- Email: [email protected]
Practical aspects of mutagenicity testing strategy: an industrial perspective
Mutation Research 455 (2000) 21–28
Practical aspects of mutagenicity testing strategy: an industrial perspective B. Bhaskar Gollapudi a,∗ , Gopala Krishna b a
The Dow Chemical Company, 1803 Building, Toxicology and Environmental Research and Consulting, Midland, MI 48674, USA b Department of Worldwide Preclinical Safety, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, 2800 Plymouth Road, Ann Arbor, MI 48105, USA
Abstract Genetic toxicology studies play a central role in the development and marketing of new chemicals for pharmaceutical, agricultural, industrial, and consumer use. During the discovery phase of product development, rapid screening tests that require minimal amounts of test materials are used to assist in the design and prioritization of new molecules. At this stage, a modified Salmonella reverse mutation assay and an in vitro micronucleus test with mammalian cell culture are frequently used for screening. Regulatory genetic toxicology studies are conducted with a short list of compounds using protocols that conform to various international guidelines. A set of four assays usually constitutes the minimum test battery that satisfies global requirements. This set includes a bacterial reverse mutation assay, an in vitro cytogenetic test with mammalian cell culture, an in vitro gene mutation assay in mammalian cell cultures, and an in vivo rodent bone marrow micronucleus test. Supplementary studies are conducted in certain instances either as a follow-up to the findings from this initial testing battery and/or to satisfy a regulatory requirement. Currently available genetic toxicology assays have helped the scientific and industrial community over the past several decades in evaluating the mutagenic potential of chemical agents. The emerging field of toxicogenomics has the potential to redefine our ability to study the response of cells to genetic damage and hence our ability to study threshold phenomenon. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Industrial Genetic Toxicology Testing Strategy; Test battery; Guidelines
1. Introduction Development of new products for agricultural, pharmaceutical, and consumer use is a time consuming, difficult and expensive process. Generally, thousands of potentially useful molecules are examined for efficacy and safety to find a single product for marketing. This process often takes more than 10 years and costs hundreds of millions of dollars. Toxicology studies play a central role in (1) evalu∗ Corresponding author. Tel.: +1-517-636-7179; fax: +1-517-638-9863. E-mail address: [email protected] (B.B. Gollapudi).
ating health hazards associated with the exposure of humans to these products, (2) making crucial decisions on whether or not to invest valuable resources in developing a new lead molecule, and (3) maintaining a product that is already on the market by providing data from contemporary or state of the art study designs. In addition, when necessary, data are generated to develop a mechanistic basis for an adverse finding during post-marketing surveillance. From the perspective of pharmaceutical drug development, toxicological studies are intended to identify an initial safe starting dose and subsequent dose escalation scheme to humans, to identify potential target organs of toxicity and reversibility
0027-5107/00/$ – see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 7 - 5 1 0 7 ( 0 0 ) 0 0 1 1 4 - 7
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Fig. 1. Staging of Genetic Toxicity Assessment.
B.B. Gollapudi, G. Krishna / Mutation Research 455 (2000) 21–28
of toxicity, and to identify parameters for clinical monitoring [1]. Genotoxicity is the process by which agents interact with DNA and other cellular targets that control the integrity of the genetic material. This includes induction of DNA adducts, strand breaks, point mutations and structural and numerical chromosomal changes. Tests for genotoxicity are considered short-term in nature and are an integral part of product safety assessment. The purpose of conducting these studies can be broadly divided into three categories: (1) to screen compounds for potential carcinogenic activity during early development to help select and/or prioritize potential leads; (2) to obtain a complete genotoxicity database on promising new molecules to support global regulatory requirements; and (3) to generate complementary data for better understanding of existing results. Each of these categories is described below and a flow chart listing these is shown in Fig. 1.
2. Genetic toxicology screening studies These studies are conducted in early stage of product development to predict potential carcinogenic activity of new molecules and to aid in the structural design of new chemical entities. The data generated from these relatively inexpensive and rapid screens are utilized early in the development process to prioritize promising prospects. These screening tests need to be sensitive enough to minimize false negatives and specific enough to reduce false positives such that promising prospects are not dropped from development. Prior to conducting any biological screening tests, chemical structure of the molecule is reviewed for alerts that could render the compound a mutagen. The structural alert diagrams given by Ashby and Tennant [2] and Ashby and Paton [3] are extremely helpful in assessing the potential of a chemical to react with DNA. There are also several computer-assisted modeling programs such as topkat [4], derek [5], and multicase [6] that can be used to scrutinize the molecular structure for potential mutagenicity and carcinogenicity. While the results of these analyses are very informative and accurate for some structural classes, they often have limited utility in predicting biological
23
activities of novel classes of compounds. The demand for structure-based modeling as a screening tool, however, has witnessed a steady increase over the past few years in industrial toxicology laboratories. This is partly due to large number of compounds made available through combinatorial chemistry libraries and to increase the probability of success in product development. The types of biological screening tests employed at the early stage of product development are primarily based on the physical and chemical properties of the compound. In addition, the quantity of test material available is a limiting factor in performing any biological testing at this stage. Bench chemists synthesize only small quantities of these materials at the initial stages and hence it is often difficult to obtain samples of more than 500 mg to conduct the desired initial biological testing. In addition to toxicity testing needs, there are other demands on the available test material, for example, efficacy testing, material characterization, stability analyses, etc. A bacterial reverse mutation assay using Salmonella typhimurium tester strains (Ames) test [7] is widely used in early screening studies. To accommodate limited quantity of test material and to maximize obtaining useful information, several modifications have been introduced into the standard Ames assay protocol. Examples of such modifications include: the use of limited number of tester strains (2 strains, TA98 and TA100), six-well plates, one activation condition (+S9 only), fewer replicates per concentration level (one or two plates), a lowered upper limit test dose (e.g., up to 1000 g/plate vs. 5000 g/plate), mixing two tester strains (TA1535+TA1537, TA98+TA100), spot testing, etc. These protocols can be further modified to accommodate specific chemical class activities based upon prior experience. For example, in screening compounds belonging to topoisomerase inhibitors, tester strain TA102 may be included since this class of compounds usually requires an intact excision repair system for mutagenesis similar to the intercalator, mitomycin C [8]. There are situations where the bacterial mutagenicity assay is probably not the appropriate choice for early phase screening. For example, nucleoside analogs are unlikely to elicit a positive response in bacterial strains routinely used for screening. Similarly, it is pertinent to know the mammalian geno-
