|The focus of AltTox.org is on the development, validation, and international acceptance of non-animal toxicity test methods, so that their data can be accepted by national and regional regulatory authorities as replacements for the many animal toxicity test methods currently required for regulatory submissions. This section provides an introduction to toxicology/risk assessment/toxicity testing/toxicity endpoints/test method validation to assist you in understanding and navigating the content of the website.
Toxicology is "the study of the adverse effects of chemical, physical, or biological agents on people, animals, and the environment." Most developed countries have enacted laws and regulations to control the marketing, labeling, and (in some cases) transportation of chemicals, pesticides, consumer products, medical products, food additives, and other substances of potential toxicological concern. Many of the provisions require manufacturers to conduct testing to identify potential hazards to human and animal health and to the environment, and to submit the test data to regulatory authorities.
Government agencies conduct human health and ecological risk assessments to ascertain the effects of a chemical or other substance on human health and/or the environment, respectively. The processes involved in a risk assessment for human health can be broken down into four steps as illustrated in the diagram below from the US Environmental Protection Agency (EPA).
The hazard identification and dose-response assessment steps can be based on existing data, but for a new chemical or substance, the original data are primarily obtained through tests using animals who are exposed to the chemical or test substance. These tests are called toxicity tests.
Government regulations often prescribe a specific regime of toxicity testing to generate the data that will enable regulators to determine whether the benefits of a particular substance outweigh its risks to human health and/or the environment. Typically, data for many different toxicity endpoints are needed for a regulatory submission on a new chemical or other regulated substance. Companies producing the chemical/product are responsible for the generation and submission of this “safety data” to regulatory authorities such as the US EPA, the European Chemicals Agency (ECHA), and Japan’s Ministry of the Environment (MOE).
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 or animal 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 acute (single-exposure) studies to repeat dose (multiple-exposure) studies, in which animals are administered daily doses of a test substance.
Toxicity endpoints considered within the scope of AltTox include the following:
Acute Systemic Toxicity
Adverse effects occurring within a relatively short time after administration of a single (typically high) dose of a substance via one or more of the following exposure routes: oral, inhalation, skin, or injection
Biologics, Vaccines, Medical Products
Variety of tests and endpoints related to efficacy, safety, and quality control testing of drugs, devices, biologics, diagnostics, and vaccines [of considerable interest for the development of non-animal methods due to the large numbers of animals used]
Chemically-induced cancer, whether through genotoxic or non-genotoxic (e.g., growth-promoting) mechanisms
Extent and rate by which a chemical is able to enter the body via the skin; also known as skin or percutaneous absorption
Chemically-induced adverse effects on organisms in the environment, including mammals, birds, fish, amphibians, crustaceans, other aquatic invertebrates, and even plants; common study designs include acute systemic, dietary, and reproductive (also known as life-cycle) toxicity
Substances that interact with the hormonal systems of humans and/or wildlife, and thereby disrupt normal biological functions
Chemically-induced eye damage that is reversible (irritation) or irreversible (corrosion)
Chemically-induced mutations and/or other alterations in the structure, information content, or segregation of genetic material (e.g., DNA strand breaks or a gain/loss in chromosome number)
Chemically-induced adverse effects on the brain, spinal cord, and/or peripheral nervous system (e.g., deficits in learning or sensory ability)
Pharmacokinetics & Metabolism
Study of the absorption, distribution, metabolism, and elimination (ADME) of drugs or chemicals in the body; also known as toxicokinetics
Toxic responses from a substance (applied to the body or ingested) following exposure to light or skin irradiation
Repeated Dose/Organ Toxicity
General toxicological effects occurring as a result of repeated daily exposure to a substance (via oral, inhalation, dermal, or injection 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, or birth defects); generally determined through the breeding of one or more generations of offspring
Chemically-induced skin damage that is reversible (irritation) or irreversible (corrosion)
The induction of allergic contact dermatitis following exposure to a chemical agent
There is a long history in the use of animals as models for toxicity testing. During the 20th century, as regulatory agencies were established by national governments, animal test guidelines were added to address regulatory requirements that products need to be "safe" for consumers. A review on pre-clinical testing by Parasuraman (2011) further explains the animal models and tests used for regulatory toxicity testing.
There are many advantages, including scientific, ethical, and economic ones, for replacing the animal toxicity tests with non-animal (in vitro and in silico) test systems. As science progresses, we have become aware of a number of problems with the use of animal models for assessing the safety of chemicals, drugs, and other products to human health, some of which are briefly discussed in the following paragraphs.
Product safety 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 cell culture and robotics have given birth to rapid "high throughput" in vitro test systems, while tissue engineering and stem cell technologies are providing ever more relevant in vitro tissues. Emerging technologies such as genomics, proteomics, metabolomics, computational biology, and in silico (computer-based) systems offer still more potential alternatives to animal test methods. The 2007 National Academy of Sciences report, Toxicity testing in the twenty-first century, a vision and a strategy, 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."
