to OECD Guideline 429 and in compliance with GLP, groups of female CBA/J mice (4 females/group) were topically applied with test material at the dose concentrations of 1, 2.5, 5, 10 and 25 % final concentration (25% w/v being the highest achievable concentration) in 3:1 diethyl phthalate/ethanol on dorsal surface of both ears (25 µL/ear) daily for three consecutive days. A vehicle control group (4 females/group) was treated with 3:1 diethyl phthalate/ethanol alone and a positive control group (4 females/group) was treated with hexylcinnamic aldehyde (HCA) at the dose concentration of 5, 15 and 35 % in diethyl phthalate/ethanol (3:1) in same manner to confirm the sensitivity and reliability of the test method. On Day 6, animals were injected intravenously with 20 µCi of 3H-thymidine in sterile saline. 5 h later, animals were euthanized and the draining auricular lymph nodes were removed and precipitated with 5 % trichloroacetic acid (TCA). 3H-thymidine incorporation was quantified using a β-scintillation counter and disintegrations per minute (DPM) and stimulation index (Sl) were calculated for each dose group.

No mortality, clinical signs or skin reactions were observed in any of the animals. No changes in mean body weights were observed. Mean DPM for 0 (vehicle), 1, 2.5, 5, 10 and 25 % were 285, 304, 197, 184, 286 and 280, respectively. SI calculated for test material was found to be 1.07, 0.69, 0.65, 1.00 and 0.98 for the dose concentrations of 1, 2.5, 5, 10 and 25 %, respectively, which indicates that test item did not show the potential to induce skin sensitization.

The SI for the positive control substance HCA was 5.80, which demonstrates the validity of this study.

Under the test conditions, test material is not considered as skin sensitiser according to the annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

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