Acute Systemic Toxicity: Regulatory Testing Overview

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Acute Systemic Toxicity: Regulatory Testing Overview

Last updated: September 19, 2017

Exposure to chemical substances can occur by various routes of exposure and over varying periods of time. Because all chemicals have the potential to cause toxicity at sufficiently high doses, suites of toxicity tests have been developed to estimate the hazard potential of chemicals of interest and to inform safety decisions by attempting to approximate the various routes and durations of exposure scenarios to which people, and other organisms, might be exposed. This section discusses those tests used to identify hazard potential resulting from very short exposures. They are referred to as acute toxicity tests. Not all acute toxicity tests will be considered, only those that result in systemic toxicity, not target organ specific toxicity such as eye or skin toxicity that is limited to one organ. Other AltTox pages describe those suites of tests used to evaluate target organ toxicity and toxicity that develops only after longer periods of exposure up to thse over a lifetime.

Acute systemic toxicity tests were initially developed using animal species in the belief that evolutionary similarity with humans made them predictive. Many decisions, regulations and court cases have been informed by acute systemic toxicity studies conducted in rodents and other animal species, and these have set precedents for the design and conduct of such testing as well as for how to use the test results to categorize hazard and inform decisions. Thus, while alternative acute systemic testing approaches have been developed, the progress to adopt them has been slow.

That situation is changing rapidly as the vision set forth in the landmark 2007 publication of “Toxicity Testing in the 21st Century” by the National Research Council is coming to pass as a result of efforts conducted over the past 10 years to develop the tools called for by the NRC and to test the proof of concept of the “Tox21” vision. The vision describes a strategy to take advantage of advances in high speed computing and our understanding of the biological basis of disease to develop rapid, efficient, and informative non-animal test approaches. Along with the increasing body of evidence, the scientific community recognizes that animal tests are often not very informative for human outcomes.

In 2011, the US Environmental Protection Agency (EPA) announced that it would conduct a pilot study to evaluate whether it would accept an alternative testing approach for ocular toxicity to inform its antimicrobial cleaning product evaluations, and in 2015 it finalized and expanded the guidance. In the 2011 notice, EPA stated that the Bovine Corneal Opacity and Permeability (BCOP) assay could be used to identify antimicrobial cleaning product Toxicity Category I and Category II eye irritants. In 2015, the Agency expanded the applicability of the BCOP assay to identify Toxicity Category III eye irritants for antimicrobial cleaning products.

In 2016, EPA and other US agencies announced that they are moving away from the commonly used animal toxicity tests, referred to as the “Six Pack”, for acute toxicity testing of pesticide products. The “Six Pack” refers to a battery of six acute toxicity studies that evaluate acute oral toxicity, acute dermal toxicity, acute inhalation toxicity, primary eye irritation, primary dermal irritation, and dermal sensitization. The first three tests in the Six Pack list measure acute systemic toxicity and will be discussed further in this section. The last three measure target organ toxicity and will not be considered further here.

EPA published a draft policy in March 2016 to waive all acute dermal lethality studies for pesticide products. EPA with five other federal agencies, including the Consumer Product Safety Commission, the Department of Defense, the Department of Transportation, the Food and Drug Administration, the Occupational Safety and Health Administration and the National Institute of Environmental Health Sciences, are developing a strategy to replace the “Six Pack” within three years.

These actions are expected to result in a significant reduction in animal use, signaling a pivot from animal to non-animal based testing approaches and represent a major change in how acute toxicity testing will be conducted. It is reasonable to anticipate that these are but the first of a series of major changes that will lead to a switch from largely animal based testing to alternative testing approaches for other toxicity endpoints as well. As a result, a major reduction in the use of animals for toxicity testing is expected in the near future.

This portion of the AltTox webpage will describe the traditional largely animal based approaches to acute systemic toxicity testing to provide context for the topic. The advances to move to alternative acute systemic toxicity through opportunities for bridging and waiving of test requirements, as well as through the use of Adverse Outcome Pathways (AOPs) in an Integrated Approach to Testing and Assessment (IATA) will also be briefly summarized to conclude this portion of the webpage.