Integrated testing strategies based on combinations of advanced in vitro and in in silico methods that model the mechanism(s) of action and address the adverse outcome pathway are now the focus of research and development in several prototype initiatives.
Questionable reliability and relevance of animal methods/models:
Animal testing is predicated on the assumption that adverse effects observed in one animal species could also occur in others. However, it is widely recognized within the scientific community that different species can respond differently to the same substance. Whether interspecies differences are due to genetic, biochemical, or metabolic factors—or a combination—the results of testing on rodents, rabbits, or dogs may not provide an accurate prediction of toxic effects in humans (i.e., questionable relevance).
There has also been a concern about the relevance of extrapolating from the high doses of test substances commonly administered to animals to realistic human or environmental exposure levels (i.e., questionable relevance).
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 toxicity test methods consume hundreds to thousands of animals per substance examined (Doe et al., 2006; Cooper et al., 2006). Statistics on animal use, such as the Sixth Report on the Statistics on the Number of Animals used for Experimental and Other Scientific Purposes in the Member States of the European Union (2010), indicate that toxicity testing accounts for a large percentage of the more painful procedures experienced by animals (e.g., the use of death as the experimental endpoint in acute systemic toxicity studies).
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 2010/63/EU on the protection of animals used for scientific purposes). Animal testing bans may also be sector-specific, as in the case of the EU Cosmetics Regulation (EC) No 1223/2009, which now bans the marketing of any cosmetic product within the EU containing ingredients that have been tested on animals. Earlier EU test bans on cosmetics ingredients and final formulations were also imposed under the 7th Amendment to the EU Cosmetics Directive (76/768/EEC). The EU’s 6th animals used statistics reported a decrease in animal use for toxicity testing of industry and agricultural products, and environment testing, and attributed this to implementation of laws phasing out animal testing for cosmetics in the EU.
Time and cost considerations:
Some conventional tests take months to years to conduct and analyze (e.g., 4-5 years for carcinogenicity studies), at a cost of hundreds of thousands to millions of dollars per substance examined (e.g., $2-4 million per two-species carcinogenicity study) (EPA, 2004).
In addition to the testing required for new chemicals and products, there is growing realization that the safety information we have on many existing chemicals is insufficient. While it is desirable to have only safe chemicals and products in the marketplace, the traditional means for conducting hazard assessments cannot keep pace with the demand. This has been the basis of a number of government testing programs such as EU's REACH (Registration, Evaluation, Authorisation and Restriction of Chemicals) legislation, and government research programs such as the US EPA's ToxCast™ program.
Non-animal Toxicity Test Methods
| AltTox.org is specifically interested in animal replacement methods used for toxicity testing. In cases where replacement methods are not available, reduction and refinement methods are covered.
The term alternative, in the context of toxicity testing, 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 pain, distress, and/or suffering. This 3Rs concept of alternatives is rooted in the 1959 publication of Russell and Burch, The Principles of Humane Experimental Technique. Since the publication of this seminal work, governments, industry, NGOs, and other stakeholders have invested substantial time and financial resources to advance the 3Rs in research and testing.
Non-animal methods for producing toxicological data include the following:
In vitro cell and tissue culture-based methods and models.
Toxicity assays can be conducted using models developed using primary cells, cell lines, stem cells, 3-dimensional cultured cells, excised tissues, or cultured organs. Some cell and tissue-based methods have already achieved validation and international acceptance.
In silico systems.
Computer-based methods such as (quantitative) structure-activity relationship ((Q)SAR) models and read-across can be used to predict the biological/toxicological properties of a substance.
Integrated testing and other emerging strategies.
Integrated testing strategies combine methods, such as in silico methods and multiple in vitro assays, along with appropriate statistical analysis, for the prediction of in vivo toxicity responses. An understanding of the mechanisms of toxicity and the cellular pathways involved are currently being investigated with the goal of obtaining more predictive integrated test strategies.
Other strategies for reducing animal testing requirements include:
- Human epidemiology or accidental exposures
- Human volunteer studies
- Existing toxicological information on a substance is recognized as sufficient for risk assessment purposes
- Waiving of a requirement to conduct new testing because human exposure levels are below what is considered a significant risk to human health, or because testing would be difficult, impossible, or meaningless given the nature of the substance in question
Scientific Validation, Regulatory Acceptance, International Harmonization
To facilitate the replacement of old tests with new ones, international authorities developed processes and criteria for evaluating new toxicity test methods to determine whether they can replace an existing method.
Test method validation refers to the process of determining whether a toxicity test method (or test battery or test scheme) is reliable (reproducible) and relevant for its intended purpose. Relevance in this setting means "the extent to which the test method correctly measures or predicts the (biological) effect of interest."
