Genomics and Toxic Substances: Part I—Toxicogenomics by Gary E

Genomics and Toxic Substances: Part I—Toxicogenomics by Gary E. Marchant

dvances in genomics, the study of the structure and ticle will address the application of toxicogenomics to Afunction of our genetic make-up, are fundamentally toxic torts and environmental regulation; a subsequent transforming toxicology, the science of how toxic sub- companion article will address toxicogenetic applications. stances affect our bodies. These changes will inevitably spill After first describing the scientific background of toxico- over into the legal regimes that frequently rely on toxicolog- genomics, this Article explores some potential uses of ical data, including toxic torts and environmental regula- toxicogenomic data in regulation and litigation involving tion.1 Genomic data, and the techniques with which they are toxic substances. generated, have the potential to make toxic torts and environmental regulation more effective, efficient, and fair, but Scientific Background at the same time will present many new doctrinal, evidentiary, and ethical challenges. Toxicogenomics is defined “as the study of the relationship Two key applications of genomic data for toxic torts and between the structure and activity of the genome (the cellu- environmental regulation are2: (i) the study of the expres- lar complement of genes) and the adverse biological effects sion of genes in cells or tissues in response to exposure to a of exogenous agents.”4 A major focus of toxicogenomics is toxicant, known as toxicogenomics; and (ii) the identifica- to characterize changes in gene expression in cells or tissues tion of genetic variations affecting susceptibility to toxic after exposure to toxic substances.5 Such exposure invari- agents, sometimes referred to as toxicogenetics.3 This Ar- ably results, either directly or indirectly, in characteristic changes in gene expression6 These gene expression changes The author is Associate Professor and Executive Director, Center for the Study of Law, Science, and Technology, Arizona State University College 4. Marilyn J. Aardema & James T. MacGregor, Toxicology and Ge- of Law. J.D. (1990); M.P.P. (1990); Ph.D. (Genetics) (1986). Portions of netic Toxicology in the New Era of “Toxicogenomics”: Impact of this Article were presented at a National Institutes of Environmental “-Omics” Technologies, 499 Mutation Res. 13, 15 (2002). Health Sciences conference on Toxicogenomics in December 2001, and at presentations in 2002 to the Woodrow Wilson International Center for 5. A small proportion of the genes in any cell are “turned on” or “ex- Scholars, the Environmental Law Institute, and faculty colloquia at Seton pressed” in a given cell at any one time. Although different types of Hall Law School and Arizona State University College of Law. The author cells, e.g., skin, blood, nerve cells, within a body contain identical genetic information, they have very different functional and struc- appreciates the many helpful comments and questions from the partici- tural properties primarily because they have different subsets of pants at those events, including some particularly valuable suggestions genes that are expressed. One estimate is that approximately 25% of from Andrew Askland and Michael Saks. all genes are active (turned on) in a given cell type, and that on aver- 1. See, e.g., P. Trinia Simmons & Christopher J. Portier, Toxico- age about 5% of genes that are active in one cell type are different genomics: The New Frontier in Risk Analysis,23Carcinogenesis from the genes turned on in another cell type. Toby G. Rossman, 903, 903 (2002) (“the complete sequence of the human genome will Cloning Genes Whose Levels of Expression Are Altered by Metals: cause a fundamental paradigm shift in the science of risk assess- Implications for Human Health Research,38Am. J. Ind. Med. 335, ment”); Wendy Yap & David Rejeski, Environmental Policy in the 335 (2000). In addition to these differences in gene expression be- Age of Genetics, Issues in Sci. & Tech., Fall 1998, at 33. tween cell types, the gene expression in any one cell type varies over 2. Many other types of genetic data may be useful in environmental time in response to external stimuli. A gene is expressed by a process regulation and toxic torts, including deoxyribonucleic acid (DNA) called transcription, in which a replicate of the functional DNA se- adducts, chromosomal aberrations, DNA breakage studies, reporter quence of the gene is created (known as messenger RNA (mRNA)), gene assays, and mutational spectra associated with specific chemi- which then moves from the cell nucleus into the cell cytoplasm to cals. See generally Stefano Bonassi & William W. Au, Biomarkers produce a protein, the primary functional and structural units of the in Molecular Epidemiology Studies for Health Risk Prediction, 511 cell. In addition to characterizing these gene expression changes, Mutation Res. 73 (2002). These applications are outside the scope “toxicogenomics” also generally encompasses other types of data of this Article. including profiling the proteins (proteomics) or metabolites (metabonomics) in a cell or tissue. See Aardema & MacGregor, su- 3. Emile F. Nuwaysir et al., Microarrays and Toxicology: The Advent pra note 4, at 14. of Toxicogenetics,24Molecular Carcinogenesis 153, 158 (1999); Richard J. Albertini, Developing Sustainable Studies on En- 6. See Nuwaysir et al., supra note 3, at 153 (“Almost without excep- vironmental Health, 510 Mutation Res. 317, 323 (2001). tion, gene expression is altered during toxicity, as either a direct or Toxicogenetics therefore involves the toxicological implications of indirect result of a toxicant exposure.”); Spencer Farr & Robert T. single genes, whereas the focus of toxicogenomics is on the entire Dunn, Concise Review: Gene Expression Applied to Toxicology,50 genome. A “genome” refers to the complete set of genes contained Toxicological Sci. 1, 1 (1999) (“The fundamental assumption of within a cell. This same distinction between focusing on one or a few toxicogenetics is that there are no toxicologically relevant outcomes susceptibility genes (toxicogenetics) versus studying the expression in vitro or in vivo, with the possible exception of rapid necrosis, that of the whole genome (toxicogenomics) in response to exposure to do not require differential gene expression.”); Russell S. Thomas et toxic substances also applies in the related field of genomic ap- al., Identification of Toxicologically Predictive Gene Sets Using proaches to pharmaceuticals. See Allen D. Roses, Pharmaco- cDNA Microarrays,60Molecular Pharmacology 1189, genetics,10Hum. Molecular Genetics 2261, 2261 (2001) 1189-90 (2001) (“[T]oxicity is commonly manifested as inflamma- (“[P]harmacognetics is defined as the study of variability in drug tion, proliferation, apoptosis, necrosis, and/or cellular differentia- responses attributed to hereditary factors in different populations. tion. All of these toxic endpoints are intimately linked to specific al- Pharmacogenomics is the determination and analysis of the ge- terations in gene expression.”); Albertini, supra note 3, at 321 (“It nome (DNA) and its products (RNA and proteins) as they relate to has long been known that cells almost always respond to noxious drug response.”). stimuli by altering gene expression.”). 33 ELR 10072 ENVIRONMENTAL LAW REPORTER 1-2003 Copyright © 2003 Environmental Law Institute®, Washington, DC. reprinted with permission from ELR®, http://www.eli.org, 1-800-433-5120. may sometimes be the cause or in other cases the consequences known as complementary DNA (cDNA) that can quence of the early stages of a toxic response.7 be “tagged” with a flourescent marker.14 The cDNA sample Gene expression changes can be analyzed by collecting is then added to the microarray, and cDNA sequences will and characterizing messenger ribonucleic acid (mRNA) us- bind (or hybridize) to sites on the microarray that contain ing a deoxyribonucleic acid (DNA) microarray. A DNA DNA with a matching sequence. A laser scans the micro- microarray (sometimes also referred to as a gene chip or array and generates flourescent spots at the locations where DNA chip) consists of a set of many different single- the cDNA binds, and the intensity of the signal at each spot stranded genetic sequences fixed to a substrate, such as a will be proportional to the abundance of matching mRNA in glass slide or membrane, in a defined pattern. The genetic the original sample isolated from the treated or control markers can consist of short (500 to 2,000 base pair) DNA cells.15 Sophisticated computer programs are available to sequences that are complementary to, and thus bind to, “read” the microarrays and produce digital graphic read- genes of potential interest. Fifty thousand or more of these outs of the genes being expressed in the cells of interest. specific DNA sequences can be spotted (or printed) onto the DNA microarrays thus permit the almost instantaneous and fixed substrate in precise locations or spots on a grid, with simultaneous genome-wide detection of the expression of each spot on the array containing several million identical thousand of genes, even if the function of some of the genes copies of a DNA segment from a specific gene.8 Alterna- is unknown.16 tively, shorter synthesized DNA sequences, called This genome-wide or “global” gene expression analysis oligonucleotides, can be constructed directly onto the is a major advancement over previous methodologies which substrate using a process called photolithography.9 At only permitted analysis of the expression of one or two indi- least 20 companies have commercialized DNA micro- vidual genes at a time.17 Toxicity usually involves the induc- array products,10 and at least 1 manufacturer has already re- tion (up regulation) and repression (down regulation) of leased a commercial gene chip containing the entire human genome.11 14. Jennifer Medlin, Array of Hope for Gene Technology, 109 Envtl. DNA microarrays can be used to identify and character- Health Persp. A34, A35-36 (2001). ize the response of a cell or tissue to an external stimulus or 12 15. Id. at A36; Hisham K. Hamadeh et al., An Overview of perturbation, such as exposure to a toxic substance. The Toxicogenomics,4Curr. Issues Molecular Biol. 45, 46 (2002). complement of mRNA culled from the cytoplasm of a cell One frequently used technique is to compare gene expression in two provides a snapshot of the genes that are being expressed in samples, e.g., control versus treatment cells, by making copies of the 13 mRNA isolated from the two samples using modified nucleotides the cell at that time. The mRNA from treated or control carrying flourescent tags. For one sample, a tag called Cy3 which cells can be collected and then copied to form DNA se- fluoresces green could be used to synthesize the cDNA from the control, i.e., untreated, cells while Cy5 which fluoresces red is used to produce the cDNA from the treated cells. The fluorescently labeled 7. See Christine Debouck & Peter N. Goodfellow, DNA Microarrays in sequences from the control and treated cells are then mixed together Drug Discovery and Development, 21 (Suppl.) Nature Genetics and hybridized to the DNA microarray and scanned by a laser to pro- 48, 49 (1999). duce flourescent patterns. If a gene is expressed in equal amounts in both the control and treatment samples, then the spot on the 8. See Hisham K. Hamadeh & Cynthia A. Afshari, Gene Chips and microarray corresponding to that gene will fluoresce yellow. If a Functional Genomics,88Am. Sci. 508, 510-11 (2000); Nuwaysir gene was expressed more in the treated cells than the control cells, et al., supra note 3, at 153-54; Stephen H. Friend & Roland B. i.e., “up-regulated,” the spot will be red. If the gene is expressed at Stoughton, The Magic of Microarrays, Sci. Am., Feb. 2002, at 44, relatively lower levels in the treated cells, i.e., “down-regulated,” the 46-47. spot will be green. The intensity of the signal will provide a quantita- 9. Nuwaysir et al., supra note 3, at 154; Timothy J. Aitman, DNA tive estimate of the extent by which a particular gene is up- or Microarrays in Medical Practice, 323 Brit. Med. J. 611, 612 (2001). down-regulated in the treated cells. Id.; see also Hisham K. These types of microarrays are particularly useful for detecting small Hamadeh et al., Discovery in Toxicology: Mediation by Gene Ex- pression Array Technology,15J. Biochem. Molecular Toxicol- changes in DNA sequences, such as point mutations in a gene, be- ogy cause it is possible to synthesize many closely related sequences that 231, 232 (2001). differ by only a single base pair. Microarrays can generally be used 16. See W.D. Pennie & I. Kimber, Toxicogenomics; Transcript Pro- both for characterizing gene expression and identifying variations in filing and Potential Application to Chemical Allergy,16Toxicol- a DNA sequence of genes independent of their gene expression. In ogy in Vitro 319, 320 (2002); Hamadeh & Afshari, supra note 8, the former application, the mRNA is collected from the cell and hy- at 509. bridized to the microarray as described infra, while in the latter the cell’s DNA is itself collected and hybridized to the microarray. 17. See Hamadeh & Afshari, supra note 8, at 509 (“Traditional assays measure RNA transcripts from one gene at a time over a three-day 10. See Friend & Stoughton, supra note 8, at 46. period. Gene chips can measure transcripts from thousands of genes in a single afternoon.”). As one scientific review of this tech- 11. Affymetrix, News Release: Affymetrix Launches First Commercial nology commented, “a global analysis of gene expression has the Human DNA Array to Use Draft of Human Genome, Jan. 21, 2002, potential to provide a more comprehensive view of toxicity than at http://www.corporate-ir.net/ireye/ir_site.zhtml?ticker=AFFX& has been possible previously, since toxicity generally involves script=410&layout=-6&item_id=248283 (last visited Aug. 29, change not only in a single or few genes but rather is a cascade of 2002). gene interactions.” Aardema & MacGregor, supra note 4, at 14. See also Hisham K. Hamadeh et al., Gene Expression Analysis Reveals 12. There are other important medical and public health applications of Chemical-Specific Profiles,67Toxicological Sci. 219, 219 DNA microarrays in addition to toxicogenomics, including differen- (2002). Gene expression signatures for identifying particular tiating similar appearing tumors with respect to prognosis and treat- classes of toxicants ment based on gene expression patterns, and rapidly identifying potentially dangerous microorganisms in the context of food safety, cannot be defined using classical methods where genes are biowarfare defense, and other applications. See, e.g., Edward K. investigated individually for potential association to chemi- Lobenhofer et al., Progress in the Application of DNA Microarrays, Envtl. Health Persp. cal exposure. This is because the most highly characterized 109 887 (2001); Charles M. Perou et al., chemical-responsive genes, such as genes encoding proteins Molecular Portraits of Human Breast Tumours, 406 Nature 747 (2000); Friend & Stoughton, supra note 8, at 44-53; Aitman, supra or enzymes that regulate metabolism, tend to be frequently note 9. modulated by many compounds, and therefore do not provide a solid footing for providing specificity for distinguishing 13. See Hamadeh & Afshari, supra note 8, at 509. multiple classes. 1-2003 NEWS & ANALYSIS 33 ELR 10073 Copyright © 2003 Environmental Law Institute®, Washington, DC. reprinted with permission from ELR®, http://www.eli.org, 1-800-433-5120. many different genes,18 and thus only the genome-wide as- studies for carcinogenicity, mutagenicity, reproductive tox- says made possible by DNA microarrays can evaluate the icity, teratogenicity, immunotoxicity, neurotoxicity, and entire cascade of gene responses to toxic exposure.19 Expo- endocrine disruption.26 sure to chemicals that cause toxicity through a mechanism Yet another advantage of studying gene expression involving DNA alkylation, for example, results in changes changes to assess toxicity is that such alterations can occur in the expression of over 2,000 genes.20 Other chemicals in- almost immediately following exposure, whereas the clini- duce changes in the expression of a more modest but still cal manifestation of toxicity may take days, months, or even substantial number of different genes.21 years to develop.27 Because these toxicological endpoints The use of DNA microarrays to study global gene expres- are the end result of earlier molecular events that can be sion provides “a tool of unprecedented power for use in toxi- monitored by microarrays, it is possible to screen for toxic- cology studies.”22 Gene expression changes measured by ity much more quickly and earlier using microarrays than microarrays have the potential to provide a more sensitive, with traditional toxicological methods.28 Moreover, be- characteristic, and earlier indicator of a toxic response than cause they represent an earlier step in a toxic response, gene typical toxicological endpoints such as morphological expression changes will be detectable in a larger percentage changes, carcinogenicity, or reproductive toxicity.23 Micro- of the exposed animal or human population than will ulti- array data promise greater specificity because while “there mately go on to develop clinical disease, thereby providing are a limited number of cellular, organ, and organismal man- a more statistically robust measure of effect. For these rea- ifestations of chemically-induced toxicity, the possible sons, gene expression changes assayed using DNA micro- number of gene expression patterns for encoding those man- arrays have the potential to provide both an earlier and more ifestations is enormous.”24 Many different toxic agents may sensitive biomarker of a toxic response. be capable fo causing the same toxicological endpoint, e.g., Of particular interest is the rapidly growing body of evi- a liver tumor, in many cases by different mechanisms, dence demonstrating that specific chemicals or classes of whereas each chemical will produce a unique gene expres- chemicals with similar toxicological properties produce a sion profile, thus providing a higher resolution tool with characteristic gene expression “fingerprint” or signature much greater specificity than simply monitoring the toxico- profile. Initial “proof-of-principle” experiments have suc- logical endpoint.25 Microarrays also permit evaluation of all cessfully identified the identity or toxicological mechanism toxicological endpoints in a single assay, whereas tradi- of chemicals based on their gene expression profiles.29 The tional toxicological methods generally require separate 26. Pennie & Kimber, supra note 16, at 319 (microarrays can “characterize simultaneously an unprecedented number of bio- 18. For example, most of the differential gene expression in cultured logical endpoints”). liver cells exposed to ethanol involve down-regulation, whereas similar treatment with carbon tetrachloride results in both up-regula- 27. See Farr & Dunn, supra note 6, at 1. tion and down-regulation of different genes. H.M. Harries et al., The Use of Genomics Technology to Investigate Gene Expression 28. See Rodi et al., supra note 25, at 107. Toxicology in Vitro Changes in Cultured Human Liver Cells,15 29. E.g., Thomas et al., supra note 6, at 1193 (a microarray analysis us- 399, 401-02 (2001). ing only 12 diagnostic genes was able to correctly classify the toxi- 19. Hamadeh et al., supra note 17, at 219 (“DNA microarrays enable the cological class of 24 toxicants with 100% predictive accuracy); Mi- study of levels of expression of thousands of genes at the mRNA chael E. Burczynski et al., Toxicogenomics-Based Discrimination of level. The concerted expression pattern across those genes consti- Toxic Mechanisms in HepG2 Human Heatoma Cells,58Toxico- tutes the expression profile of a compound at a certain dose and logical Sci. 399 (2000) (gene expression patterns used to success- time.”); Aardema & McGregor, supra note 4, at 14; fully discriminate compounds based on toxic mechanism); Jeffrey F. Waring et al., Microarray Analysis of Hepatotoxins in Vitro Reveals 20. See William E. Bishop et al., The Genomic Revolution: What Does It Risk Analysis a Correlation Between Gene Expression Profiles and Mechanisms Mean for Risk Assessment?,21 983, 986 (2001). of Toxicity, 120 Toxicology Letter 359 (2001) (microarray analy- 21. For example, 24-hour treatment of cultured liver cells with ethanol sis of the effects of 15 known liver toxins on cultured cells showed produces changes in the expression of approximately 85 different that each compound produced a unique signature, and compounds genes. Harries et al., supra note 18, at 402. Dioxin appears to change with similar toxic mechanisms had the same distinct patterns (“clus- the expression of approximately 300 genes by a factor of 2 or more. ters”) of gene expression); Matthew Bartosiewicz et al., Applica- Felix W. Frueh et al., Use of cDNA Microarrays to Analyze Di- tions of Gene Arrays in Environmental Toxicology: Fingerprints of oxin-Induced Changes in Human Liver Gene Expression, 122 Toxi- Gene Regulation Associated With Calcium Chloride, Ben- cology Letter 189, 198-99 (2001). zo(a)pyrene, and Trichloroethylene, 109 Envtl. Health Persp. 