A variety of governmental, commercial, and non-governmental organizations employ acute systemic toxicity testing to inform decisions as well as to meet regulatory requirements. Although there have been international efforts to harmonize regulatory approaches, such as through the efforts of the Organization for Economic Cooperation and Development (OECD) and the Globally Harmonized System of Classification and Labeling of Chemicals (GHS), regulatory requirements often vary between offices within a regulatory agency, between regulatory agencies within a country, as well as between countries because of differences in enabling legislation, court decisions, politics, and culture. It is important to understand both the formal legislative requirements for toxicity testing and the manner in which the respective regulatory authorities evaluate and apply testing results for those interested in why there are different requirements as well as for those wish to help facilitate the move from the traditional largely animal based testing approaches to the newer more efficient and informative alternative non-animal testing approaches.

Acute systemic toxicity testing is the estimation of the hazard potential of a substance following short-term exposure by determining its systemic toxicity. Typically, one, or more, species of animal is exposed to very high doses of the test substance once (although an acute exposure can sometimes include dosing multiple times within a 24-hour period) via the exposure route humans or another species of interest are expected to encounter during intended use or as a result of some reasonably anticipated accidental exposure. Three routes of exposure are possible: oral, dermal, and/or inhalation.

GHS defines acute toxicity as “those adverse effects occurring following oral or dermal administration of a single dose of a substance, or multiple doses given within 24 hours, or an inhalation exposure of 4 hours” (UNECE, Part 3.1, 2015). The European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) defines acute toxicity as “an assessment of the general toxic effects of a single dose or multiple doses of a chemical or product, within 24 hours by a particular route (oral, dermal, inhalation), and that occur during a subsequent 21-day observation period.” There are other definitions as well, but they are practically the same as those from GHS and the EU and are essentially “variations on a theme.”

The results from acute systemic toxicity tests are traditionally expressed as the median lethal dose (LD50) value for acute and oral exposures or as the median lethal concentration (LC50) value for inhalation exposures – both values are estimates of the amount of a test substance that kills 50% of the test animals. LD50s and LC50s are often used to define substances according to their hazard and to inform decisions about whether restrictions should be placed on exposure to the substance. They are also used to evaluate any precautions that need to be taken during manufacture, use, and disposal. LD50 and LC50 values are used to prepare a variety of information materials such as product use and warning labels, material safety data sheets, transportation hazard placards, farm worker reentry interval restriction warnings, protective clothing requirements, and the like.

While this will not be discussed in detail, it is important to note that for a substance to have systemic toxic effects it must be absorbed from the site of exposure and then be distributed by the circulation to the sites in the body where it exerts toxic effects. The liver and sometimes other organs may metabolize a circulating non-toxic drug or chemical to another form through a process also referred to as biotransformation, and this new metabolite may be the one causing the observed toxicity.

GHS uses cut off points to define categories of hazard. Category I is the most toxic and Category IV the least toxic. By virtue of its placement within a category a chemical is assigned a signal word (e.g., “danger” for categories I-III), a statement (e.g., “fatal if swallowed” for categories I and II), a symbol (e.g., skull and crossbones for categories I-III), and a hazard statement (e.g., “fatal if swallowed” for categories I and II). There are variations on the GHS categories used by certain agencies in the US (e.g., EPA’s Pesticide Program) and other countries, but they are similar to those used by the GHS (summarized in Table 1) and will not be discussed further.

Table 1. Hazard Classifications Used By Some Regulatory Authorities

Regulatory Agency (Authorizing Act)

Category Classification

Environmental Protection Agency (FIFRA)I – LD50 <50 mg/kg
II – LD50 >50 and <500 mg/kg
III – LD50 <5000 mg/kg
IV – LD50 >50 mg/kg
Consumer Product Safety Commission (Federal Hazardous Substances Act)Highly toxic – LD50 <50 mg/kg
Toxic – LD50 <500 mg/kg
Occupational Safety and Health Administration (Occupational Safety and Health Act)Highly toxic – LD50 <50 mg/kg
Toxic – LD50 <500 mg/kg
Department Of Transportation (Federal Hazardous Substances Act)Packing Group I – LD50 <5 mg/kg
Packing Group II – LD50< 50 mg/kg
Packing Group III – LD50 < 500 mg/kg (liquid) / LD50<200 mg/kg (solid)
OECD Guidance for Use of GHSI – LD50 <5 mg/kg
II – LD50 <50 mg/kg
III – LD50 <300 mg/kg
IV – LD50 <2000 mg/kg
V – LD50 <5000 mg/kg
Unclassified LD50>5000 mg/kg

The Organization for Economic Cooperation and Development (OECD) is comprised of 35 countries that work together to promote policies that will improve the economic and social well being of people around the world. A major OECD emphasis is to reduce non-tariff trade barriers by harmonizing regulatory approaches among the member states and adhering non-member countries. As part of the harmonization process OECD produces a variety of guidance documents, including those for evaluating chemical safety, that are accepted by member states through a Mutual Acceptance of Data policy (MAD). MAD states that “data generated in the testing of chemicals in an OECD member country in accordance with OECD Test Guidelines and OECD Principles of Good Laboratory Practice (GLP), shall be accepted in other member countries. Adhering non-member countries, of which there are an increasing number, should also accept this data.”

