Local Lymph Node Assays

Rebecca J. Dearman1, Ian Kimber1

1 Syngenta Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 18.2
DOI:  10.1002/0471140856.tx1802s20
Online Posting Date:  June, 2004
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Abstract

The murine local lymph node assay (LLNA) allows identification of chemicals that have the potential to cause skin sensitization and allergic contact dermatitis. In this test, contact allergens are identified as a function of events occurring during the induction phase of skin sensitization; specifically, stimulation of proliferative responses in draining lymph nodes measured as a function of radiolabeled thymidine incorporation in situ. Two methods are presented. In one, lymph nodes are pooled on an experimental group basis, while in the other, lymph nodes of individual animal are pooled, which enables statistical analyses. These methods have been developed for contact sensitization hazard assessment and provide a robust and reliable method as an alternative to guinea pig tests which measure skin sensitizing potential as a function of challenge‐induced skin reactions in previously sensitized animals. It is also possible to use LLNA data for measurement of relative potency in terms of risk assessment.

Keywords: contact allergy; local lymph node assay; hazard identification; potency

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol: Standard Local Lymph Node Assay
  • Alternate Protocol: Local Lymph Node Assay: Assessment of Proliferation for Individual Animals
  • Support Protocol: Local Lymph Node Assay: Potency Assessment
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol: Standard Local Lymph Node Assay

 Materials
  • Young adult (6‐ to 16‐week‐old) female CBA mice: maintain in semibarriered conditions
  • Chemicals to be tested
  • Vehicle—e.g., 4:1 (v/v) acetone olive oil (AOO), methylethyl ketone, dimethylformamide, or dimethyl sulfoxide (DMSO)
  • 2 Ci/mmol [3H]thymidine: pass through a 0.45‐µm filter to sterilize
  • PBS, pH 7.2 to 7.4 (appendix 2A)
  • 5% (w/v) trichloroacetic acid (TCA)
  • Scintillation fluid (e.g., Hi‐Safe Optiphase)
  • Temperature‐controlled hot box (Harvard Apparatus)
  • Mouse restraining tube with outlet for tail
  • 1‐ml graduated syringes with 25‐G, 1‐in. needles, sterile
  • 200‐G stainless steel mesh: cut into ∼3‐cm squares, wash in 70% ethanol, autoclave, and turn edges up to form a box
  • 600‐mm plastic petri dish
  • 5‐ml syringe plungers, sterile
  • 10‐ml plastic centrifuge tubes
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Figures

  •  FigureFigure 18.2.1 Typical results derived using standard LLNA (see Basic Protocol). CBA strain mice (n = 4) received 25 µl of various concentrations of (A and C) the potent contact allergen DNCB or (B and D) the nonsensitizing skin irritant pABA, or vehicle (AOO) alone on the dorsum of both ears daily for 3 consecutive days. Five days after the initiation of exposure, all mice received an intravenous injection of radiolabeled thymidine. Five hours later, draining auricular lymph nodes were excised, pooled on a treatment group basis, and processed for β‐scintillation counting. Results are expressed as group dpm/node in (A) and (B) or as a stimulation index (SI) relative to vehicle treated control groups in (C) and (D). An SI of 3 (current threshold value for a positive in the LLNA) is illustrated as a broken line in (C) and (D).
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  •  FigureFigure 18.2.2 Typical results derived using the individual animal–based LLNA (see Alternate Protocol). CBA strain mice (n = 5) received 25 µl of various concentrations of (A and C) the potent contact allergen DNCB, (B and D) the nonsensitizing skin irritant pABA, or vehicle (AOO) alone on the dorsum of both ears daily for 3 consecutive days. Five days after the initiation of exposure, all mice received an intravenous injection of radiolabeled thymidine. Five hours later, draining auricular lymph nodes were excised, pooled on an experimental animal basis, and processed for β‐scintillation counting. Results are expressed as mean ± SE dpm/node per group in (A) and (B) or as a stimulation index (SI) relative to vehicle treated control groups in (C) and (D). An SI of 3 (current threshold value for a positive in the LLNA) is illustrated as a broken line.
    View Image
  •  FigureFigure 18.2.3 Typical results derived using LLNA for the assessment of relative skin sensitizing potency (see Support Protocol). CBA strain mice received 25 µl of various concentrations of (A) the potent contact allergen DNCB, (B) the more moderate allergen HCA, or vehicle (AOO) alone on the dorsum of both ears daily for 3 consecutive days. Five days after the initiation of exposure, all mice received an intravenous injection of radiolabeled thymidine. Five hours later, draining auricular lymph nodes were excised, pooled on a treatment group basis and processed for β‐scintillation counting. Results are expressed as a stimulation index (SI) relative to vehicle treated control groups. The EC3 value (illustrated by an arrow) is calculated by linear interpolation.
    View Image

