Immunotoxicology Testing Past and Future

Chapter 1Immunotoxicology Testing: Past and FutureMichael I. Luster and G. Frank GerberickAbstractA brief historical perspective of immunotoxicology is presented describing the early development of predictive screening tests to identify xenobiotics that may cause immunosuppression or skin sensitization. This includes a discussion of the evolution of the discipline to support a better understanding of basic s cience and improvement of human risk assessment. The last section describes the need for additional validated screening tests and recent efforts to address this gap in the other areas of immunotoxicology including food and respiratory allergy, autoimmunity and immunostimulation.Key words: Testing, Guidelines, Immunosuppression, Hypersensitivity, Risk assessment1. IntroductionThe identification and regulation of xenobiotic agents that inadver-tently alter the immune system and affect human health have beenof concern to the chemical/agricultural, pharmaceutical andc onsumer product industries, as well as to the federal regulatoryagencies for over 40 years. Initial interest originated in the area ofsensitization from the observations made by Landsteiner and Jacobs(1) that low molecular weight chemicals or drugs can be antigenicand capable of producing organ-specific (i.e., skin, lung or gastroin-testinal tract) allergic responses. Subsequently, other studies reportedthat certain xenobiotics, such as halogenated a romatic hydrocar-bons, could suppress, or in rare instances s timulate, the immunesystem resulting in an increased risk of infectious or neoplasticd iseases, or in the l atter case, exacerbate autoimmune disease.Of particular concern have been the x enobiotic effects in the neo-nate as increasing e vidence suggests that the developing immunesystem is p articularly sensitive to damage. Other materials, particu-larly certain p harmaceuticals, cause autoimmune-like syndromesR.R. Dietert (ed.), Immunotoxicity Testing: Methods and Protocols, Methods in Molecular Biology,vol. 598DOI 10.1007/978-1-60761-401-2_1, © Humana Press, a part of Springer Science + Business Media, LLC 201034Luster and Gerberickin which the disease dissipates following cessation of exposure while other chemicals appear to exacerbate existing autoimmune disease.The development and adoption of appropriate experimental methods to assess the influence of xenobiotics to cause these various toxicities were for many years the major focus of immunotoxicology, and for some effects such as autoimmunity, respiratory allergy and the so-called systemic allergies still remain an issue. The fol-lowing provides a brief historical review on the development of immunotoxicity testing and a perspective of what testing strate-gies are needed in the future.As it is relatively difficult to determine the contribution of chronic low-level immunosuppression or the cumulative effects of modest changes in immune function to the background incidence of dis-ease in the human population, efforts have been made to examine the relationships between laboratory measures of immune response and disease resistance in experimental animal models. Although the experimental methods initially adopted by immu-notoxicologists to assess immune function were those common to immunology laboratories, the tests that were commonly per-formed and the experimental design by which they were con-ducted were performed ad hoc . Even the experimental species that have been selected varied with the earliest studies using rabbits and guinea pigs and later studies conducted using rats and mice. While rodents became the test species of choice, debate occurred on species selection with those trained in toxicology usually pre-ferring the rat to allow comparison with other toxicology studies, and those trained in immunology preferring the mouse as the mouse immune system was well studied.The lack of standardized testing made it difficult to compare the chemical-specific effects and led Dean et al. (2), to suggest a “Tier” approach with the idea that each subsequent tier provided identification of a more defined effect on the immune system. Subsequently, the National Toxicology Program (NTP) organized a series of workshops composed of experts in immunotoxicology, basic immunology, toxicology, risk assessment, epidemiology and clinical medicine to help identify the most appropriate tests for immunotoxicology testing (3). Two major points were agreed upon from these workshops: First, since the immune system is not fully operational until it is challenged, the most appropriate tests would be those that incorporate an antigen challenge. Second, since it may be construed that an inadequate response to antigenic challenge does not represent an “adverse effect,” tests should also be added that could be readily identified with