Whole-animal Testing Research Articles

International Consortium to Advance Cross-Species Extrapolation of the Effects of Chemicals in Regulatory Toxicology.

International Consortium to Advance Cross-Species Extrapolation of the Effects of Chemicals in Regulatory Toxicology.

Reevaluation of the Embryonic Stem Cell Test

In recent years there has been a heightened interest in developing alternative toxicity testing methods that enhance the current system that relies almost exclusively on whole-animal testing to include in vitro assays focused on defined pathways. The embryonic stem cell test (EST) is one alternative that has been suggested for use in developmental toxicity testing. The EST utilizes the D3 mouse embryonic stem cell line to assess sensitivity of chemicals on differentiating cardiomyocytes. Additionally, a BALB/3T3 line is used to monitor cytotoxicity and compare sensitivities between adult and embryonic cells. We have reevaluated the EST nearly 10 years after formal validation by the European Center for the Validation of Alternative Methods (ECVAM) to test the stability and reliability of the cell lines in predicting developmental toxicity. Eight compounds from the ECVAM validation including the positive control, 5-fluorouracil, and the negative control, penicillin G, were tested. All eight compounds matched the classification reported during validation, indicating comparable responses and transferability of the experimental protocol. However, an increased sensitivity of the cell lines, identified by lower ID50 and IC50 values, was observed for many of the chemicals when compared to the results from the ECVAM validation.

Behavioral toxicology in the 21st century: Challenges and opportunities for behavioral scientists: Summary of a symposium presented at the annual meeting of the Neurobehavioral Teratology Society, June, 2009

The National Research Council (NRC) of the National Academies of Science recently published a report of its vision of toxicity testing in the 21st century. The report proposes that the current toxicity testing paradigm that depends upon whole-animal tests be replaced with a strategy based upon in vitro tests, in silico models and evaluations of toxicity at the human population level. These goals are intended to set in motion changes that will transform risk assessment into a process in which adverse effects on public health are predicted by quantitative structure–activity relationship (QSAR) models and data from suites of high-throughput in vitro tests. The potential roles for whole-animal testing in this futuristic vision are both various and undefined. A symposium was convened at the annual meeting of the Neurobehavioral Teratology Society in Rio Grande, Puerto Rico in June, 2009 to discuss the potential challenges and opportunities for behavioral scientists in developing and/or altering this strategy toward the ultimate goal of protecting public health from hazardous chemicals. R. Kavlock described the NRC vision, introduced the concept of the ‘toxicity pathway’ (a central guiding principle of the NRC vision), and described the current status of an initial implementation this approach with the EPA's ToxCast® program. K. Crofton described a pathway based upon disruption of thyroid hormone metabolism during development, including agents, targets, and outcomes linked by this mode of action. P. Bushnell proposed a pathway linking the neural targets and cellular to behavioral effects of acute exposure to organic solvents, whose predictive power is limited by our incomplete understanding of the complex CNS circuitry that mediates the behavioral responses to solvents. B. Weiss cautioned the audience regarding a pathway approach to toxicity testing, using the example of the developmental toxicity of phthalates, whose effects on mammalian sexual differentiation would be difficult to identify based on screening tests in vitro. Finally, D. Rice raised concerns regarding the use of data derived from toxicity screening tests to human health risk assessments. Discussion centered around opportunities and challenges for behavioral toxicologists regarding this impending paradigm shift. Opportunities include: identifying and characterizing toxicity pathways; informing the conditions and limits of extrapolation; addressing issues of susceptibility and variability; providing reality-checks on selected positives and negatives from screens; and performing targeted testing and dose-response assessments of chemicals flagged during screening. Challenges include: predicting behavior using models of complex neurobiological pathways; standardizing study designs and dependent variables to facilitate creation of databases; and managing the cost and efficiency of behavioral assessments. Thus, while progress is being made in approaching the vision of 21st century toxicology, we remain a long way from replacing whole-animal tests; indeed, some animal testing will be essential for the foreseeable future at least. Initial advances will likely provide better prioritization tools so that animal resources are used more efficiently and effectively.