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B.B. Gollapudi, G. Krishna / Mutation Research 455 (2000) 21–28
toxicity of halogenated quinolone antibiotics that target primarily bacterial topoisomerases because they also have cross reactivity to mammalian topoisomerases leading to clastogenic effects [9]. An in vitro cytogenetic screening assay in mammalian cell cultures is the test of choice for assessing the genotoxic hazard posed by such agents. The standard protocol for conducting the cytogenetic assays with cell cultures can be modified to accommodate limited test material availability, e.g., reduced number of doses and replicates, use of multiwell plates to facilitate reduced treatment volume, etc. The in vitro micronucleus test with mammalian cell cultures is gaining considerable popularity in recent years as a screening assay [10]. This assay may be adapted for high throughput screening by culturing cells on glass microscope slides with multi-well chambers and examining in situ processed cells for the incidence of micronuclei. Solubility is a key consideration at an early stage of development. For example, the free acid form of a newly discovered molecule might not be soluble enough to enable in vitro testing. In such cases, a suitable salt or ester form of the material may be synthesized to facilitate testing. Highly volatile compounds are not easily amenable to screening tests as they require special exposure systems for both bacterial and cell culture based assays. Glass dessicators are generally used for exposing bacterial cultures to volatile samples. In cell culture based assays, the headspace of the culture vessel is minimized to avert the escape of vapors of volatile test materials into the headspace. The miniaturized versions of Ames and/or in vitro micronucleus assays are generally used either in sequence or in parallel depending on the need, resources, and the amount of information desired. Automation of data acquisition and analysis in these assays (for example — application of flow cytometry to in vitro micronucleus evaluation) would further enhance their utility in early screening. However, the screening tests are not designed to provide definitive answers on the mutagenicity of test material. If a positive response is observed with a given chemical in the absence of any structural alerts, it is important to rule out the contribution of an impurity to the observed response. This can be done either by purifying the available sample or resynthesizing it with a higher purity. Similarly, a negative response in these limited assay protocols does
not necessarily preclude the possibility of triggering a positive response in subsequent studies using a more rigorous test protocol.
3. Standard battery of tests to obtain robust genotoxicity data These studies are usually conducted with a short list of chemicals selected as the most promising prospective molecules from among the compound libraries based upon, among other criteria, favorable efficacy and toxicity screening data. The timing to conduct these studies, in relation to product development, depends on the intended use of the chemical. For pharmaceutical compounds, such studies are usually carried out prior to first human exposure (Phase I/II clinical trials). For agrochemicals, the timing of these studies precedes subchronic (90-day) and chronic (2-year) animal toxicity studies. With other types of industrial chemicals, data from such studies are usually obtained prior to petitioning for regulatory authorities for review prior to manufacture, import, and/or marketing. The extent of testing is usually predicated by the intended use of chemical. A standard battery of tests consisting of one or more of the following assays is usually conducted: a bacterial reverse mutation assay; an in vitro cytogenetic assay in mammalian cell cultures, and/or an in vitro gene mutation assay in mammalian cell cultures; and an in vivo cytogenetic assay in rodent bone marrow cells. In addition to providing a robust database of genotoxicity, this test battery, when conducted in accordance with applicable study guidelines (see below), can also satisfy the requirements of various global regulatory bodies. Several national and international regulatory authorities have published the minimum data requirements for regulatory reviews on chemicals in commerce [11–16]. A number of these have also published detailed guidelines for the conduct of the above studies [12,17,18]. Although there are still some country-specific requirements, there has been an enormous effort in recent years to harmonize several of the regional guidelines. As a result, studies conducted in accordance with the OECD and ICH guidelines [12,18] can reasonably be expected to satisfy the requirements of global regulatory bodies.
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Although these guidelines are described in detail, there is enough ambiguity in these guidelines for subjective interpretation by individual researchers and thus these assays are conducted somewhat differently among various laboratories. For example, the OECD guideline (#473) for the conduct of in vitro mammalian chromosome aberration test states that “negative results with metabolic activation need to be confirmed on a case-by-case basis. In those cases where confirmation of negative results is not considered necessary, justification should be provided.” Based on this, some laboratories routinely repeat the negative results to avoid the possibility that the regulatory body will reject the study in the event that the justification provided for not repeating the experiment was not acceptable to the reviewer. Since studies rejected by the regulatory agency may result in unnecessary delay in bringing the product to the market, it is important to design, conduct, and interpret study results carefully. These definitive studies, sometimes referred to as the regulatory guideline studies, are routinely conducted with well characterized test chemicals under Good Laboratory Practice (GLP) standards [19–21]. GLP studies require well-defined protocols. All changes and revisions to the protocol are properly documented. The laboratory procedures used in conducting these studies are described in protocol and/or in standard operating procedures (SOPs) of the laboratory. The conduct of study as well as the accuracy of final report relative to raw data is audited by a Quality Assurance team. Study files containing protocol, report, raw data, and data storage and retrieval are also subject to audit by regulatory agencies. The reader is referred to [19–21] for a complete description of the applicable requirements to conduct studies under GLP standards.
