* Howard University College of Medicine, 520 W Street, N. W., Washington, District of Columbia 20059; and
Johns Hopkins Center for Alternatives to Animal Testing, 111 Market Place, Suite 840, Baltimore, Maryland 21202
Received November 29, 2000; accepted February 22, 2001
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ABSTRACT |
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Introduction |
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The Johns Hopkins Center for Alternatives to Animal Testing (CAAT) coordinated an effort to examine the status of alternative methods for the SIDS endpoints listed above.
In October 1998, CAAT hosted a meeting to discuss ways to impact animal usage. The HPV challenge was identified as one such opportunity. Also in October of 1998, Vice President Gore announced a cooperative agreement among the Environmental Defense Fund (EDF), the US Environmental Protection Agency (EPA), and the Chemical Manufacturers Association, now known as the American Chemistry Council, to test the approximately 2800 chemicals using the SIDS battery. At a December 1998 stakeholders' workshop called by the EPA, the agency heard comments/concerns about the role of structure-activity relationships (SARs), what to test, and most importantly, the almost exclusive use of animals in the testing requirements.
The TestSmart group, established in January of 1999, convened a meeting of industrial, governmental, and academic scientists to examine the status of alternative methods for SIDS endpoints and to make recommendations about the use of these methods in the HPV Challenge Program. This program became known as TestSmartHPV. At this meeting, endpoints associated with the SIDS battery (for mammalian studies) were discussed. Green et al. (2001), in addition to reviewing the SIDS endpoints, also suggested alternative tests to those endpoints.
Following discussions, the participants reconvened to report to the entire group. A series of recommendations was distilled from the group summaries and subsequent discussions. These recommendations were as follows:
A time frame for implementation of these recommendations was also established. In the short-term, reduction and refinement alternatives for acute toxicity testing should be incorporated, including the fixed-dose procedure, the up-and-down procedure, the acute toxic class method, and the limit test. Existing in vitro tests for genetic toxicity should also be employed immediately. These include the Ames-Salmonella and mouse lymphoma assays for bacterial and mammalian point mutations, respectively, and the Chinese hamster ovary cell assay for chromosomal aberrations. In vitro tests evaluating sperm motility and sperm morphology should be used to screen for reproductive toxicity and SAR to establish chemical categories and to select specific chemicals within categories. Finally, it was emphasized that protocols should be combined as much as possible to reduce animal numbers. It was suggested that the required repeat-dose study (the 28- or 90-day) be combined with the inclusion of reproductive and developmental endpoints.
In the long term, other promising in vitro tests could be validated and, if successful, could be incorporated into the testing process. Such tests include the in vitro micronucleus assay for genetic toxicity, limb bud and whole embryo culture assays for developmental toxicity and basal cytotoxicity and neural red uptake assays for acute toxicity, and the frog embryo teratogenicity assay, xenopus (FETAX), which has just been reviewed by ICCVAM, (Interagency Coordinating Committee on the Validation of Alternative Methods). The results of that review are presented in the section on developmental toxicity.
Finally, areas requiring additional research were identified. Among those mentioned were: (1) the development of organ cell cultures for determining organ-specific acute and repeat-dose toxicity; (2) a general need for the identification of mechanistic endpoints for toxicity screening; (3) further development of high throughput screening methods for identifying specific genes and/or proteins involved in toxicological pathways; (4) increased effort into culturing human cells and tissues; and (5) development of whole animal refinement approaches, such as noninvasive imaging.
Based on the recommendations of the participants, the list of tests was modified and presented at a second CAAT TestSmart meeting from April 2627, 1999. Table 1 summarizes this information.
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At the April 1999 TestSmart workshop, the EPA signaled that they could accept many of the approaches not previously accepted in order to reduce animal usage. The acute oral toxicity test (OECD 401) would be eliminated. It its place the "up-and-down" procedure (OECD 425) would be conducted, reducing animal usage by 12 per chemical. The requirement for an in vivo assay for cytogenetics (OECD 474) would also be eliminated and in its place an in vitro test accepted (OECD 473). There would be a reduction of 50 animals per chemical. A single test for teratogenicity (OECD 414) and a 1-generation reproductive test (OECD 415) would be eliminated as well. A reproductive/developmental toxicity screen (OECD 421) would replace both. This would result in a saving of 240 animals per chemical. Also eliminated would be the repeat-dose 28-day toxicity test (OECD 407). The endpoints of this test would be included in a reproductive/developmental toxicity screen (OECD 422), saving 40 animals per chemical. The total number of animals without the reduction would be 430 and with the above reductions 88138. Thus, there would be a potential saving of from 292342 animals, representing a 6880% reduction. This reduction would be accomplished before quantitative structure-activity relationship (QSAR) and in vitro methods for identifying and validating categories of chemicals. Additional savings in terms of animals would be accomplished by the use of in vitro methods as range-finding approaches in determining dosages for in vivo studies.
