Non-animal Methods for Toxicity Testing

Toxicity Testing Overview

Last Updated: September 8, 2011
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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. 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 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 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 and environmental 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")
  • Carcinogenicity: Chemically induced cancer, whether through genotoxic or non-genotoxic (e.g., growth-promoting) mechanisms
  • 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 "percutaneous absorption")
  • 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
  • Eye Irritation/Corrosion: Chemically induced eye damage that is reversible (irritation) or irreversible (corrosion)
  • 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)
  • Immunotoxicity: Chemically induced adverse effects on the immune system (e.g., thymus, white blood cell number, and viability)
  • Neurotoxicity: Chemically induced adverse effects on the brain, spinal cord, and/or peripheral nervous system (e.g., deficits in learning or sensory ability)
  • Pharmacokinetics & Metabolism: The study of the absorption, distribution, metabolism, and elimination ("ADME") of chemicals in the body (also known as "toxicokinetics")
  • Repeated Dose/Organ 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
  • Skin Irritation/Corrosion: Chemically induced skin damage that is reversible (irritation) or irreversible (corrosion)
  • Skin Sensitization: The induction of allergic contact dermatitis following exposure to a chemical agent

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 (i.e., acute systemic toxicity studies) remain the most prevalent animal tests even to this day.

Two decades after the introduction of the LD50 test, US Food and Drug Administration scientist John Draize developed standardized tests for eye and skin irritation using albino rabbits, which are now known simply as "Draize tests." A few years later, the US 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 cell culture and robotics have given birth to rapid "high throughput" in vitro test systems, while tissue engineering is providing ever more relevant in vitro tissues. 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 differently to the same substance (Ekwall, et al., 1998; Hurtt, et al., 2003; Gold & Slone, 1993). Whether interspecies differences are products of genetic, biochemical, or metabolic 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 humans (i.e., questionable relevance) (Robinson, et al., 2001; Schardein, 2000; Cohen, 2002 & 2004; Haseman, et al., 1998). There is also much debate concerning the relevance of extrapolating from high doses administered to animals to realistic human or environmental exposure levels (Muller, 1948; ACSH, 1997). 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) (Weil & Scala, 1971; Bremer, et al., 2007; Gottmann, et al., 2001).
  • Animal welfare considerations: Some conventional toxicity test methods consume hundreds or thousands of animals per substance examined (Doe, et al., 2006; Cooper, et al., 2006). In addition, some countries' statistics on animal use indicate that toxicity testing accounts for up to 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) (USEPA, 2004).
  • 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 US state laws in California [pdf], New Jersey [pdf] and New York [pdf]). Animal testing bans may also be sector-specific, as in the case of the 7th Amendment to the EU Cosmetics Directive, which since 2004 has banned the marketing of any formulated cosmetic products that have been animal tested, and as of March 2009, additionally prohibits (i) animal testing of cosmetic ingredients within the EU, and (ii) with a few exceptions, also the marketing of cosmetic products whose ingredients have been tested on animals on or after that date.

Alternative Methods

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 potential pain, distress, and/or suffering. This 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 potential 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 eye irritation studies); cell lines (e.g., mouse 3T3 cell line for evaluating the potential for sunlight-induced phototoxicity); stem cells (e.g., stem cells for embryotoxicity testing); and complex reconstructed tissue models (e.g., EpiDermTM human skin corrosion test). These and other cell and tissue-based methods have already achieved international acceptance as full or partial replacement methods for their animal-based counterparts
  • In silico systems include computer structure-activity relationship (SAR) or Quantitative SAR [(Q)SAR] models, which predict the biological/toxicological properties of a substance based on its chemical structure and knowledge of similar structures (e.g., MultiCASE and TOPKAT); and expert systems programs that predict toxicological or metabolic activity (e.g., DEREK and METEOR)

Other strategies for reducing toxicity testing requirements include:

  • Human epidemiology and volunteer studies (most often to confirm to adverse effect of products, e.g., human patch tests for skin irritation and sensitization)
  • Integrated testing strategies
  • 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) human exposure levels are below what is considered a significant risk to human health, and therefore toxicological testing is not needed [Threshold of Toxicological Concern (TTC)]; 3) information on a structurally similar substance can be used to fill a knowledge gap (a process known as "read-across" or "bridging"); or 4) 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)

Other AltTox pages with information on alternative methods include:

Scientific Validation & Regulatory Acceptance

In general, government regulators will accept alternative toxicity testing methods only after they have been scientifically "validated"––that is, determined 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.