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Literature Cited

Literature Cited
    Balls, M. and Hellsten, E. 2000. Statement on the validity of the local lymph node assay for skin sensitisation testing. Altern. Lab. Anim. 28:366‐367.
    Basketter, D.A., Lea, L.J., Cooper, K., Stocks, J., Dickens, A., Pate, I., Dearman, R.J., and Kimber, I. 1999. Threshold for classification as a skin sensitizer in the local lymph node assay: A statistical evaluation. Food Chem. Toxicol. 37:1167‐1174.
    Basketter, D.A., Blaikie, L., Dearman, R.J., Kimber, I., Ryan, C.A., Gerberick, G.F., Harvey, P., Evans, P., White, I.R., and Rycroft, R.J.G. 2000. Use of the local lymph node assay for estimation of relative contact allergenic potency. Contact Dermatitis 42:344‐348.
    Basketter, D.A., Evans, P., Fielder, R.J., Gerberick, G.F., Dearman, R.J., and Kimber, I. 2002. Local lymph node assay – validation and use in practice. Food Chem. Toxicol. 40:593‐598.
    Basketter, D.A., Glimour, N.J., Briggs, D., Ludwig, U., Gerberick, G.F., Ryan, C.A., Dearman, R.J., and Kimber, I. 2003. Utility of historical control data in the interpretation of the local lymph node assay. Contact Dermatitis 49:37‐41.
    Buehler, E.V. 1985. A rationale for the selection of occlusion to induce and elicit delayed contact hypersensitivity in the guinea pig. A prospective test. In Contact Allergy Predictive Tests in Guinea Pigs: Current Problems in Dermatology. (K.E. Andersen and H.I. Maibach, eds) pp. 39‐58. S. Karger AG, Basel, Switzerland.
    Cronin, E. 1980. Contact Dermatitis. Churchill Livingstone, London.
    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 local lymph node assay. Contact Dermatitis 44:357‐361.
    Descotes, J. 1988. Identification of contact allergens: The mouse ear sensitization assay. J. Toxicol., Cutaneous and Ocular Toxicol. 7:263‐ 272.
    De Sousa, M.A.B, and Parrott, D.M.V. 1969. Induction and recall in contact sensitivity, changes in skin and draining lymph nodes of intact and thymectomized mice. J. Exp. Med. 130:671‐686.
    Gad, S.C., Dunn, B.J., Dobbs, D.W., Reilly, C., and Walsh, R.D. 1986. Development and validation of an alternative dermal sensitization test: The mouse ear swelling test (MEST). Toxicol. Appl. Pharmacol. 8:93‐114.
    Gerberick, G.F., Ryan, C.A., Kimber, I., Dearman, R.J., Lea, L.J., and Basketter, D.A. 2000. Local lymph node assay: Validation assessment for regulatory purposes. Am. J. Contact Dermatitis 11:3‐18.
    Gerberick, G.F., Robinson, M.K., Ryan, C.A., Dearman, R.J., Kimber, I., Basketter, D.A., Wright, Z., and Marks, J.G. 2001. Contact allergenic potency: Correlation of human and local lymph node assay data. Am. J. Contact Dermatitis 12:156‐161.
    Interagency Coordinating Committee on the Validation of Alternative Methods 1999. The Murine Local Lymph Node Assay: A Test Method for Assessing the Allergic Dermatitis Potential of Chemicals/Compounds. NIH No. 99‐4494.
    Kimber, I., and Dearman, R.J. 1991. Investigation of lymph node cell proliferation as a possible immunological correlate of contact sensitizing potential. Food Chem. Toxicol. 29:125‐129.
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    Kimber, I., Mitchell, J.A., and Griffin, A.C. 1986. Development of a murine local lymph node assay for the determination of sensitizing potential. Food Chem. Toxicol. 24:585‐586.
    Kimber, I., Dearman, R.J., Basketter, D.A., Ryan, C.A., and Gerberick, G.F. 2002. The local lymph node assay: Past, present and future. Contact Dermatitis 47:315‐328.
    Kimber, I., Basketter, D.A., Butler, M., Gamer, A., Garrigue, J.‐L., Gerberick, G.F., Newsome, C., Steiling, W., and Vohr, H.‐W. 2003. Classification of contact allergens according to potency: Proposals. Food Chem. Toxicol. 41:1799‐ 1809.
    Loveless, S.E., Ladics, G.L., Gerberick, G.F., Ryan, C.A., Basketter, D.A., Scholes, E.W., House, R.V., Hilton, J., Dearman, R.J., and Kimber, I., 1996. Further evaluation of the local lymph node assay in the final phase of an international collaborative trial. Toxicology 108:141‐152.
    Magnusson, B. and Kligman, A.M. 1970. Allergic Contact Dermatitis in the Guinea Pig. Charles C. Thomas, Springfield, Ill.
    Organization for Economic Cooperation and Development 2002. Guidelines for testing of Chemicals. Guideline No. 429. Skin Sensitization: The Local Lymph Node Assay. OECD, Paris.
    Parrott, D.M.V. and De Sousa, M.A.B. 1966. Changes in the thymus dependent areas of lymph nodes after immunological stimulation. Nature 212:1316‐1317.
    Thorne, P.S., Hawk, C., Kaliszewski, S.D., and Guiney, P.D. 1991. The noninvasive mouse ear swelling assay. I. Refinements for detecting weak contact sensitizers. Fundam. Appl. Toxicol. 17:790‐806.
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