The Local Lymph Node Assay (LLNA)

Costanza Rovida1, Cindy Ryan2, Serena Cinelli3, David Basketter4, Rebecca Dearman5, Ian Kimber5

1 CAAT Europe, University of Konstanz, Konstanz, Germany, 2 Procter & Gamble Company, Cincinnati, Ohio, 3 Research Toxicology Centre, Pomezia, Rome, Italy, 4 DabMed Consultancy Ltd., Bedfordshire, United Kingdom, 5 University of Manchester, Manchester, United Kingdom
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 20.7
DOI:  10.1002/0471140856.tx2007s51
Online Posting Date:  February, 2012
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Abstract

The murine local lymph node assay (LLNA) is a widely accepted method for assessing the skin sensitization potential of chemicals. Compared with other in vivo methods in guinea pig, the LLNA offers important advantages with respect to animal welfare, including a requirement for reduced animal numbers as well as reduced pain and trauma. In addition to hazard identification, the LLNA is used for determining the relative skin sensitizing potency of contact allergens as a pivotal contribution to the risk assessment process. The LLNA is the only in vivo method that has been subjected to a formal validation process. The original LLNA protocol is based on measurement of the proliferative activity of draining lymph node cells (LNC), as determined by incorporation of radiolabeled thymidine. Several variants to the original LLNA have been developed to eliminate the use of radioactive materials. One such alternative is considered here: the LLNA:BrdU‐ELISA method, which uses 5‐bromo‐2‐deoxyuridine (BrdU) in place of radiolabeled thymidine to measure LNC proliferation in draining nodes. Curr. Protoc. Toxicol. 51:20.7.1‐20.7.14. © 2012 by John Wiley & Sons, Inc.

Keywords: skin sensitization; in vivo method; LLNA; potency assessment

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Radioactive LLNA
  • Alternate Protocol 1: BrdU‐ELISA LLNA
  • Support Protocol 1: Prescreening for Highest Subtoxic Dose
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Radioactive LLNA

  Materials
  • Female CBA/J or CBA/Ca mice, nulliparous and non‐pregnant, ∼17‐26 g and 8‐12 weeks old at initiation of experiment
  • Test substance solutions (see recipe) and vehicle for negative control
  • Recommended positive control (optional): 25% (v/v) hexyl cinnamic aldehyde (α‐hexylcinnamaldehyde; HCA; CAS 101‐86‐0) in same vehicle as test substance
  • 80 µCi/ml [methyl‐3H]thymidine (3HTdR) in 1‐ml syringes (see recipe)
  • Carbon dioxide for asphyxiation
  • 70% (v/v) ethanol or 70% (v/v) isopropanol
  • Phosphate‐buffered saline (PBS) without Ca2+ or Mg2+, sterile and non‐sterile ( appendix 2A; also commercially available)
  • 5% (w/v) trichloroacetic acid (TCA; CAS 76‐03‐9) in distilled H 2O
  • Aqueous‐compatible scintillation cocktail (e.g., Ultima Gold by Packard)
  • Indelible marking pen
  • Heating pad or heat lamp (optional)
  • 25‐ to 27‐G, 3/8‐inch hypodermic needles
  • Dissecting scissors
  • Small forceps
  • 12 × 75−mm round‐bottom tubes
  • 60‐mm tissue culture plates
  • 2.5 × 2.5−cm nylon or stainless steel mesh filter (200‐mesh, 100‐µm pore size)
  • Plunger from 1‐ml syringe
  • 15‐ml conical centrifuge tubes
  • 25‐ml glass scintillation vials
  • Liquid β‐scintillation counter
NOTE: Vehicle for the positive control should be the same as for test solutions, unless the selected vehicle does not dissolve HCA. In that case, 4:1 (v/v) acetone/olive oil is recommended.

Alternate Protocol 1: BrdU‐ELISA LLNA

  • 10 mg/ml BrdU solution: 5‐bromodeoxyuridine (CAS 59‐14‐3; store at –20°C) in physiological saline (0.9% NaCl)
  • BrdU cell proliferation ELISA kit (e.g., Roche or equivalent)
  • 1‐ml syringes
  • 25‐ to 27‐G, 3/8‐inch hypodermic needles
  • 96‐microwell flat‐bottom plates
  • Centrifuge with microtiter plate holders
  • Multichannel pipettor
  • Microwell spectrophotometer (ELISA plate reader)
NOTE: As an alternative positive control, the mild sensitizer eugenol (CAS 97‐53‐0) may be used at an concentration of 25% (v/v) in 4:1 (v/v) acetone/olive oil.