4. Supplementary genetic toxicology studies These studies are triggered either by the findings of initial battery of genetic toxicology studies or by other toxicity data. Chemicals causing positive in vitro genotoxicity results but without such activity in the rodent bone marrow cytogenetic assay often require evaluation in other somatic tissues (e.g., in vivo/in vitro unscheduled DNA synthesis or micronuclei in rodent liver cells) and/or germ cell interaction studies
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(e.g., rodent spermatogonial chromosome aberrations or micronucleus analysis, dominant lethal assay in rodents, single cell gel (comet) assay in rodent sperm or testes, etc.). In certain cases, investigative work on organ specific genotoxicity (e.g., kidney, lung, nasal epithelium, etc.) is also needed to collect mode of action data in chemical carcinogenesis. Mutation assays using transgenic animals, analysis of DNA for adducts, determination of DNA strand breaks (e.g., comet assay), apoptosis, p53 gene expression, and micronuclei are a few of the frequently used tools in investigating genotoxicity in any tissue of interest. Assays for heritable mutations in the germ cells of rodents (specific locus and heritable translocation assays) are usually needed for mutagenicity risk assessment only when the weight of evidence indicates that the chemical in question is genotoxic to germ cells. Genetic toxicology tests are often used to obtain mechanistic information pertaining to the toxicity of chemicals. For example, in vitro assays can be used to compare the metabolism of promutagens among different species and different target organs by incorporating S9 derived from the species and/or organs of interest. Incorporation of S9 derived from human tissues such as the liver is also of significant value in instances where unique human metabolites are identified. Unique positive findings in in vitro chromosomal aberration assay often requires extensive follow up work to determine whether such an activity is relevant to the expected human exposure levels. Standard protocols used for conducting the mutagenicity assays need to be modified in certain instances to detect unique modes of mutagenic action. For example, with certain classes of compounds such as fluoroquinolone type of antibiotics, photogenotoxicity is of significant concern and hence, appropriate tests to address this issue need to be conducted [22]. If a given test compound is positive in the in vitro chromosome aberration assay at low concentrations without being positive in the in vivo micronucleus assay up to the limit dose, then an in vivo chromosome aberration assay may be conducted to replicate same cytogenetic endpoint. In this case, animals may be euthanized under conditions which maximize detection of such an effect in vivo, for example: appropriate postdose euthanasia times considering bone marrow toxicity (cell cycle kinetics) and peak plasma drug/specific metabolite concentration levels.
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5. Resource needs for genetic toxicology testing and research A number of major industrial toxicology laboratories have in-house capabilities to meet their needs. These laboratories are usually staffed to meet the steady demand for research, testing, and consultation. There are also contract research organizations (CROs) in various parts of the world that provide standard guideline testing services. Costs for these services vary depending upon the region, e.g., US$2000–7000 for a bacterial reverse mutation assay and US$15,000–30,000 for conducting an in vitro chromosomal aberration assays utilizing human lymphocytes or in vivo rodent micronucleus assay. In the year 2000, the total cost for conducting a four-test robust battery could add up to US$100,000. This estimate includes the costs associated with performing analytical verifications on dosing solution as required by GLPs.
6. Industrial genetic toxicologist Genetic toxicologists in the industrial toxicology laboratories benefit immensely from their association with other toxicologists and this interaction enables them to integrate other pertinent data (e.g., pharmacodynamics, pharmcokinetics, and target organ toxicity) in the design, conduct, and interpretation of mutagenicity studies. Further, when appropriate, the genotoxicity assessment can be successfully integrated into routine toxicological studies [23]. This approach utilizes: (i) the general principles of toxicology that govern the overall toxicity profile of a test substance, (ii) factors such as the dose and/or route of drug administration, drug metabolism, principles of toxicokinetics, and saturation of defense mechanisms are considered in evaluating genotoxicity, and (iii) the concept of administering multiple tolerable doses achieving steady state plasma drug concentrations — which are more relevant in human risk assessment compared to high acute doses. This approach also minimizes the amount of test compound, number of animals and other resources. Genetic toxicologists also work closely with the chemists in structurally based designing of molecules with favorable mutagenicity profile, as part of early
toxicology testing. They are also responsible for translating findings of the genetic toxicology studies, originating both internally and externally, into potential risk posed to human health and environment and present such data to the global regulatory agencies during their review of products. Particularly, with pharmaceuticals, the concept of threshold phenomenon, the age of intended treatment population, and the alternative therapies available at the time, play a key role in calculating risk-benefit ratio. Although the standard battery of regulatory genetic toxicology tests includes a well-defined set of studies, there are dozens of other genotoxicity tests practiced in various laboratories around the world. Results from all these tests often tend to distill down to a “yes” or “no” answer without any consideration to key experimental variables. For example, the doses used in a vast number of in vivo studies are near lethal levels and are often several orders of magnitude higher than the expected levels of human exposures to industrial and agricultural chemicals. The genetic toxicologists have to deal with the relevance of any positive findings given the fact that such high doses also tend to overwhelm the body’s natural defense mechanisms such as the detoxification pathways and/or lead to the generation of metabolites that might not result at sub-lethal doses.