71, 73-74 (2001) (three important environmental contaminants be- 22. Nuwaysir et al., supra note 3, at 153. See also Hamadeh et al., supra longing to different chemical classes produced unique patterns of note 15, at 231 (DNA microarrays “provide a revolutionary platform gene expression in mice); Hamadeh et al., supra note 17, at 219, to perform genome-wide gene expression analyses through compar- 228-29 (structurally unrelated compounds from the same general ison of virtually any two biological samples”); Pennie & Kimber, su- class of toxicants produce similar, but distinguishable, gene expres- pra note 16, at 319 (microarrays “represent nothing short of a revolu- sion profiles); Hisham K. Hamadeh et al., Prediction of Compound tion in our ability to characterize simultaneously an unprecedented Signature Using High-Density Gene Expression Profiling,67Toxi- number of biological endpoints”). cological Sci. 232 (2002) (microarray analysis of gene expression 23. See Harries et al., supra note 18, at 399 (“Changes in gene expression profiles from liver samples of chemical-treated rats was able to cor- often provide a far more sensitive, characteristic and measurable rectly predict the toxicological mechanism of 22 of 23 blinded endpoint than the toxicity itself.”); Hamadeh et al., supra note 15, at chemicals); Christopher M.L.S. Bouton et al., Microarray Analysis 231; Nuwaysir et al., supra note 3, at 154-55. of Differential Gene Expression in Lead-Exposed Astrocytes, 176 Toxicology & Applied Pharmacology 34, 44-45 (2001) (micro- 24. See Farr & Dunn, supra note 6, at 2. There are generally less than 100 arrays used to distinguish cells exposed to lead from nontoxicant ex- toxicological outcomes evaluated in human or animal studies, 70 posed cells); Steven J. Bulera et al., RNA Expression in the Early whereas hypothetically there are an estimated 10 different gene ex- Characterization of Hepatotoxicants in Wistar Rats by High-Den- pression patterns per cell, although the number of toxicologically sity DNA Microarrays,33Hepatology 1239 (2001) (microarray relevant gene expression patterns actually observed is obviously analysis of mRNA able to successfully distinguish six different liver much smaller than the possible number of patterns. Id. toxicants). See also Raymond W. Tennant, The National Center for 25. Charles P. Rodi et al., Revolution Through Genomics in Investiga- Toxicogenomics: Using New Technologies to Inform Mechanistic tive and Discovery Toxicology,27Toxicological Pathology Toxicology, 110 Envtl. Health Persp. A8, A8 (2002); Nuwaysir 107, 109 (1999). et al., supra note 3, at 154-56. 33 ELR 10074 ENVIRONMENTAL LAW REPORTER 1-2003 Copyright © 2003 Environmental Law Institute®, Washington, DC. reprinted with permission from ELR®, http://www.eli.org, 1-800-433-5120. potential applications of these findings are significant. The conversely to help defendants demonstrate the absence of observation that groups of chemicals with a common toxi- exposure or causation when they are lacking. cological mechanism produce a characteristic pattern of gene expression changes means that it may be possible to Exposure predict the toxicological nature and mechanism of an untested chemical in a quick and inexpensive gene expression One of the first hurdles a plaintiff must surmount in a toxic assay.30 The finding that it is possible to discern exposure to tort lawsuit is to prove that he or she was exposed suffi- an individual chemical based on unique gene expression ciently to a toxic agent associated with the defendant’s prod- changes suggests that it may be possible to use microarrays uct or conduct.37 In many toxic tort cases, direct evidence of to measure exposure or toxic responses to specific chemi- the fact or quantity of exposure is limited or lacking alto- cals in individuals or populations.31 Finally, the capability to gether.38 Examples include plaintiffs who allege they were identify the genes that are affected by a particular chemical injured by chemicals that had leached into their groundwa- may be useful for discovering the toxicological mechanism ter, pesticides that were sprayed in their homes or work- of a toxicant, which would be very helpful in characterizing places, or pollutants emitted into the air nearby. In these the risks associated with that toxic agent.32 types of cases, courts have frequently dismissed plaintiffs’ Toxicogenomics not only has tremendous potential, but cases for failure to meet their burden of proof to demonstrate this potential is unfolding at an unprecedented rate. As de- (or quantify) exposure.39 scribed by one set of reviewers, “[u]nlike other new ap- Gene expression data using microarrays may assist plain- proaches or methods in toxicology that have been adopted tiffs in demonstrating exposure, or in other cases to support slowly,” toxicogenomic methods “are being evaluated and a defendant’s argument that there was not sufficient expo- adopted rapidly by all sectors of industry, academia, and sure.40 Gene expression assays of the plaintiffs’ blood or regulatory agencies at an unprecedented rate.”33 Others skin cells may demonstrate the presence (or absence) of have referred to the rapid emergence of toxicogenomics as gene expression “fingerprints” that are characteristic of the “the microarray stampede.”34 In a mere five years, toxico- toxic substance to which the plaintiff was allegedly ex- genomics has transformed the scientific study of toxic sub- posed. Such an assay might even be capable of quantifying stances, as microarray analysis has become one of the most the level and duration of plaintiff’s exposure. common and informative methodological approaches of A number of uncertainties and questions would need to be toxicology.35 The toxicogenomic revolution will have many addressed to establish the reliability of this exposure evi- important potential applications for both toxic torts and en- dence. For example, how well characterized and validated is vironmental regulation. Some of these applications are dis- the gene expression “fingerprint”? In other words, how cer- cussed below. tain can we be that a particular gene expression pattern is indeed representative of a particular toxic exposure?41 Can Toxicogenomic Applications for Toxic Torts other potential sources of exposure to that same toxic substance (or other substances that cause similar responses) be Plaintiffs often face significant obstacles in toxic tort cases excluded? Are the gene expression changes in the easily as- in satisfying their burden of proof to demonstrate exposure and causation.36 Gene expression data may help plaintiffs to 37. See, e.g., Allen v. Pennsylvania Eng’g Corp., 102 F.3d 194, 199 (5th Cir. 1996) (“Scientific knowledge of the harmful level of exposure demonstrate exposure and causation when they exist, and to a chemical, plus knowledge that the plaintiff was exposed to such quantities, are minimal facts necessary to sustain the plaintiffs’ burden in a toxic tort case.”). 30. See Hamadeh et al., supra note 17, at 228-29; supra notes 139-40 and 38. See generally Susan R. Poulter, Science and Toxic Torts: Is There a accompanying text. Rational Solution to the Problem of Causation?,7High Tech. L.J. 31. See supra notes 118-19 and accompanying text. 189, 236-41 (1992). 32. See supra notes 105-15 and accompanying text. 39. E.g., Wright v. Williamette Indus., Inc., 91 F.3d 1105, 1107 (8th Cir. 1996) (“at a minimum, we think that there must be evidence from 33. Aardema & MacGregor, supra note 4, at 15, 22. See also David Stipp, Fortune which the fact finder can conclude that the plaintiff was exposed to Gene Chip Breakthrough, , Mar. 31, 1997, at 56 (“Micro- levels of that agent that are known to cause the kind of harm that the processors have reshaped our economy, spawned vast fortunes and plaintiff claims to have suffered”); Mitchell v. Gencorp Inc., 165 changed the way we live. Gene chips could be even bigger.”). F.3d 778, 781 (10th Cir.1999) (“[g]uesses, even if educated, are in- 34. Elizabeth Pennisi, Recharged Field’s Rallying Cry: Gene Chips for sufficient to prove the level of exposure in a toxic tort case”); Allen, All Organisms, 297 Science 1985, 1986 (2002). 102 F.3d at 197 (“Scientific knowledge of the harmful level of expo- 35. One measure of the rapid development of toxicogenomics is the sure to a chemical, plus knowledge that the plaintiff was exposed to number of published scientific studies containing the term such quantities, are minimal facts necessary to sustain the plaintiffs’ “microarray” indexed in the National Library of Medicine’s burden in a toxic tort case.”). But see Donaldson v. Central Ill. Pub. “PubMed” database, at http://www.ncbi.nlm.nih.gov/entrez/query. Serv. Co., 767 N.E.2d 314, 332 (Ill. 2002) (plaintiffs are “not re- fcgi?db=PubMed). Only 2 published studies mentioned microarrays quired to show the exact amount of exposure” in cases involving en- in 1997, but this quickly increased to 21 studies in 1998, 83 in 1999, vironmental toxics because such cases “do not afford litigants the 285 in 2000, 805 in 2001, and as of August 30, 2002, 874 in the first opportunity to specify with such certainty the exact level of and dose eight months of 2002. of exposure”). 40. See Gary E. Marchant, Toxicogenomics and Toxic Torts,20Trends 36. See, e.g., Troyen A. Brennan, Causal Chains and Statistical Links: in Biotech. the Role of Scientific Uncertainty in Hazardous-Substance Litiga- 329, 330 (2002). tion,73Cornell L. Rev. 469, 469 (1988) (proving causation is the 41. This inquiry will involve evaluating both the specificity and sensi- “paramount obstacle to just resolution of tort claims based on injury tivity of the gene expression assay. Specificity refers to the capabil- from toxic substances”); Steve Gold, Causation in Toxic Torts: Bur- ity of the assay to identify only exposures that are indeed associated dens of Proof, Standards of Persuasion, and Statistical Evidence,96 with the toxic agent of interest, i.e., avoiding false positives, while Yale L.J. 376, 376 (1986) (“Proving the cause of injuries that re- sensitivity refers to the capability of the assay to detect all exposures main latent for years, are associated with diverse risk factors, and oc- to the relevant toxic agent, i.e., avoiding false negatives. See gener- cur at background levels even without any apparent cause, is the ally Ralph R. Cook, The Importance of Test Validity and Predictive ‘central problem’ for toxic tort plaintiffs.”) (footnotes omitted). Values to Screening Programs,41Jurimetrics 111, 112 (2000). 1-2003 NEWS & ANALYSIS 33 ELR 10075 Copyright © 2003 Environmental Law Institute®, Washington, DC. reprinted with permission from ELR®, http://www.eli.org, 1-800-433-5120. sayed tissues such as blood or skin cells representative of the disease manifests itself many years later.47 On the other changes in the less accessible target tissue in which the hand, some exposures to a toxic substance may produce per- plaintiff’s disease is more likely to have occurred?42 What is manent changes in gene expression, perhaps as a result of the quantitative relationship between the level of exposure gene mutations, gene amplifications, or changes in DNA and the magnitude of gene expression changes? Over what methylation patterns, all of which control gene expres- range of exposure is this relationship valid? Do interindi- sion.48 To the extent that these types of changes in gene ex- vidual differences in susceptibility (genetic or nongenetic) pression can be verified, they may produce a much more du- affect gene expression patterns in different individuals?43 rable marker of exposure. How does gene expression vary with single, acute exposures The legal significance of the temporal component with versus long-term chronic exposures? respect to the use of genetic biomarkers to quantify expo- A critical set of issues relates to the timing of gene expres- sure is demonstrated in an analogous context by the litiga- sion changes. Specifically, what is the time course of the tion brought by residents near the Three Mile Island (TMI) gene expression changes following toxic exposure, and are nuclear reactor, who claimed that a radioactive plume from these changes transient or longer term?44 Very little existing the 1979 reactor accident caused their cancer. The plaintiffs data address the issue of the duration of gene expression lacked direct measurements or adequate modeling evidence changes in cells, and how those changes either progress or to prove that they received sufficient radiation exposure to diminish over time.45 The limited data that are available sug- cause their cancers, which the court described as the “criti- gest that gene expression changes may only provide a valid cal issue” in the case.49 They attempted to overcome this measure of exposure within a few days of exposure.46 If that problem by introducing evidence that they had an increased is the case, then gene expression data will have little or no frequency of a particular type of chromosome aberration utility for latent diseases unless the gene expression data is (dicentric chromosomes)50 in their blood cells (lympho- collected close to the time of exposure, rather than when the cytes), which they claimed provided a quantitative biomarker of radiation exposure.51 The U.S. Court of Appeals 42. See Tennant, supra note 29, at A9 (“it is important to determine for the Third Circuit held that such applications of genetic whether or not serum/blood cells can be used as an alternative to spe- markers “is an accepted method, not simply for determining cific target organ tissue”). The NIEHS is currently testing the hy- if the subject of the analysis was irradiated, but also for es- pothesis that blood cells can serve as a surrogate for tissue-specific timating radiation dose to the individual.”52 The court chemical effects. Id. See also John C. Rockett et al., DNA Arrays to Monitor Gene Expression in Rat Blood and Uterus Following found, however, that while “[r]adiation dose estimation 17B-Estradiol Exposure: Biomonitoring Environmental Effects based on dicentric enumeration is a valid and reliable sci- Using Surrogate Tissues,69Toxicological Sci. 49 (2002) (pilot entific methodology,” the “validity and reliability decrease study finding that peripheral blood lymphocytes can serve as ade- as the time gap between the alleged irradiation and the quate surrogate for uterus for monitoring gene expression changes in 53 response to endocrine disrupting chemicals). dicentric count increases.” The court concluded that di- 43. If gene expression samples are available for an individual before and centric chromosomes could only provide an accurate indi- after exposure, then comparison of those two results might provide a cator of dose within one or two years of exposure, and thus valid measure of exposure regardless of any unique genetic charac- plaintiff’s reliance on dicentric chromosome levels assayed teristics of the individual affecting gene expression levels. In the ab- 15 years after the TMI accident were no longer a reliable sence of pre-exposure data for an individual, however, the effect of 54 individual susceptibility would likely have to be considered to quan- measure of exposure. tify exposure if such susceptibilities affect the relationship between The lesson from the TMI litigation is that litigants who the quantum of exposure and the magnitude of the gene expression seek to rely on gene expression changes to quantify expo- changes. However, one study examining gene expression changes after exposure to ionizing radiation found only slight variation in sure will need to evaluate the gene expression changes in the gene expression levels in peripheral blood lymphocytes from differ- plaintiffs’ cells as soon as possible after exposure. In addi- ent donors, suggesting that interindividual genetic variability had a tion, it will be necessary to lay a proper foundation for using relatively minor effect on gene expression changes for at least this response. Sally A. Amundson et al., Identification of Potential such evidence to quantify exposure by providing data on mRNA Biomarkers in Peripheral Blood Lymphocytes for Human Ex- how such gene expression changes change over time from posure to Ionizing Radiation, 154 Radiation Res. 342, 343, 346 exposure. Notwithstanding these limitations, the use of (2000). DNA microarrays to monitor gene expression changes 44. See Bonassi & Au, supra note 2, at 76 (“For many types of within a plaintiff’s cells has the potential to provide a very biomarkers the most important consideration is the stability of the biomarkers with respect to time after exposure.”); Carol J. Henry et 47. See Henry et al., supra note 44, at 1049 (“Recently examined gene al., Use of Genomics in Toxicology and Epidemiology: Findings and Envtl. Health Persp. expression products may not be relevant to disease that have long la- Recommendations of a Workshop, 110 tency periods.”). 1047, 1049 (2002) (“An additional challenge [of toxicogenomic methods] is to examine gene expression at the time period that is rel- 48. See Rossman, supra note 5, at 336 (suggesting that some changes in evant to the health outcome of interest.”). gene expression may be permanent). 45. Most toxicogenomic data produced to date only examines the short- 49. In re TMI Litig., 193 F.3d 613, 622 (3d Cir. 1999), cert. denied, 120 term effect of toxic exposure to cells isolated in tissue culture or ro- S.Ct. 2238 (2000). dents exposed for 24 to 72 hours. But see Hamadeh et al., supra note 50. Dicentric chromosomes contain two rather than the normal one 17, at 225, 227 (finding quantitative and qualitative differences in centromere. A centromere is the structural center of a chromosome gene expression between one-time exposure to toxicant and after at which the left and right chromosome arms are joined. Dicentric two weeks of daily exposure to same toxicant). chromosomes are formed when chromosomes are broken by an agent such as radiation and then rejoin in an aberrant manner. Id. at 46. One study evaluated the time course of gene expression changes in 688 n.24. human peripheral blood lymphocytes grown in tissue culture and exposed to a single dose of ionizing radiation. Amundson et al., supra 51. Id. at 688. note 43, at 342-46. The maximal response for most of the marker 52. Id. at 690. genes occurred 24 hours after irradiation, and then gradually de- clined, but remained significantly above background levels at 72 53. Id. at 692. hours. Id. at 344. 54. Id. 33 ELR 10076 ENVIRONMENTAL LAW REPORTER 1-2003 Copyright © 2003 Environmental Law Institute®, Washington, DC. reprinted with permission from ELR®, http://www.eli.org, 1-800-433-5120. specific and informative quantitative molecular dosimeter The challenge facing plaintiffs in proving general causa- that can be used by plaintiffs to demonstrate and quantify tion is thus much more daunting than, for example, the chal- their exposure to toxic substances or by defendants to dem- lenge facing a regulatory agency attempting to regulate the onstrate the lack of sufficient exposure.55 same substance. The regulatory agency need only show that the chemical might cause any adverse health effect in some General Causation people. In contrast, a toxic tort plaintiff has the burden of proof to show that the chemical did cause a specific adverse Plaintiffs who are able to adequately quantify their exposure effect, i.e., that from which plaintiff suffers, in a particular must next prove that this exposure caused their injuries. person. “Toxic ignorance,”60 or the lack of adequate testing Causation analysis typically involves two steps.56 First, the data for many potential toxic substances, thus severely lim- plaintiff must prove general causation, which requires a its a plaintiff’s ability to introduce the required data on a spe- demonstration that the toxic agent produced by the defen- cific chemical-health effect relationship, given that data eval- dant is capable of causing the health effect incurred by the uating many such relationships will often be nonexistent.61 plaintiff.57 Most courts have required relevant evidence that Toxicogenomic data may provide some opening for is specific to both the toxic substance and the health effect in plaintiffs to proceed when no data are available for the spe- question for demonstrating general causation. Thus, courts cific toxicant-health effect combination relevant to their have generally excluded evidence that the same chemical case. For example, consider a case in which plaintiffs have can cause diseases perhaps related to but different than the been exposed to agent A and had developed kidney cancer, condition afflicting the plaintiff, such as tumors in other but there is no toxicological data directly linking agent A types of tissues in the case of a plaintiff with cancer.58 Plain- and kidney cancer. Plaintiffs may nevertheless be able to tiffs have likewise often been precluded from relying on evi- rely on data showing that agent A stimulates gene expres- dence showing that chemicals related to the one which the sion changes that are similar to those induced by agent B, individual plaintiff has been exposed can cause the specific which has been found to cause kidney cancer. Alternatively, health effect for which the plaintiff has been diagnosed.59 plaintiffs may be able to introduce evidence that agent A causes liver cancer by a mode of action that involves a char- 55. The potential of genomic data to verify exposure to specific products is provided by the recent actions of a prominent personal injury law acteristic gene expression profile, and that agent A has also firm specializing in foodborne illness. The firm has posted on the been observed to cause a similar gene expression change in Internet at http://www.fsis-pfge.org the genetic fingerprints of kidneys in animal studies, even though such studies have pathogenic E.Coli 0157:H7 strains associated with recalls of ground not detected a statistically significant increase in kidney tu- beef. Individuals who contract food poisoning from E. Coli 0157:H7 62 can check if the genetic fingerprints of the strain that has infected mors. These types of toxicogenomic data may provide a them matches one of the genetic profiles from the contaminated molecular link between agent A and kidney cancer even in meet. Given the variability between different bacterial strains, such a the absence of data directly showing such a relationship. match would provide strong evidence of causation in a personal in- 63 jury case. Allison Beers, Marler Clark Posting E. Coli Genetic Fin- A Texas case, Austin v. Kerr-McGee Refining Corp., gerprints From Recalls, Food Chem. News, June 3, 2002, at 1. can be used to further illustrate the potential role of toxico- 56. See, e.g., Sterling v. Velsicol Chem. Corp., 855 F.21d 1188, 1200, 19 genomics to demonstrate general causation. In that case, the ELR 20404, 20408 (6th Cir. 1988); Raynor v. Merrell Pharmaceuti- spouse of a deceased worker who had worked with mineral cals, Inc., 104 F.3d 1371, 1376 (D.C. Cir. 1997); Merrell Dow Phar- spirits claimed that benzene in the mineral spirits caused maceuticals, Inc. v. Havner, 