Development of a Modified Local Lymph Node Assay using ATP Measurement as an Endpoint

Several proposed modified local lymph node assays (LLNAs) use a non-RI method, but such methods tend to yield lower stimulation indices (SIs) than those obtained with the standard LLNA when used to assess moderate sensitizers. To address this issue, we propose a modified LLNA in which mice are exposed to the test material four times, instead of three times, and adenosine triphosphate (ATP) content is used as a measure of proliferation of lymph node cells (LNCs). The chemicals tested were 2.4dinitrochlorobenzene (DNCB), eugenol, and -hexyl cinnamic aldehyde (HCA), which are classified as strong-to-moderate sensitizers in the LLNA. Methyl salicylate (MS) was used as an example of a non-sensitizer. Using an SI of 3 as a cut-off, as recommended in the standard LLNA, DNCB, eugenol and HCA were classified as positive, while MS was classified as negative using the ATP-based non-RI method. Furthermore, there were no marked differences in SI levels for 10% eugenol and 10% HCA (a moderate sensitizer) between the non-RI method and the standard LLNA. Thus, it is reasonable to conclude that the non-RI method proposed in the present study could be a valid alternative to the standard LLNA.

[1]  B H Margolin,et al.  ICCVAM evaluation of the murine local lymph node assay. Data analyses completed by the National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods. , 2001, Regulatory toxicology and pharmacology : RTP.

[2]  I. Kimber,et al.  The murine local lymph node assay for identification of contact allergens: a preliminary evaluation of in situ measurement of lymphocyte proliferation , 1989, Contact dermatitis.

[3]  R J Fielder,et al.  Local lymph node assay - validation, conduct and use in practice. , 2002, Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association.

[4]  W Slob,et al.  A quantitative method for assessing the sensitizing potency of low molecular weight chemicals using a local lymph node assay: employment of a regression method that includes determination of the uncertainty margins. , 2000, Toxicology.

[5]  J Hilton,et al.  An international evaluation of the murine local lymph node assay and comparison of modified procedures. , 1995, Toxicology.

[6]  N. Tsutsui,et al.  Local lymph node assay with non-radioisotope alternative endpoints. , 2002, The Journal of toxicological sciences.

[7]  A. Kligman,et al.  The Identification of Contact Allergens by Animal Assay. the Guinea Pig Maximization Test , 1969 .

[8]  I. Kimber,et al.  Cytokine endpoints for the local lymph node assay: consideration of interferon‐γ and interleukin 12 , 1999, Journal of applied toxicology : JAT.

[9]  W S Stokes,et al.  ICCVAM evaluation of the murine local lymph node assay. The ICCVAM review process. , 2001, Regulatory toxicology and pharmacology : RTP.

[10]  I. Kimber,et al.  Development of a murine local lymph node assay for the determination of sensitizing potential , 1986 .

[11]  S Kato,et al.  A modification of the local lymph node assay for contact allergenicity screening: measurement of interleukin-2 as an alternative to radioisotope-dependent proliferation assay. , 1995, Toxicology.

[12]  A M Kligman,et al.  The identification of contact allergens by animal assay. The guinea pig maximization test. , 1970, The Journal of investigative dermatology.

[13]  M Hatao,et al.  Development of a non-radioactive endpoint in a modified local lymph node assay. , 1999, Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association.

[14]  Ian Kimber,et al.  Assessment of statistic analysis in non-radioisotopic local lymph node assay (non-RI-LLNA) with alpha-hexylcinnamic aldehyde as an example. , 2003, Toxicology.

[15]  I Kimber,et al.  The local lymph node assay: a viable alternative to currently accepted skin sensitization tests. , 1996, Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association.

[16]  I Kimber,et al.  Local lymph node assay: validation assessment for regulatory purposes. , 2000, American journal of contact dermatitis : official journal of the American Contact Dermatitis Society.

[17]  I Kimber,et al.  Development of non-radio isotopic endpoint of murine local lymph node assay based on 5-bromo-2'-deoxyuridine (BrdU) incorporation. , 2001, Toxicology letters.

[18]  R. Kozłowski,et al.  The use of ATP bioluminescence as a measure of cell proliferation and cytotoxicity. , 1993, Journal of immunological methods.

[19]  W S Stokes,et al.  ICCVAM evaluation of the murine local lymph node assay. Conclusions and recommendations of an independent scientific peer review panel. , 2001, Regulatory toxicology and pharmacology : RTP.

[20]  Miguel Cámara,et al.  Development of a bioluminescent ATP assay to quantify mammalian and bacterial cell number from a mixed population. , 2003, Biomaterials.

[21]  G F Gerberick,et al.  Local lymph node assay: differentiating allergic and irritant responses using flow cytometry. , 1999, Methods.

[22]  D A Basketter,et al.  Comparison of the local lymph node assay with the guinea-pig maximization test for the detection of a range of contact allergens. , 1992, Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association.