7. Future industrial genetic toxicology needs Much of the emphasis over the past 3 decades in genetic toxicology community has been to identify the most appropriate battery of tests, from among dozens of useful assays, to screen chemicals for mutagenic potential. These tests have served the scientific community and the public fairly well, thus far, in evaluating potentially highly mutagenic compounds before they are introduced into commerce. Revolutionary advances in molecular biology are offering exciting possibilities for the development of new products. For example, genetically modified crops with characteristics such as resistance to insect infestation, tolerance to herbicide usage, and increased nutritional value, offer the greatest promise of successfully tackling the world’s nutritional problems. These
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technologies are also being investigated for producing chemicals and oils with renewable plant based raw materials. The safety of biotechnology derived products is becoming a significant issue of interest to many different stakeholders. Concerns regarding the potential pleiotrophic effects of the inserted transgenes on the safety of foods derived from such sources have been raised. Strategies for genetic toxicology testing of whole foods need to be thoroughly validated in order to address such concern. Foods such as vegetables contain naturally occurring mutagens and hence, it becomes more important to develop approaches that could quantitatively address the substantial equivalence of foods before and after genetic manipulation. Molecular biology has also offered the exciting new DNA microarray technology to monitor changes in gene expression [24]. This technology, if deployed correctly, has the potential to facilitate the definition and identification of threshold phenomenon in genetic toxicology. The inadequate sensitivity of the existing mutagenicity assays has hampered efforts to generate data to challenge the default assumption of lack of thresholds for DNA-reactive chemicals. There is also an urgent need for additional assays that can be used as early screens for potential mutagens, clastogens, and aneugens. Future advances in DNA micro-array technology will undoubtedly result in the availability of affordable DNA chips that can be used to profile dozens and even hundreds of compounds in a short period of time as high throughput assays. References [1] J. Nath, G. Krishna, Safety screening of drugs in cancer therapy, Acta Haematol. 99 (1998) 138–147. [2] J. Ashby, R. Tennant, Chemical structure, Salmonella mutagenicity and extent of carcinogenicity as indicators of genotoxic carcinogenesis among 222 chemicals tested in rodents by the US NCI/NTP, Mutat. Res. 204 (1988) 17– 115. [3] J. Ashby, D. Paton, The influence of chemical structure on the extent and sites of carcinogenesis for 522 rodent carcinogens and 55 different human carcinogen exposures, Mutat. Res. 286 (1993) 3–74. [4] K. Enslein, V.K. Gombar, B.W. Blake, Use of SAR in computer-assisted prediction of carcinogenicity and mutagenicity of chemicals by the topkat program, Mutat. Res. 305 (1994) 47–61.
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[5] J.E. Ridings, M.D. Barratt, C.G. R.Cary, C.E. Earnshaw, M.K. Eggington, P.N. Ellis, J.J. Judson, C.A. Langowski, M.P. Marchant, W.P. Payne, T.D. Watson, Computer prediction of possible toxic action from chemical structure: an update on the derek system, Toxicology 106 (1996) 267–279. [6] G. Klopman, H.S. Rosenkranz, Approaches to SAR in carcinogenesis and mutagenesis: prediction of carcinogenicity/mutagenicity using multicase, Mutat. Res. 305 (1994) 33–46. [7] D.M. Maron, B. Ames, Revised methods for the Salmonella mutagenicity test, Mutat. Res. 113 (1983) 173–215. [8] S. Albertini, A.A. Chetelat, B. Miller, W. Muster, E. Pujadas, R. Strobel, E. Gocke, Genotoxicity of 17 gyrase-and four mammalian topoisomerase II-poisons in prokaryotic and eukaryotic test systems, Mutagenesis 10 (1995) 343–351. [9] R.D. Anderson, N.A. Berger, Mutagenicity and carcinogenicity of topoisomerase-interactive agents, 309 (1994) 109–142. [10] M. Kirsch-Volders, T. Sofuni, M. Aardema, S. Albertini, D. Eastmond, M. Fenech, M. Ishidate Jr., E. Lorge, H. Norppa, J. Surralles, W. von der Hude, A. Wakata, Report from the in vitro micronucleus assay working group, Environ. Mol. Mutagen. 35 (2000) 167–172. [11] K.L. Dearfield, A.E. Auletta, M.C. Cimino, M.M. Moore, Considerations in the US Environmental Protection Agency’s testing approach for mutagenicity, Mutat. Res. 258 (1991) 259–283. [12] L. Muller, Y. Kikuchi, G. Probst, L. Schechtman, H. Shimada, T. Sofuni, D. Tweats, ICH-Harmonized guidances on genotoxicity testing of pharmaceuticals: evolution, reasoning and impact, Mutat. Res. 436 (1999) 195–225. [13] Y. Shirasu, The Japanese mutagenicity studies guidelines for pesticide registration, Mutat. Res. 205 (1988) 393–395. [14] M. Ishidate Jr., A proposed battery of tests for the initial evaluation of the mutagenic potential of medicinal and industrial chemicals, Mutat. Res. 205 (1988) 397–407. [15] Japan Ministry of Health and Welfare, Guidelines for Toxicity Studies of Drugs Manual, Yakuji Nippo, Tokyo, Japan, 1991. [16] Health and Welfare Canada, Guidelines on the Use of Mutagenicity Tests in the Toxicological Evaluation of Chemicals, Public Affairs Directorate, Ottawa, Canada, 1986. [17] U.S. EPA, Health Effects Test Guidelines, 870 Series, Office of Prevention, Pesticides, and Toxic Substances, United States Environmental Protection Agency, Washington, DC. [18] OECD, Guidelines for Testing of Chemicals, Organization of Economic Cooperation and Development Publication Service, 2 rue Andre Pascal, Paris Cedex-16, France, 1997. [19] U.S. EPA, Good Laboratory Practice Standards, United States Environmental Protection Agency, Washington, DC, 1989, 40 CFR, Parts 160 and 792. [20] U.S. FDA, Good Laboratory Practice Regulation, 21 CFR Part 58, Department of Health and Human Services, Washington, DC. [21] OECD, Principles of Good Laboratory Practice and Compliance Monitoring, Organization of Economic Cooperation
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and Development Publication Service, 2 rue Andre Pascal, Paris Cedex-16, France, 1997. [22] E. Gocke, L. Muller, P.J. Guzzie, S. Brendler-Schwaab, S. Bulera, C.F. Chignell, L.M. Henderson, A. Jacobs, H. Murli, R.D. Snyder, N. Tanaka, Considerations on photochemical genotoxicity: report of the International Workshop on Genotoxicity Test Procedures Working Group, Environ. Mol. Mutagen. 35 (2000) 173–184.