While each OECD member state does accept data generated by other member states and adhering non-member countries, they may require additional information as well. Thus, there are testing requirements that go beyond that which is specified in the OECD test guidelines. Nonetheless, because the vast majority of testing complies with OECD guidance, the OECD guidance for acute systemic toxicity testing is described here.

Animal welfare is an important concern in safety testing and is a guiding principle in the development, validation, and use of toxicity tests. Regarding animal welfare, OECD states that “the welfare of laboratory animals is important; it will continue to be an important factor influencing the work in the OECD Chemicals Programme. The progress in OECD on the harmonisation of chemicals control, in particular the agreement on Mutual Acceptance of Data (MAD), by reducing duplicative testing, will do much to reduce the number of animals used in testing. Such testing cannot be eliminated at present, but every effort should be made to discover, develop and validate alternative testing systems.”

The GHS animal welfare position is contained in section “Animal Welfare” of the 6th revised edition (GHS, 2015). It states that “the welfare of animals is a concern. This ethical concern includes not only the alleviation of stress and suffering but also, in some countries, the use and consumption of animals. Where possible and appropriate, tests and experiments that do not require the use of live animals are preferred to those using live experimental animals. To that end, for certain hazards non-animal observationsmeasurements are included as part of the classification system. Additionally, alternative animal tests, using fewer animals or causing less suffering are internationally accepted and should be preferred.”

Five OECD Test Guidelines (TGs 402, 403, 420, 423, and 425) comprise the universe of acute systemic testing guidelines (Table 2). The OECD TG 401 for Acute Oral Toxicity was deleted in December 2002 because of concerns about excessive animal usage. OECD has been committed to the 3Rs since the publication of “The Principles of Humane Experimental Technique” by Russell and Burch in 1959. Thus, a variety of actions have been taken by OECD to refine, reduce, and ultimately replace the use of animals in testing, including the development and validation of the Fixed Dose Procedure (OECD TG 420), the Acute Toxic Class method (OECD TG 423), and the Up-and-Down Procedure (OECD TG 425) as a means to replace TG401 with procedures that reduce the use of animals.

Each of these methods uses preset doses, with the starting dose based on a small range-finding study, cytotoxicity screens, or preexisting data (Whitehead & Stallard, 2004). Following dosing (typically by gavage), the animals are monitored for overt toxicological signs until death (Acute Toxic Class or Up and Down Method) or “evident toxicity” (Fixed Dose Procedure).

Because the Fixed Dose Procedure (OECD 420) does not rely on death as an endpoint, it is a refinement as well as a reduction alternative to the LD50 test (OECD TG 401). In recent years, using death as an endpoint has been discouraged in all testing contexts, so the use of the reduction alternatives is now mandatory for acute systemic toxicity testing (Schlede et al., 2005).

For the OECD TG 402, Acute Dermal Toxicity, a test substance is applied to no less than 10% of the area of the skin of rats, rabbits, or guinea pigs, followed by 14 days of observation. Death of the animals is used to determine an LD50 value. Also, gross pathological changes are used to estimate the relative toxicity of a substance. The test may be useful in indicating dermal absorption, and thus it can be used to determine the dose levels for other studies such as subchronic dermal testing. Stallard, et al. (2004) described a reduction/refinement alternative method and its validation, the dermal fixed dose procedure (dermal FDP), which is based on using only moderately toxic doses and a nonlethal endpoint.

Acute inhalation toxicity is assessed according to OECD TG 403. Rats are typically exposed by inhalation of the test substance for no less than four hours and are then monitored for 14 days to determine the LC50. The Acute Toxic Class method for inhalation testing (TG 436) was adopted in 2009. OECD Draft Guideline 433, Acute Inhalation Toxicity – Fixed Concentration Procedure is a reduction/refinement to TG 403 because it uses only one sex of rat (females) and moderate doses that do not result in lethality. This draft Test Guideline is not part of Mutual Acceptance of Data (MAD) and those wishing to follow the procedure are advised by OECD to consult with national authorities before using this draft Test Guideline. Stallard, et al. (2003) described the FCP and its validation using statistical simulation.