953 S.W.2d 706, 714 (Tex. 1997). her deceased husband’s chronic myelogenous leukemia 57. Havner, 953 S.W.2d at 714 (“General causation is whether a sub- (CML). The plaintiff’s causation expert relied primarily on stance is capable of causing a particular injury or injury in the general population....”). studies showing that benzene caused acute myelogenous leukemia (AML), and argued that these and other data show 58. See, e.g., General Elec. Co. v. Joiner, 522 U.S. 136, 144, 28 ELR 64 20227, 20228 (1997) (study finding that polychlorinated biphenyls that benzene causes all types of leukemia, including CML. (PCBs) cause alveologenic adenomas at high concentrations in mice cannot be used to show that PCBs caused plaintiff’s small-cell carci- vents); Lynch v. Merrell-National Labs., 830 F.2d 1190 (1st Cir. noma); Allen v. Pennsylvania Eng’g Corp., 102 F.3d 194, 197 (5th 1987) (rejecting expert’s reliance on toxicological studies with Cir. 1996) (“Evidence has been found that suggests a connection be- “analogous chemicals” to show causation). tween [ethylene oxide] exposure and human lymphatic and hema- 60. Environmental Defense Fund, Toxic Ignorance (1997). topoietic cancers, but this is not probative on the causation of brain cancer.”); Christophersen v. Allied-Signal Corp., 939 F.2d 1106, 61. See Richard J. Pierce Jr., Causation in Government Regulation and 1115-16 (5th Cir. 1991) (evidence that defendant’s chemicals may Toxic Torts,76Wash. U. L.Q. 1307, 1308 (1998) (“There are thou- cause small-cell carcinoma of the lung inadmissible to show that sands of regulated substances that rarely, if ever, could be the subject same chemicals may cause small-cell carcinoma of the colon). See of a successful tort action. The available evidence is sufficient to generally Poulter, supra note 38, at 227 (“it is not uncommon for support a finding that they probably cause nontrivial injuries of some plaintiffs’ experts to assert that evidence that a substance causes any types, but it is insufficient to support a finding that they probably cancer is evidence that it can and has caused other cancers. Although caused any particular injury.”). substances that are discovered to cause one type of cancer may cause 62. For example, in Christophersen v. Allied-Signal Corp., 939 F.2d other types of cancer as well, that possibility does not permit a pre- 1106 (5th Cir. 1991) the plaintiff argued that evidence showing de- diction of what those other cancers, if any, are likely to be.”) (foot- fendant’s chemicals can cause small-cell carcinoma of the lung may notes omitted). But see Donaldson v. Central Ill. Pub. Serv. Co., 767 be relevant to show chemicals can also cause small-cell carcinoma of N.E.2d 314, 327 (Ill. 2002) (Illinois permits extrapolation between the colon based “on the nature of the biochemical reaction that re- similar but not identical cause and effect relationships in the “limited sults in the development of small cell carcinoma.” Id. at 1116 n.10. instances” where science unable to directly establish cause of disease). The court ruled that the plaintiff’s expert had failed to adequately 59. E.g., Glastetter v. Novartis Pharmaceuticals Corp., 252 F.3d 986, substantiate this connection, but microarray data may be able to 990 (8th Cir. 2001) (“Even minor deviations in molecular structure show that a particular type of cancer, e.g., small-cell carcinoma, in can radically change a particular substance’s properties and propen- two different tissues is (or is not) caused by the same molecular sities.”); Amorgianos v. National Rd. Passenger Corp., 137 F. Supp. events, and thus perhaps the same chemical agent. 2d 147, 190 (E.D.N.Y. 2001) (too great of an “analytical gap” be- 63. 25 S.W.2d 280 (Tex. Ct. App. 2000). tween plaintiff’s short-term exposure to xylene and studies relied on by plaintiff’s experts involving longer term exposure to other sol- 64. Id. at 288. 1-2003 NEWS & ANALYSIS 33 ELR 10077 Copyright © 2003 Environmental Law Institute®, Washington, DC. reprinted with permission from ELR®, http://www.eli.org, 1-800-433-5120. The court held that the plaintiff’s expert must demonstrate Plaintiffs generally try to overcome this specific causa- “that all types of leukemia are related or interchangeable,” tion hurdle using one of two approaches.70 The statistical which the expert attempted to demonstrate by alleging that approach attempts to show that exposure to defendant’s benzene causes a common genetic mutation in bone marrow agent more than doubles the background risk of the health cells that can result in all types of leukemia.65 The court con- effect at issue, i.e., relative risk >2.0, thus making it sta- cluded that the expert failed to adequately support his argu- tistically more likely than not that the plaintiff’s health ment that all types of leukemia derive from a common ge- effect was caused by defendant’s product.71 This approach netic mutation.66 has two shortcomings. First, relatively few toxic agents Gene expression data might have provided the missing double background risk, especially for health effects that link required by the court to treat AML and CML as related. are relatively common in the general population.72 Second, If microarray data were available showing that benzene some commentators have argued that even if a plaintiff causes the same changes in gene expression in patients who demonstrates a doubling of background risk, such purely ultimately developed either AML or CML, a persuasive statistical evidence is insufficient to establish specific cau- case could be made that the two types of leukemia shared a sation in the absence of “particularized” data relating to common mechanism. Alternatively, DNA microarrays the specific plaintiff, although few courts have adopted could be used to compare the DNA mutations in patients this suggestion.73 with AML and CML, rather than their gene expression pro- The second approach for establishing specific causation files.67 Microarray evidence that AML and CML tumors is through differential diagnosis, in which a physician rules contain similar mutations would also be probative of a com- out other known possible causes of the health effect based mon mechanism. If microarray data can provide such a mo- on the case history and other clinical evidence.74 The courts lecular link between AML and CML, the available epidemi- have been inconsistent on whether and under what circum- ology studies showing that benzene increased the incidence stances they will allow differential diagnosis evidence to be of AML would therefore arguably be relevant to whether introduced to prove specific causation.75 Some courts have benzene can cause CML. Alternatively, if benzene exposure held that expert testimony based on differential diagnosis results in different patterns of gene expression changes in AML and CML patients, the defendant’s case would be 70. For a few diseases, such as mesothelioma caused by asbestos or clear strengthened, because such a finding would indicate that cell adenocarcinoma caused by the drug DES, almost every case of benzene does not cause a similar response in patients who disease is caused by only one known cause, in which case the spe- develop AML and CML. In that situation, the question of cific causation inquiry becomes elementary. Such examples of “signature” diseases are rare. See Daniel A. Farber, Toxic Causation,71 whether benzene indeed does cause CML would require di- Minn. L. Rev. 1219, 1251-52 (1987). rect evidence of such a causal relationship, and could not be 71. E.g., Daubert v. Merrell Dow Pharmaceuticals, Inc., 43 F.3d 1311, based on the AML findings. 1321, 25 ELR 20856, 20860 (9th Cir. 1995); Hall v. Baxter Health- care Corp., 947 F. Supp. 1387, 1403-04 (D. Or. 1996); Allison v. Specific Causation McGhan Med. Corp., 184 F.3d 1300, 1315 (11th Cir. 1999). Other courts have rejected the requirement to show a doubling of relative risk. See generally Russellyn S. Carruth & Bernard D. Goldstein, The second step of the causation inquiry is specific causa- Relative Risk Greater Than Two in Proof of Causation in Toxic Tort tion. While general causation refers to the question of Litigation,41Jurimetrics 195 (2001). whether an agent can cause the disease from which the 72. See Frederica P. Perera, Environment and Cancer: Who Are Suscep- plaintiff suffers, specific causation asks whether the agent tible?, 278 Science 1068, 1072 (1997) (“In epidemiology, it has 68 been difficult to detect relative risks of 1.5 or even 2.0.”); Gary did in fact cause the disease in that specific individual. Taubes, Epidemiology Faces Its Limits, 269 Science 164, 165 Proving specific causation is perhaps the most formidable (1995) (noting only a handful of carcinogenic agents have produced challenge facing a plaintiff, because human or animal stud- relative risk greater than two in epidemiology studies). ies of toxic risks evaluate the overall rate of disease in an 73. In re Agent Orange Prod. Liab., 597 F. Supp. at 835 (describing (but not adopting) the requirement for particularistic evidence in addition exposed group versus a control population, but generally to a relative risk of at least two as the “strong” version of the prepon- have no way of discerning which individuals in the ex- derance rule); Michael Dore, A Commentary on the Use of Epidemi- posed group developed the disease from the toxic exposure ological Evidence in Demonstrating Cause-in-Fact,7Harv. as opposed to background factors. As one court succinctly Envtl. L. Rev. 429, 434 (1983) (epidemiological evidence alone cannot establish causation). stated it, “science cannot tell us what caused a particular 69 74. See Glastetter v. Novartis Pharmaceuticals Corp., 252 F.3d 986, 989 plaintiff’s injury.” (8th Cir. 2001): In performing a differential diagnosis, a physician begins by 65. Id. “ruling in” all scientifically plausible causes of the plaintiff’s injury. The physician then “rules out” the least plausible 66. Id. at 290-91. causes of injury until the most likely cause remains. The final 67. See supra note 9. result of a differential diagnosis is the expert’s conclusion that a defendant’s product caused (or did not cause) the plain- 68. See Merrell Dow Pharmaceuticals, Inc. v. Havner, 953 S.W.2d 706, tiff’s injury. 714 (Tex. 1997) (“specific causation is whether a substance caused a particular individual’s injury”). 75. See Joseph Sanders & Julie Machal-Fulks, The Admissibility of Dif- ferential Diagnosis Testimony to Prove Causation in Toxic Tort 69. Id. at 715. See also In re Agent Orange Prod. Liab. Litig., 597 F. Cases: The Interplay of Adjective and Substantive Law,64Law & Supp. 740, 834 (E.D.N.Y. 1984) (“it may be impossible to pinpoint Contemp. Probs. 107, 1201-29 (2001) (reviewing case law); Mar- which particular person’s cancer would have occurred naturally and garet A. Berger, The Supreme Court’s Trilogy on the Admissibility which would not have occurred but for exposure to the substance”); of Expert Evidence, in Federal Judicial Center, Reference Gold, supra note 36, at 379 (“Cancers and mutations provide no Manual on Scientific Evidence 9, 34 (2d. ed. 2000) (“Judges physical evidence of the inducing agent, so direct observation of in- disagree on whether a physician relying on the methodology of dividual plaintiffs provides little or no evidence of causation in clinical medicine can provide adequate proof of causation in a toxic many instances.”). tort action.”). 33 ELR 10078 ENVIRONMENTAL LAW REPORTER 1-2003 Copyright © 2003 Environmental Law Institute®, Washington, DC. reprinted with permission from ELR®, http://www.eli.org, 1-800-433-5120. can be used to establish specific causation,76 but other courts able direct evidence of specific causation in toxic tort cases, have been more skeptical of this approach.77 there may no longer be a need to evaluate general causation, Given the limitations of the two existing approaches for as direct evidence that a particular substance did or did not in establishing specific causation, there is likely to be signifi- fact cause a given plaintiff’s illness moots the issue of cant potential and interest in the use of DNA microarrays for whether the substance is capable of causing such disease.83 proving that a particular agent did or did not cause the dis- Toxicogenomic data thus offers the potential of an unprece- ease process in an individual plaintiff. At least two potential dented advance in directly demonstrating causation or the applications of microarrays are relevant to specific causa- lack thereof in toxic tort cases. tion. First, a plaintiff could assay for gene expression changes in his or her cells that are characteristic of the spe- Recovery for Latent Risks cific toxic agent associated with the defendant. Here, the types of gene expression changes that would be most rele- In recent years, plaintiffs exposed to hazardous substances vant are not those of the initial cellular response to exposure frequently seek recovery for their latent risks that have not to the toxic agent, but rather the subsequent gene expression yet manifested into clinical disease. Such claims usually changes that are typical of the developing disease process. seek damages for the increased risk of future disease as well Preliminary studies have demonstrated that different toxic as recovery for the present fear associated with the increased compounds produce a “unique expression profile.”78 risk. To prevent a flood of latent risk claims, yet at the same A second potential use of DNA microarrays would be to time providing the possibility of recovery for the most com- assay not for changes in gene expression, but rather for pelling claims, courts have imposed stringent threshold re- changes in the DNA sequence that represent chemical-spe- quirements for such claims.84 For example, most courts re- cific mutations in genes relevant to the disease process.79 quire proof of a “present injury” for increased risk and fear For example, the p53 tumor suppressor gene is mutated in of disease claims,85 as well as a demonstration (and often over 50% of human tumors, and several important human quantification) of a sufficient quantum of increased risk.86 carcinogens appear to induce chemical-specific “mutational Most plaintiffs exposed to hazardous substances are unable fingerprints” at precise sites in the p53 gene.80 Microarrays to meet these threshold requirements, at least with the types could be used to detect these specific mutations in a plaintiff of scientific evidence presently available.87 with cancer, and used to establish specific causation by showing that the p53 mutation in their tumor was character- 81 There would appear to be little harm in retaining the require- istic of the specific agent produced by the defendant. Con- ment for “particularistic” evidence of causation in sporadic versely, the absence of such biomarkers would support the accident cases since such evidence is almost always available argument of defendants that there was no specific causation. in such litigation. In mass exposure cases, however, where By allowing scientists to “peer” inside cells and look for the chance that there would be particularistic evidence is in chemical-specific genetic markers of disease processes, most cases quite small.... whether they be changes in gene expression or mutational 83. Similarly, there is no general causation requirement in most traumatic injury cases because the general propensity of the technology spectra, toxicogenomics offers to provide the first direct evi- or action involved is beyond dispute, and the only contested issue is dence of specific causation. The lack of such direct evidence whether it did cause the injury in the specific case. See American explains in large part why toxic tort cases have generally Law Institute, Restatement of the Law Torts: Liability for been much more controversial and difficult than traditional Physical Harm (Basic Principles), Tentative Draft No. 2 §28, at 102 (2002) (“In cases involving traumatic injuries, such as a personal injury cases involving traumatic injury, such as au- broken bone following an automobile accident, the absence of other tomobile accident cases, where the issue of “specific causa- causal sets and better understanding of the causal mechanisms in- tion” is obvious.82 If toxicogenomic data can provide reli- volved moots the necessity for independent proof of general causation beyond the ‘specific causation’ evidence in the case.”). 76. E.g., Westberry v. Gislaved Gummi AB, 178 F.3d 257, 262-66 (4th 84. See, e.g., Metro-North Commuter R.R. Co. v. Buckley, 521 U.S. Cir. 1999); Turner v. Iowa Fire Equip. Co., 229 F.3d 1202, 1208 (8th 424, 433 (1997) (courts generally deny recovery for latent risks Cir. 2000); McCullock v. H.B. Fuller, 61 F.3d 1038, 1043-44 (2d because of policy considerations including: (i) preventing defen- Cir. 1995). dants from being subjected to “unlimited and unpredictable lia- 77. E.g., Daubert v. Merrell Dow Pharmaceuticals, Inc., 43 F.3d 1311, bility”; (ii) protecting courts from having to sift through meritorious 1319, 25 ELR 20856, 20859 (9th Cir. 1995); Glastetter, 252 F.3d at versus frivolous claims; and (iii) avoiding a “flood of comparatively 989-92; Meister v. Medical Eng’g Corp., 267 F.3d 1123, 1129, 1131 unimportant claims”). (D.C. Cir. 2001). 85. E.g., Adams v. Johns-Manville Sales Corp., 783 F.2d 589, 591-93 78. See, e.g., Waring et al., supra note 29, at 367. See also generally su- (5th Cir. 1986); Anderson v. W.R. Grace & Co., 628 F. Supp. 1219, pra note 29 and accompanying text. 1226-27, 16 ELR 20577, 20579-80 (D. Mass. 1986). 79. See supra note 9. 86. E.g., Ayers v. Township of Jackson, 525 A.2d 287, 308, 17 ELR 20858, 20862 (N.J. 1987) (rejecting claim for unquantified en- 80. S. Perwez Hussain et al., Tumor Suppressor Genes: At The Cross- hanced risk of disease because of speculative nature of unquantified roads of Molecular Carcinogenesis, Molecular Epidemiology, and Lung Cancer risk); Abuan v. General Elec. Co., 3 F.3d 329, 334 (9th Cir. 1993) Human Risk Assessment,34 S7 (2001); Ian C. (recovery for increased risk only where plaintiff shows that toxic ex- Semenza & Lisa H. Weasel, Molecular Epidemiology in Environ- posure will more likely than not result in disease); Gideon v. Johns- mental Health: The Potential of Tumor Suppressor Gene p53 as a Envtl. Health Persp. Manville Sales Corp., 761 F.2d 1137-38 (5th Cir. 1985) (increased Biomarker, 105 (Suppl. 1) 155, 155-56 risk of cancer must be more likely than not to occur for claim to (1997). be recognized). 81. See Gary Marchant, Genetics and Toxic Torts,31Seton Hall L. Rev. 87. See, e.g., Brafford v. Susquehanna Corp., 586 F. Supp. 14, 18 (D. 949, 971-72 (2001). Col. 1984) (“the inability to precisely quantify the extent of present 82. See Daubert, 43 F.3d at 1320 n.13, 25 ELR at 20860 n.13 (“unfair- damage to the chromosomes is a function of medical technology’s ness is inevitable when our tools for detecting causation are imper- inability to make such a measure”); Andrew R. Klein, A Model for fect and we must rely on probabilities rather than more direct Enhanced Risk Recovery in Tort,56Wash. & Lee L. Rev. 1173, proof”); In re Agent Orange Prod. Liab. Litig., 597 F. Supp. 740, 836 1179 (1999) (threshold requirements imposed by courts create “a (E.D.N.Y. 1984): nearly insurmountable barrier for enhanced risk plaintiffs”). 1-2003 NEWS & ANALYSIS 33 ELR 10079 Copyright © 2003 Environmental Law Institute®, Washington, DC. reprinted with permission from ELR®, http://www.eli.org, 1-800-433-5120. Gene expression data may assist plaintiffs in appropriate courts and legislatures) are likely to be confronted with the cases to demonstrate both an existing injury and a sufficient difficult question of whether to allow recovery for a much increase in risk to trigger recovery. By providing a highly larger number of latent risk claims that meet the existing sensitive and specific assay of toxicological response at the threshold requirements using toxicogenomic data.92 molecular level, microarrays may demonstrate subcellular effects that may qualify as a “present physical injury” in at Medical Monitoring least those jurisdictions that permit asymptomatic conditions to satisfy the present injury requirement.88 Other juris- Courts in at least 17 states and the District of Columbia dictions require symptomatic disease to satisfy the present have recognized claims for medical monitoring, in which injury requirement, motivated in large part by the difficulty exposed at-risk plaintiffs can recover for future periodic in objectively proving alleged subcellular injuries.89 Even in medical tests intended to detect the onset of latent diseases those jurisdictions, a plaintiff that can objectively show resulting from exposure to toxic substances.93 The precise gene expression changes that have been validated as a reli- formulation of the standard for awarding medical monitor- able marker of developing toxicological injury may be able ing costs varies somewhat between different States, but to assert a credible argument for relaxing the legal insistence most courts have required that plaintiffs must suffer from on symptoms to establish present injury. Likewise, gene ex- an increased risk of contracting a serious latent disease as a pression data may provide objective quantitative evidence proximate result of defendant’s negligent act, that this in- of increased risk, which, if of adequate magnitude, could creased risk makes periodic diagnostic medical examina- satisfy the other pre-condition for recovery for latent risks tions reasonably necessary, and that monitoring and diag- which is that the plaintiff demonstrate a sufficiently en- nostic methods exist that make early detection and treat- hanced risk. ment of the disease both possible and beneficial.94 Unlike The issue of whether and when to allow recovery for la- increased risk and fear of disease claims, medical monitor- tent risks has been described as the most difficult problem confronting toxic torts.90 Most jurisdictions are still grap- mutations throughout their lives that would eventually progress to pling with this issue, often seeking to balance the competing cancer if the person lived indefinitely. Robert A. Weinberg, One policy considerations for and against recovery for latent Renegade Cell: How Cancer Begins 156 (1998) (“given risks by imposing restrictive threshold requirements that un- enough time cancer will strike every human body”); Donald T. Ramsey, The Trigger of Coverage for Cancer: When Does Genetic til now have excluded most latent risk claims. Toxico- Mutation Become “Bodily Injury, Sickness, or Disease?,” 41 Santa genomics offers a tool of unprecedented power for satisfy- Clara L. Rev. 293, 298 (2001) (“even people who die from some ing the evidentiary requirements for latent risk claims, and other cause, before they can develop cancer, carry many thousands will likely make recovery for latent risk both more and less of cells bearing mutations to key genes throughout most of their exis- tence”). Except for those caused by random copying errors, these problematic. It will be less problematic to the extent that mutations will have been caused by some exogenous or endogenous toxicogenomic data provide some objective, scientific evi- agent. New genetic technologies that make possible the detection of dence of future risk that can better inform cases that are to- these mutations, and perhaps their cause, would therefore create the dilemma that every person could be classified as injured if such day litigated with almost complete ignorance of an individ- mutations are determined to be a “present injury.” See id. at 329 ual’s actual future risk. On the other hand, gene expression (“according to the view that brands the mere initiation of a DNA assays may bring to fruition the fears that latent risk claims mutation in a cell as injury, everyone is injured all the time.”). See could flood the courts with an almost unlimited number of also Andrew R. Klein, Fear of Disease and the Puzzle of Futures 91 Cases in Tort,35U.C. Davis L. Rev. 965, 966 n.2 (2002) (collect- new, asymptomatic litigants. Legal decisionmakers (both ing statistics on large percentages of population who have been exposed to various toxic agents); Arvin Maskin et al., Medical Moni- 88. For example, in Anderson, 628 F. Supp. at 1226-27, 16 ELR at toring: A Viable Remedy for Deserving Plaintiffs or Tort Law’s 20579-80, the court required plaintiffs seeking to recover for emo- Most Expensive Consolation Prize? 