The LLNA-BrdU ELISA is conducted at MB Research Labs and is based upon the OECD 442B Testing Guideline. The LLNA is an alternative toxicology assay for dermal sensitization using CBA/J mice. The LLNA is an alternative to guinea pig studies that require more animals and long test durations.
To determine the sensitizing potential of topically applied test material utilizing the LLNA, and measurement of lymphocyte proliferation by BrdU incorporation detected by ELISA.
Irritation Screen Test:
An initial Irritation Screen is performed using 3-5 highest achievable concentrations. The screen consists of 2 animals per concentration that will be dosed in an identical manner to that of the main test. Ear measurements will be taken prior to dosing on Study Days 1 and 3, and prior to euthanasia on Study Day 6. Measurements taken on Study Days 3 and 6 will be compared to pre- dosing measurements. If there is no effect, or an increase of <25% in ear thickness, the same concentrations will be assayed in definitive test.
However, if significant ear swelling (ES) is present (>25% over control), then an extended screen (Quantitative Irritation Test; QIT) is recommended. The QIT is similar to the Irritation Screen except 4-6 concentrations will be assayed to determine the highest non-irritating concentration.
Definitive Sensitization Test:
The main test will be conducted by topically applying test material to the dorsum of the ears at the 3-5 highest achievable concentrations (100 [liquid only], 50, 25, 10, 5, 2.5, 1, 0.5, etc.), which do not cause severe irritation or overt systemic effects. Ear measurements will be taken on Study Days 1 (prior to first dosing), 3 (approximately 48 hours after initial dosing) and 6 (prior to euthanasia). Animals will be injected with BrdU on Study Day 5, approximately 24 hours prior to euthanasia. Auricular lymph nodes will be collected on Study Day 6, a single-cell suspension will be created and lymph node cells (LNC) will be assayed for BrdU incorporation via ELISA.
- A Positive Response is when a Stimulation Index (SI) is greater than or equal to 1.6 (relative to that of control lymph nodes).
- A “Borderline Positive Response” is defined as an SI from 1.6 to 1.9; additional information and testing may be needed.
- A Negative Response is when the SI is <1.6.
For more information about this and other studies, please see: www.mbresearch.com