National Toxicology Program

Female BALB/c mice are used in a majority of NTP studies which evaluate contact hypersensitivity. Historically, the guinea pig has been used as a model for evaluating drug and chemical hypersensitivity. The high cost associated with those studies and an increasing awareness of animal welfare issues, however, have led to the development of more cost effective and humane hypersensitivity models. The murine model fits these criteria, and is now being used extensively to study chemical hypersensitivity. Recent acceptance of the Local Lymph Node Assay (LLNA) as a stand-alone alternative to the Guinea Pig Maximization Test/Beuhler Assay by the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) demonstrates the commitment of using more humane testing models for screening chemicals that may elicit contact hypersensitivity in humans.

Species Maintenance

Female BALB/c mice are obtained from National Cancer Institute or Charles River Laboratories. Mice are hepatitis and Sendai virus free and are obtained at 45-60 days of age (17-20 grams). Upon arrival, animals are examined for clinical evidence of disease by a trained representative from the Division of Animal Resources (DAR) and quarantined for approximately one week prior to commencement of treatment. At the end of the quarantine period, animals are re-examined by a DAR representative and released from quarantine. All mice are randomized using a computer-generated randomization procedure and individually identified by tattoo and cage card in accordance with standard operating procedure. Mice are housed no more than 4 animals per cage in plastic shoe box cages. Animals are housed in a dedicated facility where the temperature is maintained at 18-26°C and the relative humidity between 40-70%. The light/dark cycle is maintained on 12-hour intervals.

Experimental Design

The design of these studies is based on the optimization of a number of endpoints that are reported to be altered during contact sensitization in the guinea pig and mouse.

Primary Irritancy & Cumulative Percutaneous Toxicity Study: A primary irritancy study of potential sensitizers is performed in order to establish the respective concentrations for induction (sensitization) and challenge in the LLNA and Mouse Ear Swelling Test (MEST).

On the first day of the treatment period (considered day 1) prior to the first application of test material, measurements of the ear thickness are made. Animals are restrained and two measurements of ear thickness are made on each ear at the designated sites using a modified Mitutoyo micrometer (shown below).

Designated Areas for Mouse Ear Measurements

Four mice are used to examine each concentration of test article (specified as % w/v or % v/v dependent on the test article and positive control). Each mouse receives 12.5 ml of either the test article, vehicle, positive control, or positive control vehicle on both sides of each ear. Mice are treated in this manner one time per day for four consecutive days. At 24 hours (+/- 2 hrs) after the final treatment, mice are restrained and measurements of ear thickness are made in the same manner as the pre-treatment measurements.

Major Events for the Primary Irritancy Study

The percent ear swelling is calculated as the [(mean thickness of the post treated ear / mean thickness of the pre-treated ear) X 100]-100. The percent ear swelling for the test article is compared to the percent swelling for the VH using a one-way analysis of variance followed by the Dunnett's t Test to determine the minimal irritating concentration (MlC), and the maximal non-irritating concentration (MNC). The MIC is defined as the lowest concentration of test article to elicit a significantly greater ear swelling response than that observed in the vehicle control group; the MNC, on the other hand, is the highest concentration producing ear swelling that is not significantly different from the vehicle control.

Contact Hypersensitivity Studies

Mouse Ear Swelling Test (MEST): Based on the irritancy studies described above, concentrations are selected for selected potentially sensitizing doses and challenge. The major events for the MEST are shown on below:

Major Events of the Mouse Ear Swelling Test

Prior to sensitization, mice are weighed, tattooed and anesthetized so that the dorsal thorax of each mouse may be shaved. Beginning on day 1, exposures are accomplished by the direct application of 50 ml of test article, DNFB (as a positive control) or the respective vehicles to the prepared site with a pipette. Following application, the animals are restrained long enough to allow the vehicle to start to volatilize. The sensitization procedures of day 1 are repeated on days 2 and 3. Mice are then rested for days 4-7. On day 8, the pre-treatment thickness of both ears on each mouse is measured.

Following this measurement, mice are challenged with 25 ml of either the vehicle, test article, the DNFB positive control or the positive control vehicle on both sides of each ear. The same ears are then measured 24 and 48 hours after challenge. Recorded raw data included pre-treatment measurements, 24 and 48 hours post treatment measurements of the thickness of 2 sites on the right ear of all mice. The percent ear swelling is calculated as follows: [(mean thickness of both ears (24 or 48 hrs post treatment) / mean thickness of both ears measured before treatment) X 100]-100. The percent ear swelling for the test article is compared to the percent ear swelling for the vehicle for significance and dose response.

Local Lymph Node Assay (LLNA): Doses are selected from the concentration series 100%, 50%, 25%, 10%, 5%, 2.5%, 1%, 0.5%

Prior to initiating the study, treatment mice are weighed and tattooed. On day 1, mice are treated with the appropriate concentration of test article by applying 25 ml of the appropriae dilution to the doesum of both ears. Identical treatments are repeated for the next two days. On day 4 and 5, mice are rested and receive no treatment. On day 6, all mice are injected with 0.2 ml (20 mCi) ³H- thymidine i.v. via the tail vein. Approximately 5 hours after injection, mice are sacrificed and both auricular Iymph nodes from each animal are excised and put into tubes with 4 ml cold PBS. All Iymph nodes are dissociated by grinding between the frosted ends of two microscope slides. Cells are washed twice in 10 ml of PBS and then re-suspended in 3 ml of 5% trichloroacetic acid (TCA) (w/v, distilled H₂O). Cells are allowed to set overnight (approximately 18 hours) at 4°C before being resuspended in TCA, transferred into scintillation cocktail, and counted in a Beta counter for 5-10 minutes. Data are presented as DPM, calculated as the (CPM — background count) / the efficiency of the Beta counter.

Major Events of the Local Lymph Node Assay

Immunofluorescent Staining of Lymph Node Subpopulations.

Mice (4 per treatment group) are treated with either test article, vehicle, the positive control, DNFB (0.15%), or the positive control vehicle using the same dosing regimen described in the irritancy assay. On the fifth day, mice are euthanized and the auricular lymph nodes removed. The right and left nodes from each animal are pooled and pressed into a single cell suspension between two-frosted microscope slides. Cells are labeled in 96-well culture plates with 0.10 ml of the appropriate anti-mouse monoclonal antibodies, diluted in phosphate buffered saline (PBS). CD3m (145-2C11) conjugated to FITC is used to label T cells. B cells are dual stained with CD45R/B220 (RA3-6B2) conjugated to PE, and FITC conjugated affinity purified anti-mouse immunoglobulin. T cell subpopulations are dual stained with a PE conjugated anti-mouse CD4⁺ monoclonal antibody, and a FITC conjugated anti-mouse CD8⁺a monoclonal antibody. The appropriate isotope controls are included in the analysis. All cell-staining combinations are incubated on ice in the dark for at least 30 minutes, washed, then re-suspended in 0.10 ml of a 1 ug/ml solution of propidium iodide (PI) for 5 minutes prior to flow cytometric enumeration. Dead cells are eliminated from the analysis by gating out the PI stained cells; the forward scatter threshold is set to eliminate red blood cells.