Commentary on ‘‘Toxicity Testing in the 21st Century: A vision and a Strategy’’

The vision of the NAS Committee represents a transition in toxicology from an emphasis on the study of apical endpoints in whole animals to a focus on the study of pathway perturbations, based primarily on in vitro data, from which dose-response models will ultimately be developed. It should be emphasized that this vision does not seek to eliminate whole-animal testing altogether; however, it does aim to eventually use animal testing to a far less extent than in our current system. Such a change to the current method of toxicity testing represents an important goal for many reasons. For example, toxicity testing needs to incorporate the knowledge that has been developed through the most recent advances in research, particularly at the molecular level. Through the application of mechanistic information resulting from the testing of chemicals by the proposed in vitro methods, it may be possible to predict the toxicity of similar chemicals that have not yet been tested in whole animals. The number of chemicals in commercial use for which we have adequate toxicity information is limited, and higherthroughput assays have the potential to increase this number. While the vision’s approach would result in a loss of information from a reduction in testing at high doses, the NAS Committee has proposed that this lack of information would be compensated by a greater understanding of effects at low doses, and that such an understanding can eventually be applied in a practical context. Clearly, however, there are multiple challenges to the actual implementation of the vision. First, we need to consider the vast biological complexity of whole organisms. Will in vitro assays for pathway perturbations, in the absence of some understanding from in vivo studies, be able to identify all of the possible toxic endpoints of a particular chemical? The application of in vitro methods may be a slow and uncertain process without some initial hypotheses about likely critical apical endpoints for a particular chemical. In contrast, with animal bioassays, we can identify toxic endpoints that we may not have originally thought to look for, through the analysis of different target organs. An understanding of how pathway activation and the corresponding dose-response relationships change with differences in the duration of exposure will be necessary, and yet it may be difficult to distinguish between acute, subchronic, and chronic effects at the pathway level. In addition, in vitro assays cannot mirror the metabolism of a whole animal, and the NAS Committee has agreed that much research will be needed to ensure that the new testing methods associated with their vision fully evaluate the effects of both chemicals and their metabolites. The development of ‘-omic’ technologies (such as genomics, proteomics, and metabolomics) is a key element for advancing the vision of the NAS Committee; however, there are challenges to the use of these approaches for the prediction of in vivo toxicity. Rhomberg et al. state that:

TOX 21: New Dimensions of Toxicity Testing

On the ground floor of the National Institutes of Health Chemical Genomics Center (NCGC) in Rockville, Maryland, a $10-million automated laboratory spends all day and night screening chemicals at speeds no team of human researchers could ever match. In a week, depending on the nature of the assay, it can yield up to 2.2 million molecular data points derived from thousands of chemicals tested at 15 concentrations each.

The challenge of reproductive and developmental toxicology under REACH

The European Union’s REACH regulation has explicit requirements for reproductive and developmental toxicity data on all substances manufactured in or imported into the European Union at ⩾10 metric tons/year. Meeting the data requirements with whole-animal testing could result in the use of almost 22 million vertebrate animals for the registration of existing chemicals and cost up to several hundred thousand dollars per registered substance. The requirement for financial and animal resources can be reduced by the use of in vitro testing, quantitative structure–activity relationship models, and grouping of related substances. Although REACH strongly encourages these methods of avoiding vertebrate animal testing, it does not appear that in vitro testing or quantitative structure–activity relationship analysis will be able to replace whole-animal reproductive and developmental toxicity testing. Grouping of related compounds offers the possibility, perhaps in conjunction with in vitro testing and structure–activity analysis, of reducing vertebrate animal testing provided there is sufficient information on the related compounds and sufficient reason to believe that the related compounds will have similar toxicological properties. The designation of a substance as a reproductive or developmental toxicant follows criteria that do not consider the dose level of the substance at which reproductive or developmental effects occur, as long as excessive generalized toxicity does not occur. This method of labeling substances without consideration of effective dose level does not provide information on the actual risk of the chemical. Designation of a substance as a reproductive or developmental toxicant may have important implications under REACH and can be expected to result in the need to obtain authorization for marketing of the substance in the European Union.