27 Wm. Mitchell L. Rev. tional distress associated with an increased risk from toxic exposure 521, 528 (2000) (listing toxic exposures which most Americans to demonstrate a present injury “manifested by objective have experienced). symptomatology,” and held that subcellular injuries could meet this standard provided they were “objectively evidenced.” See also 92. The current controversy over latent risk claims relating to asbestos Brafford, 586 F. Supp. at 17-18 (denying summary judgment against exposure gives a flavor of the difficult issues to be faced by the pro- plaintiff who relied on an inference that he must have incurred sub- liferation of latent risk claims. See, e.g., James A. Henderson Jr. & cellular chromosomal damage from radiation exposure); Bryson v. Aaron D. Twerski, Asbestos Litigation Gone Mad: Exposure-Based Pillsbury Co., 573 N.W.2d 718, 720-21 (Minn. Ct. App. 1998) Recovery for Increased Risk, Mental Distress, and Medical Moni- (asymptomatic, subcellular injury may constitute a legally recog- toring,53S.C. L. Rev. 815 (2002) (discussing how the “massive, nized present injury). never-ending que of claimants” litigating latent risk claims for as- 89. See, e.g., Dodge v. Cotter Corp., 203 F.3d 1190, 1202 (10th Cir. bestos exposure has “become a tragic chapter in American jurispru- 2000) (requiring evidence of “a chronic objective condition caused dence” and “will remain so unless courts put an end to the mad- by their increased risk of developing cancer” to permit recovery for ness”). Even some plaintiffs’ counsel are now advocating restricting emotional distress damages); In re Hawaii Fed. Asbestos Cases, 734 claims for unimpaired plaintiffs who have been exposed to asbestos F. Supp. 1563, 1567 (D. Haw. 1990) (requiring “an objectively veri- because their latent risk claims are consuming a disproportionate fiable functional impairment”); Schweitzer v. Consolidated Rail share of the available funds from bankrupt defendants. Mark P. Goodman et al., Plaintiffs’ Bar Now Opposes Unimpaired Asbestos Corp., 758 F.2d 936, 942 (3d Cir. 1985) (subclincial injury insuffi- Nat’l L.J. cient for recovery). Suits, , Apr. 1, 2002, at B14, B15; Alex Berenson, A Surge in Asbestos Suits, Many by Healthy Plaintiffs, N.Y. Times, Apr. 10, 90. Geoffrey C. Hazard, The Futures Problem, 148 U. Pa. L. Rev. 2002, at A1, C4. 1901, 1901 (2000) (“Perhaps the most difficult problem in address- ing mass torts is that of future claimants.”); Richard W. Wright, Cau- 93. See Badillo v. American Brands, Inc., 16 P.3d 435, 438-39 (Nev. sation, Responsibility, Risk, Probability, Naked Statistics, and 2001) (surveying case law). Proof: Pruning the Bramble Bush by Clarifying the Concepts,73 Iowa L. Rev. 94. See, e.g., In re Paoli R.R. Yard PCB Litig., 916 F.2d 829, 852, 21 1001, 1067 (1988) (liability for risk exposure is “the cert. denied most problematic area of current tort practice”). ELR 20184, 20196 (3d Cir. 1990), , 499 U.S. 961 (1991); Hansen v. Mountain Fuel Supply Co., 858 P.2d 970, 979 91. For example, approximately one-third of all humans will contract (Utah 1993); In re Asbestos Cases, 265 F.3d 861, 866 (9th Cir. cancer at some point in their lives, and every person incurs numerous 2001). 33 ELR 10080 ENVIRONMENTAL LAW REPORTER 1-2003 Copyright © 2003 Environmental Law Institute®, Washington, DC. reprinted with permission from ELR®, http://www.eli.org, 1-800-433-5120. ing claims in most jurisdictions do not require proof of pres- Enhancing Risk Assessment ent injury.95 Plaintiffs may soon seek medical monitoring expenses to Several major uncertainties limit the confidence in and util- conduct gene expression assays on exposed individuals at ity of risk assessment for informing regulatory decisions.99 increased risk of developing future disease. Such assays, if These uncertainties include extrapolating from animal re- properly validated, may provide a much more reliable as- sults to humans, extrapolating from high-dose experimental sessment of pre-clinical disease progression in such indi- results to more typical low-dose human exposures, under- viduals, in many cases perhaps leading to timely medical standing the mechanism of action of a toxicant and its impli- intervention. By providing a more sensitive and specific cations for risk assessment, determining the shape of the diagnosis of the disease process before it manifests into dose-response curve, and estimating the exposure levels for clinical symptoms, gene expression assays have the poten- actual human populations.100 Gene expression data may tial to greatly expand the number of valid medical monitor- help to overcome many of these limitations.101 ing claims as well as to prevent or mitigate many new dis- In the summer of 2002, the U.S. Environmental Protec- ease cases. tion Agency (EPA) issued an Interim Policy on Genomics On the other hand, microarrays may have the potential to which stated that “EPA believes that genomics will have an produce too much information and too many putative enormous impact on our ability to assess the risk from expo- plaintiffs. Every American has certainly been exposed to sure to stressors and ultimately to improve our risk assess- toxic substances in some form or level, whether it be from ments.”102 EPA’s Interim Policy states that genomics can be living near a hazardous waste site or polluting facility, ex- used “to explore the possible link between exposure, mech- posure to pesticides, inhaling second-hand smoke, or using anism(s) of action, and adverse effects,” and may also be products with hazardous constituents.96 Microarrays may useful to EPA “in setting priorities, in ranking of chemicals for the first time provide the scientific capability to directly for further testing, and in supporting possible regulatory ac- monitor molecular changes in the exposed population. As tions.”103 While the Interim Policy states that genomic data mentioned above, most courts do not require plaintiffs to may be considered in current regulatory decisions, it cau- demonstrate a significant quantum of increased risk as a tions that for at least the time being, such data alone are “in- threshold to recover medical monitoring damages.97 To the sufficient as a basis for decisions,” and “EPA will consider extent that gene expression changes resulting from toxic genomics information on a case-by-case basis.”104 exposures meet the criteria for medical monitoring tests, There are several potential ways toxicogenomic data can every person could conceivably be entitled to medical improve risk assessment. First, gene expression data, by monitoring damages.98 Hence, the scientific expansion of providing a characteristic “fingerprint” of different toxico- the capability to test for disease development may force logical mechanisms,105 can be used to characterize the the legal contraction of the right to recover for the costs of mechanism or mode of action of a toxicant.106 Regulatory such testing. 99. See, e.g., Donald T. Hornstein, Reclaiming Environmental Law: A Normative Critique of Comparative Risk Analysis,92Colum. L. Toxicogenomic Applications for Environmental Rev. 562 (1992); Mark Eliot Shere, The Myth of Meaningful Risk Regulation Assessment,19Harv. Envtl. L. Rev. 409 (1995). 100. National Research Council, Risk Assessment in the Fed- The revolutionary impact of toxicogenomics for the science eral Government: Managing the Process 29-33 (1983) (listing of toxicology will translate into equally fundamental over 50 uncertainties in risk assessment) 101. See generally Bishop et al., supra note 20, at 986-987; Henry et al., changes in regulatory risk assessment and decisionmaking. supra note 44, at 10478 (increased understanding from toxico- Some of these transitions have already begun. Several po- genomic data could reduce need to apply default uncertainty factors tential applications of toxicogenomic data to environmental in risk assessment). regulation are discussed below. 102. U.S. EPA, Science Policy Council, Interim Policy on Genomics (2000) at 1, available at http://epa.gov/osp/spc/genomics. pdf. See also Pat Phibbs, EPA Scientist Expects Genomic Informa- tion to Improve Analyses of Chemical Effects, Daily Env’t Rep. 95. See Maskin et al., supra note 91, at 532-33 (most courts have re- (BNA), Nov. 8, 2001, at A2 (reporting on presentation by high-rank- quired plaintiffs to demonstrate exposures that have increased their ing EPA scientist claiming that EPA has already evaluated or is de- risks, but impose no requirement with respect to the quantification or veloping gene expression data from exposure to dioxins, formalde- magnitude of such increased risk). For example, in the landmark hyde, disinfection byproducts, phthalates, and other chemicals); medical monitoring case of Ayers v. Jackson Township, 525 A.2d John C. Rockett & David J. Dix, Application of DNA Arrays to Toxi- 287, 312 (N.J. 1987), the New Jersey Supreme Court held that plain- cology, 107 Envtl. Health Perspect. 681, 681 (1999) (two EPA tiffs could recover medical monitoring costs, even though the lower scientists write that “[EPA] is interested in applying DNA array court had found that plaintiffs’ experts could not exclude the possi- technology to ongoing toxicologic studies.”). bility that plaintiffs’ increased risk was “so microscopically small 103. Interim Policy, supra note 102, at 2. as to be meaningless.” See also Badillo v. American Brands, Inc., 16 P.3d 435, 441 (Nev. 2001) (surveying the requirements of dif- 104. Id. ferent states). 105. See supra note 29 and accompanying text. 96. See supra note 91. 106. See Aardema & MacGregor, supra note 4, at 16-17; Interim Pol- icy, supra note 102, at 3 (genomics will “likely provide a better un- 97. See supra note 95. derstanding of the mechanism or mode of action of a stressor and 98. See, e.g., Metro-North Commuter R.R. Co. v. Buckley, 521 U.S. thus assist in predictive toxicology, in the screening of stressors, and 424, 442 (1997) (“tens of millions of individuals may have suffered in the design of monitoring activities and exposure studies”). While exposure to substances that might justify some form of substance- “mode of action” and “mechanism” are often used interchangeably, exposure-related medical monitoring”). The potential for a flood the former involves a more generalized level of knowledge than the of new claims would be particularly pronounced in those jurisdic- latter. “Mode of action” refers to the critical events caused by a par- tions that recognize medical monitoring as a separate cause ticular substance that leads to toxicity, whereas “mechanism” refers of action that can be brought in the absence of any other injury to the precise molecular changes involved in the toxicity response. or claim. See Robert J. Golden et al., Chloroform Mode of Action: Implica- 1-2003 NEWS & ANALYSIS 33 ELR 10081 Copyright © 2003 Environmental Law Institute®, Washington, DC. reprinted with permission from ELR®, http://www.eli.org, 1-800-433-5120. agencies such as EPA have recently focused on mode of ac- low doses.112 A finding that gene expression changes char- tion as a central factor in risk assessment, because this infor- acteristic of the carcinogenic response at high doses are also mation is critical for estimating the shape of the dose-re- observed in low-dose groups, even though those low-dose sponse curve, extrapolating of results from animals to hu- animals may not develop tumors, may indicate that low- mans, and deciding whether or not the agent is likely to ex- dose exposures present a carcinogenic risk in large popula- hibit a threshold below which there is no significant toxic- tions. Alternatively, the absence of any characteristic gene ity.107 Gene expression assays “provide a new and powerful expression response in low-dose animals may suggest that way of determining the mode of action,” in that “association the carcinogenic response only occurs at high doses.113 of a given toxic endpoint (e.g. carcinogenicity, Third, comparing gene expression changes in rodent and genotoxicity, hapatoxicity) with a particular pattern of human cells after a similar exposure may provide informa- gene/protein expression . . . may provide a ‘fingerprint’ that tion on the relevance of rodent tumor responses for human is characteristic of a specific mechanism of induction of that health risk.114 Most toxicology studies are necessarily con- toxicity.”108 Indeed, DNA microarray analyses have already ducted in animal species, and the extrapolation of animal re- been used to identify unique mechanistic pathways through sults to humans raises an additional important element of which the body reacts to certain classes of toxic expo- uncertainty in risk assessment. While most chemicals that sures.109 cause cancer in mice or rats are also carcinogenic in hu- Second, gene expression data will be useful in extrapolat- mans, there are now a number of examples where an agent ing results obtained in animal and epidemiology studies that causes toxicity in rodents but not humans, or humans but not typically involve high-dose levels to lower doses more rele- rodents.115 By providing a quick and inexpensive test of vant for the general human population.110 Until now, low- whether a chemical is causing a similar response in rodents dose effects have generally been refractory to empirical and humans, gene expression assays can help detect and analysis, and risk assessors have had to rely on models to ex- prevent what would otherwise be false positives for chemi- trapolate results from high- to low-dose levels.111 For exam- cals that cause toxicity in rodents but not humans, and false ple, the risks of low-dose ionizing radiation have long been a negatives for chemicals that cause toxicity in humans but subject of controversy, but direct testing of the dose-re- not rodents.116 Gene expression profiling can thus provide a sponse relationship at low doses has been beyond the reach of existing toxicological methods. Direct detection of low- 113. See Farr & Dunn, supra note 6, at 1 (“measurement of gene expres- dose effects from ionizing radiation and other agents by as- sion may allow us to identify threshold concentrations below which sessing gene expression changes will provide much needed there is little health risk”) information to better characterize and quantify risk levels at 114. See Aardema & MacGregor, supra note 4, at 19-20. The Presidential/Congressional Commission on Risk tions for Cancer Risk Assessment,26Reg. Toxicology & Pharma- 115. See cology Assessment and Risk Management,2Risk Assessment and 142, 143 (1997) (“The mode of action for a carcinogenic Risk Management in Regulatory Decision-Making, Final substance refers to the primary obligatory step(s) in the carcinogenic Report process (e.g., DNA reactivity resulting in mutations), whereas the 64 (1997) (while the results of most animal studies were mechanism of action refers to the myriad of primary and secondary found to be relevant to humans, “some chemicals elicit tumors in ro- effects, interactions, and biochemical alterations that can occur in dents only through mechanisms or at doses that have been clearly conjunction with chemical carcinogenesis.”) (citations omitted). demonstrated to be very different from mechanisms and exposures in humans”); E. Dybing et al., Hazard Characterisation of Chemi- 107. U.S. EPA, Proposed Guidelines for Carcinogen Risk Assessment, cals in Food and Diet: Dose Response, Mechanisms, and Extrapola- 61 Fed. Reg. 17960, 17980-81 (Apr. 23, 1996); Lewis L. Smith, Key tion Issues,40Food & Chem. Toxicology 237, 259 (2002) (listing Challenges for Toxicologists in the 21st Century,22Trends in examples). For example, saccharin causes bladder tumors in rats by a Pharmacol. Sci. 281, 282 (2001) (“It is increasingly recognized species-specific mechanism that does not apply to humans, and that an understanding of the mechanism of toxicity of a chemical in based on these findings saccharin has recently been delisted as a hu- experimental animals, together with a knowledge of the biochemis- man carcinogen. See National Toxicology Program, 9th Re- try and physiology of humans, provides a more reliable basis to pre- port on Carcinogens B3-B4 (2000) (explaining decision to delist dict whether a chemical is likely to prove harmful.”). saccharin as reasonable anticipated to be a carcinogen). 108. Aardema & MacGregor, supra note 4, at 17. 116. For example, one recent study compared gene expression changes in 109. See, e.g., Scott A. Jelinsky & Leona D. Samson, Global Response of the livers of rodents with cancer resulting from exposure to arsenic Saccharomyces cerevisiae to an Alkylating Agent,96Proc. Nat’l with gene expression patterns in human liver cells removed by biopsy Acad. Sci. 1486, 1490 (1999) (gene expression profiling of yeast from humans exposed to arsenic. The finding that the gene expression cells following exposure to alkylating agent reveals role of protein changes in the rodent and human tissues were largely in agreement in- degradation in responding to toxic damage); Hamadeh et al., supra dicated that arsenic was producing a similar toxicological response in note 17, at 227 (microarray data provides new insights on molecular humans and rodents. Tong Lu et al., Application of cDNA Microarray mechanism of liver toxicity caused by phenobarbital); Ahmet Zeytun to the Study of Arsenic-Induced Liver Diseases in the Population of et al., Analysis of 2,3,7,8-Tetrachlorodibenzo-p-Dioxin-Induced Guizhou, China,59Toxicological Sci. 185, 190 (2001). Gene Expression Profile in Vivo Using Pathway-Specific cDNA Ar- 117. See Aardema & MacGregor, supra note 4, at 19. This use of gene ex- rays, 178 Toxicology 241 (2002) (microarray analysis provided pression data to extrapolate from animals to humans, while provid- useful information on mechanism of toxicity of dioxin based on the ing additional data for a more informed risk assessment judgment, nature of genes up- and down-regulated following exposure). will nevertheless create its own set of uncertainties and issues. For 110. See Aardema & MacGregor, supra note 4, at 18. example, how much weight should be given to the lack of a gene expression response in human cells for extrapolating a positive re- 111. The Director of the National Institute of Environmental Health Sci- sponse in animals to humans? If a particular chemical produces both ences, the nation’s preeminent center of environmental toxicological tumors and gene expression changes in mice, but no gene expression research, concedes that “[w]e have no idea what kinds of risks are changes (and no indications of carcinogenicity) in rats or human cell posed by low-dose exposures . . . because testing to this point has, out cultures, would this provide regulators sufficient confidence to con- of necessity, focused on higher exposure levels.” Kenneth Olden et clude that the chemical is likely not a human carcinogen? Con- al., A Bold New Direction for Environmental Health Research,91 Am. J. Pub. Health versely, if another chemical does not increase tumors in a chronic ro- 1964, 1965 (2001). dent study, but does induce a significant alteration in gene expres- 112. The Department of Energy’s Low-Dose Radiation Program is thus uti- sion that is characteristic of a carcinogenic response for other com- lizing gene expression data using microarrays to study empirically the pounds, should regulators disregard the absence of tumors in the ro- shape of the dose-response curve for ionizing radiation at low levels of dent study and nevertheless classify the chemical as a possible hu- exposure. See http://www.er.doe.gov/production/ober/lowdose.html. man carcinogen based solely on the gene expression changes? 