As the next step in investigating alternatives to the SIDS battery, an evaluation of the data available for the recommended alternative was conducted. This evaluation was undertaken to provide a firmer basis for the categorization of the alternative tests as ready for immediate use, in need of validation, or currently needing research/developmental work. This article reports the results of that evaluation.
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Acute Toxicity |
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Replacement Alternatives to the Classic LD50
Basal cytotoxicity is more an approach to cytotoxicity than an actual test per se, for it can be applied to almost any mammalian cell culture system. It is defined or thought to be damage as a result of chemical injury to 1 or more of the basic cellular structures or functions common to mammalian cells (Barile, 1998). Numerous studies prior to this publication have been conducted demonstrating a correlation between in vitro cytotoxicity and in vivo lethality (Clemedson et al., 1996; Clothier et al., 1987
; Walum and Peterson, 1984
). One of the most extensive early studies conducted was by Clemedson et al. (1996), which involved 68 methods with 50 reference chemicals. The chemicals were selected on the basis of available human acute toxicity data. The results from 68 methods with 30 of the 50 chemicals showed that similar results were obtained, regardless of cell type and regardless of whether cell viability or cell proliferation was used as an endpoint. In a recent report on this approach, Ekwall et al. (1998) concluded the following:
The authors also performed a "correlative/mechanistic" study. In this study IC50 values from 10 human cell lines were compared with lethal blood concentrations in humans. The 50 chemicals were separated into 3 categories: (1) fast-acting, brain-barrier restricted; (2) slow-acting, brain-barrier restricted; and (3) those that cross the blood-brain barrier freely while inducing a nonspecific excitation/depression of the central nervous system. The r2 values were 0.98 (category 1), 0.85 (category 2), and 0.82 (category 3). The authors concluded that these results supported the basal cytotoxicity theory (good in vitro/in vivo correlations), and further pointed to nonspecific CNS suppression as the primary reaction of humans to cytotoxic concentrations of chemicals, once they traverse the blood-brain barrier.
Rasmussen (1999) evaluated the cytotoxicity of MEIC (Multicenter Evaluation of in Vitro Cytotoxicity) chemicals Nos. 1130 in Balb/c3T3 cells with and without metabolic activation. The cytotoxicity data was compared to in vivo toxicity data and to cytotoxicity data from human and rat hepatocytes. She also investigated the correlation of the data to human and animal cell lines used in the MEIC program. She reported "moderately good correlations" between the cytotoxicity obtained with and without metabolic activation and rat and mouse LD50 data. She cautioned, however, that the approach had limitations due to the inadequacies of the metabolic activating systems. She concluded that there is limited evidence to support the relevance of in vitro/in vivo correlations obtained in the MEIC project primarily due to the high degree of variability of the in vitro and in vivo data used.
In considering basal cytotoxicity as a means of replacing LD50 testing in animals, careful thought must be given to the above studies. First, one has to recognize that the purpose of the studies was to provide additional evidence of the existence of the basal cytotoxicity phenomenon, not to recommend use of the approach in a hazard determination or regulatory context. Some of the correlations, however, are relatively poor. For example, those of the rat and mouse LD50 data compared to human acute lethal dosages (correlation coefficients of 0.61 and 0.65 respectively). The best correlations were achieved with grouping certain chemicals in terms of their ability to penetrate the blood-brain barrier, and comparing IC50 values of those chemicals with human lethal blood concentrations (correlation coefficients of 0.98 for fast-acting nonrestricted, 0.85 for slow-acting restricted, and 0.82 for those crossing freely). This argues for knowledge beforehand of the types of agents that can confidently be tested in this approach, particularly if one considers making hazard determinations on the basis of these results.
In considering use of the basal cytotoxicity approach for hazard determination, the following questions or issues need to be raised. What can we conclude on the basis of a result in this approach? Without companion human data, we could suggest this result indicates this chemical may be lethal to humans at this dose. Is there a satisfactory level of confidence in the approach for a governmental agency, pharmaceutical firm, or other industrial organization to accept that conclusion? Would the usual next step be taken; i.e., that of repeat-dose toxicity based on the dosages of the basal cytotoxicity study? What about chemicals that cause acute toxicity by mechanisms other than basal cytotoxicity? It is certain that additional questions/issues can be raised in considering how data from this approach should be used in making hazard determinations and facilitating regulatory decisions. It seems that much work remains to be done focusing on how and where basal cytotoxicity fits into the regulatory decision-making process. Thus, we have included this approach in the correct category of research and development. After this article was written, ICCVAM initiated a review of this method.
