1. Animal Models for Predicting Sensitization Potential Judith C. Stadler Haskell Laboratory, DuPont Company Newark, DE

2. Dermal Sensitization n Regulatory Acceptance –Guinea Pig Buehler Maximization Test –Mouse Local Lymph Node Assay

3. Dermal Sensitization (continued) n Other Tests –Guinea Pigs Open Epicutaneous Mauer Optimization Test Split Adjuvant Technique Freund’s Complete Adjuvant Test Draize Sensitization –Mice Mouse Ear Swelling Test

4. Guinea Pig Tests n Based on common principles –Chemical induction –Rest phase –Dermal Challenge –Skin Reaction Assessment Erythema, edema,

5. Guinea Pig Tests

6. n Advantages –Measures skin reactions similar to human response –Large data base for comparison of many chemicals –Correlates well to human response n Disadvantages –Requires large numbers of animals –Subjective evaluation –Assessment of colored materials difficult

7. Mouse Local Lymph Node Assay n Based on responses during induction rather than elicitation phase of sensitization n Measures induction of the T- lymphocyte proliferative response in skin draining lymph nodes

9. Local Lymph Node Assay n Sequence of Events –Topical application, generally to the ear –Epidermal cells release cytokines –Mobilize Langerhans’ cells –Antigen transported from skin to draining lymph nodes –Responsive T-cells activated and divide –Radioactivity incorporated during clonal expansion

10. Local Lymph Node Assay n Advantages –Smaller numbers of animals required –Quantitative assay –Several doses can be evaluated –Colored materials can be assayed n Disadvantages –Insoluble or systemically toxic materials may not test well –Tests not predictive for metals

11. Local Lymph Node Assay Regulatory Status n U.S. EPA has approved its use as a stand-alone assay n Europe has approved for registrations if the assay is positive. If the assay is negative, a follow-up guinea pig assay is required –This requirement expected to change by the end of 2000

12. Respiratory Sensitization n No tests have been validated for regulatory testing n Use of animal models primarily for research and mechanistic studies n Evaluation of models for predictive assays continues

13. Animal Models of Respiratory Sensitization n Some use as predictive assays –Mouse –Guinea pig –Rat n Primarily used in asthma research –rabbit –dog –sheep –primate

14. Occupational respiratory allergic disorders n Wide spectrum of disease –asthma –hypersensitivity pneumonitis –Pulmonary infiltrates with eosinophilia –Pulmonary institial fibrosis n Mechanisms –IgE, IgG4, cell-mediated immunity n Extent and duration of exposure related to likelihood of sensitization

15. Guinea pig n Similar to humans –Histaminic bronchoconstriction at antigen challenge –Exhibits airways hyperreactivity –Late-phase responses can be elicited –Lung eosinophilia

16. Guinea pig use with chemical and protein workplace allergens n Isocyanates –Various methods of induction –Inhalation challenge with chemical or the protein conjugates n Detergent enzymes –Inhalation induction model –Intratracheal test

17. Guinea pig n Advantages –Ease of sensitization –Immunologically robust –Multifactorial n Disadvantages –IgG-dependent mechanism –Hyper eosinophilia –Dissociation between airway hyperreactivity and eosinophilia –Biological variability (outbred strains) –Cost prohibitive

18. Mouse n Similarities of the model to human –Influx of eosinophils and T-cells –histological features following acute exposures similar to human Epithelial denudation Interstitial airway edema Thick basement membrane Goblet cell hyperplasia –Mucus hypersecretions –Repeated exposure results in histological features of chronic asthma

19. Mouse n Advantages –Molecular and immunological assays available –Acute bronchoconstriction and airway hyperreactivity can be elicited from antigen challenge n Disadvantages –Large doses required for antigen challenge –Difficult to measure eosinophil degranulation, rarely in airways

20. Brown Norway Rat n Similarities to human sensitization –Serotonergic bronchoconstriction –IgE mediated –Early and late phase reactions –Airway hyperreactivity –Tissue + BAL accumulation of neutrophils, eosinophils, lymphocytes

21. Brown Norway Rat n Advantages –Good inflammatory responses n Disadvantages –Induction of airway hyperreactivity difficult

22. Regulatory Status n No one model has been validated for prediction of respiratory sensitization n New concerns may place higher priority on this area and encourage methods validation –Children’s Health issues –Food allergy

23. Future direction n Emphasis on structure-activity relationships n Assay development –molecular biology –other in vitro screening

24. References/Suggested Reading n OECD Guidelines for the Testing of Chemicals 406 Skin Sensitisation (17-JUL- 92) n OPPTS Health Effects Test Guidelines. OPPTS 870.2600 Skin Sensitization (5- AUG-98) n Dearman, R.J. D.A. Basketter, and I. Kimber (1999) Local lymph node assay: use in hazard and risk assessment REVIEW. J. Appl Toxicol. 19, 299-306. n Kimber, I, I.L. Bernstein, M.H. Karol, M.K. Robinson, K. Sarlo, and M.K. Selgrade (1996) Identification of respiratory allergens. Fund. and Appl Tox 33, 1-10. n Padrid, Philip. Animal models of asthma. in Lung Biol. Health Dis. (1996), 96(Genetics of Asthma), 211-233. n Selig, William M.; Chapman, Richard W. Asthma. in In Vivo Models of Inflammation (1999), 111-135.