Table 2. Adopted OECD test guidelines on short-term toxicity testing, with 3R relevance



Original Adoption

3R Relevance and Most Recent Update

401Acute Oral Toxicity (deleted)12 May 1981Date of Deletion: 20 December 2002 (Replaced by TGs 420, 423 and 425, introducing refinement and reduction).
402Acute Dermal Toxicity12 May 198124 February 1987: Animal test introducing reduction compared to the original TG (lowering of the dose level).
403Acute Inhalation ToxicityDraft updated Test Guideline (Original adoption: 12 May 1981)Animal test introducing potential reduction in animal usage compared to original 403, if one sex is more susceptible. Refinement by applying humane endpoints.
420Acute Oral toxicity – Fixed Dose Procedure (FDP)17 July 199217 December 2001: Animal test (reduction/ and refinement method in comparison with the conventional TG 401), less suffering, smaller number of animals.
423Acute Toxic Class Method (ATC)22 March 199617 December 2001 Animal test (reduction method compared to the conventional TG 401), much smaller number of animals (10% of that required for TG 401).
425Acute Oral Toxicity – Acute Oral Toxicity: Up-and- Down Procedure21 September 199817 December 2001: Animal test (reduction method compared to the conventional TG 401), smaller number of animals, provides a closer estimate of the LD50 than TGs 420 and 423.
436Acute Inhalation Toxicity: Acute Toxic Class (ATC) Method7 September 2009Animal test introducing reduction in animal usage compared to TG 403, and refinement by applying humane endpoints.

In addition to the OECD Test Guideline methods, the European Union has focused on the use of in vitro cytotoxicity assays as a means to develop alternatives to animal tests. The EURL ECVAM strategy to replace, reduce and refine the use of animals in the assessment of acute mammalian systemic toxicity employs the 3T3 neutral red uptake (NRU) assay to estimate starting doses for acute oral systemic toxicity tests and to categorize substances not requiring classification for acute oral toxicity (LD50 > 2000 mg/kg bw). They also utilize embryo-larval fish models and quantitative structure activity relationships (QSAR) modeling to predict metabolism and to test the parameters of the assay. Other approaches include the Colony Forming Unit-Granulocyte/Macrophage (CFU-GM) Assay to predict acute neutropenia in humans.

Table 3. In vivo animal reduction approaches for assessing acute toxicity for regulatory testing purposes.



Validation Authority*

International Acceptance*

Regional Acceptance

Acute mammalian toxicity (oral)Acute oral toxicityESAC (2000)OECD TG 401; Deleted in 2002
Acute toxic class methodESAC (2007)OECD TG 423 (2001)
Fixed dose procedureESAC (2007)OECD TG 420 (2001)
Up-and-down procedureICCVAM (2001)
ESAC (2007)
OECD TG 425 (2006)EPA OPPTS 870.1100 (2002)
Acute mammalian toxicity (inhalation)Acute toxic class methodOECD TG 436 (2009)
OECD GD No. 153 (2011)
Fixed concentration procedureDraft OECD TG 433 (2015)
Acute toxicity testing of pesticidesGuidance for waiving or bridging of mammalian acute toxicity tests for pesticides (acute oral, dermal, inhalation; primary eye and dermal; dermal sensitization)EPA OPP (2012)

*Links for accessing documents from organizations mentioned in the table:

Waivers from test requirements can be granted by regulatory agencies if a case can be made that it does not make sense to conduct the testing because the outcome is known, because the chemical’s properties or the product’s design make testing impossible or impracticable, or because there is no need to conduct the testing. For example, factors such as the physical state and/or properties of the chemical (e.g., volatility or extreme pH) may make if unnecessary to test the material. Other factors considered in waiver requests include such things as the product size or packaging design that prevent exposure to the material, the study not technically feasible (e.g., aerosol generation) or the properties of the substance are known (e.g., it is a sensitizer). “Bridging” refers to a process where requests to use existing toxicity data from a related chemical to estimate the toxic potential of a closely related substance can be evaluated and granted.

