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The efficacy of a lymph node cell proliferation assay in the guinea pig as a first stage screening method of predicting sensitizing potentials of chemicals was studied by using several haptens. Animals were sensitized by a single 24-hr occlusive patch (24 cp), intradermal injection (id) and a combination of id and 24 cp, at a concentration used for guinea pig conventional contact hypersensitivity assay methods. Control animals were treated with vehicle(s) only. Suspensions of the lymph node cells (LNC) were individually prepared and cultured with [3H]methyl thymidine ([1H]TdR). [1H]TdR incorporation was measured and a stimulation index (SI) was calculated as a ratio of the mean [3H]TdR incorporation in sensitized animals and the mean [3H]TdR incorporation in control animals. LNC sensitized by 24 cp with 2,4-dinitrochlorobenzene proliferated maximally and significantly at day 5, whereas this occurred at day 7 after id sensitization. Significant LNC proliferation and high SI values were obtained successively by a combination of 24 cp and id. Moreover, strongly sensitizing chemicals increased significant LNC proliferation (SI > 2.0); weakly to moderately sensitizing chemicals also induced significant LNC proliferation (SI = 1.3 1.7). On the other hand, a primary irritant, sodium dodecyl sulfate, failed to encourage LNC proliferation (SI ≈ 1.0).

A new method for delayed contact hypersensitivity assay of chemical compounds in guinea pigs, a short-period method (14 days) with a high detection sensitivity, has been developed. The new method was as follows; a combination of a Freund's complete adjuvant (FCA, undiluted) intradermal injection and a 24-h occlusive patch on a guinea pig was performed 2x at an interval of 4 days and challenged by non-occlusive topical application 11 days after the first sensitization (with benzyl alcohol during test development). Acanthosis and spongiosis in the epidermis and mononuclear cell infiltration into the dermis were observed histopathologically at the skin reaction site. This newly developed method (adjuvant and 24-h occlusive patch 2x test: AP2 test) could equally and/or better detect the allergenicities of 6 other chemical compounds (bromostyrol, citronellal, benzyl salicylate, p-aminobenzoic acid ethyl ester, p-phenylenediamine and formaldehyde) as compared with the cumulative contact enhancement test (CCET) and the guinea pig maximization test (GPMT).

Simple and efficient ex vivo/in vitro screening systems for contact allergens are developed for alternative to conventional animal tests. We have previously proposed an ex vivo/in vitro proliferation assay as a first stage screening method with advantages over existing alternatives, using lymph node cells (LNC) from sensitized guinea pigs of the Hartley strain. In this study, we have first confirmed, by histochemical analysis using in vivo bromodeoxyuridine (BrdU) and pyronin staining, that the ex vivo/in vitro LNC proliferation reflects in vivo response of lymph nodes to contact allergens. Furthermore, to improve the LNC assay, we then have investigated several experimental conditions for their influences on the LNC assay, demonstrating that, (1) the subscapular and the cervical LNC responded highly to contact allergens, (2) among three cervical lymph nodes the superficial dorsal cervical lymph nodes were the most reactive, (3) several vehicles alone used for animal sensitization exhibited little influence on the LNC proliferation, (4) employment of stimulation index offset the inter-batch fluctuation of the LNC proliferation in the control animals as baseline proliferation. Under optimized experimental conditions as above, experimentally determined stimulation indexes of several contact allergens correlated well with their sensitizing potential estimated by conventional animal tests. Therefore, the ex vivo/in vitro LNC proliferation assay should be a simple and efficient alternative to conventional guinea pig testings including the guinea pig maximization test (GPMT).

The enhancement effect of cyclophosphamide on the delayed contact hypersensitivity reaction of chemical compounds was studied in Hartley albino guinea pigs. A series of assay procedures, combining the AP2 test (adjuvant and 24-h occlusive patch 2x test, as previously reported) with intraperitoneal cyclophosphamide administration, were examined. The newly developed method was as follows; cyclophosphamide 200 mg/kg intraperitoneal administration 3 days before the 1st sensitization of the AP2 test (cyclophosphamide, adjuvant and 24-h occlusive patch 2x test: CAP2 test). Comparing the CAP2 test with the AP2 test, the cumulative contact enhancement test (CCET) and the guinea pig maximization test (GPMT), the CAP2 test equally and/or better enabled the detection of allergenicities not only of strong allergens such as bromostyrol, citronellal, p-phenylenediamine and formaldehyde, but also of weak allergens such as benzyl salicylate and p-aminobenzoic acid ethyl ester. Acanthosis and spongiosis in the epidermis and mononuclear cell infiltration into the dermis at the skin reaction site were histopathologically observed. Cyclophosphamide effectively enhanced the delayed contact hypersensitivity reaction of weak allergens.