33 ELR 10082 ENVIRONMENTAL LAW REPORTER 1-2003 Copyright © 2003 Environmental Law Institute®, Washington, DC. reprinted with permission from ELR®, http://www.eli.org, 1-800-433-5120. “bridging biomarker” that can connect toxicological re- certainty factor for children under the Food Quality Protec- sponses in animals and humans.117 tion Act (FQPA).125 Fourth, gene expression data will also be useful in quanti- These potential applications of gene expression data may fying human exposure, a key input to risk assessment. Some help reduce many of the most important uncertainties in risk experts consider that the limited information available on assessment, although by no means eliminating such uncer- exposure is perhaps the most serious problem afflicting risk tainties altogether. The major existing uncertainties in risk assessment.118 Without reliable data on human exposure, it assessment and the resulting controversies they have is not possible to estimate accurately the relationship be- spawned have produced significant delays in implementing tween dose and response that underlies risk assessment esti- risk-based regulation, and have resulted in a shift away from mates. By characterizing gene expression patterns in ex- risk-based standard setting in recent years to other ap- posed persons, microarrays have the potential to provide proaches such as setting standards based on the best avail- more precise quantitative estimates of exposure to specific able technology.126 Perhaps these frustrations with risk as- toxic substances in contemporaneous and prospective hu- sessments were best expressed by one prominent senator’s man studies.119 declaration during the 1990 Clean Air Act Amendments that Fifth, gene expression profiling may be particularly use- he “would be glad to declare risk assessment dead.”127 Toxi- ful for evaluating the toxicity of chemical mixtures,120 cogenomics has the potential to help restore confidence in which are the most typical human exposure scenarios, but risk assessment by reducing many of the most important un- which are hard to evaluate using traditional toxicological certainties, and may thereby open the door to a renewed em- methods.121 The combined effects of exposure to several phasis on risk-based standards, since after all it is an accept- different toxic substances present in a mixture may not be able level of risk and not best technology per se that provides additive, but may instead be greater, e.g., synergistic, or the most direct measure of what environmental regulation lesser, e.g., antagonistic, than expected from simply adding seeks to protect, i.e., human and environmental health. the predicted effects of the individual compounds.122 Be- cause DNA microarrays permit the simultaneous monitor- High Throughput Toxicity Screening of Chemicals ing of all gene expression changes within a cell in a single experiment, they “are particularly suitable to evaluate any The majority of chemicals in commercial use in the United kind of combinational effect resulting from combined expo- States have not been comprehensively tested for human tox- sure to toxicants.”123 The National Institute of Environmen- icity and carcinogenicity potential.128 Other than pharma- tal Health Sciences has made the study of mixtures its “top ceuticals and pesticides, there is no legal duty imposed on priority,” in significant part because the availability of manufacturers to pre-market test their products for toxicity. microarrays will for the first time make the toxicological as- EPA and the chemical industry have begun to address this sessment of mixtures feasible.124 data gap for chemical risk assessment with the High-Pro- Finally, gene expression assays may also provide a more duction Volume (HPV) chemical testing initiative.129 How- sensitive methodology for examining other risk assessment ever, given that there are now some 80,000 chemicals in issues such as the differential sensitivity of children versus commerce,130 it is not feasible to conduct traditional toxico- adults to specific environmental exposures. For example, if logical testing for all or even most chemicals in commerce gene expression changes show that a pesticide induces a with existing test methods. greater relative response in neonatal rodents than in adult ro- For example, the “gold standard” assay for carcinogenic- dents, there would be good reason to suspect that human children might be more susceptible to a toxic response than 126. See Wendy E. Wagner, The Triumph of Technology-Based Stan- dards, 2000 U. Ill. L. Rev. 83 (2000). For example, the 1990 Clean adults. Conversely, if there are no differences in gene ex- Air Act Amendments replace the previous risk-based approach for pression between neonatal and adult rodents after exposure regulating hazardous air pollutants with primarily a technol- to a particular pesticide, there would be less concern about ogy-based approach based on “maximum available control technol- differential susceptibility of human children, and perhaps ogy” or “MACT.” 42 U.S.C. §7412(d), ELR Stat. CAA §112(d); see Arnold W. Reitze Jr., Control of Hazardous Air Pollution,inAir grounds for not applying the default additional tenfold un- Pollution Control Law: Compliance and Enforcement 123 (Envtl. L. Inst. 2001). 118. See Olden et al., supra note 111, at 1966. 127. 136 Cong. Rec. S16895, S16932 (Oct. 27, 1990) (statement of Sen. Durenberger). 119. See Nuwaysir et al., supra note 3, at 157. 128. See Bernard D. Goldstein & Mary Sue Henifin, Reference Guide on 120. See Aardema & MacGregor, supra note 4, at 20-21; V.J. Feron & Toxicology,inFederal Judicial Center, Reference Manual J.P. Groten, Toxicological Evaluation of Chemical Mixtures,40 on Scientific Evidence Food & Chem. Toxicology Interim Policy 401, 412 (2d ed. 2000) (“less than 1% of 825, 834 (2002); , su- the 60,000-75,000 chemicals in commerce have been subjected to a pra note 102, at 3 (“Genomic analysis . . . holds promise to evaluate full safety assessment, and there are significant toxicological data on the cumulative impacts resulting from the interplay of factors such as only 10%-20%”); National Research Council, Toxicity genetic diversity, health status, and life stage in responding to expo- Testing: Strategies to Determine Needs and Priorities sures(s) to multiple stressors.”). (1984); Environmental Defense Fund, supra note 60. 121. See Feron & Groten, supra note 120, at 825 (“Mixtures are tough for 129. Under the HPV Challenge program, chemical manufacturers have everybody.”) (quoting Jonathan Samet). volunteered to collect and make public by 2005 basic toxicity and 122. Id. at 826. environmental fate data on chemicals produced or imported into the United States in volumes exceeding one million tons annually. See 123. Id. at 834. http://www.epa.gov/opptintr/chemrtk/volchall.htm. 124. See Olden et al., supra note 111, at 1966. 130. National Toxicology Program, Current Directions and 125. 21 U.S.C. §346a(b)(2)(C); Cheryl Hogue, Toxicogenomics Uses Evolving Strategies 2 (2002) (“More than 80,000 chemicals are Genes as a Toxic Screen, Chem. Eng. News, Mar. 19, 2001, at 33, registered for use in commerce in the United States, and an estimated 34 (“microarrays using DNA from babies and youngsters may shed 2,000 new ones are introduced annually for use in everyday items light on whether they are more sensitive or more resilient to pesticide such as foods, personal care products, prescription drugs, household toxicity than adults are”). cleaners, and lawn care products.”). 1-2003 NEWS & ANALYSIS 33 ELR 10083 Copyright © 2003 Environmental Law Institute®, Washington, DC. reprinted with permission from ELR®, http://www.eli.org, 1-800-433-5120. ity is the chronic rodent bioassay, in which rats or mice are genotoxic responses.137 The Director of the NIEHS predicts exposed to a potentially carcinogenic substance over a two- that by using DNA microarrays the time it takes to test po- year period, followed by a comprehensive pathological ex- tential carcinogens will be reduced from several years to “a amination.131 The largest chemical testing program in the few days,” while the costs will be reduced from millions of United States, conducted by the National Toxicology Pro- dollars to conduct a chronic bioassay “to less than $500 dol- gram of the National Institute of Environmental Health Sci- lars” to test each chemical using DNA microarrays.138 Mi- ences (NIEHS), has recently completed its 500th chronic ro- croarrays can be used to interrogate the gene expression of dent bioassay for evaluating carcinogenicity after 30 years cells either in tissue culture or in living mice or rats that of testing.132 A chronic rodent bioassay can take five or more have been treated with chemical candidates, with the re- years to complete from start to finish, and costs $2 to $6 mil- sulting gene expression profiles used to classify those lion per chemical.133 Given these time and cost require- chemicals to specific toxicological categories and to char- ments, it is simply not feasible to conduct chronic bioassays acterize their likely risks.139 It may be possible to success- for all or even most chemicals in commerce. Moreover, fully and accurately make such classifications within 24 even for those relatively few chemicals that are evaluated in hours of an initial exposure, long before any physical man- chronic bioassays, additional tests may be needed to assess ifestation of toxicity.140 other toxicological endpoints such as reproductive toxicity, In addition to providing a cheaper and quicker toxicity developmental toxicity, immunotoxicity, neurotoxicity, en- screen than existing test methods, microarrays offer several docrine disruption, and other possible effects. The Director other important advantages for toxicity screening. Because of the NIEHS recently testified to the U.S. Congress that a microarrays simultaneously monitor all changes in the ex- large number of commercial products require additional pression of all genes within a cell, all toxicological end- testing, but “we can never satisfy this testing requirement points can be evaluated in a single microarray assay, using traditional technologies.”134 whereas today separate tests are currently needed to evalu- There is a pressing need for rapid, inexpensive, and reli- ate carcinogenicity, genotoxicity, developmental toxicity, able assays that can be used to screen a large number of reproductive toxicity, immunotoxicity, neurotoxicity, and chemicals for toxicity. Genotoxicity assays and structure- endocrine disruption.141 Microarrays are also more sensi- activity relationship (SAR) analyses are currently used to tive than current methods, because they can detect immedi- screen many chemicals relatively quickly and cheaply, but ate changes within every exposed cell or organism, where- these assays are limited in their utility and predictiveness.135 as existing methods can only detect observable toxicity Alternative testing models, such as transgenic mice, are be- that develops many weeks, months, or even years after ex- ing developed to provide less expensive and more rapid as- posure, and in only some cells or organisms within the says, but these models have not yet been fully validated, and study population. moreover still involve a considerable expenditure of time Initially, gene expression assays will need to be con- and resources.136 ducted in association with traditional toxicity testing until a Gene expression assays have tremendous potential for sufficiently robust and validated data set has been accumu- providing a rapid, inexpensive and high throughput screen- lated to reliably correlate specific gene expression profiles ing of chemicals for a wide range of genotoxic and non- with particular toxicological mechanisms and endpoints.142 Used in conjunction with traditional toxicology tests, gene 131. See Elaine M. Faustman & Gilbert S. Omenn, Risk Assessment,in expression data have the potential to improve the sensitivity Casarett & Doull’s Toxicology: The Basic Science of Poi- 143 sons 83, 88 (Curtis D. Klansmen ed., 6th ed. 2001); Ernest E. and interpretability of the standard tests. Once such a da- McConnell, Historical Review of the Rodent Bioassay and Future tabase has been established, gene expression assays might Directions,21Reg. Toxicology & Pharmacology 38 (1995). replace some or all of the current toxicological screening 132. Press Release, National Institute of Environmental Health Sciences, and testing assays, or at least to narrow and select the spe- NTP Completes 500th Two-Year Rodent Study and Report; series is cific assays that are indicated by the observed gene expres- the Gold Standard of Animal Toxicology (Release #01-03, Jan. 25, 144 2001). sion pattern. 133. NIH Environmental Health Prevention Research, Prepared Testi- mony of Kenneth Olden, Director of the National Institute of Envi- 138. Press Release, National Institute of Environmental Health Sciences, ronmental Health Sciences, before the Subcomm. on Public Health, National Center for Toxicogenomics to Study Genetic Basis of Dis- Senate Comm. on Health, Education, Labor, and Pensions, at 6 (Mar. ease Caused by Environmental Pollution (Dec. 7, 2000), available at 6, 2002). http://www.niehs.nih.gov/nct/pr07de00.htm. 134. Id. See also Olden et al., supra note 111, at 1965 (“Without new, 139. See supra note 29; Thomas, supra note 6, at 1194 (initial promising high-throughput technologies, ...wewill not be able to assess the results using microarrays “open[] the door to a new era of toxicologi- toxicity of the thousands of chemicals on which there are inadequate cal testing where relatively short and inexpensive studies using tran- toxicity data.”). script expression as an endpoint allow the prioritization of untested chemicals based upon their classification”). Potential toxicity mech- 135. See Joseph Sanders, From Science to Evidence: The Testimony of Stan. L. Rev. anisms that may induce characteristic gene expression “finger- Causation in the Bendectin Cases,46 1, 19 (1993) prints” include DNA alkylation, inflammation, oxidative stress, (“Molecules with minor structural differences can produce very dif- peroxisome proliferators, estrogenic action, and many others. Id. at ferent biological effects.”); Goldstein & Henefin, supra note 39, at 1189-90. 421 (reliability of SAR “has a number of limitations”); R. Julian Preston & George R. Hoffman, Genetic Toxicology,inCasarett & 140. See, e.g., Hamadeh et al., supra note 17, at 225. Doull’s Toxicology: The Basic Science of Poisons 321 141. See Bartosiewicz et al., supra note 29, at 73. (Curtis D. Klaassen ed., 6th ed. 2001); James D. McKinney et al., The Practice of Structure Activity Relationships (SAR) in Toxicol- 142. See Aardema & MacGregor, supra note 4, at 18 (“it will be necessary ogy,56Toxicological Sci. 8 (2000). to characterize multiple classes of agents with well-defined mechanisms of action before expression profiles for new biomarkers can be 136. See Raymond W. Tennant, Evaluation and Validation Issues in the used reliably in regulatory decision-making.”). Development of Transgenic Mouse Carcinogenicity Bioassays, 106 (Suppl. 2) Envtl. Health Persp. 473 (1998). 143. See Nuwaysir et al., supra note 3, at 157. 137. See Aardema & MacGregor, supra note 4, at 17-18. 144. See Aardema & MacGregor, supra note 4, at 18. 33 ELR 10084 ENVIRONMENTAL LAW REPORTER 1-2003 Copyright © 2003 Environmental Law Institute®, Washington, DC. reprinted with permission from ELR®, http://www.eli.org, 1-800-433-5120. One possible initial regulatory application of this gene mechanism of toxicity in a single cumulative risk assess- expression technology, which would also help to build the ment.149 Toxicogenomic data may indicate which pesticides necessary database to validate gene expression data, would should be grouped together for such assessments.150 be to require companies submitting premanufacturing no- Gene expression data may also be useful for prioritizing tices (PMNs) for new chemical substances under §5 of the contaminated sites. Indeed, in the foreseeable future EPA Toxic Substances Control Act (TSCA)145 to include the re- might want to consider adding gene expression assays to its sults of a gene expression assay in their submission. Cur- hazard ranking scheme for establishing the national priori- rently, companies are not required to generate any new data ties list (NPL) under Superfund.151 Gene expression data to support PMNs; they are only required to submit relevant may also be useful in assessing risks and selecting appropri- data already in their possession.146 Requiring a manufac- ate cleanup options at individual waste sites. Many aban- turer of a new substance to conduct and submit a gene ex- doned waste disposal sites contain large quantities of soils pression assay would not be unduly burdensome, and would and sediments with moderate or low levels of contamination begin to build an experiential database of chemical-specific which present uncertain risks but very large cleanup gene expression data that would then be available to EPA. costs.152 In most cases, the primary potential hazard is to lo- Such a database would only be useful to the extent that the cal ecosystems and species rather than to human health. As- submitted data were roughly consistent in the genes and sessment of these ecological risks using standard toxicolog- methods used, and so some form of standardization of ical tests can be very expensive and highly uncertain due to microarray platforms and methods would be required be- factors such as incomplete information on the speciation of fore such a program could be implemented. metals and other toxic substances present in the material, a In addition to potential regulatory applications in the lack of data on the bioavailability and interaction of con- screening of new chemicals under statutes such as TSCA, taminants, and the limited ability to test the contaminants screening of chemicals using DNA microarrays have a num- in the most sensitive species that may be affected by such ber of other potential regulatory applications. For example, contamination.153 In the face of such uncertainties, EPA the chemicals included on the toxic release inventory (TRI) generally applies conservative default assumptions that list of reportable substances might be based at least in part will often overestimate risks and result in unnecessarily on the results of DNA microarray analyses.147 Similarly, the stringent and costly remediation.154 In other cases, however, identification of listed hazardous wastes or hazardous the risk assessment may fail to recognize synergistic inter- wastes based on the characteristic of toxicity could be based actions between different contaminants in a mixture, result- on a quick and inexpensive microarray assay evaluating ing in underestimation of risk and inadequate health and whether the waste induces a gene expression profile that is ecological protection.155 characteristic of a known toxicity mechanism.148 Under the Differential expression of stress-response and other FQPA, EPA must combine all pesticides that share the same genes known to be involved in toxicity response could be evaluated and used to rank and prioritize contaminated soils 145. 15 U.S.C. §2604, ELR Stat. TSCA §5. See Biotechnology and sediments for cleanup. High throughput, automated Deskbook 43 (Envtl. L. Inst. 2001). DNA microarray systems are being developed for the direct, Stat. TSCA Deskbook rapid, and affordable assessments of soil and sediment tox- 146. 15 U.S.C.§2604(d), ELR TSCA §5(d). See 156 (Envtl. L. Inst. 1999); Carolyne R. Hathaway et al., A Practitioner’s icity based on changes in gene expression levels. In one Guide to the Toxic Substances Control Act: Part I, 24 ELR 10207 initial test of such a system, which have been described as (May 1994). “genosensors,” the microarray system was capable of assay- 147. Section 313 of the Emergency Planning and Community ing 672 environmental samples for effects on the expression Right-To-Know Act of 1996 establishes a list of chemicals for which 157 certain facilities are required to report annual releases into the envi- of 64 different stress response genes in a two-hour period. ronment. 42 U.S.C. §11023, ELR Stat. EPCRA §313. EPA can add This represents a major advancement in the cost and speed chemicals to this list that are “known to cause or can reasonably be anticipated to cause” major types of toxicity “based on generally ac- 149. 21 U.S.C. §346a(b)(2)(D)(v). cepted scientific principles or laboratory tests.” Id. §11023(d)(2), ELR Stat. TSCA §313(d)(2). EPA has established a toxicity screen- 150. See Hogue, supra note 38, at 34. ing methodology for determining the addition or deletion of chemi- 151. EPA has developed a “Hazard Ranking System” (HRS) to determine cals from this TRI list. See U.S. EPA, Addition of Certain Chemi- which hazardous waste sites should be placed on the NPL, which is cals; Toxic Chemical Release Reporting; Community Right-to- EPA’s list of sites that are priorities from long-term evaluation and Know, 59 Fed. Reg. 61432, 61432-33 (Nov. 30, 1994). Gene expres- remediation under the Comprehensive Environmental Response, sion assays may be a quick and inexpensive addition to this screen- Compensation, and Liability Act (CERCLA). U.S. EPA, Hazard ing procedure. Ranking System; Final Rule, 55 Fed. Reg. 51532 (Dec. 14, 1990). See also Terry C. Clarke, A Practitioner’s View of the National Pri- 148. There are two ways that EPA can designate hazardous wastes—ei- Envtl. Law. ther by listing wastes by regulation based on their toxicity, or by orities List,2 57 (1995). The HRS uses a scoring sys- finding that they exhibit one or more hazardous “characteristics,” in- tem that attempts to assess the relative potential of a site to pose a cluding the “toxicity” characteristic. See generally U.S. EPA, threat to human health or the environment. Gene expression data RCRA Orientation Manual, III-3 to III-22 (EPA/530-R-00-006, from nearby species could be factored into this scoring system. June 2000). EPA conducts a screening toxicity assessment for 152. Herbert L. Frederickson et al., Towards Environmental Toxico- adding listed wastes, and tests for the toxicity characteristic using genomics—Development of a Flow-Through, High-Density DNA an assay called the Toxicity Characteristic Leaching Procedure Hybridization Array and Its Application to Ecotoxicity Assessment, (TCLP). This assay, which simply measures the leaching poten- 274 Sci. Total Env’t 137, 138 (2001). tial of wastes, has been strongly criticized. See, e.g., David Mont- 153. Id. at 139. gomery Moore, The Toxicity Characteristic Rule for Hazardous Waste Determination: Has EPA Satisfied Congress’ Mandate?,7 154. Id. Tul. Envtl. L.J. 467 (1994). Microarray analysis of gene ex- 155. Id. pression changes in test cells or organisms treated with a candi- 156. Id. date waste have the potential to substantially improve the scientific determination of both listed wastes and wastes exhibiting the 157. Id. at 141. toxicity characteristic. 