Other AltTox pages with information on validation and regulatory acceptance include:


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


ACSH (American Council on Science and Health). (1997). Of Mice and Mandates: Animal Experiments, Human Cancer Risk and Regulatory Policies. New York: ACSH.

Bremer, S., Pellizzer, C., Hoffmann, S., Seidle, T. & Hartung, T. (2007). The development of new concepts for assessing reproductive toxicity applicable to large scale toxicological programmes. Curr. Pharm. Des. 13, 3047-3058.

Cohen, S.M. (2002). Bioassay bashing is bad science: Cohen's response. Environ. Health Perspect. 110, A737.

Cohen, S.M. (2004). Human carcinogenic risk evaluation: an alternative approach to the two-year rodent bioassay. Toxicol. Sci. 80, 225-229.

Cooper, R.L., Lamb, J.C., Barlow, S.M., Bentley, K., Brady, A.M., Doerrer, N.G., et al. (2006). A Tiered Approach to Life Stages Testing for Agricultural Chemical Safety Assessment. Crit. Rev. Toxicol. 36, 69-98.

Doe, J.E., Boobis, A.R., Blacker, A., Dellarco, V., Doerrer, N.G., Franklin, et al. (2006). A Tiered Approach to Systemic Toxicity Testing for Agricultural Chemical Safety Assessment. Crit. Rev. Toxicol. 36, 37-68.

Ekwall, B., Barile, F.A., Castano, A., et al. (1998). MEIC evaluation of acute systemic toxicity. Part VI. The prediction of human toxicity by rodent LD50 values and results from 61 in vitro methods. Altern. Lab. Anim. 26(2), 617-658.

Gold, L.S. & Slone, T.H. (1993). Prediction of carcinogenicity from two versus four sex-species groups in the carcinogenic potency database. J. Toxicol. Environ. Health. 39, 143-157.

Gottmann, E., Kramer, S., Pfahringer, B., et al. (2001). Data quality in predictive toxicology: Reproducibility of rodent carcinogenicity experiments. Environ. Health Perspect. 109, 509-514.

Haseman, J.K., Hailer, R.J. & Morris, R.W. (1998). Spontaneous neoplasm incidences in Fischer 344 rats and B6C3F1 mice in two-year carcinogenicity studies: A National Toxicology Program update. Toxicol. Pathol. 26, 428-441.

Hurtt, M.E., Cappon, G.D. & Browning, A. (2003). Proposal for a tiered approach to developmental toxicity testing for veterinary pharmaceutical products for food-producing animals. Food Chem. Toxicol. 41, 611-619.

Müller, R. (1948). Vergleich der im Tierexperiment und beim Menschen tödlichen Dosen wichtiger Pharmaka. Diss. Univ. Frankfurt/Main.

Robinson, M.K., McFadden, J.P. & Basketter, D.A. (2001). Validity and ethics of the human 4-h parth test as an alternative method to assess acute skin irritation potential. Contact Derm. 45, 1-12.

Schardein, J.L. (2000). Chemically Induced Birth Defects, 3rd Ed. Rev. New York: Marcel Dekker.

USEPA (U.S. Environmental Protection Agency). (2004). Economic Analysis of the Proposed Change in Data Requirements Rule for Conventional Pesticides. Washington, DC: USEPA.

Weil, C.S. & Scala, R.A. (1971). Study of intra- and interlaboratory variability in the results of eye and skin irritation tests. Toxicol. Appl. Pharmacol. 19, 276-360.