Toxicity Testing Overview

Toxicology is defined as "the study of the adverse effects of chemical, physical or biological agents on living organisms and the ecosystem" and is based on the 16th century principle that any substance can be toxic if consumed in sufficient quantity.

Today, most developed countries have enacted laws and regulations to control the marketing of drugs, vaccines, food additives, pesticides, industrial chemicals, and other substances of potential toxicological concern. Such regulations often prescribe a specific regime of toxicity testing to generate information that will enable government regulators to determine whether the benefits of a particular substance outweigh its risks to human health and/or the environment. This process of regulatory risk assessment can be broken down into three main phases:

• Hazard identification: Determination of a substance's intrinsic toxicity (e.g., eye irritation, birth defects, or cancer) through the use of toxicity tests. Test results are then analyzed to determine what, if any, adverse effects occur at different exposure levels (known as a "dose-response" assessment) and, where possible, to identify the lowest exposure level at which no adverse effects are observed (known as the "no observed adverse effect level" or "NOAEL").
• Exposure assessment: Determination of the extent of human and/or environmental exposure to a substance, including the identification of specific populations exposed, their composition and size, and the types, magnitudes, frequencies, and durations of exposure.
• Risk characterization: A composite analysis of the hazard and exposure assessment results to arrive at a "real world" estimate of health and/or ecological risk.

AltTox.org focuses primarily on toxicity tests used in the hazard identification step of risk assessment. However, exposure information can impact hazard identification strategies, and this will be discussed in sections of AltTox dealing with integrated testing strategies and criteria for waiving testing requirements.

Toxicity Tests

A test method is a definitive procedure that produces a test result. A toxicity test, by extension, is designed to generate data concerning the adverse effects of a substance on human health or the environment. Many toxicity tests examine specific types of adverse effects, known as "endpoints," such as eye irritation or cancer. Other tests are more general in nature, ranging from single-exposure ("acute") studies to multiple-exposure ("repeat dose") studies, in which animals are administered daily doses of a test substance to calculate NOAELs and determine whether one or more organ or system is adversely affected following exposures of one-month ("subacute"), three-month ("subchronic"), and/or two-year ("chronic") duration. Tests aimed at identifying hazards to humans are generally referred to as "safety" or "health effects" studies, whereas wildlife-oriented tests are known as "ecotoxicity" studies.

Toxicity endpoints considered within the scope of AltTox include the following:

• Acute Systemic Toxicity: Adverse effects occurring within a short time after administration of a single (usually extremely high) dose of a substance via one or more of the following exposure routes: oral ("gavage"), inhalation, skin ("dermal"); or injection into the bloodstream ("intravenous"), the abdomen ("intra-peritoneal"), or the muscles ("intra-muscular")
• Skin Irritation/Corrosion: Chemically induced skin damage that is reversible (irritation) or irreversible (corrosion)
• Eye Irritation/Corrosion: Chemically induced eye damage that is reversible (irritation) or irreversible (corrosion)
• Skin Sensitization: The induction of allergic contact dermatitis following exposure to a chemical agent
• Dermal Penetration: The extent and rate by which a chemical is able to enter the body via the skin (also known as "skin absorption" or "percutaneuos absorption")
• Repeated Dose Toxicity: General toxicological effects occurring as a result of repeated daily exposure to a substance (via oral, inhalation and/or dermal routes) for a portion of the expected life span (i.e., subacute or subchronic exposure) or for the majority of the life span (i.e., chronic exposure)
• Reproductive & Developmental Toxicity: Chemically induced adverse effects on sexual function, fertility and/or normal offspring development (e.g., spontaneous abortion, premature delivery, and birth defects), generally determined through the breeding of one or more generations of offspring
• Genotoxicity: Chemically induced genetic mutations and/or other alterations of the structure, information content, or segregation of genetic material (e.g., DNA strand breaks or a gain/loss in chromosome number in cells)
• Carcinogenicity: Chemically induced cancer, whether through genotoxic or non-genotoxic (i.e., growth-promoting) mechanisms
• Neurotoxicity: Chemically induced adverse effects on the brain, spinal cord, and/or peripheral nervous system (e.g., deficits in learning or sensory ability)
• Immunotoxicity: Chemically induced adverse effects on the immune system (e.g., thymus, white blood cell number, and viability)
• Pharmacokinetics & Metabolism: The study of the absorption, distribution, metabolism, and elimination ("ADME") of chemicals in the body (also known as "toxicokinetics")
• Ecotoxicity: Chemically induced adverse effects on organisms in the wild, including mammals, birds, fish, amphibians, crustaceans, and other aquatic invertebrates; common study designs include acute systemic, dietary, and reproductive (also known as "life-cycle") toxicity