Support Protocol 1: Prescreening for Highest Subtoxic Dose

  • Thickness gauge/micrometer (e.g., Oditest D‐1000 or equivalent)
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Figures

Videos

Literature Cited

   Basketter, D.A. and Kimber, I. 2011. Skin irritation, false positives and the local lymph node assay: A guideline issue. Regul. Toxicol. Pharmacol. 61:137‐140.
   Basketter, D.A., Selbie, E., Scholes, E.W., Lees, D., Kimber, I., and Botham, P.A. 1993. Results with OECD recommended positive control sensitizers in the maximisation, Buehler and local lymph node assays. Food Chem. Toxicol. 31:63‐67.
   Basketter, D.A., Gilmour, N.J., Briggs, D., Ullmann, L.G., Gerberick, G.F., Ryan, C.A., Dearman, R.J., and Kimber, I. 2003. Utility of historical vehicle‐control data in the interpretation of the local lymph node assay. Contact Dermatitis 49:37‐41.
   Basketter, D.A., Andersen, K.E., Lidén, C., van Loveren, H., Boman, A., Kimber, I., Alanko, K., and Berggren, E. 2005. Evaluation of the skin sensitizing potency of chemicals using existing methods and considerations of relevance for elicitation. Contact Dermatitis 52:39‐43.
   Dearman, R.J., Hilton, J., Evans, P., Harvey, P., Basketter, D.A., and Kimber, I. 1998. Temporal stability of local lymph node assay responses to hexyl cinnamic aldehyde. J. Appl. Toxicol. 18:281‐284.
   Dearman, R.J., Wright, Z.M., Basketter, D.A., Ryan, C.A., Gerberick, G.F., and Kimber, I. 2001. The suitability of hexyl cinnamic aldehyde as a calibrant for the murine local lymph node assay. Contact Dermatitis 44:357‐361.
   European Union, 2011. Commission Regulation (EU) No 286/2011 of 10 March 2011, amending, for the purposes of its adaptation to technical and scientific progress, Regulation (EC) No 1272/2008 of the European Parliament and of the Council on classification, labelling and packaging of substances and mixtures. Official Journal of the European Union, L83/1‐53.
   ICCVAM (Interagency Coordinating Committee for the Validation of Alternative Methods) and NICEATM (National Toxicology Program Center for the Evaluation of Alternative Toxicological Methods). 1999. The Murine Local Lymph Node Assay: A Test Method for Assessing the Allergic Contact Dermatitis Potential of Chemicals/Compounds. The Results of an Independent Peer Review Evaluation Coordinated by the ICCVAM and the NICEATM. NIH Publication No. 99‐4494, http://iccvam.niehs.nih.gov/docs/immunotox_docs/llna/llnarep.pdf, February 1999 (accessed September 12, 2011).
   ICCVAM and NICEATM. 2008. Independent Scientific Peer Review Panel Report: Validation Status of New Versions and Applications of the Murine Local Lymph Node Assay: A Test Method for Assessing the Allergic Contact Dermatitis Potential of Chemicals and Products. http://iccvam.niehs.nih.gov/docs/immunotox_docs/LLNAPRPRept2008.pdf, May 2008 (accessed September 12, 2011).
   ICCVAM and NICEATM. 2010. ICCVAM Test Method Evaluation Report on the Murine Local Lymph Node Assay: BrdU‐ELISA, A Nonradioactive Alternative Test Method to Assess the Allergic Contact Dermatitis Potential of Chemicals and Products. NIH Publication No. 10‐7552, http://iccvam.niehs.nih.gov/docs/immunotox_docs/LLNA‐ELISA/TMER.pdf, March 2010 (accessed September 12, 2011).
   Jowsey, I.R., Clapp, C.J., Safford, B., Gibbons, B.T., and Basketter, D.A. 2008. The impact of vehicle on the relative potency of skin‐sensitizing chemicals in the local lymph node assay. Cutan. Ocul. Toxicol. 27:67‐75.
   Loveless, S.E., Api, A.‐M., Crevel, R.W.R., Debruyne, E., Gamer, A., Jowsey, I.R., Kern, P., Kimber, I., Lea, L., Lloyd, P., Mehmoodj, Z., Steiling, W., Veenstra, G., Woolhiserm, M., and Hennes, C. 2010. Potency values from the local lymph node assay: Application to classification, labelling and risk assessment. Regul. Toxicol. Pharmacol. 56:54‐66.
   OECD (Organisation for Economic Co‐operation and Development). 1992. OECD TG 406. Skin Sensitization. OECD Guideline for Testing of Chemicals. Adopted July 17, 1992.
   OECD. 2010a. OECD TG 429. Skin Sensitization: Local Lymph Node Assay. OECD Guideline for Testing of Chemicals. Adopted July 22, 2010.
   OECD. 2010b. OECD TG 442a. Skin Sensitization: Local Lymph Node Assay: DA. OECD Guideline for Testing of Chemicals. Adopted July 22, 2010.
   OECD. 2010c. OECD TG 442b. Skin Sensitization: Local Lymph Node Assay: BrdU‐ELISA. OECD Guideline for Testing of Chemicals. Adopted July 22, 2010.
   Rovida, C. 2011. Local lymph node assay: How testing laboratories apply OECD TG 429 for REACH purposes. ALTEX 28:117‐129.
   Ryan, C.A., Cruse, L.W., Skinner, R.A., Dearman, R.J., Kimber, I., and Gerberick, G.F. 2002. Examination of a vehicle for use with water soluble materials in the murine local lymph node assay. Food Chem. Toxicol. 40:1719‐1725.
Internet Resources
  http://iccvam.niehs.nih.gov/methods/immunotox/immunotox.htm
  The immunotoxicity page of the NICEATM‐ICCVAM website contains all documents related to the validation process of the LLNA. The site is constantly updated as soon as any new method variations are available.
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