Use of In Vitro Absorption, Distribution, Metabolism, and Excretion (ADME) Data in Bioaccumulation Assessments for Fish

ABSTRACT A scientific workshop was held in 2006 to discuss the use of in vitro Absorption, Distribution, Metabolism, and Excretion (ADME) data in chemical bioaccumulation assessments for fish. Computer-based (in silico) modeling tools are widely used to estimate chemical bioaccumulation. These in silico methods have inherent limitations that result in inaccurate estimates for many compounds. Based on a review of the science, workshop participants concluded that two factors, absorption and metabolism, represent the greatest sources of uncertainty in current bioaccumulation models. Both factors can be investigated experimentally using in vitro test systems. A variety of abiotic and biotic systems have been used to predict chemical accumulation by invertebrates, and dietary absorption of drugs and xenobiotics by mammals. Research is needed to determine whether these or similar methods can be used to better predict chemical absorption across the gills and gut of fish. Scientists studying mammals have developed a stepwise approach to extrapolate in vitro hepatic metabolism data to the whole animal. A series of demonstration projects was proposed to investigate the utility of these in vitro–in vivo extrapolation procedures in bioaccumulation assessments for fish and delineate the applicability domain of different in vitro test systems. Anticipating research progress on these topics, participants developed a “decision tree” to show how in vitro information for individual compounds could be used in a tiered approach to improve bioaccumulation assessments for fish and inform the possible need for whole-animal testing.

In vitro and in vivo assessment of the effect of impurities and chirality on methamidophos-induced neuropathy target esterase aging.

In vitro and in vivo studies evaluated neuropathy target esterase (NTE) inhibition and aging (i.e., loss of reactivation potential) by analytical and technical grade racemic and resolved L-(-) and D-(+) isomers of methamidophos (O,S-dimethyl phosphoramidothioate). For studies in vitro, microsomal protein from phenobarbital-induced livers was isolated from chick embryos and NTE inhibition assays were performed using chick embryo brain homogenate treated with 1 or 5 mM methamidophos (with and without metabolic enzymes); for studies in vivo, hens received 30 to 35 mg/kg methamidophos injected into the pectoral muscle. NTE aging in hens was assessed 24 h later or after 30 min to 1 h incubation in vitro using solutions of potassium fluoride (KF) reactivator. Technical methamidophos produced significantly higher levels of aged-inhibited NTE than analytical methamidophos or isolated optical isomers. In vivo, technical methamidophos produced 61% total NTE inhibition with 18% aged and 43% unaged NTE; hens receiving analytical grade averaged 6% aged, 52% unaged, and 58% total NTE inhibition. Results for 1 mM analytical methamidophos in vitro were 5% aged, 54% unaged, and 59% total inhibition; for 1 mM technical methamidophos, values averaged 11% aged, 50% unaged, and 60% total NTE inhibition. The degree of NTE aging obtained both in vivo and in vitro for the isolated D-(+) and L-(-) isomers never exceeded that obtained using analytical grade. These data indicate that impurities in methamidophos could contribute to OPIDN potential. The in vitro methodology described could be applied to first tier screening for detection of NTE inhibition and aging, thus reducing the need for whole-animal testing for OPIDN.

CULTURED KERATINOCYTES IN IN VITRO DERMATOTOXICOLOGICAL INVESTIGATION: A REVIEW

The keratinocyte is responsible for the architecture of the epidermis, that portion of the skin that forms the environmental barrier necessary for survival. It also interacts with other cell types in the epidermis in response to various environmental influences. This cell type is used frequently for in vitro cutaneous toxicological investigations as an alternative to whole-animal studies. Several areas of cutaneous research using cultured keratinocytes are germane as regards the scope of this journal. The following areas of biomedical research were reviewed: (1) dermatotoxicology, including environmental chemicals, antiseptics, drugs, metals, and pesticides; (2) immunotoxicology, including inflammation and allergic dermatitis; (3) radiation, including ultraviolet and x-irradiation; and (4) the development of assays as alternatives to whole-animal testing. Due to the abundance of such investigations reported in the last 30 years, this review is limited mainly to reviewing reports published in this decade.

Conference summary

Although the Draize test will not yet pass into disuse, the conference attendees were exposed to a number of alternative methodologies that can reduce the number of animals used in whole-animal testing. These alternatives have reached the status of screening tests. Many participants thought that in vitro tests can replace in vivo experiments only when there is a greater understanding of the mechanism or underlying biology involved in the in vitro test. With respect to refinement and reduction, the strongest consensus was related to use of two-stage testing in which only one or two animals were studied initially, and the nature and consistency of the findings dictated further testing. The low-volume test provoked some of the sharpest debate, and participant consensus was not evident. Much information is lost when the number of grades or classes of irritants is reduced. Consequently the elimination of a ‘corrosive’ class will require more discussion. International practices and conventions prohibited a consensus regarding the definition of a positive test, either as number of animals responding or as the mean score. The use of total acidity or alkalinity as a substitute for pH requires further study. The opinion of the participants was that regulatory consensus on the use and interpretation of irritation data from updated methods would be highly desirable.