History of Animal Use in Toxicity Testing

Following the birth of the synthetic chemical industry in the late 1800s, the field of toxicology grew in response to the need to understand how tens of thousands of new substances might affect the health of workers and consumers involved in their production and use. The use of living animals to study the potential adverse effects of new drugs, food additives, pesticides, and other substances began in earnest during the 1920s, when British pharmacologist J.W. Trevan proposed the "lethal dose fifty percent" or "LD₅₀" test to determine the single dose of a chemical that would kill half the animals exposed to it. The idea of a comparative toxicity index offered instant appeal to government regulators––so much so that variants of the LD₅₀ remain the most prevalent animal tests even to this day (i.e., acute systemic toxicity studies).

Two decades after the introduction of the LD₅₀ test, US Food and Drug Administration scientist John Draize developed a standardised test for eye and skin irritation using albino rabbits, which is now known simply as the "Draize test." A few years later, the USToxicity Tests Overview 2007.11.26b FINAL National Cancer Institute developed a standardized test for the identification of chemical carcinogens through the daily dosing of rats and mice for up to two years. Then in the early 1960s, as thousands of babies worldwide were born with debilitating birth defects caused by the drug thalidomide, a number of new and more complex animal breeding studies were developed (i.e., reproductive and developmental toxicity studies), in which large numbers of animals are dosed with a test agent before they mate, throughout their pregnancy, and after giving birth, to evaluate effects on reproductive performance and/or developing offspring.

As chemical and pharmaceutical markets became more global during the 1980s, animal tests became entrenched in "internationally harmonized test guidelines" of multinational bodies such as the Organisation for Economic Co-operation and Development (OECD) and the International Conference on Harmonization (ICH). Today, more than 50 such animal-based test guidelines exist representing the default method for testing everything from drugs and food additives to industrial chemicals and pesticides.

Why Deviate from the Status Quo?

• Testing methods have not kept pace with scientific progress: Between the time that most commonly used toxicity tests were conceived and today, there has been a revolution in biology and biotechnology. Advances in tissue engineering and robotics have given birth to rapid "high throughput" in vitro (cell culture) systems, while emerging technologies such as bioinformatics, genomics, proteomics, metabonomics, systems biology, and in silico (computer-based) systems offer still more potential alternatives to animal use. In June 2007, the US National Academy of Sciences called for a major paradigm shift in toxicology that would "rely less heavily on animal studies and instead focus on in vitro methods that evaluate chemicals' effects on biological processes using cells, cell lines, or cellular components, preferably of human origin. The new approach would generate more-relevant data to evaluate risks people face, expand the number of chemicals that could be scrutinized, and reduce the time, money, and animals involved in testing."
• Questionable reliability and relevance of current testing methods: Animal testing is predicated on the assumption that adverse effects observed in one animal species could also occur in others. However, it is also recognized that different species may respond very differently to the same substance (e.g., penicillin is toxic to guinea pigs, aspirin can be fatal to cats, and arsenic—which is deadly to humans—is relatively nontoxic to sheep). Whether these striking interspecies differences are products of genetic, biochemical, metabolic, or physiological factors—or a combination—it is virtually impossible to know whether the results of testing on rodents, rabbits, or dogs will provide an accurate prediction of toxic effects in people (i.e., questionable relevance). There is also much debate concerning the relevance of extrapolating from high doses administered to animals to realistic human or environmental exposure levels. In addition, despite efforts to standardize procedures, the results of some animal tests can be highly variable and difficult to reproduce (i.e., poor reliability).
• Animal welfare considerations: Some conventional test methods consume hundreds or thousands of animals per substance examined (e.g., carcinogenicity studies consume approximately 400 rats and 400 mice, a standalone developmental toxicity study consumes 1,300 rats and/or 900 rabbits, and a multigenerational reproductive toxicity study consumes upwards of 2,600 rats). In addition, some countries' statistics on animal use indicate that toxicity testing accounts for upwards of 70% of the most painful procedures to which animals are subject for all experimental purposes (e.g., the continued use of death or moribundity (near death) as the experimental endpoint in acute systemic toxicity studies).
• Time and cost considerations: Some conventional tests take months or years to conduct and analyze (e.g., 4-5 years, in the case of carcinogenicity studies), at a cost of hundreds of thousands––and sometimes millions––of dollars per substance examined (e.g., US $2-4 million per two-species carcinogenicity study).
• Legal obligations: As public opposition towards animal testing has grown, some parts of the world have broadly prohibited testing on animals where alternative methods are "reasonably and practicably available" (e.g., EU Directive 86/609/EEC and State of California Administrative Code, Chapter 476). Animal testing bans may also be sector-specific, as in the case of the 7^th Amendment to the EU Cosmetics Directive, which currently bans the marketing of any formulated cosmetic products that have been animal tested and will soon culminate in an EU-wide marketing ban of cosmetic products whose ingredients have been animal tested following 2009 and 2013 cut-off points.

