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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:
2019-07-31 - 2019-08-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
Deviations:
not specified
Qualifier:
according to
Guideline:
other: EPA OCSPP 870.2600
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA): BrdU-ELISA

Test material

Reference
Name:
Unnamed
Type:
Constituent

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
- Age at study initiation: Approx. 1-2 months
- Weight at study initiation: 19.0 - 21.4 g (screen animals); 18.3 -24.5 g (main animals); The weight variation of the animals at study start did not exceed ± 20% of the mean body weight in each phase.
- Housing: 1/cage in suspended wire-bottom cages during the study. Bedding was placed beneath the cages and changed at least three times per week
- Diet (e.g. ad libitum): Fresh PMI Rodent Chow (Diet #5002) ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

thirty-one nulliparous, non-pregnant female mice were assigned to the test groups of the preliminary dermal irritation screen, and the main test without conscious bias.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): controlled and continuously monitored
- Humidity (%): continuously monitored
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
5%, 10%, and 25% (w/v) in acetone/olive oil (4:1 v/v; AOO
No. of animals per dose:
2 animals per group (Preliminary Dermal Irritation Screen)
5 animals per group (Main Test)
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: Test Sample was tested for solubility at 50% (w/v) in vehicle AOO
Three groups of CBA/J mice (two animals per group) were treated with increasing concentrations of the test article: 5%, 10%, and 25% (w/v) in AOO. Treatment was via topical application of the test article concentrations to the dorsum of each ear once daily for three consecutive days. The test article was spread over the entire dorsal surface of the ear using a micropipette to deliver 25 μL/ear.

Sample preparation:
250 mg of Test Sample were added to 1000 μL of AOO and mixed to yield a 25% test article formulation. 400 μL of the 25% (w/v) formulation were added to 600 μL in AOO and mixed to yield a 10% test article formulation. 500 μL of the 10% formulation were added to 500 μL in AOO and mixed to yield a 5% (w/v) test article formulation.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: animal were assigned randomly
- Criteria used to consider a positive response:
An increase in ear thickness of 25% indicates a positive dermal irritation response.
A stimulation index SI > 3 indicates a sensitizing response.

TREATMENT PREPARATION AND ADMINISTRATION:
The test article concentrations used in the main LLNA study were chosen such that the maximum concentration tested avoided both overt systemic toxicity and excessive local dermal irritation. Concentrations of 10%, 25% and 50% (w/v) were used. 500 mg of Test Sample were added to 1000 μL with AOO and mixed to yield a 50% test article formulation. 500 μL of the 50% formulation were added to 500 μL of AOO and mixed to yield a 25% (w/v) test article formulation. 400 μL of the 25% formulation were added to 600 μL in AOO and mixed to yield a 10% (w/v) test article formulation.
Five groups of CBA/J mice (5 animals per group) were treated by topical application of the test article concentrations, vehicle control or positive control in the same manner as in the screen. AOO was chosen as the vehicle, based on solubility. Historically, the AOO vehicle has produced consistent results with the positive control 25% HCA, as well as with other sensitizers and non-sensitizers.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
For each test article-treated group, the individual animal SI values along with the mean group SI and standard deviation were calculated, and ANOVA followed by the Student-Newman-Keuls test was performed to statistically compare each test article dose group to the vehicle control group. Although specified in the test guidelines, these calculations and results were not incorporated into the interpretation of the data. An SI value of 1.6 or more is the sole determinant for a positive sensitization response.

Results and discussion

Positive control results:
The positive control 25% HCA (alpha-Hexylcinnamaldehyde) gave positive results, i.e. an increase in ear thickness of 17.6% and a Stimulation Index of 3.2, which is greater than 1.6 and indicates a sensitizing response.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
1
Test group / Remarks:
10% (w/v)
Parameter:
SI
Value:
0.7
Test group / Remarks:
25% (w/v)
Parameter:
SI
Value:
1
Test group / Remarks:
50% (w/v)

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The skin sensitizing potential of the test item was determined in a well-documented guideline study compliant to GLP. Therefore, it can be considered as reliable without restrictions (Reliability 1) and the obtained results can be used for classification.
Topical application of Test Sample at 10%, 25%, and 50% (w/v) resulted in SI values that were less than 1.6 (SI < 1.6); therefore, the test article was not a dermal sensitizer in the Local Lymph Node Assay in Mice.
Executive summary:

The purpose of this GLP study was to determine the sensitizing potential of topically applied test articles. This LLNA protocol, utilizing the BrdU ELISA method, was designed to be a stand-alone assay for dermal sensitization as described in the NIH report “The Murine Local Lymph Node Assay: A Test Method for Assessing the Allergic Contact Dermatitis Potential of Chemicals/Compounds,” NIH No. 99-4494, and the LLNA test guidelines as defined in EPA OCSPP 870.2600 and OECD Guideline for the Testing of

Chemicals No. 442B.

The results of this preliminary screen indicated that none of the test article treatments resulted in significant dermal irritation. Based on these results, test article concentrations of 10%, 25% and 50% (w/v) were chosen for the main Local Lymph Node study.

The main test was conducted with five separate groups of healthy female CBA/J mice (five animals per group). Three groups were treated with increasing concentrations of the test article. A vehicle control group was treated with AOO and another group was treated with the positive control, alpha-Hexylcinnamaldehyde in AOO (25% HCA), in the exact same manner. The test article, vehicle control, and positive control solutions were administered by topical application to the dorsum of each ear, once daily for three consecutive days.

The mean SI and standard deviation were calculated for each group from the individual animal data. If any test article treatment group yielded an SI value of 1.6 or more, then the

test article was considered to be a sensitizer.

All animals survived the in-life phase of the study and were observed to be normal. There were no difficulties noted in administration of the test article or with its adherence to the dosed ears. Body weight losses (less than 2 grams) were noted but were not considered to affect the interpretation of the study. Ear thickness measurements and individual animal observations indicated that none of the test article treatments in the screen or the main test resulted in more-than-moderate local dermal irritation. No erythema was observed in any test article treatment group.

The SI of the positive control group, 25% HCA, was 3.2. The group SI values for the test article were as follows:

Test article concentration   10% (w/v)  25% (w/v)  50% (w/v)
 SI value  1.0  0.7  1.0

Topical application of Test Sample at 10%, 25%, and 50% (w/v) resulted in SI values that were less than 1.6 (SI < 1.6); therefore, the test article was not a dermal sensitizer in the Local Lymph Node Assay in Mice.