Developmental toxicity of four model alkylating agents on japanese medaka fish (Oryzias latipes) embryos

As public and scientific interest increases in identifying in vitro alternatives to whole-animal testing, there is a need to characterize the responses of in vitro systems to defined chemical agents. In this paper we have utilized the medaka (Oryzias latipes) embryo culture system to examine the developmental toxicity of a series of chemically defined model alkylating agents. The medaka embryo culture system was used because it offers numerous advantages over other in vitro embryo systems. This study has examined the developmental toxicity of four model alkylating agents: methylnitrosourea (MNU), ethylnitrosourea (ENU), methyl methanesulfonate (MMS), and ethyl methanesulfonate (EMS). These alkylating agents represent chemical models for compounds that are environmental and occupational pollutants. The embryotoxic parameters that were monitored include embryolethality and specific malformations. Medaka embryos in early organogenesis (stage 20) were exposed for 2 h to the test compound at concentrations ranging from 0.75 to 120 mM. The embryos were cultured at 20°C in Petri dishes and were assessed daily for viability and gross malformations until hatching. All agents tested produced concentration-dependent decreases in viability and increases in malformations. The developmental toxicity of the four alkylating agents examined is, in order of decreasing potency, MNU > ENU > MMS > EMS. A similar spectrum of malformations was produced following exposure to each alkylating agent; however, agents undergoing first-order nucleophilic substitution reactions were more potent developmental toxins than those agents which undergo second-order nucleophilic substitution reactions. Observed abnormalities included flexure, cardiac, optic, and cephalic malformations.

Reproductive Toxicity Testing: Evaluating and Developing New Testing Systems

Reproductive toxicity testing systems are used by national and international regulatory agencies. Protocols have not been standardized between agencies or even within certain agencies. Although there have been efforts at standardization, a certain amount of the differences between testing protocols is a reflection of the needs of the particular agency. New developments in in vitro techniques might lead to new test systems, but reproductive function is dependent upon the interaction of various cells and organs that cannot presently be copied in the test tube; this makes whole-animal testing systems a necessity. The present whole-animal models used by the Food and Drug Administration include the 3 segment reproduction studies used for testing drug safety and the multigeneration studies used for food additives. The Environmental Protection Agency has adopted 2 similar versions of a 2-generation study for the Office of Pesticide Programs and the Office of Toxic Substances. The National Toxicology Program, although not a regulatory agency, has taken a prominent role in reproductive toxicity testing, test system development, and test system evaluation. A new testing system, Fertility Assessment by Continuous Breeding (FACB), is currently being studied as a cost-effective and reliable alternative test system. The FACB protocol houses male and female mice as breeding pairs and removes offspring as soon as they are born during the first 14 weeks to allow continuous mating. Each breeding pair normally has up to 5 litters, and the last litter is saved to evaluate the second generation. The efficiency, reliability, and expense of the protocol are being compared to the existing testing systems.

Use of chick embryo in screening for embryotoxicity.

In vitro systems afford a unique opportunity for investigating the direct interaction of substances with developing morphogenetic systems (MGSs), since maternal influences are excluded. As a carrier of a complete set of MGSs, the chick embryo in ovo manifests an advantage over those in vitro systems that employ isolated embryos or embryonic tissues that have only limited survival. Under controlled experimental conditions including standardization of subjects, administration technique and mode of evaluation according to the basic principles of teratology, the chick embryo test was demonstrated to be reliable and to afford quantifiable endpoints for evaluation (e.g., specific embryotoxicity effect level, positive dose response, and a stage response and effect). Individual compounds, mixtures of compounds, and even poorly identified extracts can easily be tested. The chick embryo possesses its own basic enzyme-catalyzed drug-transformation capacity and, moreover, it can be used for screening specific human metabolites. Comparative studies have indicated a high predictive value of the chick embryo system relative to other in vitro systems as well as to whole-animal testing.