disease.2. Immunosup-pression5Immunotoxicology Testing: Past and FutureThe former recommendation highlighted several common assays including measurement of an antibody response following antigen challenge as a measure of humoral immunity and quantifi-cation of delayed hypersensitive response (DHR) or cytotoxic T lymphocyte response (CTL) as a measure for cell-mediated immu-nity. These assays were based upon the measurement of a primary immune response rather than secondary since it is generally thought that memory responses are less sensitive to inhibition than primary responses. To address the need to identify a clear adverse effect, a set of tests, usually referred to as “host resistance assays,” was sug-gested. These tests would also be used to validate the usefulness of other methods and extrapolate the potential for environmental agents to alter host susceptibility in the human population.In these assays, groups of experimental animals are challenged with either an infectious agent or transplantable tumor at a chal-lenge level sufficient to produce disease in control animals and increased incidence is examined in the treated groups. As the end-points in these tests have evolved from relatively non-specific (e.g., animal morbidity and mortality) to quantitative, such as tumor numbers, viral titers or bacterial cell counts, the sensitivity of these models has significantly increased. However, they are still somewhat limited by the need to use relatively large numbers of animals.Eventually, a three tier approach emerged in which Tier 1 included screening assays that would likely detect immunotoxic xenobiotics, Tier 2 allowed for defining the immune component(s) effected as well as establish effects on host resistance and Tier 3 provided, in very general terms, approaches that could be used to identify the mode of action.An interlaboratory validation effort involving four laboratories1 and sponsored by the NTP was conducted using Tier 1 and 2 tests (4). In addition to the demonstration of interlaboratory reproduc-ibility, this effort helped identifying the relative sensitivity of the various immune tests and the degree to which they agreed with the commonly employed host susceptibility tests. This effort was followed several years later in which the concordance between various histological, hematological and immune function tests to identify immunotoxicity and host susceptibility changes were determined in a large dataset (5, 6). These latter studies were important, not only as a validation exercise for tier testing, but for providing a basis for moving immunotoxicology assessment forward. The analyses indicated that inclusion of a functional test, most notably the T-dependent antibody response (TDAR) to1The four participating laboratories were the National Institute of Environmental Health Sciences (Research Triangle Park, NC), Chemical Industry Institute of Toxicology (Research Triangle Park, NC), Virginia Commonwealth University (Richmond, VA) and IIT Research Institute (Chicago, IL).6Luster and Gerbericksheep red blood cells, along with a non-functional test, such asthymus weights, allowed achieving concordance, with respect toidentifying potential immunotoxic agents, of well over 90%,although a number of other immune test groupings providedexcellent levels of accuracy. These studies also provided evidenceof a linear relationship between many of the immune tests andhost resistance assays. In the Unites States, the preferred speciesfor testing was the mouse, but subsequent validation studies wereconducted in rats (7–9) and results from either rodent species arenow equally accepted.Tiered screening panels have been the basis for several riskassessment guidelines and most regulatory agencies in theUnited States, European Union and Japan have established orare developing requirements or guidelines (reviewed by(10)).However, the Office of Prevention, Pesticides and ToxicSubstances (OPPTS), US Environmental Protection Agency(EPA) was responsible for developing the first immunotoxicol-ogy test guideline (11) and over the years has taken the lead intheir development. It should be noted that the configurationsof these testing panels vary depending upon the agency/organization/program under which they are conducted. Themost notable difference is whether a functional immune test(i.e., incorporates antigen challenge) is included in Tier 1 orTier 2. Although, as indicated earlier, it is generally agreedthat functional testing provides the greatest sensitivity foridentifying immunosuppression, it has been argued that ac areful histological and hematological evaluation, particu-larly inclusion of extended histopathology endpoints, wouldi dentify a large proportion of potential immunotoxic agents(12–14). This is reflected in published and proposed immuno-toxicity testing guidelines by the Committee for ProprietaryMedicinal