Reduction/Refinement Alternatives to the Classic LD50
Four animal tests also have been recommended as alternatives to the classic LD50 These tests are the limit test, fixed-dose procedure, toxic class method, and the up-and-down method. These tests do not represent the first attempt to explore alternatives to the classic LD50. Weil (1952) developed a mathematical model for determining the LD50 using fewer animals than the classic approach.
The limit test, which uses 5 animals per dose level, is used when there is evidence that a chemical is of low toxicity. It uses 3 dosages, the highest of which is 2000 mg/kg. This is the dose that is tested first and if no toxicity is observed, the substance is considered nontoxic. If toxicity is observed, a more definitive study of its toxicity is conducted. The fixed-dose procedure uses 5 males and 5 females that are administered 1 of 4 fixed dosages (5, 50, 500, or 2000 mg/kg). Morbidity defined by signs of toxicity is the endpoint rather than mortality. If death occurs, a lower dosage is tested. Depending on the dosage at which morbidity occurs, the chemical is classified as very toxic, 5 mg/kg; toxic, > 5
50; harmful, > 50
500; or not labeled, > 500
2000 mg/kg. In the acute toxic class method, 3 animals are dosed at 1 of 3 OECD classification dose limits and mortality is observed. The lowest dose at which mortality is observed determines the classification of the chemical.
The up-and-down procedure usually uses 1 animal per exposure. If the animal dies, a dosage reduced by a factor of 1:3 is used. If this second animal survives, the LD50 is then determined. If the first animal survives, the dose for the second is increased by a factor of 1:3 and this continues until death is observed, or the dosage of 2000 mg/kg is reached. These procedures have been in use for several years and can be considered validated. For a review of these procedures, see Walum (1998). Their status as being ready for immediate use, as reflected in Table 1, is supported by available scientific data.
The neutral red uptake assay is a method that can be used to determine cytotoxicity. (Borenfreund and Puerner, 1984). The principle of the method involves penetration of the cellular membrane by a vital dye neutral red, (3-amino-7-dimethylamino-2-methylphenazine hydrochloride). Once in the cell the lysosome takes up the dye. The uptake of neutral red can be used as a quantitative measure of cell viability. Much work has been done in assessing the performance of this assay for ocular irritation. Keratinocytes have been the cells of choice. The assay theoretically could be conducted with any cell line or possibly primary cell culture. In order to provide information about acute in vivo systemic effects as opposed to local effects, as in the case of ocular irritation, metabolic activation would need to be added. There would also be the need to conduct studies to establish its reliability and reproducibility in predicting acute toxicity. In essence an assessment for validation needs to be accomplished to determine the applicability of this method to acute systemic toxicity.
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Non-LD50 Acute Toxicity |
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Liver cultures have been used for years to study toxicity and metabolism. These cells, primary or continuous cell lines, conceivably could be part of a battery of cell cultures designed to assess acute systemic toxicity. Cell lines seem to be best in terms of technical ease and longevity. The ability of cell lines to reflect accurately the in vivo situation is questionable at this time. For example, they do not possess the full spectrum of liver metabolizing enzymes as do primary cultures (Silber, 1999). Additional work needs to be done to improve retention of in vivo qualities in these cell lines. Guillouzo (1998) provides a review of the application of liver cell cultures to toxicity testing. Similarly, kidney cells have been used for some time in the generation of toxicity information. Freshly isolated cells, kidney fragments, precision-cut kidney slices, and cell lines have been used. As in the case of liver, cell lines seem best in terms of technical ease and longevity, but suffer the same major deficiency, the inability to reflect accurately the in vivo situation. Some endpoints that could be examined in kidney cells are suggested by Spielmann et al. (1998) and a review of the application of kidney cells to toxicity testing is given by Pfaller and Gstraunthaler (1998).
Epithelial cells from the trachea and bronchioles of rat, rabbit, human, guinea pig, and hamster have been cultured. Squamous type II pneumocytes and cuboidal Type II pneumocytes have also been cultured. The cuboidal Type II cells usually retain most of the functions found in the alveolar region. Alveolar macrophages obtained from bronchial lavage have been used for some time in studying toxicity of gases and particles. Most of these have been used as primary cultures. While this tends to insure relevance to the source and to other animals, longevity and ease of use can represent concerns. The addition of metabolic activation to in vitro cultures of cells of the respiratory tract must also be considered. Lambre et al. (1996) provides an excellent review of the types of in vitro systems for respiratory toxicology investigations. It is clear that additional research needs to be conducted to develop a tier or battery approach to the use of in vitro alternatives for this purpose.
