Toxicity Testing Overview

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Toxicity Testing Overview

Last updated: February 19, 2016

The focus of is on the development, validation, and international acceptance of non-animal toxicity test methods, so that data from these “alternative” methods 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 of AltTox provides an introduction to toxicity testing, toxicity endpoints, and 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 living organisms and the ecosystem… .” 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 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 are primarily based on a number of different tests where animals are exposed to the chemical or test substance. These tests are called toxicity tests.

Government regulations often prescribe a specific regimen of toxicity testing to generate the data that enable regulators to determine the chemical’s 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).

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 acute (single-exposure) studies to repeat dose (multiple-exposure) studies, in which animals are administered daily doses of a test substance.

Toxicity endpoints covered AltTox include the following:

Acute Systemic ToxicityAdverse 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
CarcinogenicityChemically-induced cancer, whether through genotoxic or non-genotoxic (e.g., growth-promoting) mechanisms
Dermal PenetrationExtent and rate by which a chemical is able to enter the body via the skin; also known as skin or percutaneous absorption
EcotoxicityChemically-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, and bioaccumulation
Endocrine DisruptorsSubstances that interact with the hormonal systems of humans and/or wildlife, and thereby disrupt normal biological functions
Eye Irritation/CorrosionChemically-induced eye damage that is reversible (irritation) or irreversible (corrosion)
GenotoxicityChemically-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)
NeurotoxicityChemically-induced adverse effects on the brain, spinal cord, and/or peripheral nervous system (e.g., deficits in learning or sensory ability)
Pharmacokinetics & MetabolismStudy of the absorption, distribution, metabolism, and elimination (ADME) of drugs or chemicals in the body; also known as toxicokinetics
PhototoxicityToxic responses from a substance (applied to the body or ingested) following exposure to light or skin irradiation
Repeated Dose/Organ ToxicityGeneral 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 ToxicityChemically-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
Skin Irritation/CorrosionChemically-induced skin damage that is reversible (irritation) or irreversible (corrosion)
Skin SensitizationThe induction of allergic contact dermatitis following exposure to a chemical substance

In addition to the traditional toxicity endpoints used for regulatory testing, toxicity test information for the following product categories are (or will be) covered on AltTox:

Product or Topical CategoryDescription
Biologics, Vaccines, Medical ProductsA variety of tests and endpoints related to efficacy, safety, and quality control testing of drugs, devices, biologics, diagnostics, and vaccines.

This category of testing is of considerable interest for the development of non-animal methods due to the large numbers of animals used.

NanomaterialsA variety of endpoints and tests related to the safety and environmental impact of nanomaterials and products containing nanomaterials.

This category of testing is of considerable interest because of the uncertainty with the traditional animal toxicity test methods in providing hazard assessment data relevant to humans.

CosmeticsUnder development
Green ChemistryUnder development

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 developed 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.

Animal welfare considerations

Some conventional toxicity test methods consume hundreds to thousands of animals per substance examined. Statistics on animal use, such as the Seventh Report on the Statistics on the Number of Animals used for Experimental and Other Scientific Purposes in the Member States of the European Union (2013), indicate that toxicity testing accounts for a substantial portion of the more painful procedures experienced by animals (e.g., the use of death as the experimental endpoint in acute systemic toxicity studies).

Large government testing program requirements, such as REACH and HPV, have further increased the animal testing already conducted by companies. While REACH requires that animal testing is used “only when there are no other scientifically reliable ways of assessing the potential effects on humans or the environment,” a report by the European Chemicals Agency in 2014 on the use of alternatives in testing for the REACH regulation explains that companies were not fully implementing the use of animal alternative methods, and that animal tests increased due to new registrations (ECHA, 2014).

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 had been imposed under the 7th Amendment to the EU Cosmetics Directive (76/768/EEC). Most recently, several additional countries have adopted bans on animal testing for cosmetic products, including India and Israel.

Time and cost considerations

Some conventional toxiciy tests take months to years to conduct and analyze (e.g., 4-5 years for carcinogenicity studies), and can cost 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.

Non-animal Toxicity Test Methods is a source of information on animal replacement methods for toxicity testing. This is only one of the 3Rs. In cases where Replacement methods are not available, Reduction and Refinement methods may be mentioned, but not covered in detail.

The term “alternative,” in the context of toxicity testing, has been used to describe new or revised test methods that result in the replacement of animal test methods, or that reduce the numbers of animals used, or refine the 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.

At this time, the terms “non-animal” or “animal-free” are commonly used to describe Replacement methods. This avoids confusion due to the multiple uses of “alternatives.”

Non-animal methods for producing toxicological data include the following:
In vitro cell and tissue-based methods and models: Toxicity assays can be conducted using models developed with primary cells, cell lines, stem cells, 3-dimensional cultured cells, excised tissues, or cultured organs. Some cell-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: It is often necessary to use more than one non-animal test to assess a single biological endpoint; it is therefore necessary to develop strategies for optimally combining test methods to address specific information needs. Integrated testing strategies combine methods, such as in silico methods and 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.

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.

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 (European Centre for the Validation of Alternative Methods or ECVAM), the US (Interagency Coordinating Committee on the Validation of Alternative Methods or ICCVAM), and by the international Organisation for Economic Cooperation and Development (OECD).

By 2005, the validation criteria established by each 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); the “principles and criteria for test method validation” are listed in Table 1, page 23, and the regulatory acceptance criteria are explained on page 48. This Guidance Document provides vital information for any test developer planning to conduct a validation study of a new of revised toxicity test method.

Each validation authority, however, still has some distinct processes, and provides their own test method submission guidance:

To optimize success, the requirements for regulatory acceptance should be considered during the design and conduct of a validation study. The validation authorities emphasize the importance of the involvement of regulatory authorities in all stages of the validation process.

According to the OECD, formal validation “contributes strongly to the international acceptance of any proposed test method,” however, validation in not a requirement for the development of a method as a TG (OECD, 2005). The OECD recommends that the acceptance of any test method in the absence of validation should be accompanied by a written justification.

The OECD seeks to promote the “harmonization of international regulatory acceptance of adequately validated test methods” (OECD, 2005). The OECD notes that regulatory acceptance practices differ by country and even among agencies within a particular country, so acceptance by one authority does not indicate universal acceptance. National regulatory authorities may accept a test that has not undergone formal validation, or they may not accept a test that has undergone formal validation. The OECD provides guidance on international regulatory acceptance for its member countries, and encourages worldwide mutual acceptance of data.

Formal validation as described in these documents may not be necessary for use of all test methods. All methods should be evaluated for reliability, reproducibility, sensitivity and specificity; however, the need for additional formal evaluation is context and use-dependent. In this light, OECD has developed a guidance document for describing non-standard test methods (OECD, 2014).

OECD, ECVAM, and ICCVAM also provide guidance on quality assurance and quality control issues relevant to the conduct of inter-laboratory validation studies, such as test substance coding/blindness, data audits, and the use of Good Laboratory Practice (GLP) (OECD, n.d.) and Good Cell Culture Practice (GCCP) (Bal-Price & Coecke, 2011; Coecke, et al., 2005) guidelines.

Further information on historical developments for toxicity test method validation, regulatory acceptance, and international harmonization can be found in these archived pages:

The AltTox Table of Validated and Accepted Alternative Methods provides information on current internationally endorsed non-animal toxicity test methods.

Sherry L. Ward, PhD, MBA, AltTox Contributing Editor

AltTox Editorial Board reviewer(s):
George Daston, PhD
Catherine E. Willett, PhD