Products (CPMP), Organization of EconomicCooperation and Development (OECD) and InternationalConference on Harmonization of Technical Requirements forRegistration of Pharmaceuticals for Humans Use (ICH)(reviewed by(10)).While testing for potential immunotoxicty in experimentalanimals has gained increased acceptance, few systematic epide-miological immunotoxicological studies had been undertaken.This is due to a number of difficulties in working with humanpopulations (15):(a) Lack of validated immunological assays of sufficientsensitivity(b) Difficulty in accurately determining infectious diseaseincidence(c) Large cost and difficulty of sample acquisition at sites geo-graphically distant from the investigator7Immunotoxicology Testing: Past and Future The US National Academy of Sciences (NAS) in understanding these challenges, proposed a three tier testing scheme to be used for study of populations known or suspected to have been exposed to an immunotoxicant (16). As with experimental animals, it was proposed that tests be conducted from the first Tier and the results used to consider proceeding to the next Tier. The tests included in the first Tier are shown in Table 1.1. The International Programme on Chemical Safety of the World Health Organization (IPCS/WHO) issued a report on principles and methods for assessing direct immunotoxicity associated with exposure to chemicals (17). Although many assays overlapped, that report recommended a larger number of assays that can be used to evaluate possible immu-notoxicity than the NRC tier system (Table 1.1). A symposium on Epidemiology of Immunotoxicity remarked on the need for well-designed studies of immunotoxicity in humans and supported the application of the NRC three Tier approach (18). Over the years, a number of immunotoxicology population studies were conducted based on a selection of immune biomarkers from both the WHO and NRC Tier 1 recommendations (e.g., 19–21).For many decades, the guinea pig has been the animal of choice for predictive studies of skin sensitization potential. This arose largely as3. SkinSensitization TestingTable 1.1Tier 1 immunotoxicty testing recommendations for human studiesNRC recommendationsWHO recommendations8Luster and Gerbericka result of the use of the guinea pig in the pioneering investigationsinto mechanisms of skin sensitization to chemicals (1, 22).The first definition of a real predictive test came from the work ofDraize more than 60 years ago (23). Since that time, numerousprotocols have been described whose aims have been, in one wayor another, to make improvements to the sensitivity and predic-tivity of the guinea pig as a surrogate for man.In essence, all the test protocols follow similar principles.Typically, a combination of intradermal and/or epicutaneoustreatments is administered to 10–20 guinea pigs, with or withoutadjuvant, over a 2–3 week period in an attempt to induce skinsensitization, then a 1–2 week rest period to allow any immuneresponse to mature, followed finally by a topical challenge toassess the extent to which the skin sensitization might have beeninduced. A set of 5–10 sham treated controls is also challenged.Evaluation of the skin reactions is usually by subjective visualassessments 24–48 h after the challenge application, the mainreaction element being erythema. The protocol of Magnussonand Kligman (24) and that of Buehler (25, 26) are the two moststudied and accepted guinea pig methods used for regulatorypurposes worldwide (27).The Local Lymph Node Assay (LLNA) is a validated alterna-tive approach to the traditional guinea pig test methods for skinsensitization testing that provides important animal welfare ben-efits (28, 29). In this method, skin sensitizing potential is mea-sured as a function of lymph node cell proliferative responsesinduced in mice following repeated topical exposure to the testchemical (30, 31). Not least due to the improved animal welfarebenefits, the LLNA has become the preferred method for assessingskin sensitization hazard by various regulatory authorities (32, 33).The OECD test guideline 429 for the LLNA indicates that aminimum of 3 test concentrations and a vehicle control groupwith a minimum of 4 animals per group are needed (27).A chemical is classified as a skin sensitizer if, at one or more testconcentrations, it induces a three-fold or greater increase in draininglymph node cell proliferation compared with concurrent vehicle-treated controls (Stimulation Index [SI] ≥3). In the standardLLNA, lymph nodes are pooled and processed on an experimentalgroup basis using 4 mice per group. Alternatively, using 5 mice pergroup, lymph nodes are pooled on an individual animal basis pro-viding the opportunity to employ statistical analyses and appropri-ate power (34). The LLNA has been evaluated extensively in bothnational and international inter-laboratory