[23] G. Krishna, G. Urda, J. Theiss, Principles and practices of integrating genotoxicity evaluation into routine toxicology studies: a pharmaceutical industry perspective, Environ. Mol. Mutagen. 32 (1998) 115–120. [24] E.F. Nuwaysir, M. Bittner, J. Trent, J.C. Barrett, C.A. Afshari, Microarrays and toxicology: the advent of toxicogenomics, Mol. Carcinog. 24 (1999) 153–159.
Practical aspects of mutagenicity testing strategy: an industrial perspective B. Bhaskar Gollapudi a,∗ , Gopala Krishna b a
The Dow Chemical Company, 1803 Building, Toxicology and Environmental Research and Consulting, Midland, MI 48674, USA b Department of Worldwide Preclinical Safety, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, 2800 Plymouth Road, Ann Arbor, MI 48105, USA
Abstract Genetic toxicology studies play a central role in the development and marketing of new chemicals for pharmaceutical, agricultural, industrial, and consumer use. During the discovery phase of product development, rapid screening tests that require minimal amounts of test materials are used to assist in the design and prioritization of new molecules. At this stage, a modified Salmonella reverse mutation assay and an in vitro micronucleus test with mammalian cell culture are frequently used for screening. Regulatory genetic toxicology studies are conducted with a short list of compounds using protocols that conform to various international guidelines. A set of four assays usually constitutes the minimum test battery that satisfies global requirements. This set includes a bacterial reverse mutation assay, an in vitro cytogenetic test with mammalian cell culture, an in vitro gene mutation assay in mammalian cell cultures, and an in vivo rodent bone marrow micronucleus test. Supplementary studies are conducted in certain instances either as a follow-up to the findings from this initial testing battery and/or to satisfy a regulatory requirement. Currently available genetic toxicology assays have helped the scientific and industrial community over the past several decades in evaluating the mutagenic potential of chemical agents. The emerging field of toxicogenomics has the potential to redefine our ability to study the response of cells to genetic damage and hence our ability to study threshold phenomenon. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Industrial Genetic Toxicology Testing Strategy; Test battery; Guidelines
1. Introduction Development of new products for agricultural, pharmaceutical, and consumer use is a time consuming, difficult and expensive process. Generally, thousands of potentially useful molecules are examined for efficacy and safety to find a single product for marketing. This process often takes more than 10 years and costs hundreds of millions of dollars. Toxicology studies play a central role in (1) evalu∗ Corresponding author. Tel.: +1-517-636-7179; fax: +1-517-638-9863. E-mail address: [email protected] (B.B. Gollapudi).
ating health hazards associated with the exposure of humans to these products, (2) making crucial decisions on whether or not to invest valuable resources in developing a new lead molecule, and (3) maintaining a product that is already on the market by providing data from contemporary or state of the art study designs. In addition, when necessary, data are generated to develop a mechanistic basis for an adverse finding during post-marketing surveillance. From the perspective of pharmaceutical drug development, toxicological studies are intended to identify an initial safe starting dose and subsequent dose escalation scheme to humans, to identify potential target organs of toxicity and reversibility
0027-5107/00/$ – see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 7 - 5 1 0 7 ( 0 0 ) 0 0 1 1 4 - 7
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Fig. 1. Staging of Genetic Toxicity Assessment.
B.B. Gollapudi, G. Krishna / Mutation Research 455 (2000) 21–28
of toxicity, and to identify parameters for clinical monitoring [1]. Genotoxicity is the process by which agents interact with DNA and other cellular targets that control the integrity of the genetic material. This includes induction of DNA adducts, strand breaks, point mutations and structural and numerical chromosomal changes. Tests for genotoxicity are considered short-term in nature and are an integral part of product safety assessment. The purpose of conducting these studies can be broadly divided into three categories: (1) to screen compounds for potential carcinogenic activity during early development to help select and/or prioritize potential leads; (2) to obtain a complete genotoxicity database on promising new molecules to support global regulatory requirements; and (3) to generate complementary data for better understanding of existing results. Each of these categories is described below and a flow chart listing these is shown in Fig. 1.