Alternative Methods

The term "alternative" in the context of toxicology is used to describe any change from present procedures that will result in the replacement of animals, a reduction in the numbers used, or a refinement of techniques to alleviate or minimize potential pain, distress, and/or suffering. This so-called 3Rs concept of alternatives is rooted in the 1959 publication The Principles of Humane Experimental Technique. During the subsequent half-century, tens of millions of dollars have been invested by corporations, governments, and other stakeholders with the goal of advancing the 3Rs in research and testing.

Examples of practical replacement methods include the following:

• In vitro cell and tissue cultures, such as freshly harvested "primary" cells, tissues, or organs (e.g., liver slices for metabolism studies; corneas from slaughtered cow or chicken eyes for irritation studies); self-sustaining "cell lines" (e.g., the mouse 3T3 cell line for evaluating the potential for sunlight-induced "phototoxicity"); and complex reconstructed tissue models (e.g., the EpiDerm™ human skin corrosion test). The examples above and many others have already achieved worldwide regulatory acceptance as full or partial replacement methods for their animal-based counterparts
• In silico systems, including computerized structure-activity relationship (SAR or Quantitative SAR) models, which predict the biological/toxicological properties of a substance based on its chemical structure and knowledge of similar structures (e.g., TOPKAT and MultiCASE), as well as "expert systems," which rely on rules based on human experience to predict toxicological or metabolic activity (e.g., DEREK and METEOR)
• Predictions based on the physico-chemical properties of a substance (e.g., estimating the potential for skin absorption based on the octanol-water partition coefficient, or log K_ow)
• Embryos and fetal stages before sentience develops (e.g., embryonic stem cell test for embryotoxicity)
• Organisms of lower neurological sensitivity (e.g., use of Salmonella bacteria in genetic toxicity tests and the creation of new models of developmental toxicity and neurotoxicity using the microscopic roundworm C. elegans)
• Human epidemiology and volunteer studies (most often to confirm to adverse effect of products, e.g., human patch tests for skin irritation and sensitization)
• Waiving of a requirement to conduct new testing because 1) existing toxicological information on a substance is recognized as sufficient for risk assessment purposes (e.g., the 30 member countries of the OECD have agreed to recognize one another's testing results); 2) information on a structurally similar substance can be used to fill a knowledge gap (a process known as "read-across" or "bridging"); or 3) testing would be difficult, impossible, or meaningless given the nature of the substance in question (e.g., conducting aquatic toxicity studies using a substance that does not dissolve in water)