collaborative trials andhas been the subject of comparisons with guinea pig predictive testmethods and human sensitization data. An important point is thatthe LLNA was subjected to rigorous independent scrutiny andvalidated by the International Coordinating Committee on theValidation of Alternative Methods (ICCVAM) (29). There soon9Immunotoxicology Testing: Past and Future followed a similar endorsement by the European Centre for the Validation of Alternative Methods (ECVAM) (28).Thus, the tests traditionally used for the identification of chemicals possessing the intrinsic ability to cause skin sensitiza-tion are the guinea pig maximization test, the Buehler occluded patch test and the LLNA. The capacity of these methods to iden-tify skin sensitization hazard has only been formally validated for the LLNA. However, both within this validation and via the pub-lication of other datasets, the guinea pig methods are also recog-nized to be of sufficient sensitivity and specificity.As described above, the most attention to development of validated methods for immuntoxicity testing thus far has been devoted to identify xenobiotics that have the potential to produce skin sensiti-zation or immunosuppression. Although not validated, opportuni-ties exist to improve the current testing schemes in terms of improved sensitivity, less reliance on experimental animals and cost reduction, such as cytokine and gene expression profiling (35–37). These procedures offer the additional opportunity to make direct comparisons with humans using samples from serum or isolated leuokocytes. The use of in vitro systems, such as dendritic cell acti-vation, peptide reactivity and T-cell activation are also being applied to hypersensitivty testing (38, 39).Unfortunately, these traditional testing paradigms are inadequate for many issues relevant to immunotoxicity testing that are of signi-ficant current importance. For example, food allergies, which are often life-threatening, are common and affect 6–8% of children under the age of 4 and 1–4% of adults (40). While considerable attention has been given to the types of food products that can produce an allergic response, few studies have addressed development of appro-priate test methods for identifying sensitizers in food. Rodent models employed for the evaluation of food allergy have utilized strains inherently skewed towards a Th2 allergic phenotype and high IgE production such as the Brown Norway rat or BALB/c and C3H/HeJ mouse (41). Rodent models tend to replicate the IgE response to food a llergens seen in humans but often fail to present similar clinical symptoms. Other animal models, including dogs and pigs, demonstrate many clinical characteristics of human food allergy including respiratory involvement, digestive problems and even ana-phylaxis (41). However, lack of standardization due to variable use of adjuvant, varying routes of exposure, not to mention most a ppropriate test model, make assessing chemical-induced modulation of responses to protein allergens difficult. Importantly, however, dialog on stan-dardized testing protocols for food allergens is continuing (42).4. Needs in Immunotoxicity Screening Testing10Luster and GerberickRespiratory sensitizers can be identified in inhalation studiesusing the guinea pig (43). However, the technical difficulty, par-ticularly as it relates to conducting inhalation, does not lend thisprocedure to a routine screening test. Most of the issues relatedto screening tests for food sensitizers, particularly as it relates tothe Th2 phenotype, also apply for respiratory sensitizers. A numberof studies have and still continue to address screening tests forrespiratory sensitizers. These include among others, monitoringIgE or IgG1 levels in various test species and employing bron-chial associated lymphoid tissues for immunophenotyping,cytokine profiling, gene expression and a modified LLNA (43).The observation that many drugs produce autoimmune-likesyndromes and environmental chemicals induce onset and modu-late autoimmune disease severity, has led the efforts to identify reli-able screening tests for xenobiotic-induced autoimmunity. Animalmodels of autoimmunity have been used to explore both molecularmechanisms and therapeutic interventions for a variety of autoim-mune diseases (44). However, while a number of syndromes thatare similar to those clinically observed in humans can be mimickedin animal models, the diversity of autoimmune diseases limits theutility of any single model as a screening tool. The popliteal lymphnode assay, which measures non-specific stimulation and prolifera-tion in the lymph nodes draining chemically exposed tissues, hasbeen used in conjunction with reporter antigens as a tool to screenfor immunostimulating compounds (45). However, this assay fallsshort of measuring the potential to produce