2. Genetic toxicology screening studies These studies are conducted in early stage of product development to predict potential carcinogenic activity of new molecules and to aid in the structural design of new chemical entities. The data generated from these relatively inexpensive and rapid screens are utilized early in the development process to prioritize promising prospects. These screening tests need to be sensitive enough to minimize false negatives and specific enough to reduce false positives such that promising prospects are not dropped from development. Prior to conducting any biological screening tests, chemical structure of the molecule is reviewed for alerts that could render the compound a mutagen. The structural alert diagrams given by Ashby and Tennant [2] and Ashby and Paton [3] are extremely helpful in assessing the potential of a chemical to react with DNA. There are also several computer-assisted modeling programs such as topkat [4], derek [5], and multicase [6] that can be used to scrutinize the molecular structure for potential mutagenicity and carcinogenicity. While the results of these analyses are very informative and accurate for some structural classes, they often have limited utility in predicting biological
23
activities of novel classes of compounds. The demand for structure-based modeling as a screening tool, however, has witnessed a steady increase over the past few years in industrial toxicology laboratories. This is partly due to large number of compounds made available through combinatorial chemistry libraries and to increase the probability of success in product development. The types of biological screening tests employed at the early stage of product development are primarily based on the physical and chemical properties of the compound. In addition, the quantity of test material available is a limiting factor in performing any biological testing at this stage. Bench chemists synthesize only small quantities of these materials at the initial stages and hence it is often difficult to obtain samples of more than 500 mg to conduct the desired initial biological testing. In addition to toxicity testing needs, there are other demands on the available test material, for example, efficacy testing, material characterization, stability analyses, etc. A bacterial reverse mutation assay using Salmonella typhimurium tester strains (Ames) test [7] is widely used in early screening studies. To accommodate limited quantity of test material and to maximize obtaining useful information, several modifications have been introduced into the standard Ames assay protocol. Examples of such modifications include: the use of limited number of tester strains (2 strains, TA98 and TA100), six-well plates, one activation condition (+S9 only), fewer replicates per concentration level (one or two plates), a lowered upper limit test dose (e.g., up to 1000 g/plate vs. 5000 g/plate), mixing two tester strains (TA1535+TA1537, TA98+TA100), spot testing, etc. These protocols can be further modified to accommodate specific chemical class activities based upon prior experience. For example, in screening compounds belonging to topoisomerase inhibitors, tester strain TA102 may be included since this class of compounds usually requires an intact excision repair system for mutagenesis similar to the intercalator, mitomycin C [8]. There are situations where the bacterial mutagenicity assay is probably not the appropriate choice for early phase screening. For example, nucleoside analogs are unlikely to elicit a positive response in bacterial strains routinely used for screening. Similarly, it is pertinent to know the mammalian geno-
24
B.B. Gollapudi, G. Krishna / Mutation Research 455 (2000) 21–28
toxicity of halogenated quinolone antibiotics that target primarily bacterial topoisomerases because they also have cross reactivity to mammalian topoisomerases leading to clastogenic effects [9]. An in vitro cytogenetic screening assay in mammalian cell cultures is the test of choice for assessing the genotoxic hazard posed by such agents. The standard protocol for conducting the cytogenetic assays with cell cultures can be modified to accommodate limited test material availability, e.g., reduced number of doses and replicates, use of multiwell plates to facilitate reduced treatment volume, etc. The in vitro micronucleus test with mammalian cell cultures is gaining considerable popularity in recent years as a screening assay [10]. This assay may be adapted for high throughput screening by culturing cells on glass microscope slides with multi-well chambers and examining in situ processed cells for the incidence of micronuclei. Solubility is a key consideration at an early stage of development. For example, the free acid form of a newly discovered molecule might not be soluble enough to enable in vitro testing. In such cases, a suitable salt or ester form of the material may be synthesized to facilitate testing. Highly volatile compounds are not easily amenable to screening tests as they require special exposure systems for both bacterial and cell culture based assays. Glass dessicators are generally used for exposing bacterial cultures to volatile samples. In cell culture based assays, the headspace of the culture vessel is minimized to avert the escape of vapors of volatile test materials into the headspace. The miniaturized versions of Ames and/or in vitro micronucleus assays are generally used either in sequence or in parallel depending on the need, resources, and the amount of information desired. Automation of data acquisition and analysis in these assays (for example — application of flow cytometry to in vitro micronucleus evaluation) would further enhance their utility in early screening. However, the screening tests are not designed to provide definitive answers on the mutagenicity of test material. If a positive response is observed with a given chemical in the absence of any structural alerts, it is important to rule out the contribution of an impurity to the observed response. This can be done either by purifying the available sample or resynthesizing it with a higher purity. Similarly, a negative response in these limited assay protocols does
not necessarily preclude the possibility of triggering a positive response in subsequent studies using a more rigorous test protocol.