The nervous system is arguably the most complex organ system of the body. Developing cell culture methods to detect motor and cognitive effects may be exceptionally difficult. Thus, it may not be possible to use in vitro methods to assess toxicity in such areas at this time. We have listed the autonomic nervous system as a major concern regarding acute toxicity testing. The autonomic nervous system consists of the sympathetic and parasympathetic divisions. Autonomic dysfunction could be responsible for some acute deaths. However, the deaths observed would be the result of failure of the cardiovascular, respiratory, or other organ system. To specifically focus on the autonomic nervous system may be somewhat premature given the state of in vitro neurotoxicity testing. Currently the emphasis is on methods that can provide information about the nervous system in general, and not on specific segments such as central or peripheral. Organotypic explants, primary cell cultures, brain slices, reaggregate cultures, and established cell lines have been used. Although established cell lines are easier to use, they are derived from tumors of the mouse, rat, or human. For the most part these methods have been used to study mechanisms of action and not specifically to identify toxic agents. Consequently more research is needed to determine how these methods can be applied to toxicity testing. Costa (1998) discusses the state of in vitro neurotoxicity testing.
We have also listed the vascular system as an area deserving of attention in the development of in vitro methods. It would be of interest to know the number of chemicals that have a direct action on the walls of blood vessels and the types of acute or other types of toxicity they produce. This would be the obligatory step in determining the need for as well as the direction of alternative research in this area.
Cell cultures have been used for some time in determining the mechanism of action of toxic chemicals. For certain organ systems, techniques have become even routine. However, it is equally clear that further studies are needed in the use of these techniques in determining the acute systemic target organ toxicity of xenobiotics. To propose their use singly or in a battery approach in a hazard assessment scheme at this time would not be prudent. Thus the listing of these alternative tests under the category "research" is an accurate reflection of their current status.
We have also listed the mitochondria as an organelle deserving of attention when considering systems that if adversely affected, could lead to systemic acute toxicity. Mitochondria are involved in cellular energy production. Aerobic metabolism, which produces this energy, is primarily conducted in these organelles. In fact the mitochondria produce approximately 95% of the energy needed by the cell. Therefore, chemicals that adversely affect the mitochondria could have serious consequences for the cell and the organism. There is a dearth of information on attempts to develop cultures of mitochondria although some cell culture endpoints are a direct measure of mitochondrial function, e.g., MTT. This clearly is a subject for future research.
Binding at the Ah receptor has recently been suggested as a possible surrogate for the LD50. Rosenkranz and Cunningham (2000) conducted an analysis of several endpoints including binding to the Ah receptor and found that the LD50 was significantly related to binding at the receptor. Validation of this association needs to be conducted. Thus the Ah receptor has been placed in the category of needing validation.
It is evident that most of the suggested alternative tests for non-LD50 testing are deserving of additional research before even validation activities should be considered. They have been characterized in Table 1 as in the research stage.
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Repeat-Dose Toxicity |
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One endpoint that is always measured in repeat-dose toxicity studies is that of rate of growth. There is some thought that the in vitro measurement of the rate of protein synthesis could provide insight to this parameter. Protein synthesis is easily measured in cells but the correlation of this measurement to rate of growth in the whole animal has rarely been attempted. Research is needed to explore such a relationship. Similar complications in regard to duration and frequency also apply here.
In vitro studies of cell signaling and apoptosis are also suggested as alternatives in providing information about repeat-dose systemic toxicity. Apoptosis is regulated cell death and is thought to be a counterbalance to mitosis. Cell signaling is thought to be involved in determining whether a cell undergoes mitosis or apoptosis. An assessment of these endpoints could give insight to mechanisms of cell and thus organ toxicity. The application of these endpoints in a battery approach together with information from in vitro toxicity to cell cultures of liver, kidney, etc could represent an integrated approach to determining systemic toxicity. As before, the issue of duration and frequency of dose need to be taken into account. Corcoran et al. (1994) discuss these endpoints and the theory as to how they interact in producing toxicity. Further research along these lines could represent a novel approach to alternative testing.
We can conclude that all proposed alternative tests for repeat-dose systemic toxicity are in the research stage. Factors complicating the development of alternative tests in this area are how to model duration and frequency of dose as well as interplay between and among organ systems in the animal. These conclusions would also apply to the 28-day repeat-dose toxicity studies.