158. Id. at 147. 1-2003 NEWS & ANALYSIS 33 ELR 10085 Copyright © 2003 Environmental Law Institute®, Washington, DC. reprinted with permission from ELR®, http://www.eli.org, 1-800-433-5120. of analyzing environmental samples.158 More importantly, certainty factors to the no observed adverse effect level this technology also offers a more accurate prediction of risk (NOAEL),164 or in the absence of a NOAEL, the lowest ob- than conventional toxicology methods, because it directly served adverse effect level (LOAEL).165 In the past, the assesses the toxicity of contaminated soils or sediments in a terms no observed effect level (NOEL) and lowest observed manner that eliminates critical uncertainties relating to con- effect level (LOEL) were used in many regulatory pro- taminant bioavailability, speciation of metals, and interac- grams, but these terms were modified to LOAEL and tive effect of contaminant mixtures.159 The major limitation NOAEL to require an effect to be “adverse” before it has of the genosensor is in selecting which genes to include in regulatory significance.166 If studies show that a chemical the microarray to provide the most accurate and comprehen- induces gene expression changes at levels below the ex- sive assay for toxicity that encompasses a variety of poten- isting NOAEL or LOAEL, should these changes be con- tially affected species, but notwithstanding this issue rapid sidered “adverse” and used to establish a lower NOAEL progress is being made in developing this technology and or LOAEL, which in turn will mean a more stringent RfC overcoming the remaining limitations.160 or RfD? Finally, in addition to these potential screening applica- Industry groups are understandably concerned that gene tions of microarrays by regulatory agencies, industry will be expression changes that are not adequately validated as true able to use the technology to screen potential future prod- markers of a toxic response will be used to establish more ucts for toxicity. By providing an earlier and more specific stringent reference values.167 Many changes in gene expres- biomarker of toxicity, DNA microarrays have the potential sion in response to chemical exposures will merely be an to create significant cost and time savings for product devel- adaptive response of the cell or organism to exposures that opment by screening out potentially harmful products early help the organism maintain homeostasis, or represent a tem- in the developmental cycle.161 porary aberration that would not normally progress to toxicity.168 Such adaptive responses will not be indicative of a Calculation of Reference Dose true toxicological response. It will often be difficult to distinguish whether a particular response is adverse or adap- EPA traditionally uses reference doses (RfDs) or reference tive.169 There is generally no bright line that can be used to concentrations (RfCs) that are listed in the Agency’s Inte- differentiate adverse and adaptive responses, and often grated Risk Information Systems (IRIS) in making regula- there is a continuum in which the same general category of tory decisions for noncarcinogenic chemicals.162 An RfD or response may be adverse in some circumstances and adap- RfC is defined as “[a]n estimate (with uncertainty spanning tive in others.170 Another complication is that a similar ex- perhaps an order of magnitude)” of an ongoing exposure “to 164. EPA defines a NOAEL as the “highest exposure level at which there the human population (including sensitive subgroups) that are no statistically or biologically significant increases in the fre- is likely to be without an appreciable risk of deleterious ef- quency or severity of adverse effect between the exposed population fects during a lifetime.”163 EPA attempts to set risk-based and its appropriate control; some effects may be produced at this regulatory standards for noncarcinogens at a level of expo- level, but they are not considered adverse, nor precursors to adverse sure that does not exceed the applicable RfD or RfC. In ef- effects.” Id. 165. A LOAEL is defined by EPA as the “lowest exposure level at which fect then, the RfC or RfD is used as a “safe” threshold value there are statistically or biologically significant increases in the fre- for noncarcinogenic chemicals, recognizing that there will quency or severity of adverse effects between the exposed popula- always be some uncertainty about any such value. tion and its appropriate control group.” Id. RfDs and RfCs are calculated by applying a series of un- 166. Richard W. Lewis et al., Recognition of Adverse and Nonadverse Ef- fects inToxicity Studies,30Toxicological Pathology 66, 67 (2002). 167. ECETOC, supra note 161, at 1 (“There is...thereal danger that in- 159. Id. Because the genosensor tests soil and sediment samples directly, discriminate application of these technologies will lead to the gener- unlike conventional toxicology methods which test individual con- ation of misleading data. Furthermore, the current (relative) lack stituents separately, many of the uncertainties associated with con- of reference data could easily lead to mis- or over-interpretation ventional test methods do not apply to genosensor technology. and subsequently to undue concern by regulatory agencies.”); 160. Id.; National Research Council, Bioavailability of Con- Pennie & Kimber, supra note 16, at 321 (“Where the results are taminants in Soils and Sediments: Processes, Tools, and likely to influence the derivation of No-Observed-Adverse-Ef- Applications 243 (2002) (“Using microarray techniques, it is pos- fect Levels (NOAELs) the relevance to the in vivo situation must sible to develop a sensitive and inclusive snapshot of the response of be established by correlating the observed changes with ‘classi- cells, tissues, and organisms to a contaminant without the time re- cal’ adverse effects.”). quirements, labor, or subjectivity of more traditional analyses. Vali- 168. See Dybing et al., supra note 115, at 254 (adaptive responses are dating these techniques, and increasing their practicality for specifi- “stress reactions to environmental influences whereby the organism cally assessing contaminant bioavailability from soils and sedi- tries to maintain homeostasis. Enzyme induction, changes in hor- ments, should occur in the near future.”). mone levels, and indicators of slightly altered cellular functions are examples of such adaptive responses. In many instances, these re- 161. See William D. Pennie et al., Application of Genomics to the Defini- Toxicology Letter sponses do not lead to clinically significant altered structure or function of the Molecular Basis for Toxicity, 120 tion, namely adverse reactions.”). 353, 354 (2001) (gene expression profiling “could highlight potential toxicity earlier in a new compound’s development”); European 169. Id. at 254-55 (“it will be a great challenge to clarify whether such Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC), [gene expression] changes simply represent non-adverse alterations ECETOC White Paper on Genomics, Transcript Profiling, of physiological function or if they predict impending develop- Proteomics and Metabonomics (GTPM) (Mar. 2001). ment of more serious irreversible injury, should exposure to the chemical continue”). 162. The IRIS database is available online at http://www.epa.gov/iris/in- dex.html. RfCs are used when the route of exposure for a chemical is 170. For example, the induction of increased enzyme activity, a frequent inhalation, while RfDs are used for oral routes of exposure (e.g., via response to exposure to foreign substances, “may in some situations drinking water). be present as an adaptive response without any biological significance; sometimes it may be beneficial in that it leads to more rapid 163. U.S. EPA, Integrated Risk Information System, Glossary metabolism and elimination of potentially toxic compounds; or it of IRIS Terms (rev. 1999), available at http://www.epa.gov/iris/ may be a truly adverse response in that it may lead to increases in re- gloss8.htm [hereinafter U.S. EPA IRIS Glossary]. active intermediates and thus potentiate toxic effects.” Id. at 254. 33 ELR 10086 ENVIRONMENTAL LAW REPORTER 1-2003 Copyright © 2003 Environmental Law Institute®, Washington, DC. reprinted with permission from ELR®, http://www.eli.org, 1-800-433-5120. posure may produce a response that is adverse in some indi- an “adverse effect” in a particular case, the next question is viduals but not others based on various factors affecting sus- whether EPA should adjust the traditional uncertainty fac- ceptibility, including genetics, health, weight, other toxic tors it uses to calculate the RfC or RfD based on this new exposures, age, gender, or nutritional status.171 critical adverse effect. The standard set of uncertainty fac- While some attempts have been made to define when an tors that EPA applies to calculate an RfD or RfC do not take effect is “adverse,”172 there is no single accepted defini- into account the severity of the adverse effect that defines tion.173 In a recent draft report on the process for determin- the LOAEL.177 EPA has occasionally applied on a case-by- ing RfDs and RfCs, EPA stated that “[p]rofessional judg- case basis a reduced overall uncertainty factor when the rel- ment is required to decide, on the basis of a thorough review evant “adverse effect” is of low severity, such as minor irri- of all available data and studies, whether any observed ef- tation lesions in the nasal cavity after inhalation of a chemi- fect is adverse and how the results fit with what is known cal,178 but there is no general requirement for such an adjust- about the underlying mode of action.”174 The draft report in- ment in the IRIS methodology. dicates that “biological significance” will be the critical fac- Should EPA reduce the uncertainty factors applied to cal- tor in determining whether an effect is adverse, and pro- culate an RfD or RfC based on gene expression effects to ceeds to define biological significance as “the determina- compensate for the low severity of this “adverse” effect? tion that the observed effect (a biochemical change, a func- There is a case to be made that the availability of a more sen- tional impairment, or a pathological lesion) is likely to im- sitive test to detect a toxic response provided by microarrays pair the performance or reduce the ability of an individual to should not necessarily result in more stringent standards, function or to respond to additional challenge from the but rather should be used to only provide a more precise and agent. Biological significance is also attributed to effects certain assay for characterizing whether a toxicant does in- that are consistent with steps in a known mode of action.”175 deed cause a toxic response in humans. Under this view, Under this limited available guidance, the determination EPAshould compensate for the more sensitive test by reduc- of whether a particular gene expression change is “adverse” ing the otherwise applicable uncertainty factors to compen- will require expert judgment on a case-by-case basis.176 sate for the low severity of the critical adverse effect. Again, Gene expression changes per se are unlikely to “impair the this is a new issue for which there is currently no applica- performance” of an individual, although they conceivably ble guidance. may be indicative in some cases of a reduced capability to accommodate additional exposures to the same or a similar Real-Time Surveillance toxic agent. The more relevant inquiry in most cases will be whether a specific gene expression change is consistent with In many cases, environmental risks are not discovered until a “known mode of action.” This will require validated data they manifest in human disease or death. Microarray assays showing that the particular gene expression change is a con- may provide an early warning of potentially dangerous ex- sistent biomarker for a known toxicological response. posures before adverse health effects occur by providing “a If EPA does determine that changes in gene expression is rapid means of assessing the bioavailability and potential toxicity of complex mixtures of chemicals released into the 171. Id. at 255. air and into groundwater.”179 Pre-symptomatic detection of 172. Lewis et al., supra note 166, at 68-74 (proposing multi-factor frame- hazardous exposures would permit early intervention to work for defining adverse effects); Dybing et al., supra note 115, at 254 (the National Academy of Sciences has defined an adverse ef- monitor and treat affected persons in a more timely and effect as a change in morphology, growth, development, or life span, fective manner, as well as to minimize further exposure to an impairment of the organism’s capacity to compensate for additional stress, or an increased susceptibility to additional toxic expo- 177. See George V. Alexeeff et al., Characterization of the sures). EPA defines an “adverse effect” as “[a] biochemical change, LOAEL-to-NOAEL Uncertainty Factor for Mild Adverse Effects functional impairment, or pathological lesion that affects the perfor- From Acute Inhalation Exposures,36Reg. Toxicology & Phar- mance pf the whole organism, or reduces the organism’s ability to re- macology 96 (2002); A.G. Renwick, The Use of an Additional spond to an additional environmental challenge.” U.S. EPA IRIS Safety or Uncertainty Factor for Nature of Toxicity in the Estimation Glossary, supra note 163. This definition obviously leaves much of Acceptable Daily Intake and Tolerable Daily Intake Values,22 room for subjectivity. Reg. Toxicology & Pharmacology 250 (1995); John D. Graham, Historical Perspective on Risk Assessment in the Federal Govern- 173. Lewis et al., supra note 166, at 67. ment, 102 Toxicology 29, 33 (1995): 174. U.S. EPA, Risk Assessment Forum, A Review of the Refer- ence Dose and Reference Concentration Processes, Exter- The severity of adverse effects caused by toxic agents can nal Review Draft 4-9 (May 2002), available at http://www.epa. vary from mild cases of reversible skin irritation to death. gov/ncea/raf/pdfs/RfDRfC/rfdrfcextrevdrft.pdf. Historically, the severity of the adverse health effect caused by an agent has not played an explicit role in the choice of 175. Id. safety factors, although some extremely mild biological re- 176. See Lewis et al., supra note 166, at 72 (“often the distinction between sponses have on occasion not been considered “adverse.” adverse and nonadverse effects is not clearly defined and interpretation needs scientific judgment on a case-by-case basis”); William D. 178. See Alexeef et al., supra note 177, at 103 (“The value of 3 is often Pennie et al., The Principles and Practice of Toxicogenomics: Appli- used when the adverse effect at the LOAEL is considered mild in se- cations and Opportunities,54Toxicological Sci. 277, 282 (2000): verity.”). For example, EPA reduced the standard uncertainty factors in calculating the RfC for acrylic acid “because the effect is con- It must be recognized that the interaction of xenobiotics with sidered mild.” U.S. EPA, Integrated Risk Information Sys- biological systems will in many instances result in some tem, Acrylic Acid (CASRN 79-10-7), available at http://www. changes in gene expression, even under circumstances where epa.gov/iris/subst/0002.htm (last visited Sept. 9, 2002). In Chemical such interactions are benign with respect to adverse effects. Manufacturers Association v. EPA, 28 F.3d 1259, 24 ELR 21210 The challenge again is to ensure that sound judgment and the (D.C. 1994), the D.C. Circuit rejected EPA’s reliance on the RfC for appropriate toxicological skills and experience are brought to methylene diphenyl diisocyanate in promulgating a regulation be- bear on the data generated, so that toxicologically relevant cause EPA failed to give any weight to the seriousness of the health effect upon which the RfC was based. changes in gene expression are distinguished from those that are of no concern. 179. Bartosiewicz et al., supra note 29, at 71. 1-2003 NEWS & ANALYSIS 33 ELR 10087 Copyright © 2003 Environmental Law Institute®, Washington, DC. reprinted with permission from ELR®, http://www.eli.org, 1-800-433-5120. those and other persons. Such a surveillance program could lutant effects on nonhuman organisms, such as aquatic spe- be applied to individuals living or working near a polluting cies.188 For example, one recent study demonstrated that an facility or hazardous waste site,180 or it could be applied to a estrogenic compound produced a characteristic pattern of cohort of individuals exposed to a potentially hazardous changes in the expression of estrogen responsive genes in substance such as consumers using a particular household sheepshead minnows.189 The minnow can thus be used as a product that is suspected of toxicity.181 It could also be used living sensor for the presence and effect of endocrine dis- to monitor citizens living near the site of an environmental ruptive chemicals in coastal habitats.190 Another study accident such as TMI or Bhopal. showed that characteristic changes in gene expression in A recent study demonstrated the potential of microarrays tadpoles can be detected from laboratory exposure to the to provide real-time surveillance of potentially exposed in- herbicide acetochlor prior to development of overt morpho- dividuals or populations. The study involved exposing pe- logical changes brought about by the endocrine disruption ripheral blood lymphocytes from several different human effect of the herbicide.191 This information could be used to donors to ionizing radiation while the cells grew in tissue provide a more sensitive and earlier indicator of potential culture.182 The study found that 48 genes were significantly toxicity in sensitive species, as well as to help understand up-regulated and 7 genes significantly down-regulated after the nature and mechanism of the toxicological response in radiation exposure.183 These changes in gene expression particular species. Tester animal or plant species could be were reproducible, peaked at 24 hours after exposure, and placed intentionally near a hazardous facility or environ- still remained significantly above background levels 72 mental accident to monitor for changes in their gene expres- hours after exposure.184 Importantly, the quantitative re- sion, which could serve as a “sentinel” for nearby residents sponse was very similar in cells from different donors, or natural species.192 which greatly enhances the practical utility of such genes as Gene expression assays have several advantages over tra- potential markers of exposure across an exposed popula- ditional toxicological endpoints for real-time surveillance tion, without having to account for individual differences in of potentially at-risk populations. Microarrays have the po- background levels of the marker.185 These findings suggest tential to provide real-time, on-site estimates of both expo- that it “may be possible to establish normal ranges for ex- sure and risk.193 In particular, it is possible, using high pression levels of these genes to distinguish irradiated indi- throughput gene expression screening to quantify the poten- viduals, whose expression levels of these genes would fall tial exposures of a large number of people in a quick and outside the normal range.”186 Ongoing or targeted screening minimally intrusive manner.194 Another important advan- of an exposed population using microarrays could detect tage of microarrays for real-time surveillance is that micro- such abnormal gene expression profiles in individuals, fa- arrays provide a more sensitive and earlier indication of a cilitating both individualized intervention to assist those potential risk than traditional methods.195 Of course, one of at-risk people and also population-wide risk assessment and the inevitable consequences of using a more sensitive assay risk management measures.187 is that the results will require careful interpretation and the Microarray technology may also be used to monitor pol- exercise of judgment by both regulators and companies to avoid false alarms while recognizing truly significant early 180. Philip M. Iannaccone, Toxicogenomics: “The Call of the Wild Chip,” 109 Envtl. Health Persp. A8, A10 (2001) (using toxicological responses. microarrays, “[i]t may be possible to screen biological samples ob- The potential for real-time surveillance provided by tained from workers at Superfund sites for the adverse effects of ex- microarrays may trigger or raise questions about some exposure to compounds present in the site”). Some studies have reported an increased frequency of chromosomal anomalies in resi- isting regulatory requirements for product safety surveil- dents living near hazardous waste sites. E.g., M. Vrijheid et al., lance by manufacturers. For example, §8(e) of TSCA re- Chromosomal Congenital Anomalies and Residence Near Hazard- quires the manufacturer of a substance or mixture to report ous Waste Landfill Sites, 359 Lancet 320 (2002). Gene expression assays have the potential to provide a monitoring assay of such resi- 188. See Nuwaysir et al., supra note 3, at 157 (bioassays based on stan- dents that is both more sensitive (by producing a positive response in dard ecotoxicity model systems could be improved by the addition a higher proportion of exposed individuals) and informative (by proof microarray analysis). viding more specific information on the class of toxicant causing the response and the risk associated with that response) than currently 189. Patrick Larkin et al., Array Technology as a Tool to Monitor Expo- available methods. sure of Fish to Xenoestrogens,54Marine Envtl. Res. 395 (2002). 181. See Bishop et al., supra note 20, at 986. 190. Id. 182. Amundson et al., supra note 14, at 342. 191. Doug Crump et al., Exposure to the Herbicide Acetochlor Alters Thyroid Hormone-Dependent Gene Expression and Metamorphosis 183. Id. at 343. in Xenopus Laevis, 110 Envtl. Health Persp. 