Criteria and processes for toxicity test method validation were developed in the mid-1990’s by validation authorities in the EU and US, ECVAM (European Centre for the Validation of Alternative Methods) and ICCVAM (Interagency Coordinating Committee on the Validation of Alternative Methods), respectively, and by the international Organisation for Economic Cooperation and Development (OECD). By 2005, the validation criteria of these organizations was harmonized and published as the Guidance Document on the Validation and International Acceptance of New or Updated Test Methods for Hazard Assessment (OECD, 2005).
Key steps in the pathway from test method development to widespread use and acceptance of a new toxicity test method are the following – in their typical chronological order:
- Research & development consists of the basic and applied research involved in the genesis of a new test method. It might be undertaken and/or funded by biotech companies, contract testing laboratories, research institutes, regulated industry, or government bodies.
- Prevalidation is the process that aims to establish the mechanistic basis of a test, standardize and optimize the test protocol, evaluate within-lab variability, and define a model or procedure by which test results are used to predict the in vivo toxicological endpoint.
- Validation is the process to evaluate a defined set of criteria that includes a test method’s transferability to other laboratories, between-lab variability and reproducibility, and reliability.
- Peer review or independent assessment is the formal evaluation process of the results from a validation study. Recommendations on the validity of the test method for the indicated purpose (as well as other aspects of the study) are made to the sponsoring validation authority.
- Regulatory acceptance processes differ region by region. In Europe, ESAC endorsement can lead to EU-wide acceptance, considering the legal requirement under Directive 86/609/EEC that non-animal alternatives be used preferentially. In the US, agencies individually decide whether to accept an ICCVAM recommendation. Most government agencies have the authority to accept whatever methods they find valid to meet their particular needs.
- International harmonization involves collaborative efforts through established organizations such as the OECD, ICH, and VICH to adopt the same testing requirements and test protocols, so that different countries and regions require and accept the same types of data for regulatory decisions. International acceptance in the form of an OECD Test Guideline is an important milestone for a validated non-animal test method, and can have a significant impact on reducing animal use in regulatory assessments.
Progress: Milestones Timeline
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; CAAT sponsors the first World Congress on Alternatives & Animal Use in the Life Sciences in Baltimore, MD, which remains the primary international scientific conference series dedicated to the 3Rs
- 1996: The second World Congress on Alternatives & Animal Use in the Life Sciences is sponsored by the University of Utrecht; the OECD convenes the first international validation conference
- 1997: ICCVAM is established as an ad hoc standing committee; ECVAM endorses first cell-based toxicity test method, the 3T3 neutral red uptake phototoxicity test
- 1998: Three in vitro skin corrosion test methods endorsed by ECVAM
- 1999: The third World Congress on Alternatives & Animal Use in the Life Sciences is held in Italy
- 2000: Passage of ICCVAM Authorization Act; ICCVAM endorses its first in vitro method, the Corrositex® assay for assessing skin corrosion
- 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; the fourth World Congress on Alternatives & Animal Use in the Life Sciencesis held in New Orleans
- 2003: The 7th 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; EU ban on animal testing of finished cosmetic products as of September 11, 2004; OECD test guidelines for in vitro 3T3 NRU Phototoxicity Test and for in vitroskin dermal penetration test methods
- 2005: The US National Toxicology Program (NTP) adopts a 21st Century Roadmap emphasizing mechanistic, non-animal studies; the fifth World Congress on Alternatives & Animal Use in the Life Sciences is held in Berlin; 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 (NAS) panel calls for a fundamental paradigm shift in regulatory toxicology in its report, Toxicity Testing in the 21st Century: a Vision and a Strategy; ECVAM endorses the EPISKINTM skin irritation test as a full replacement for rabbit skin irritation tests; ICCVAM and ECVAM endorse two enucleated eye methods for classifying ocular severe/corrosive materials; the 6th World Congress on Alternatives & Animal Use in the Life Sciences is held in Japan
- 2008: ICCVAM releases its five-year plan which identifies four priority areas for alternatives test method development; US Federal agencies announce collaboration on new high throughput toxicity screening initiative; two additional in vitro methods endorsed for skin irritation testing
- 2009: EU ban on animal-based acute testing of cosmetic ingredients for all human health effects as of March 11, 2009; US Environmental Protection Agency (EPA) adopts the NAS vision for evaluating the toxicity of chemicals in its new strategic plan; new international agreement to coordinate recommendations on alternative methods should speed their adoption and reduce animal testing; 50th anniversary of the Russell and Burch book that launched the 3Rs, The Principles of Humane Experimental Technique; 7th World Congress on Alternatives & Animal Use in the Life Sciences (WC7) in Rome, Italy
- 2011: 8th World Congress on Alternatives & Animal Use in the Life Sciences (WC8) in Montreal, Canada.