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Genetic Toxicity |
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With respect to chromosomal abnormalities we have recommended the in vitro micronucleus assay. The principles underlying the in vivo micronucleus assay have been documented by Heddle et al. (1983) and also apply to the in vitro assay. The in vitro micronucleus assay is currently undergoing validation in a number of countries and its utility for detecting chromosomal abnormalities seems promising (Gibson et al.1998; Miller et al.1998
). We have also recommended the Syrian hamster embryo in vitro transformation assay as an additional method to the genetic toxicology panel of tests. It is thought that information regarding potential carcinogenicity in a mammalian cell would be exceptionally useful at the screening stage of chemicals. The principles underlying this method are documented in LeBoeuf et al. (1999) and this method is ready for validation.
In summary the discipline of genetic toxicology presents an excellent opportunity to demonstrate use of in vitro alternatives in screening for mutations and for potential carcinogenicity.
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Developmental Toxicity |
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Embryonic stem cells have the possibility of developing into any tissue of the body. This capability offers obvious advantages to the assessment of developmental toxicity. Many research studies are in progress. For this reason, these assays also are placed in the category of "undergoing validation." Primary limb bud mesenchymal cell isolated from 10-day mouse embryos can be cultured as micro-mass cultures. Inhibition of chondrogenesis as measured by alcian blue staining and the uptake of 35S are the endpoints assessed. This assay is also undergoing validation by ECVAM and is placed in that category. Zebrafish may also be used as an alternative for developmental toxicity. In this test fertilized eggs are exposed for 48 h and coagulation, development of the blastula, gastrulation, termination of gastrulation, development of somites, extension of the tail, and development of eyes, heartbeat, circulation, pigmentation, and edema are assessed as endpoints (Walker et al., 1998). This approach is undergoing additional validation and is thus placed in that category. In gene chip technology, DNA variants can be detected in an efficient manner. In regard to developmental toxicity, this means screening DNA variants that have been linked to abnormal developmental effects. In this technique, the DNA to be screened is labeled with a fluorescent dye and then applied to the chip. It binds more strongly to the sequence that is its complement. It is thus identified and its sequence determined. To our knowledge very little has been done in investigating the application of this technology to developmental toxicology. This is an entirely new area of investigation undergoing significant development at the National Institute of Environmental Health Sciences laboratories and in commercial ventures. The major question to be addressed is the functional outcome of gene alterations as measured by gene-chip technology. Thus it is placed in the category of "research."
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Reproductive Toxicity |
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The concern over the possibility of chemicals in our environment having the capability to disturb endocrine balance has stimulated development of receptor binding assays. Many individuals have recommended estrogen and androgen binding. Although much work has been done in developing these assays, there remains a degree of uncertainty as to their repeatability, reliability, and interpretability. Therefore additional research should be conducted to demonstrate their utility for broad reproductive screening purposes.
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Repeat-Dose Reproductive/Developmental Screen |
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Other Applications of Alternatives to the HPV Challenge Program |
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There is yet another step in the development of a category. Assuming the characterization is successful, the sponsor would then need to select representative chemicals, some from the low, middle and high end of the category, for testing. These would then represent the category. Testing these chemicals and evaluating the results should allow a decision as to whether the group could be viewed as a category. There should be some pattern of results from the low to the high end that demonstrates a relationship between structure and effect. Interpolation can then be accomplished for those chemicals that were not tested. This step in the development of a category could be called validation. Validated alternatives could provide the data to permit such conclusions to be made. Use of alternatives at this point in the Challenge Program would facilitate familiarity with in vitro methods and make the transition from whole animal toxicity methods more efficient.
In vitro assays could also be used as range-finding approaches to acute systemic toxicity testing. Careful consideration would need to be given to the database supporting an in vitro assay for this purpose. Validation studies would be needed to establish the qualitative and quantitative correlation between such an assay and animal tests. Given that most acute toxicity data are gathered because little is known about a chemical, structural similarity to a known toxicant that has been tested in the in vitro assay could serve as the first step in screening an unknown. Spielmann et al. (1999) recently illustrated use of in vitro assays in determining the starting dose for acute oral toxicity data (up-and-down procedure).
We have outlined what we consider one approach to the development of alternatives to the SIDS approach to high production volume chemical testing. We have called this program "TestSmart, A Humane and Efficient Approach to Regulatory Toxicological Data." Progress along these lines would not only apply to SIDS, but to toxicology in general. We recognize there may be other approaches and welcome input from the scientific community at large. Based on our analysis much work remains to be done to develop scientifically defensible alternatives (screens and/or replacements). We urge industry, government, and academia to marshal resources in order to continue the progress made thus far.
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ACKNOWLEDGMENTS |
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NOTES |
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2 Present address: National Academy of Sciences, Washington, D.C.
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