disease.Finally, an ongoing need in immunotoxicology testing is todevelop screening tests to identify adverse health consequencesfrom xenobiotics that produce immunostimulation or modulateinflammatory responses. Although these may include some indus-trial chemicals, they primarily represent therapeutics designed totreat immune-mediated diseases, such as asthma, autoimmunity, orchronic inflammatory diseases. Some general examples includeToll-like receptor (TLR) agonists, cytokine agonists or antago-nists, modulators of adhesion molecules, angiogenic therapies,novel vaccine adjuvants and arachidonic acid modulators. Sincethe immune system represents a vast network of regulatory loops,altering the production or expression of one regulatory immunemediator to treat a disease would likely influence other mediators,the consequences of which may have adverse effects that out-weigh the benefits of its intended use.5. ConclusionIn this survey, we have provided the reader with a brief historicalperspective of immunotoxicology conveying that its foundationwas in the development of predictive screening tests to identify11Immunotoxicology Testing: Past and Futurexenobiotics that may cause immunosuppression or skin sensitization.The discipline, however, continues to evolve to include contri-butions to basic science as well as in improving human risk assess-ment. It is essential, however, that we understand the fact thatimmunotoxicology represents the study of a number of distinctdiseases associated with perturbances of the immune system, andthat there is a critical need to develop standardized and validatedscreening tests for all these immunotoxicities.References1. Landsteiner K, Jacobs J (1935) Studies on thesensitization of animals with simple chemical compounds I. J Exp Med 61:643–6572. Dean JH, Padarathsingh ML, Jerrells TR(1979) Assessment of immunobiological effects induced by chemicals, drugs or food additives. I Tier testing and screening approach. Drug Chem Toxicol 2:5–163. Dean JH, Luster MI, Boorman GA, LauerLD (1982) Procedures available to examine the immunotoxicity of chemicals and drugs.Pharmacol Rev 34:137–1484. Luster MI, Munson AE, Thomas P, HolsappleMP, Fenters J, White K, Lauer LD, Dean JH (1988) Development of a testing battery to assess chemical-induced immunotoxicity.Fund Appl Toxicol 10:2–195. Luster MI, Portier C, Pait DG, White KL,Gennings C, Munson AE, Rosenthal GJ (1992) Risk assessment in immunotoxicology.I. Sensitivity and predictability of immunetests. Fund Apppl Toxicol 18:200–2106. Luster MI, Portier C, Pait DG, Rosenthal GJ,Germolec DR, Corsini E, Blaylock BL, Pollock P, Kouchi Y, Craig W, White KL, Munson AE, Comment CC (1993) Risk assessment in immunotoxicology. II.Relationship between immune and host resis-tance tests. Fund Appl Toxicol 21:71–827. van Loveren H, Vos J (1989)Immunotoxicological considerations: a practi-cal approach to immunotoxicity testing in the rat. In: Dayan A, Paine A (eds) Advances in applied toxicology. Taylor & Francis, New York, NY, pp 143–1648. White K, Jennings P, Murray P, Dean J (1994)International validation study carried out in 9 laboratories on the immunological assessment of cyclosporin A in the Fisher 344 rat. Toxicol In Vitro 8:957–9629. Ladics GS, Smith CE, Elliott GS, Slone TW,Loveless SE (1998) Further evaluation of the incorporation of an immunotoxicologi-cal functional asay for assessing humoralimmunity for hazard identification purposes in rats in a standard toxicology study.Toxicology 126:137–15210. House RV, Luebke RW (2007)Immunotoxicology: thirty years and count-ing. In: Luebke R, House R, Kimber I (eds) Immunotoxicology and immunopharmacol-ogy, 3rd edn. CRC, Boca Raton, FL, pp 3–2011. EPA, Biochemical Test Guidelines (1996)OPPTS 880.3550 immunotoxicity. US/EPA, Washington DC12. Kuper CF, Harleman JH, Richter-ReichelmHB, Vos JG (2000) Histopathologic approaches to detect changes indicative of immunotoxicity. Toxicol Pathol 2:454–466 13. Germolec DR, Nyska A, Kashon M, KuperCF, Portier C, Kommineni C, Johnson KA, Luster MI (2004) The accuracy of extended histopathology to detect immunotoxic chemi-cals. Toxicol Sci 82:504–51414. Haley P, Perry R, Ennulat D, Frame S,Johnson C, Lapointe JM, Nyska A, Snyder P, Walker D, Walter G (2005) STP Immunotoxicology Working Group. Best practice guideline for the routine patholgy evaluation of the immune system. Toxicol Pathol 33:404–40815. Descotes J, Nicolas B, Pham E (1997) Sentinelscreening for human immunotoxicity. In: Environment and immunity. Proceedings of a Workshop held in Brussels on 20–21 May 1996. Air Pollution Epidemiology Reports Series. S. R16. NRC (1992) Biologic markers in immuno-toxicology. A report by the US National Research Council. National Academy Press, Washington DC17. WHO (1996) Principles and methods forassessing direct immunotoxicity associated with exposure to