Evaluation of Sex Sensitivity in Local Lymph Node Assay Using Acephate and α- Hexylcinnamaldehyde

Abstract

The current OECD Test Guideline for the conduct of the Local Lymph Node Assay (LLNA) recommends the use of only female mice for the assessment of skin sensitization potential for a given chemical. The NIH publication N° 99-4494 recommends that only female CBA mice be used, as they reportedly develop a stronger contact dermatitis response when compared to males however if male to be used, systematic studies evaluating potential sex differences should be conducted. Moreover, males were reported to display a larger variation in response due to a greater tendency to fight and to be involved in 'social ranking' behavior when group housed. However, there are several advantages to consider with the inclusion of male mice in LLNA testing including a more refined and responsible use of animals. Therefore, to begin to systematically assess the appropriateness of using male mice in the LLNA a comparative guideline study was conducted with individual housing of mice using a non-sensitizer and a sensitizer viz., Acephate 97 DF and α-hexylcinnamaldehyde (HCA) respectively. We have conducted an in-house study using a vehicle (dimethyl formamide) that is recommended by OECD 429 for the testing of mixtures in the LLNA for assessing skin sensitisation. We conducted the test according to the methods described by OECD 429 (Skin Sensitization: Local Lymph Node Assay). One common pesticide formulation, Acephate 97 DF and one known skin sensitizer - HCA, were each applied to the dorsum of both ears (25 µL per ear) of groups of 5 female CBA/J mice (male and female). Acephate 97 DF was applied at 3 separate concentrations and one concentration for HCA for 3 consecutive days (days 0, 1 and 2). A further group was given the vehicle (dimethyl formamide), alone. On day 5, all mice were injected intravenously (tail vein) with approximately 20 (±1) µCi of tritiated methyl thymidine. Five hours post-administration the uptake of 3H-thymidine into the auricular (local) lymph nodes draining the site of chemical application was measured in order to assess the proliferative response of the lymph node. The DPM values were measured individually for each mouse. Stimulation Index (SI Value) was calculated and are given below: - Acephate 97 DF (Male) (10%) 1.16 (25%) 1.58 (50%) 1.59 - Acephate 97 DF (Female (10%) 1.39 (25%) 1.55 (50%) 1.56 -