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Skin sensitisation

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skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Not applicable
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference Type:
Detection of contact sensitivity of metal salts using the murine local lymph node assay
Ikarashi Y, Tsuchiya T & Nakamura A
Bibliographic source:
Toxicol. Lett. 62: 53-61

Materials and methods

Test guideline
no guideline followed
Principles of method if other than guideline:
The basic principle underlying the LLNA method used is to induce a primary proliferation of lymphocytes in the lymph node draining the site of test material application. An ear abrasion technique is employed which enhances penetration of the metal ion by removing the horny layer of the epidermis. It enables the detection of ear-swelling responses of weak contact allergens by increasing their LNC proliferation. This proliferation is proportional to the dose applied (and to the potency of the allergen) and provides a simple means of obtaining an objective and quantitative measurement of sensitisation. The LLNA assesses this proliferation, wherein the proliferation in test groups is compared to that in vehicle treated controls. The ratio of the proliferation in treated groups to that in vehicle controls (Stimulation Index), is determined, and must be at least three. The methods described here are based on the use of radioactive labelling to measure the proliferation of the lymph node cells.
GLP compliance:
not specified
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Zinc sulphate
EC Number:
EC Name:
Zinc sulphate
Cas Number:
Molecular formula:
zinc sulfate
Details on test material:
- Name of test material (as cited in study report): Zinc sulfate
- Other: Source - Wako Pure Chemical Industries, Ltd., Osaka, Japan

In vivo test system

Test animals

Details on test animals and environmental conditions:
- Age at study initiation: 6-8 wk

Study design: in vivo (LLNA)

other: 20 % ethanol
10 % test material in 20 % ethanol solution
No. of animals per dose:
Details on study design:

- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: Stimulation index ≥ 3

TREATMENT PREPARATION AND ADMINISTRATION: 25 µL of test material was applied to the dorsum of both ears for three consecutive days. Prior to the test material treatment, the ears of each mouse were gently abraded using a 19 g needle. Four days following the initial application, draining lymph nodes were excised.

Preparation of cell suspension: A single cell suspension of LNC was prepared by mechanical disaggregation through sterile 200 mesh steel gauge. The cells were washed twice with an excess phosphate-buffered saline (PBS) and resuspended in RPMI-1640 culture medium supplemented with 10 % fetal calf serum (FCS), 25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes), 100 µg/mL penicillin and 100 U/mL streptomycin. The cell concentration was adjusted to give 5 x 10E6 cells/mL.
Lymphocyte suspensions were seeded into 96 well microtiter plates at a concentration of 1 x 10E6 cells/well (5 wells per animal), and cultured with 0.5 µCi [3H] methyl thymidine ([3H]TdR) for 18 h at 37 °C in a humidified atmosphere of 5 % CO2 in air.
The incorporation of [3H]TdR was measured using a liquid scintillation counter and expressed as mean counts per min (cpm) ± standard deviation per node of three animals for each test group.

Other: Compound solubility - Completely dissolved in 20 % ethanol solution
Positive control substance(s):
not specified
Not reported

Results and discussion

Positive control results:
Not reported

In vivo (LLNA)

Resultsopen allclose all
Remarks on result:
other: 10 % zinc sulfate = 1.41
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean value cpm ± SD ( x 10-3) = 2.14 ± 0.77

Any other information on results incl. tables

Validity of ear abrasion technique: It does not affect the LNC response in the vehicle treated group.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Migrated information
Under the conditions of the test, the test material was determined to be non-sensitising to mice.
Executive summary:

A study was conducted to evaluate the skin sensitisation potential of the test material in mouse using a modified Local Lymph Node Assay. No guideline or GLP compliance was documented in the study report.

Groups of BALB/c mice (n=3) were treated with 10% concentration of test material or vehicle (20% ethanol solution) by applying 25 µL to the dorsum of both abraded ears for three consecutive days. Four days following the initial application, draining lymph nodes were excised. A single cell suspension of LNC was prepared and the incorporation of [3H]TdR was measured using a liquid scintillation counter.

[3H]TdR incorporation (expressed as mean counts per min (cpm) ± standard deviation per node x 10-3) was 2.14 ± 0.77 and the ratio of the proliferation in treated group to that in vehicle control (stimulation index) was 1.41.

Hence, under the conditions of the test, the test material was determined to be non-sensitising to mice.