3. Standard battery of tests to obtain robust genotoxicity data These studies are usually conducted with a short list of chemicals selected as the most promising prospective molecules from among the compound libraries based upon, among other criteria, favorable efficacy and toxicity screening data. The timing to conduct these studies, in relation to product development, depends on the intended use of the chemical. For pharmaceutical compounds, such studies are usually carried out prior to first human exposure (Phase I/II clinical trials). For agrochemicals, the timing of these studies precedes subchronic (90-day) and chronic (2-year) animal toxicity studies. With other types of industrial chemicals, data from such studies are usually obtained prior to petitioning for regulatory authorities for review prior to manufacture, import, and/or marketing. The extent of testing is usually predicated by the intended use of chemical. A standard battery of tests consisting of one or more of the following assays is usually conducted: a bacterial reverse mutation assay; an in vitro cytogenetic assay in mammalian cell cultures, and/or an in vitro gene mutation assay in mammalian cell cultures; and an in vivo cytogenetic assay in rodent bone marrow cells. In addition to providing a robust database of genotoxicity, this test battery, when conducted in accordance with applicable study guidelines (see below), can also satisfy the requirements of various global regulatory bodies. Several national and international regulatory authorities have published the minimum data requirements for regulatory reviews on chemicals in commerce [11–16]. A number of these have also published detailed guidelines for the conduct of the above studies [12,17,18]. Although there are still some country-specific requirements, there has been an enormous effort in recent years to harmonize several of the regional guidelines. As a result, studies conducted in accordance with the OECD and ICH guidelines [12,18] can reasonably be expected to satisfy the requirements of global regulatory bodies.
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Although these guidelines are described in detail, there is enough ambiguity in these guidelines for subjective interpretation by individual researchers and thus these assays are conducted somewhat differently among various laboratories. For example, the OECD guideline (#473) for the conduct of in vitro mammalian chromosome aberration test states that “negative results with metabolic activation need to be confirmed on a case-by-case basis. In those cases where confirmation of negative results is not considered necessary, justification should be provided.” Based on this, some laboratories routinely repeat the negative results to avoid the possibility that the regulatory body will reject the study in the event that the justification provided for not repeating the experiment was not acceptable to the reviewer. Since studies rejected by the regulatory agency may result in unnecessary delay in bringing the product to the market, it is important to design, conduct, and interpret study results carefully. These definitive studies, sometimes referred to as the regulatory guideline studies, are routinely conducted with well characterized test chemicals under Good Laboratory Practice (GLP) standards [19–21]. GLP studies require well-defined protocols. All changes and revisions to the protocol are properly documented. The laboratory procedures used in conducting these studies are described in protocol and/or in standard operating procedures (SOPs) of the laboratory. The conduct of study as well as the accuracy of final report relative to raw data is audited by a Quality Assurance team. Study files containing protocol, report, raw data, and data storage and retrieval are also subject to audit by regulatory agencies. The reader is referred to [19–21] for a complete description of the applicable requirements to conduct studies under GLP standards.
4. Supplementary genetic toxicology studies These studies are triggered either by the findings of initial battery of genetic toxicology studies or by other toxicity data. Chemicals causing positive in vitro genotoxicity results but without such activity in the rodent bone marrow cytogenetic assay often require evaluation in other somatic tissues (e.g., in vivo/in vitro unscheduled DNA synthesis or micronuclei in rodent liver cells) and/or germ cell interaction studies
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(e.g., rodent spermatogonial chromosome aberrations or micronucleus analysis, dominant lethal assay in rodents, single cell gel (comet) assay in rodent sperm or testes, etc.). In certain cases, investigative work on organ specific genotoxicity (e.g., kidney, lung, nasal epithelium, etc.) is also needed to collect mode of action data in chemical carcinogenesis. Mutation assays using transgenic animals, analysis of DNA for adducts, determination of DNA strand breaks (e.g., comet assay), apoptosis, p53 gene expression, and micronuclei are a few of the frequently used tools in investigating genotoxicity in any tissue of interest. Assays for heritable mutations in the germ cells of rodents (specific locus and heritable translocation assays) are usually needed for mutagenicity risk assessment only when the weight of evidence indicates that the chemical in question is genotoxic to germ cells. Genetic toxicology tests are often used to obtain mechanistic information pertaining to the toxicity of chemicals. For example, in vitro assays can be used to compare the metabolism of promutagens among different species and different target organs by incorporating S9 derived from the species and/or organs of interest. Incorporation of S9 derived from human tissues such as the liver is also of significant value in instances where unique human metabolites are identified. Unique positive findings in in vitro chromosomal aberration assay often requires extensive follow up work to determine whether such an activity is relevant to the expected human exposure levels. Standard protocols used for conducting the mutagenicity assays need to be modified in certain instances to detect unique modes of mutagenic action. For example, with certain classes of compounds such as fluoroquinolone type of antibiotics, photogenotoxicity is of significant concern and hence, appropriate tests to address this issue need to be conducted [22]. If a given test compound is positive in the in vitro chromosome aberration assay at low concentrations without being positive in the in vivo micronucleus assay up to the limit dose, then an in vivo chromosome aberration assay may be conducted to replicate same cytogenetic endpoint. In this case, animals may be euthanized under conditions which maximize detection of such an effect in vivo, for example: appropriate postdose euthanasia times considering bone marrow toxicity (cell cycle kinetics) and peak plasma drug/specific metabolite concentration levels.
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5. Resource needs for genetic toxicology testing and research A number of major industrial toxicology laboratories have in-house capabilities to meet their needs. These laboratories are usually staffed to meet the steady demand for research, testing, and consultation. There are also contract research organizations (CROs) in various parts of the world that provide standard guideline testing services. Costs for these services vary depending upon the region, e.g., US$2000–7000 for a bacterial reverse mutation assay and US$15,000–30,000 for conducting an in vitro chromosomal aberration assays utilizing human lymphocytes or in vivo rodent micronucleus assay. In the year 2000, the total cost for conducting a four-test robust battery could add up to US$100,000. This estimate includes the costs associated with performing analytical verifications on dosing solution as required by GLPs.