Scientific Validation & Regulatory Acceptance

In general, government regulators will accept alternative toxicity testing methods only after they have been scientifically "validated"––that is, proven to be reliable (reproducible) and relevant for their intended purpose. Criteria and processes for test method validation have been developed and implemented in Europe (under the auspices of the European Centre for the Validation of Alternative Methods, or ECVAM), the US (through the Interagency Coordinating Committee on the Validation of Alternative Methods, or ICCVAM), Japan (through the Japanese Centre for the Validation of Alternative Methods (JaCVAM), and internationally through the OECD. Key steps include the following:

• Research & development, which is generally undertaken and/or funded by regulated industry or government
• Prevalidation, an approximately two-year process that aims to establish the mechanistic basis of a test; standardize and optimize the test protocol; evaluate within-lab variability using a training set of coded chemicals; and define a "prediction model" or "data interpretation procedure," which articulates the process by which test results are used to predict toxicological endpoints in vivo
• Validation, an approximately one-year process which aims to evaluate a test's transferability to a second laboratory, together with a test's between-labs variability and reproducibility (involving up to four outside laboratories)
• If a test performs well during the preceding steps, a peer review is undertaken to independently evaluate the results of the validation study. This process requires approximately one year, depending whether an existing peer review body (e.g., the ECVAM Scientific Advisory Committee, or ESAC) is used or whether a new ad hoc expert panel is convened
• Processes for regulatory acceptance differ region by region. In Europe, ESAC endorsement usually leads to EU-wide acceptance under applicable regulations, given the longstanding legal requirement under Directive 86/609/EEC that non-animal alternatives be used preferentially. In the US, ICCVAM formulates recommendations on the basis of peer review findings and in consultation with the public, and regulatory agencies are required by law to respond to ICCVAM's recommendations within six months. This process can take two years or more at the national/regional level and longer in the case of international consensus-driven bodies such as OECD, ICH, and VICH

Although the process above was initially designed with only alternative (non-animal) methods in mind, it has since been recognized that proper validation should be a requisite for all new and revised test methods.

Progress

The following are key milestones in the decades-long, global pursuit of alternatives to animal testing:

• 1969: Founding of the Fund for the Replacement of Animals in Medical Experiments (FRAME) in the UK
• 1981: OECD Council decision regarding the Mutual Acceptance of Data; founding of the Johns Hopkins University Center for Alternatives to Animal Testing (CAAT)
• 1986: EU Directive 86/609 for the protection of animals used for experimentation and other scientific purposes, which stipulates that: "An experiment shall not be performed if another scientifically satisfactory method of obtaining the result sought, not entailing the use of an animal, is reasonably and practicably available"
• 1989: Founding of the German Centre for the Documentation and Evaluation of Alternatives to Animal Experiments (ZEBET)
• 1991: Establishment of the ECVAM as part of the European Commission
• 1993: The US National Institutes of Health Revitalization Act calls for emphasis on alternatives; the First World Congress on Alternatives is held in Baltimore, MD
• 1996: The OECD convenes the first international validation conference
• 1997: ICCVAM is established as an ad hoc standing committee
• 2000: Passage of ICCVAM Authorization Act
• 2001: Congress directs the US Environmental Protection Agency (EPA) to spend $4 million on alternatives; OECD Test Guideline 401 (oral lethal dose) is deleted from international guidelines
• 2002: The OECD Test Guidelines Program adopts the first formally validated in vitro tests; OECD establishes a Validation Management Group dedicated to non-animal methods
• 2003: The 7^th Amendment of the EU Cosmetics Directive creates deadlines for banning animal testing of cosmetic products and their raw ingredients
• 2004: The UK National Centre for the 3Rs (NC3Rs) is established
• 2005: The US National Toxicology Program (NTP) adopts a 21^st Century Roadmap emphasizing mechanistic, non-animal studies; EU regulators and industry launch the European Partnership for Alternative Approaches to Animal Testing (EPAA)
• 2006: The EU provides more than 80 million euros for targeted, multiyear 3Rs research projects; an international task force of pesticide producers and regulators proposes a testing strategy that could reduce animal use in reproductive and developmental toxicity studies by up to 70 percent
• 2007: A US National Academy of Sciences panel calls for a fundamental paradigm shift in regulatory toxicology; ECVAM endorses the EPISKIN™ skin irritation test as a full replacement for rabbit skin irritation tests