Introduction: Allergic contact dermatitis (ACD) is a skin disease characterized by eczema and itching.A considerable proportion of chemicals induce ACD in humans.More than 10,000 substances should be tested for skin sensitization potential under the Registration,Evaluation,Authorization and Restriction of Chemical substances (RFACH) regulation.The Local Lymph Node Assay (LLN1A) has been designated as the first-choice in vivo assay for sensitization testing by REACH.The LLNA:BrdU-EHSA is a validated non-radioactive modification to the LLNA.For both the LLNA and the LLNA:BrdU-ELISA.CBA/JN mouse is the preferred mouse strain recommended in the regulatory guidelines.However,the availability of CBA/JN mouse in China is only limited to a few animal suppliers,which makes the mouse difficult to obtain.BALB/c mouse,which is widely commercially available,is considered for alternative use but it can only be used in the assay after it has been evaluated by formal validation study.Thus,a validation study was conducted in our laboratory to determine if BALB/c mouse could also be used in the LLNA:BrdU-ELISA.Methods: Forty-three test substances including 32 LLNA sensitizers and 11 LLNA non-sensitizers,their vehicles and each concentration used were the same as that used in the formal validation study for the LLNA:BrdUELISA using CBA/JN mouse.Female BALB/c mice of 8-10 weeks old were randomly allocated to groups (four mice per group).The test substance (25 μl) or the vehicle alone was applied to the dorsum of both ears daily for 3 consecutive days.A single intraperitoneal injection of 0.5 ml of BrdU (10 mg/ml) solution was given on day 5.On day 6,a pair of auricular lymph nodes from each mouse was excised,weighed and stored at-20 ℃C until BrdU-ELISA was conducted.Results: This validation study for the LLNA:BrdU-EUSA using BALB/c mouse correctly identified 30 of 31 sensitizers and 8 of 11 non-sensitizers.The accuracy,sensitivity,specificity,false positive rate,false negative rate,positive predictivity values and negative predictivity values in this study,which could indicate the performance of the LLNA:BrdU-EUSA using BALB/c mouse,were not different statistically from that of the validation study for the LLNA:BrdU-ELISA using CBA/JN mouse.Discussion: This validation study indicates that BALB/c mouse could be used alternatively in the LLNA:BrdU-ELISA for the prediction of the skin sensitizing potential of chemicals.