1199 (2002). 184. Id. at 344. 192. See Bartosiewicz et al., supra note 29, at 71. 185. Id. at 343. If different people with varying genetic and environmen- 193. See Iannaccone, supra note 180, at A10. tal backgrounds do indeed respond in a similar way to a particular ex- 194. Other currently available surveillance assays, such as monitoring for posure, it is not necessary to obtain unexposed control samples from chromosomal changes or electron spin resonance of dental enamel each individual in order to quantify exposures. Obtaining preexpo- (for detecting radiation exposure), are not capable of large-scale sure background gene expression samples from an entire at-risk pop- population monitoring. See Amundson et al., supra note 14, at 346. ulation would be very difficult after an accident or other exposure In addition, these methods may be more intrusive, e.g., the electron scenario had occurred. Id. spin resonance method requires extraction of a tooth. Id. 186. Id. at 346. 195. See Farr & Dunn, supra note 6, at 1 (“the detection of altered gene 187. Another recent study found that humans exposed to arsenic exhib- expression can serve as an early warning for subsequent deleterious ited gene expression changes in their liver cells removed by biopsy outcomes”); Tennant, supra note 29, at A9 (“If array data can be that closely resembled the gene expression changes in rodent livers ‘phenotypically anchored’ to conventional indices of toxicity...it that developed carcinomas from arsenic exposure. Lu et al., supra will be possible to search for evidence of injury prior to its clinical or note 116, at 190. Such assays using microarray systems could there- pathological manifestation. This approach could lead to develop- fore be used to identify individuals at increased risk of cancer from ment of early biomarkers of toxic injury....”). See also supra notes exposure to toxicants such as arsenic. 137-61 and accompanying text. 33 ELR 10088 ENVIRONMENTAL LAW REPORTER 1-2003 Copyright © 2003 Environmental Law Institute®, Washington, DC. reprinted with permission from ELR®, http://www.eli.org, 1-800-433-5120. to EPA information received “which reasonably supports EPA.201 Thus, the §6(a)(2) reporting requirement for gene the conclusion that such substance or mixture presents a expression results will depend on whether such findings substantial risk of injury to human health or the environ- represent an “adverse effect,” which as discussed above is ment.”196 If a manufacturer obtains data showing that one of not clear under existing definitions.202 Product manufactur- its chemical products induces gene expression changes in ers are thus likely to face significant uncertainties about the animal studies, but there is no other indication of toxicity, reporting of gene expression changes under both TSCA and will the company be required to report that data under §8(e)? FIFRA in the absence of more specific guidance provided Is the requirement to report stronger if the gene expression by EPA. changes are characteristic of a known mechanism of toxicity? EPA’s guide for §8(e) reporting indicates that reporting Setting Environmental Standards is generally only required for “serious” toxic effects, and thus effects of less certain significance such as organ weight Several environmental statutes require risk-based standards that protect the public health against “adverse effects” with change or in vitro genotoxicity test results need not be re- 203 ported unless other evidence or factors show that the ob- an adequate “margin of safety.” For example, §109 of the served effect is indeed predictive of the potential for a more Clean Air Act requires EPA to establish national ambient air 197 quality standards that protect the public health “with an ade- serious effect. While gene expression changes of un- 204 known toxicological significance should clearly not be re- quate margin of safety.” The legislative history of the stat- portable under those criteria, changes that are signatures of ute indicates Congress’ intent that EPA protect the public from “adverse effects” resulting from exposure to air pollut- known toxicological mechanisms present a closer question. 205 On the one hand, the gene expression changes are in and of ants, and one of the key issues EPA addresses in setting themselves not a “serious” toxicological effect, but because such standards is determining whether a particular response such changes have the potential to provide unprecedented is an “adverse effect.” In the most recent revision to the acumen in predicting toxicity, there is at least an argument ozone air quality standard, for instance, EPA concluded that “transient and reversible” effects on lungs from ozone expo- that §8(e) reporting incorporates such a nonserious but in- 206 formative effect.198 sure were not “adverse effects.” Similarly, §6(a)(2) of the Federal Insecticide, Fungicide, However, regulatory and judicial precedent hold that an and Rodenticide Act (FIFRA) requires pesticide registrants “adverse effect” need not have clinical symptoms. When to report “factual information regarding unreasonable ad- EPA first promulgated its ambient air quality standard for verse effects” associated with their products.199 Again, lead in 1978, the “adverse effect” on which the Agency agency guidance on this reporting requirement is ambigu- based the standard was elevated erythrocytein protoporphyrin (EP) levels, a “subclinical” molecular change in the ous on whether gene expression data alone can ever repre- 207 sent information on “unreasonable adverse effects.” EPA’s cell that may indicate an impairment of heme synthesis. implementing regulations require that a pesticide registrant EPA itself acknowledged that initial elevation of EP levels report any “information relevant to the assessment of risks from exposure to lead “may not be a disease state or be seen or benefits” of a registered pesticide,”200 which is further de- as a clinically detectable decline in performance,” but found fined to include “[t]he results of a study of the toxicity of a that EP elevation becomes progressively more significant as an indicator of physiological response as lead exposure in- pesticide to humans or other non-target domestic organisms 208 if they show an adverse effect” not previously reported to creases. EPA concluded that, “as with other subclinical manifestations of impaired function, it is a prudent public 196. 15 U.S.C.§2607(e), ELR Stat. TSCA §8(e). 197. EPA’s Reporting Guide states the two factors to be considered in de- 202. See supra notes 166-215 and accompanying text. termining whether an effect indicates “substantial risk” and thus Stat. should be reported are: “1) the seriousness of the adverse effect, and 203. See, e.g., 42 U.S.C. §7409(b)(1), ELR CAA §109(b)(1) (re- 2) the fact or probability of the effect’s occurrence.” U.S. EPA, quiring EPA to set national ambient air quality standards that protect TSCA Section 8(e) Reporting Guide the public health with an adequate margin of safety); 42 U.S.C. 3 (1991), available at Stat. http://www.epa.gov/oppt/tsca8e/doc/rguide91.pdf. The guide then §300g-1(b)(4)(A), ELR SDWA §1412(b)(4)(A) (requiring discusses some specific examples to illustrate these factors. For ex- EPA to set maximum contaminant level goals (MCLGs) under the ample, it states that “[s]erious in vivo genotoxicological effects (e.g., Safe Drinking Water Act at “the level at which no known or antici- gene or chromosomal mutations) are reportable in and of them- pated adverse on the health of persons occur and which allows an ad- selves,” whereas “a positive in vitro genotoxicity test, when consid- equate margin of safety”). ered alone, is usually insufficient to cause reporting under Section 204. 42 U.S.C. §7409(b)(1), ELR Stat. CAA §109(b)(1). 8(e).” Id. at 28-29. The guide continues that a positive in vitro test 205. E.g., S. Rep. 1196, 91st Cong. 86 (1970) (air quality standards must would, however, normally suggest the need for additional studies, ensure “an absence of adverse effect on the health of a statistically which if also positive, may trigger a reporting requirement. Id. at 29. related sample of persons in sensitive groups....”). Another relevant example discussed is organ weight change, “which in and of itself, may not reflect a serious or prolonged incapacita- 206. U.S. EPA, National Ambient Air Quality Standards for Ozone, Final tion,” but the reportability of such effects will depend on other fac- Rule, 62 Fed. Reg. 38856, 38868 (July 18, 1997); U.S. EPA, Na- tors such as “the biological significance of the change.” Id. at 45. tional Ambient Air Quality Standards for Ozone—Final Decision, 58 Fed. Reg. 13008, 13011 (Mar. 9, 1993). 198. EPA’s §8(e) guidance requires reporting of “[a]ny pattern of effects or evidence which reasonably supports the conclusion that the chem- 207. U.S. EPA, National Primary and Secondary Ambient Air Quality ical substance or mixture can produce cancer, mutation, birth defects Standards: for Lead; Final Rulemaking, 43 Fed. Reg. 46246, 46247 or toxic effects resulting in death, or serious or prolonged incapacita- (Oct. 5, 1978). Protoporphyrin combines with iron in the red blood tion.” U.S. EPA, Toxic Substances Control Act: Notification of Sub- cells (erythrocytes) to form heme, a critical component of hemoglo- stantial Risk Under Section 8(e), 43 Fed. Reg. 11110, 11112 (Mar. bin, which transports oxygen in the blood. Lead blocks the formation 16, 1978). of heme, resulting in elevated levels of protoporphyrin in erythrocytes that would otherwise have been incorporated into heme. Id. at 199. 7 U.S.C. §136d(a)(2). 46253. 200. 40 C.F.R. §159.158(a). 208. Id. at 46247. 201. Id. §159.165(a). 209. Id. 1-2003 NEWS & ANALYSIS 33 ELR 10089 Copyright © 2003 Environmental Law Institute®, Washington, DC. reprinted with permission from ELR®, http://www.eli.org, 1-800-433-5120. health practice to exercise corrective action prior to the ap- tute an adequate “adverse effect” to require tightening of the pearance of clinical symptoms.”209 NAAQS?216 The precedent established in the Lead Indus- EPA did not find that any amount of EP elevation that tries case suggests that an effect need not be clinically de- could be detected would qualify as an “adverse effect,” but tectable or “clearly harmful” to be considered adverse, and only elevations above a certain quantum that EPA con- that an effect can be adverse if it occurs solely at the molecu- cluded indicated that the EP elevation “has progressed to the lar level. It therefore appears that at least some gene expres- extent that it should be regarded as an adverse health ef- sion changes may be considered “adverse.” On the other fect.”210 In the case Lead Industries Ass’n v. EPA,211 the U.S. hand, EPA’s own precedent, established in its decision that Court of Appeals for the D.C. Circuit upheld EPA’s determi- *-ALAD alterations were not an adverse effect for purposes nation to base its standard on EP elevation, holding that EPA of setting the lead air quality standard, suggests that a gene need not show that an effect caused by exposure to an air expression change should only be considered adverse if it pollutant was “clearly harmful” to health. Instead, it was results in “functional impairment.” Once again, there is sufficient, according to the court, that the chemical changes likely to be considerable uncertainty and disagreement relied on by EPA indicated that “lead has begun to affect one about whether gene expression changes are an “adverse ef- of the basic biological functions of the body.”212 fect,” a reflection of the unique nature of toxicogenomic In the same lead rulemaking, EPA also considered data. Gene expression data provides information much ear- whether it should base its standard on a different subclinical lier in the disease process than toxicologists have tradition- effect, the inhibition of the enzyme *-aminolevulinic acid ally used to identify toxicity, long before any symptoms ap- dehyratase (*-ALAD) in red blood cells and other tissues.213 pear. At the same time, gene expression changes may have This enzyme catalyzes the formation of one of the compo- powerful predictive value, providing more sensitivity, spec- nents involved in the cellular synthesis of heme, and lead in- ificity, and information than much more obvious toxicolog- hibits this enzyme at a concentration significantly below ical responses. It remains to be seen how this new category that which produces any other physiological or molecular of data will impact regulatory standard setting given that the effect from lead exposure, including EP elevation. EPA con- existing criteria and precedents for such standards were es- cluded that this subclinical effect was not “adverse,” and tablished in the pre-genomic era. thus should not be used as the basis for the standard, “because of the absence of evidence that there is an impairment Toxicogenomics: Caveats and Limitations of heme synthesis” at levels below which other adverse effects, i.e., EP elevation, occur.214 The evidence before EPA Gene expression profiling using DNA microarrays, by pro- suggested that while lead reduced the activity of *-ALAD at viding a window to peer within the cell to observe the earli- low-exposure levels, this reduced enzymatic activity did not est molecular responses to toxic exposures, offers a tool of correspond to any changes in the rate of heme synthesis, and unprecedented power for understanding and predicting the thus resulted in no “functional impairment.”215 body’s response to toxic exposures.217 As such, this new If data showed that a criteria pollutant induced gene ex- technology will have numerous potential applications in pression changes that were characteristic of a known toxico- toxic torts and environmental regulation, no doubt includ- logical profile at levels below the existing national ambient ing many uses in addition to those discussed above. Some of air quality standards (NAAQS), would this finding consti- these applications will be available in the very near future, and in some cases are available now, at least in “proof of 210. Id. at 46251. See also id. at 46252 (“EPA is making a distinction between the blood lead level that is the threshold for detection of the bi- concept” form, while other applications are further into the ological effect, impaired heme synthesis, and the blood lead level at future, albeit still likely within the next decade. Scientists which this effect has progressed to an extent that it is regarded as ad- predict that “it is almost certain” that the widespread use of verse to health.”). DNA microarrays will become routine and inexpensive in 211. 647 F.2d 1130, 10 ELR 20643 (D.C. Cir.), cert. denied, 449 U.S. the near future.218 1042 (1980). Nevertheless, many obstacles and uncertainties remain to 212. Id. at 1139, 10 ELR at 20646. be resolved before toxicogenomic data can be given wide- 213. 43 Fed. Reg. at 46252. 214. Id. Health & Env’t and Oversight & Investigations, House Comm. on 215. U.S. EPA, Lead; Proposed National Ambient Air Quality Standard, Commerce (Apr. 10, 1997), available at 1997 WL 10571479. 42 Fed. Reg. 63076, 63078 (Dec. 14, 1977). 217. See supra note 22 and accompanying text. See also Tennant, supra 216. The Chairman of EPA’s Clean Air Act Scientific Advisory Commit- note 29, at A10: tee (CASAC) testified to Congress in 1997 that Given the vast numbers and diversity of drug, chemicals, and as our ability to detect subtle responses of lung tissue to O[3] environmental stressors, the diversity of species in which improves, and as we gain more refined information from es- they act, the time and dose factors that are critical to the in- pecially susceptible humans exposed in the environment and duction of beneficial and adverse effects, and the diversity of in laboratories, it is becoming increasingly apparent that phenotypic consequences of ex posures, it is only through the there will probably be no clear threshold for detecting re- development of a rich knowledge base [of microarray data] sponses at exposure concentrations within the concentration and its availability to all of the scientific community that toxi- range amenable to regulatory control. It should be understood cology and environmental health can rapidly advance. that our growing ability to detect and study responses to ex- 218. Aardema & MacGregor, supra note 4, at 23; Hamadeh & Afshari, posures to O[3] and other pollutants down to, and perhaps in- supra note 8, at 515 (“Microarray technology will undoubtedly have cluding, background (uncontrollable) concentrations is nec- a profound impact on many avenues of biological and biomedical re- essarily accompanied by an increasing demand on our judg- search, including toxicology....”). ment of...when cellular responses should be considered sufficiently adverse to warrant regulatory action.... 219. See generally Hadley C. King & Animesh A. Sinha, Gene Expres- sion Profile Analysis by DNA Microarrays; Promise and Pitfalls, U.S. EPA’s Proposed Clean Air Act Regulations, Prepared State- 286 J. Am. Med. Ass’n 2280 (2001); Rockett & Dix, supra note ment of Joe L. Mauderly, Testimony Before the Subcomms. on 102, at 684. 33 ELR 10090 ENVIRONMENTAL LAW REPORTER 1-2003 Copyright © 2003 Environmental Law Institute®, Washington, DC. reprinted with permission from ELR®, http://www.eli.org, 1-800-433-5120. spread practical and legal effect outside of the research labo- dish in an incubator or the time of day in which the assay ratory.219 In the words of one toxicological expert, micro- is performed, can cause perturbations in gene expres- arrays are “going to revolutionize science. But the technol- sion.226 Such perturbations may at least partly explain the ogy is in its infancy, so there are going to be some growing significant interlaboratory variability in results that has pains.”220 To begin with, the toxicological significance of been reported, as well as the observation that the same lab- gene expression changes must be validated,221 which is not oratory can produce significantly different results in ex- an easy undertaking given the rapid pace at which micro- periments repeated just a few weeks apart.227 Given that array technology is still evolving.222 Validation will involve each microarray use can involve assays of tens of thou- evaluating the robustness and reproducibility of toxico- sands of different genes, “hundreds of false positives are genomic assays between or across different laboratories, almost guaranteed” from the widespread use of micro- species, individuals, tissues, development stages, exposure arrays given the potential for background fluctuations in levels, and exposure durations,223 all of which could poten- gene expression.228 tially affect gene expression patterns.224 A major challenge to the successful use of microarrays “Normal” gene expression is a dynamic and ever-chang- will therefore be the capability to distinguish background ing condition.225 The smallest perturbations in the microen- “noise” caused by fluctuations in background conditions vironment of the cell, such as the position of a tissue culture from true, biologically significant cellular responses to toxic exposures. Data sets of background levels of gene expres- 220. Jonathan Knight, When the Chips Are Down, 410 Nature 860, 860 (2001) (quoting toxicologist Timothy Zacharewski). sion in unexposed persons, and how these levels vary between individuals and in response to differences in location, 221. See Stefano Bonassi et al., Validation of Biomarkers as Early Pre- Mutation Res. health status, nutritional intake, lifestyle, time of day, and dictors of Disease, 480 349 (2001); Bonassi & Au, 229 supra note 2, at 79. other potential modifiers are needed. These background 222. Bernard A. Schwetz, Toxicology at the Food and Drug Administra- data sets can then be compared to gene expression patterns tion: New Century, New Challenges,20Int’l J. Toxicology 3, 6 in exposed persons to determine if observed changes are (2001): “real” or artifacts of other external or internal factors. One possible measure to minimize the number of false positives New chips can be developed faster than chips can be validated. Validation is a significant underpinning of the quality and signal noise from changes in the expression of genes of toxicology data today. If microarray technology moves with unknown significance is to monitor only a limited set faster than the validation technology, how will we use these of genes with established associations with toxic responses, data for the development of new products and the develop- while excluding genes that are highly sensitive to external ment of data to assess safety and efficacy? perturbations.230 Of course, limiting the number of genes 223. Criteria for validation of new toxicological test methods were de- monitored runs the risk of missing relevant information, and fined in a report issued by the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), established in may also lead to disagreement between laboratories about 1997 by the National Institute of Environmental Health Sciences and which genes should be included. subsequently established as a permanent committee of representa- Microarray analyses need not only distinguish “real” tives of 15 federal agencies by the ICCVAM Authorization Act of changes in gene expression from background fluctuations, 2000, Pub. L. No. 106-545 (2000). These validation criteria include, inter alia, requirements that (i) the relationship of the test method’s but also need to discriminate between two types of changes endpoint(s) to the biological effect of interest must be described, in gene expression in response to toxic exposures. Some (ii) a detailed protocol of the test method must be available, (iii) the extent of within-test variability, and the reproducibility of the test 226. Eric S. Lander, Array of Hope, 21 (Suppl.) Nature Genetics 3, 3 within and between laboratories, must have been demonstrated, and (1999); Hogue, supra note 38, at 33. (iv) the test method’s performance must have been demonstrated using representative reference chemicals or test agents. ICCVAM, 227. See Stephen A. Bustin & Sina Dorundi, The Value of Microarray Validation and Regulatory