Animal Models for Predicting Sensitization Potential

Judith C. Stadler

Haskell Laboratory, DuPont Company

Newark, DE

Dermal Sensitization

Regulatory Acceptance– Guinea Pig

• Buehler• Maximization Test

– Mouse• Local Lymph Node Assay

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

Guinea Pig Tests

Based on common principles– Chemical induction– Rest phase– Dermal Challenge– Skin Reaction Assessment

• Erythema, edema,

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

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

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

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

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

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

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

Animal Models of Respiratory Sensitization

Some use as predictive assays– Mouse– Guinea pig– Rat

Primarily used in asthma research– rabbit– dog– sheep– primate

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

Guinea pig

Similar to humans– Histaminic bronchoconstriction at antigen

challenge– Exhibits airways hyperreactivity– Late-phase responses can be elicited– Lung eosinophilia

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

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

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

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

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

Brown Norway Rat

Advantages– Good inflammatory responses

Disadvantages– Induction of airway hyperreactivity difficult

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

Future direction

Emphasis on structure-activity relationships

Assay development – molecular biology– other in vitro screening

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.