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Animal Models for Predicting Sensitization Potential
Judith C. Stadler
Haskell Laboratory, DuPont Company
Newark, DE
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Dermal Sensitization
Regulatory Acceptance– Guinea Pig
• Buehler• Maximization Test
– Mouse• Local Lymph Node Assay
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Dermal Sensitization (continued)
Other Tests– Guinea Pigs
• Open Epicutaneous• Mauer Optimization Test• Split Adjuvant Technique• Freund’s Complete Adjuvant Test • Draize Sensitization
– Mice• Mouse Ear Swelling Test
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Guinea Pig Tests
Based on common principles– Chemical induction– Rest phase– Dermal Challenge– Skin Reaction Assessment
• Erythema, edema,
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Guinea Pig Tests
Buehler Maximization
InductionMethod:
TopicalApplication
ID FreundsAdj. +Topical
InductionDays:
0, 6-8, 13-15 5-8
ChallengeMethod:
Topical, 6 hrs Topical
ChallengeDays:
27-28 20-22
Rechallenge: If equivocal
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Guinea Pig Tests Advantages
– Measures skin reactions similar to human response– Large data base for comparison of many chemicals– Correlates well to human response
Disadvantages– Requires large numbers of animals– Subjective evaluation– Assessment of colored materials difficult
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Mouse Local Lymph Node Assay
Based on responses during induction rather than elicitation phase of sensitization
Measures induction of the T-lymphocyte proliferative response in skin draining lymph nodes
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Local Lymph Node Assay
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
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Local Lymph Node Assay
Advantages– Smaller numbers of animals required– Quantitative assay– Several doses can be evaluated– Colored materials can be assayed
Disadvantages– Insoluble or systemically toxic materials may not
test well– Tests not predictive for metals
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Local Lymph Node AssayRegulatory Status U.S. EPA has approved its use as a
stand-alone assay 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
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Respiratory Sensitization
No tests have been validated for regulatory testing
Use of animal models primarily for research and mechanistic studies
Evaluation of models for predictive assays continues
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Animal Models of Respiratory Sensitization
Some use as predictive assays– Mouse– Guinea pig– Rat
Primarily used in asthma research– rabbit– dog– sheep– primate
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Occupational respiratory allergic disorders Wide spectrum of disease
– asthma– hypersensitivity pneumonitis– Pulmonary infiltrates with eosinophilia– Pulmonary institial fibrosis
Mechanisms– IgE, IgG4, cell-mediated immunity
Extent and duration of exposure related to likelihood of sensitization
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Guinea pig
Similar to humans– Histaminic bronchoconstriction at antigen
challenge– Exhibits airways hyperreactivity– Late-phase responses can be elicited– Lung eosinophilia
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Guinea pig use with chemical and protein workplace allergens Isocyanates
– Various methods of induction– Inhalation challenge with chemical or the
protein conjugates Detergent enzymes
– Inhalation induction model– Intratracheal test
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Guinea pig Advantages
– Ease of sensitization– Immunologically robust– Multifactorial
Disadvantages– IgG-dependent mechanism– Hyper eosinophilia– Dissociation between airway hyperreactivity and
eosinophilia– Biological variability (outbred strains)– Cost prohibitive
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Mouse 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
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Mouse Advantages
– Molecular and immunological assays available– Acute bronchoconstriction and airway
hyperreactivity can be elicited from antigen challenge
Disadvantages– Large doses required for antigen challenge– Difficult to measure eosinophil degranulation,
rarely in airways
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Brown Norway Rat
Similarities to human sensitization– Serotonergic bronchoconstriction– IgE mediated– Early and late phase reactions– Airway hyperreactivity– Tissue + BAL accumulation of neutrophils,
eosinophils, lymphocytes
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Brown Norway Rat
Advantages– Good inflammatory responses
Disadvantages– Induction of airway hyperreactivity difficult
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Regulatory Status
No one model has been validated for prediction of respiratory sensitization
New concerns may place higher priority on this area and encourage methods validation– Children’s Health issues– Food allergy
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Future direction
Emphasis on structure-activity relationships
Assay development – molecular biology– other in vitro screening
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References/Suggested Reading OECD Guidelines for the Testing of Chemicals 406 Skin Sensitisation (17-JUL-
92)
OPPTS Health Effects Test Guidelines. OPPTS 870.2600 Skin Sensitization (5-AUG-98)
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.
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.
Padrid, Philip. Animal models of asthma. in Lung Biol. Health Dis. (1996), 96(Genetics of Asthma), 211-233.
Selig, William M.; Chapman, Richard W. Asthma. in In Vivo Models of Inflammation (1999), 111-135.