Acceptance of Toxicological Techniques for Quantitative Gene Profiling in Molecular Diagnos- Test Methods 21-22 (NIH Pub. #97-3981, Mar. 1997), available tics,8Trends Molecular Med. 269, 269 (2002). at http://iccvam.niehs.nih.gov/docs/guidelines/validate.pdf. 228. Kathleen McGowan, As Pipelines Wither, Pharma and FDA Ex- 224. See Hamadeh et al., supra note 15, at 239; King & Sinha, supra note plore Whether Microarrays Are Ready for Primetime, Genome- 219, at 2281 (“The ethnicity, sex, age, and genetic background of a Web.com, Apr. 15, 2002, available at http://www.genomeweb.com/ patient are likely to affect the gene expression profiles of many tis- articles/view-article.asp?Article=200241594143. sues to varying extents.”); Mark R. Fielden & Tim R. Zacharewski, 229. See Marjorie F. Oleksiak et al., Variation in Gene Expression Within Challenges and Limitations of Gene Expression Profiling in Mecha- and Among Natural Populations,32Nature Genetics 261, 263 nistic and Predictive Toxicology,60Toxicological Sci. 6, 8 (2002) (finding substantial variations in gene expression within nat- (2001) (“transcriptional responses may differ between one target ural population of fish species); Nuwaysir et al., supra note 3, at 157 cell and another, from cell culture to in vivo conditions, or from ro- (diet, health, and lifestyle factors can affect gene expression pat- dent models to humans”). terns); King & Sinha, supra note 219, at 2281-82 (same). See also 225. See Timothy R. Hughes et al., Functional Discovery via a Compen- Henry et al., supra note 44, at 1049 (“Little information is available dium of Expression Profiles, 102 Cell 109 (2000) (comparison of on the prevalence of mutations and gene expression patterns across gene expression profiles in genetically identical strains of yeast various population groups, lifestyles, and health conditions.”). grown under identical conditions finds significant fluctuations in ex- 230. See Zeytun et al., supra note 109, at 256 (“the important finding of pression of some genes, which apparently represent a form of “bio- this study was that exclusion of genes that are highly sensitive to logical noise”); King & Sinha, supra note 219, at 2281; Rockett & slight variations in the experimental condition leads to better corre- Dix, supra note 102, at 684. Most laboratories require at least a two- lation between gene expression profiles and the mode of action of the fold increase or decrease in gene expression before a change in gene toxicant”). See also Fielden & Zacharewski, supra note 65, at 8 expression will be considered significant. Id. (detection of 1.5 to (“gene expression profiles cannot be used as an explanation or pre- 2-fold changes in gene expression reported, but expressing concern dictor of toxicity unless correlated with an adverse effect”); Pat about the capability to achieve such detection levels generally); King Phibbs, Testing: Using Fewer Genes to Classify Chemicals Said to & Sinha, supra note 219, at 2284 (arguing that a twofold change in Be More Effective, Limit Liability, 25 Chem. Reg. Reptr. (BNA), expression may not always indicate a meaningful effect given the Dec. 10, 2001, at 1753 (reporting findings that a microarray contain- large natural variability in gene expression); Hamadeh & Afshari, ing only the 12 most important genes for toxicity was able to accu- supra note 8, at 513 (microarrays can “detect changes in the expres- rately classify chemicals and may “also limit liability by reducing sion level of a gene of about 1.5 times”). the number of ‘smoking guns’ in a company’s closet”). 1-2003 NEWS & ANALYSIS 33 ELR 10091 Copyright © 2003 Environmental Law Institute®, Washington, DC. reprinted with permission from ELR®, http://www.eli.org, 1-800-433-5120. changes will be nothing more than the adaptive response of tion of a toxic chemical with the critical components of a cells to external stimuli having no toxicological significance cell, there are therefore limitations on how useful it can be in or increased risk, whereas other changes will truly represent delineating toxic responses and mechanisms.238 the early stages of disease progression.231 One important There will ultimately be a need to standardize DNA mi- factor to be considered is the extent to which gene expres- croarrays, although premature standardization may carry its sion changes have been correlated with “classical” observed own risks by freezing a rapidly developing technology be- effects of toxicity.232 Some experts have suggested that fore it matures.239 Many different microarray formats (or toxicogenomic data should only be used as a “hypothe- “platforms”), methodologies, and content have been devel- sis-generating tool” in the initial years, whereby it would be oped and utilized by commercial companies and individual understood that microarray results may point to interesting laboratories, making difficult interlaboratory comparison possibilities that need to be confirmed by additional testing, and reconciliation of results.240 For example, different mi- but would not alone be used to establish any final conclu- croarray platforms often contain different gene sets, guar- sions regarding toxicity.233 anteeing variability in results. In addition, laboratories Another inherent limitation of DNA microarrays is that currently use different data analysis systems to analyze the they only measure changes in the expression of genes into massive quantity of data produced by microarrays, which mRNAs, but not the subsequent synthesis of proteins from is responsible for some of the interlaboratory variability that RNA. Proteins are the functional and structural units of in results.241 the cell that are likely to be the specific molecular target for While these various limitations and challenges associated many toxic substances, and otherwise involved directly in with microarrays are significant and in many cases formida- the toxic response in other cases.234 While gene expression ble, there is no question that toxicogenomic data from is a critical determinant of protein synthesis, it is not the only microarrays are already contributing to our understanding factor, as protein levels may also be affected by factors such of toxic substances, and will play an increasingly important as RNA and protein stability and turnover.235 The RNA:pro- role in evaluating toxicity in the future.242 It is critical to the tein abundance ratio can vary over a range of at least tenfold successful deployment of this technology that scientific and for different RNAs within the cell.236 The related fields of regulatory bodies develop standards and guidelines for the proteomics and metabonomics attempt to measure changes appropriate use of microarray data. To that end, the NIEHS in cellular proteins and metabolites, respectively, but to date established the National Center for Toxicogenomics (NCT) it has been much more difficult to accurately measure these in 2000 “to promote the evolution and coordinated use of parameters than gene expression.237 In sum, because gene gene expression technologies and to apply them to the toxi- expression is one step removed from the biological interac- cological effects in humans.”243 This undertaking will likely include an effort to move toward the establishment of stan- 244 231. See Pennie et al., supra note 161, at 356 (“The technology does not dardized procedures and data quality standards. The ef- . . . distinguish causative events from adaptive response (or even sys- forts of the NCT as well as regulatory agencies such as EPA tem noise)....”)(emphasis in original); Henry et al., supra note 4, at 1049 (necessary to determine “whether observed changes in must go beyond the use of toxicogenomic data in research gene/protein expression are causative, coincidental, or adaptive re- applications, but must also anticipate and develop guide- sponses to a chemical”); Smith, supra note 107, at 283: lines for the regulatory use of toxicogenomic data, which 245 [T]his technology is not without its dangers, at least during may also be indirectly relevant to toxic tort applications. the phase where toxicologists are beginning to understand appropriate application of toxicogenomic and proteomic 238. See Fielden & Zacharewski, supra note 65, at 7 (“our ability to de- data. It is already known that the response of organs, or in- fine the mechanism of action of a compound using gene expression deed of individual cell types, to exposure to individual toxi- profiling technologies will be highly limited in resolution”); cants can lead to an alteration in many genes in a simulta- Iannaccone, supra note 180, at A10. neous and, as yet, undefined manner. However, many of these changes are almost certainly adaptive and unrelated to 239. Henry et al., supra note 44, at 1047 (“given the speed with which the the mechanism of toxicity and/or human safety. As this field is evolving, standardization of research platforms or methods technology develops, scientists will be able to describe al- does not appear to be appropriate at this time”). tered gene expression provoked by chemicals long before 240. See Aardema & MacGregor, supra note 4, at 22; King & Sinha, su- they are able to offer valid interpretations of their meaning. pra note 219, at 2284. the potential for inadvertently raising concerns over the ef- 241. See Bustin & Dorundi, supra note 227, at 269-70. fect of chemicals in experimental animals (and hence humans), or even the intentional misrepresentation of the re- 242. See Hamadeh & Afshari, supra note 8, at 515 (“Microarrays are cer- sults to suggest that chemicals are “playing” with our genes, tainly a giant leap into the future of performing a quality biological is enormous. research that holds the promise to aid in discovery of better chemicals, diagnostics and pharmaceutical compounds and ultimately, to See also supra notes 169-71 and accompanying text. improve the quality of life of future generations.”); Pennie & 232. See Bonassi & Au, supra note 2, at 82; Pennie & Kimber, supra note Kimber, supra note 16, at 319. 16, at 321; Henry et al., supra note 44, at 1047 (“there is a critical 243. Tennant, supra note 136, at A8. need to establish relationships between gene expression data and toxicological changes, enabling an integration of ‘omics’ informa- 244. Id. tion with known toxicological measures”). 245. See Aardema & MacGregor, supra note 4, at 18 (“It is critical that 233. Hogue, supra note 38, at 33. toxicologists in industry, regulatory agencies, and academic institu- tions develop a consensus, based on rigorous experimental data, 234. See Fielden & Zacharewski, supra note 65, at 7. about the reliability and interpretation of endpoints such as global 235. Id.; Iannaccone, supra note 180, at A10. gene expression patterns prior to use in regulatory and industrial set- tings.”); Scott W. Burchiel et al., Analysis of Genetic and Epigenetic 236. See Bustin & Dorundi, supra note 227, at 271. Mechanisms of Toxicity: Potential Roles of Toxicogenomics and 237. See, e.g., Carol Ezzell, Proteins Rule, Sci. Am., April 2002, at 41; Proteomics in Toxicology,59Toxicological Sci. 193, 194 Daniel C. Liebler, Proteomic Approaches to Characterize Protein (2001) (“Whereas scientists pursuing mechanistic data may be in- Modifications: New Tools to Study the Effects of Environmental Ex- terested in identifying a broad range of genes that are affected by posures, 110 Envtl. Health Persp. 3 (2002). drugs, chemicals, and complex mixtures, the regulatory communi- 33 ELR 10092 ENVIRONMENTAL LAW REPORTER 1-2003 Copyright © 2003 Environmental Law Institute®, Washington, DC. reprinted with permission from ELR®, http://www.eli.org, 1-800-433-5120. EPA has recently taken the first steps in providing such with pesticides.251 While the Interim Policy indicates that regulatory guidance by issuing its Interim Policy on Ge- regulatory decisions will not need be based exclusively on nomics in the summer of 2002.246 This document conveys gene expression data, it does not address the issue of considerable enthusiasm for the potential usefulness of whether gene expression data must be submitted under genomic data in risk assessment and regulatory decisions.247 TSCA and FIFRA reporting requirements. Microarray data At the same time, the EPA guidance expresses appropriate are unique relative to other toxicological information in that caution with respect to some of the limitations and uncer- changes in gene expression in and of themselves are not a tainties discussed above. For example, the Interim Policy health problem, yet on the other hand are highly prognostic states that while gene expression data may provide “valu- of the development of toxicity. Most of the existing reportable insights” for predicting toxicity of environmental ing guidance and precedent on defining an “adverse effect” stressors, it cautions that “the relationship between changes do not contemplate or “fit” such a powerful predictive tech- in gene expression and adverse effects are unclear at this nology, but rather assume that observed toxicological ef- time and may likely be difficult to elucidate.”248 EPA states fects must be “serious” or “significant” before they can be that it “expects that genomics data may be received, as sup- considered predictive of toxicity. porting information for various assessment and regulatory Without specific guidance on the implications of gene ex- purposes,” and that “genomics data may be considered in pression changes for reporting requirements, regulated par- decision-making at this time” although “these data alone are ties and agency staff will face significant uncertainties with insufficient as a basis for decisions.”249 respect to whether some gene expression changes will trig- The Interim Policy therefore necessarily walks a fine line. ger reporting requirements.252 While EPA may understand- On one hand, EPA is encouraging the development and sub- ably be reluctant to “lock in” any specific policies at such an mission of genomics data by affirming that it will indeed early stage of a rapidly developing technology, the concept consider such data in making decisions. On the other hand, of “fair notice”253 requires that the Agency provide advance EPA recognizes the existing limitations and uncertainties notification if it intends to use toxicogenomic data in an en- involved with toxicogenomic data, and provides an impor- forcement context, such as penalizing a company for failure tant backstop against inappropriate use of genomic data by to submit “substantial risk” or “adverse effect” notifications taking the position that it will not make decisions at this time based on gene expression changes. EPA might be able to based solely on genomic data. In other words, gene expres- provide more guidance for these and other questions, with- sion changes must be connected to traditional toxicological out unduly tying its hands with rigid regulatory require- effects before they can be used for regulatory purposes at the ments, by producing more detailed “points to consider” or present time. “best practices” guidelines that provide specific guidance Another important aspect of the Interim Policy is that it on how microarray data should be developed and used for expressly provides that the agency will continue to monitor regulatory purposes.254 and participate in the development of this technology, and An alternative approach for the interim might be for EPA update its guidance accordingly.250 The use of the term “in- to expressly provide a “safe harbor” for toxicogenomic data, terim” policy conveys its transitional status. With such a in which the Agency encourages the development and sub- rapidly developing technology as microarrays, it is critical mission of such data, but commits not to take enforcement that the policies of agencies such as EPA stay current with action for the failure to submit gene expression data or oth- the evolving science, which requires that policies remain erwise use the data for enforcement purposes until the meth- flexible and current. odology has been adequately validated.255 Such an approach If there is one area where the Interim Policy falls short, it would be consistent with the recently enacted ICCVAM256 is the lack of guidance for industry on the relevance of Authorization Act of 2000, which requires that “any new or microarray data for various reporting requirements such as revised acute or chronic test method” should be “determined the TSCA §8(e) requirement to report “substantial risk” in- to be valid for its proposed use” prior to being required or formation or the FIFRA §6(a)(2) requirement to report in- encouraged by a federal agency.257 formation on “unreasonable adverse effects” associated In contrast to the role of EPA in the regulatory context, there is no expert agency that can provide guidance and ties will be interested in only those genes that are indicative of a critical health effect.”). 251. See supra notes 196-202 and accompanying text. 246. U.S. EPA, supra note 102. 252. See Henry et al., supra note 44, at 1047 (initially, “‘omics’ findings 247. E.g., id. at 1 (“EPA believes that genomics will have an enormous will likely be misinterpreted, because no guidelines currently exist impact on our ability to assess the risk from exposure to stressors and for correlating quantitative or qualitative changes in gene/protein/ ultimately to improve our risk assessments.”). metabolite expression with the potential for adverse effects”). 248. Id. at 2. 253. See General Elec. Co. v. EPA, 53 F.3d 1324, 1329, 25 ELR 20982, 249. Id. 20984 (D.C. Cir. 1995) (an “agency must always provide ‘fair notice’ of its regulatory interpretations to the regulated public”). 250. Id. at 3-4: 254. See, e.g., Henry et al., supra note 44, at 1047 (recommending adop- As EPA gains experience in applying genomics information tion of recommendations for best practices for microarray systems). and refines its understanding of the use of such information, it 255. See David Rejeski, Exploring the Genomics Frontier, Risk Policy will develop guidance to explain how genomics data can be Rep. better utilized in informing decisionmaking and related ethi- , Aug. 20, 2002, at 24, 25. cal, legal, and societal implications. EPA is working with 256. ICCVAM refers to the Interagency Coordination Committee on the other federal, state, and tribal organizations, as well as with Validation of Alternative Methods, an interagency coordinating academic, international, and industry groups to facilitate sci- committee established by the National Institute of Environmental entifically sound applications of genomics data. In addition, Health Sciences to improve the toxicological test methods used by EPA will continue to build partnerships and communicate federal agencies. The ICCVAM Act of 2000 made this committee with all interested stakeholders as an essential component of permanent. See supra note 223. the Agency’s future activities in genomics. 257. 42 U.S.C. §2581-4(c). 1-2003 NEWS & ANALYSIS 33 ELR 10093 Copyright © 2003 Environmental Law Institute®, Washington, DC. reprinted with permission from ELR®, http://www.eli.org, 1-800-433-5120. Conclusion oversee the appropriate introduction of toxicogenomic data For many years, environmental regulators and toxic tort fact in the toxic torts context. Although there are no cases re- finders have had to make their decisions about the risks of ported to date involving the use of gene expression data, toxic substances under conditions of paralyzing uncertainty. there has been increasing interest by practitioners in this Toxicogenomics offers a tool of unprecedented power to subject area,258 and it is only a matter of time before trial look within the black box of the cell and directly observe the lawyers and their experts seek to introduce toxicogenomic earliest stages of the toxicological response with informa- data for the many potential applications in tort cases. The tion that is both highly specific and sensitive. While the po- high-stakes and one-shot dynamics of tort litigation will tential applications and benefits of toxicogenomics for both provide strong incentives for the use of all potentially help- environmental regulation and toxic torts are immense, the ful evidence, even if some uses may be perceived as prema- use of this technology in such contexts is not without limita- ture or inappropriate by the scientific and regulatory com- tions and the need for caution. In particular, toxicogenomics munities.259 Lay judges and juries are unlikely to be in a po- has the potential to produce too much information—in that sition to screen the reliability and validity of toxicogenomic it has the potential to identify too many chemicals and prod- data on their own. Clear and carefully developed codes of ucts that are interacting with biological systems, and too practice or guidance documents produced by influential sci- many people that are experiencing gene expression changes entific and regulatory bodies are likely to be the best option as a result of exposures to environmental agents—to permit for defending against inappropriate or premature use of practical decisions and priority setting based on gene ex- toxicogenomic data in civil litigation and other nonregu- pression changes alone. Careful judgment and rigorous latory contexts.260 validation will be needed to discriminate those gene expression changes that warrant public health concern from 258. A number of recent conference workshops and newsletter articles those with no public health significance that merely reflect for trial lawyers have addressed the potential applications of innocuous adaptive responses and normal fluctuations toxicogenomics for toxic tort and product liability litigation. within dynamic cells. One thing that seems clear, however, 259. See Gary E. Marchant, Genetic Susceptibility and Biomarkers in is that environmental regulation and toxic tort litigation will Toxic Injury Litigation,41Jurimetrics 67, 106 (2000). both look very different 10 years from now, in large part 260. See Henry et al., supra note 44, at 1049 (“Because of the excite- due to the revolutionary capabilities and information pro- ment surrounding the discovery of a new technology, some peo- vided by toxicogenomics. ple may use information based on ‘omics’ data...before its effec-