6. Industrial genetic toxicologist Genetic toxicologists in the industrial toxicology laboratories benefit immensely from their association with other toxicologists and this interaction enables them to integrate other pertinent data (e.g., pharmacodynamics, pharmcokinetics, and target organ toxicity) in the design, conduct, and interpretation of mutagenicity studies. Further, when appropriate, the genotoxicity assessment can be successfully integrated into routine toxicological studies [23]. This approach utilizes: (i) the general principles of toxicology that govern the overall toxicity profile of a test substance, (ii) factors such as the dose and/or route of drug administration, drug metabolism, principles of toxicokinetics, and saturation of defense mechanisms are considered in evaluating genotoxicity, and (iii) the concept of administering multiple tolerable doses achieving steady state plasma drug concentrations — which are more relevant in human risk assessment compared to high acute doses. This approach also minimizes the amount of test compound, number of animals and other resources. Genetic toxicologists also work closely with the chemists in structurally based designing of molecules with favorable mutagenicity profile, as part of early
toxicology testing. They are also responsible for translating findings of the genetic toxicology studies, originating both internally and externally, into potential risk posed to human health and environment and present such data to the global regulatory agencies during their review of products. Particularly, with pharmaceuticals, the concept of threshold phenomenon, the age of intended treatment population, and the alternative therapies available at the time, play a key role in calculating risk-benefit ratio. Although the standard battery of regulatory genetic toxicology tests includes a well-defined set of studies, there are dozens of other genotoxicity tests practiced in various laboratories around the world. Results from all these tests often tend to distill down to a “yes” or “no” answer without any consideration to key experimental variables. For example, the doses used in a vast number of in vivo studies are near lethal levels and are often several orders of magnitude higher than the expected levels of human exposures to industrial and agricultural chemicals. The genetic toxicologists have to deal with the relevance of any positive findings given the fact that such high doses also tend to overwhelm the body’s natural defense mechanisms such as the detoxification pathways and/or lead to the generation of metabolites that might not result at sub-lethal doses.
7. Future industrial genetic toxicology needs Much of the emphasis over the past 3 decades in genetic toxicology community has been to identify the most appropriate battery of tests, from among dozens of useful assays, to screen chemicals for mutagenic potential. These tests have served the scientific community and the public fairly well, thus far, in evaluating potentially highly mutagenic compounds before they are introduced into commerce. Revolutionary advances in molecular biology are offering exciting possibilities for the development of new products. For example, genetically modified crops with characteristics such as resistance to insect infestation, tolerance to herbicide usage, and increased nutritional value, offer the greatest promise of successfully tackling the world’s nutritional problems. These
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technologies are also being investigated for producing chemicals and oils with renewable plant based raw materials. The safety of biotechnology derived products is becoming a significant issue of interest to many different stakeholders. Concerns regarding the potential pleiotrophic effects of the inserted transgenes on the safety of foods derived from such sources have been raised. Strategies for genetic toxicology testing of whole foods need to be thoroughly validated in order to address such concern. Foods such as vegetables contain naturally occurring mutagens and hence, it becomes more important to develop approaches that could quantitatively address the substantial equivalence of foods before and after genetic manipulation. Molecular biology has also offered the exciting new DNA microarray technology to monitor changes in gene expression [24]. This technology, if deployed correctly, has the potential to facilitate the definition and identification of threshold phenomenon in genetic toxicology. The inadequate sensitivity of the existing mutagenicity assays has hampered efforts to generate data to challenge the default assumption of lack of thresholds for DNA-reactive chemicals. There is also an urgent need for additional assays that can be used as early screens for potential mutagens, clastogens, and aneugens. Future advances in DNA micro-array technology will undoubtedly result in the availability of affordable DNA chips that can be used to profile dozens and even hundreds of compounds in a short period of time as high throughput assays. References [1] J. Nath, G. Krishna, Safety screening of drugs in cancer therapy, Acta Haematol. 99 (1998) 138–147. [2] J. Ashby, R. Tennant, Chemical structure, Salmonella mutagenicity and extent of carcinogenicity as indicators of genotoxic carcinogenesis among 222 chemicals tested in rodents by the US NCI/NTP, Mutat. Res. 204 (1988) 17– 115. [3] J. Ashby, D. Paton, The influence of chemical structure on the extent and sites of carcinogenesis for 522 rodent carcinogens and 55 different human carcinogen exposures, Mutat. Res. 286 (1993) 3–74. [4] K. Enslein, V.K. Gombar, B.W. Blake, Use of SAR in computer-assisted prediction of carcinogenicity and mutagenicity of chemicals by the topkat program, Mutat. Res. 305 (1994) 47–61.
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and Development Publication Service, 2 rue Andre Pascal, Paris Cedex-16, France, 1997. [22] E. Gocke, L. Muller, P.J. Guzzie, S. Brendler-Schwaab, S. Bulera, C.F. Chignell, L.M. Henderson, A. Jacobs, H. Murli, R.D. Snyder, N. Tanaka, Considerations on photochemical genotoxicity: report of the International Workshop on Genotoxicity Test Procedures Working Group, Environ. Mol. Mutagen. 35 (2000) 173–184.
[23] G. Krishna, G. Urda, J. Theiss, Principles and practices of integrating genotoxicity evaluation into routine toxicology studies: a pharmaceutical industry perspective, Environ. Mol. Mutagen. 32 (1998) 115–120. [24] E.F. Nuwaysir, M. Bittner, J. Trent, J.C. Barrett, C.A. Afshari, Microarrays and toxicology: the advent of toxicogenomics, Mol. Carcinog. 24 (1999) 153–159.