chemicals. A report of the International Programme on Chemical Safety (Environmental Health Criteria 180). World Health Organization, Geneva12Luster and Gerberick18. van Loveren H, Germolec DR, Koren HS,Luster MI, Nolan C, Repetto R, Smith E, Vos JG, Vogt RF (1999) Report of the Bilthoven Symposium: advancement of epidemiological studies in assessing the human health effects of immunotoxic agents in the environment and the workplace. Biomarkers 4:135–157 19. Weisglas-Kuperus N, Patandin S, BerbersGAM, Sas TCJ, Mulder PGH, Sauer PJJ, Hooijkaas H (2000) Immunologic effects of background exposure to polychlorinated biphenyls and dioxins in Dutch preschool children. Environ Health Perspect 108:1203–120720. Leonardi GS, Houthuijs D, Steerenberg PA,Fletcher T, Armstrong B, Antova T, Lochman I, Lochmanova A, Rudnai P, Erdei E, Musial J, Jazwiec-Kanyion B, Niciu EM, Durbaca S, Fabianova E, Koppova K, Lebret E, Brunekreef B, van Loveren H (2000) Immune biomarkers in relation to exposure to particulate matter: a cross-sectional survey in 17 cities of Central Europe. Inhalation Toxicol 12(Supp 4):1–14 21. Pinkerton L, Biagini R, Ward EM, Hull RD,Deddens JA, Boeniger MF, Schnoor TM, Luster MI (1998) Immunologic findings among lead-exposed workers. Am J Indus Med 33:400–40822. Landsteiner K, Jacobs J (1936) Studies on thesensitization of animals with simple chemical compounds II. J Exp Med 64:625–63923. Draize JH, Woodard G, Calvery HO (1944)Methods for the study of irritation and toxic-ity of substances applied topically to the skin and mucous membranes. J Pharmacol Exp Ther 8:377–39024. Magnusson B, Kligman AM (1970) Allergiccontact dermatitis in the guinea pig.Identification of contact allergens. Charles C.Thomas, Springfield IL25. Buehler EV (1965) Delayed contact hyper-sensitivity in the guinea pig. Arch Dermatol 91:171–17726. Robinson MK, Nusair TL, Fletcher ER, RitzHL (1990) A review of the Buehler guinea pig skin sensitization test and its use in a risk assessment process for human skin sensitiza-tion. Toxicology 61:91–10727. Organisation for Economic Cooperation andDevelopment (2002) Test Guideline 429: The local Lymph Node Assay. OECD, Paris 28. Balls M, Hellsten E (2000) Statement on thevalidity of the local lymph node assay for skin sensitization testing. ECVAM Joint Research Centre, European Commission, Ispra. Altern Lab Anim 28:366–36729. Dean JH, Twerdock LE, Tice RR, SailstadDM, Hattan DG, Stokes WS (2001) ICCVAM evaluation of the murine local lymph nodeassay. II. Conclusions and recommendations of an independent scientific review panel.Regul Toxicol Pharmacol 34:258–27330. Kimber I, Mitchell JA, Griffin AC (1986)Development of a murine local lymph node assay for the determination of sensitizing potential. Fd Chem Toxic 24:585–59631. Kimber I, Hilton J, Weisenberger C (1989)The murine local lymph node assay for identi-fication of contact allergens: a preliminary evaluation of in situ measurement of lympho-cyte proliferation. Contact Derm 21:215–220 32. United States Environmental ProtectionAgency Health Effects Test Guidelines (2003) OPPTS 870.2600 Skin sensitization. US/EPA, Washington, DC33. Cockshott A, Evans P, Ryan CA, GerberickGF, Betts CJ, Dearman RJ, Kimber I, Basketter DA (2006) The local lymph node assay in practice: a current regulatory perspec-tive. Hum Exp Toxicol 25:387–39434. Gerberick GF, Ryan CA, Dearman RJ, KimberI (2007) Local lymph node assay (LLNA) fordetection of sensitizing capacity of chemicals.Methods 41:54–6035. Loveless SE, Ladics GS, Smith C, HolsappleMP, Woolhiser MR, White KL, Musgrove DL, Smialowicz RJ, Williams W (2007) Interlaboratory study of the primary antibody response to sheep red blood cells in outbred rodents following exposure to cyclophosph-amide or dexamethasone. J Immunotoxicol 4:233–23836. Luebke R, Holsapple M, Ladics G, Luster MI,Selgrade M-J, Woolhiser M, Germolec DR (2006) Immunotoxicogenomics: the potential of genomics technology in the immunotoxicity risk assessment process. Toxicol Sci 94:22–27 37. Boverhof DR, Gollapudi BB, Hotchkiss JA,Osterioh-Quiroz M, Woolhiser MR (2008) Evaluation of a toxicogenomic approach to the local lymph node assay (LLNA). Toxicol Sci 107(2):427–43938. Ryan CA, Hulette BC, Gerberick GF (2001)Review: approaches for the development of cell based in vitro methods for contact sensiti-zation. Toxicol In Vitro 15:43–5539. Ryan CA, Gerberick GF, Gildea LA, HuletteBC, Betts CJ, Cumberbatch M, Dearman RJ (2005) Interactions of chemicals with den-dritic cells – a novel approach for identification of potential allergens. Toxicol Sci 88:4–11 40. Teufel M, Biedermann T, Rapps N, HausteinerC, Henningsen P, Enck P, Zipfel S (2007) Psychological burden of food allergy. World J Gastroenterol 3:3456–346541. McClain S, Bannon GA (2006) Animal modelsof food allergy: opportunities and barriers.Curr Allergy Asthma Rep 2:141–144。