Waiver and bridging options exist because test guidelines are written to cover a large universe of substances that can differ greatly with respect to chemical and physical properties. Guidelines are also written to specify standard testing approaches and cannot anticipate all possible situations where the standard testing approach may not be appropriate. Waivers and the use of bridging data lead more efficient approaches to safety evaluation, and they also reduce the numbers of animals used in testing.

EPA’s Office of Pesticide Programs issued “Guidance for Waiving or Bridging of Mammalian Acute Toxicity Tests for Pesticides and Pesticide Products (Acute Oral, Acute Dermal, Acute Inhalation, Primary Eye, Primary Dermal, and Dermal Sensitization)” in March 2012. The guidance included examples of situations where acute oral, dermal and inhalation toxicity testing may not be required for conventional, antimicrobial, and biochemical pesticides providing registrants with insights about how to craft waiver requests. It also provided guidance for crafting waiver requests for eye irritation, dermal irritation, and skin sensitization test requirements. In addition, it provided guidance for requests to bridge data for end-use products and for toxicologically similar products. The guidance clarifies that specifically, data related to dermal acute toxicity for conventional, antimicrobial, and biochemical pesticides may be waived if any of the following criteria are met:

  • The test material has been placed in Toxicity Category I for primary dermal irritation. Such products will be placed in dermal Toxicity Category I on the basis of potential dermal effects.
  • The test material is corrosive to skin, or has a pH less than 2 or greater than 11.5 (40 CFR 158.500(e)(3); 40 CFR 158.2050(e)(2); 40 CFR 158. 2083(e)(2); 40 CFR 161.340(b)(2)). Such products will be placed in dermal Toxicity Category I on the basis of potential dermal effects.
  • The product design prevents dermal exposure. Products such as childproof insect baits and rodent bait boxes typically meet these criteria.

When evaluating an untested pesticide formulation, the toxicity of the active ingredient can provide useful information to predict the toxicity of the formulated product. Also, it is often possible to predict what the results of testing will be for an untested chemical, such as a pesticide active ingredient, based on the results of existing testing that was conducted on a closely related chemical substance if the substance of concern and the comparator substance are structurally similar chemicals. Care must be taken in bridging based on related compounds because subtle differences between the chemicals can lead to big differences in how they are absorbed, distributed, metabolized, excreted and/or alter biological pathways causing toxicity. In the evaluation of pesticide products it is also important to consider such factors such as is there a similar existing formulation with definitive data, are the products in the same physical form, do they include similar concentrations of active ingredient, etc.? At this time such bridging efforts are not routine, and they require expert judgment.

In addition to the March 2012 “Guidance for Waiving or Bridging of Mammalian Acute Toxicity Tests for Pesticides and Pesticide Products (Acute Oral, Acute Dermal, Acute Inhalation, Primary Eye, Primary Dermal, and Dermal Sensitization),” Jack Housenger, Director of EPA’s Office of Pesticide Programs, issued a letter on March 16,2016 that further moved OPP towards its goal of adopting more informative alternative testing approach to pesticide evaluation. The letter stated that OPP is working to achieve three main objectives by “(1) critically evaluating the studies that form the basis of OPP decisions, (2) expanding our acceptance of alternative methods and (3) reducing barriers to adopting methods in the U.S. and internationally; these barriers include the difficulties of data sharing among companies, international harmonization, and the assurance of acceptance of submitted non-animal tests by the EPA.”

With respect to the first objective, the letter requested comment on OPP’s draft waiver guidance for acute dermal toxicity studies for formulated pesticide products (i.e., when the guidance is finalized this test would no longer be required because an analysis of acute toxicity classifications revealed that they are rarely based on the results of acute systemic dermal studies). Regarding the second objective, the letter noted that it was providing clear guidance about expanding acceptance of alternatives for animal testing for skin sensitization, and that it was working with the OECD to solicit integrated approaches to testing and assessment (IATA) case studies for skin sensitization in the hopes of accelerating international approval of non-animal IATA’s. Finally, with respect to the third objective, finding solutions to barriers to alternative methods, it noted that OPP was working to adopt GHS categories for the hazard portion of the pesticide label in favor of the current OPP categorization approach (see Table 1).

The letter also noted that EPA was conducting a pilot study to see if it could accept submission of oral and inhalation toxicity study data, paired with GHS calculations, to support the evaluation of pesticide product formulations. This purpose was to evaluate the utility and acceptability of the GHS dose additive mixtures equation as an alternative to oral and inhalation toxicity studies for pesticide formulations.

Based on the results of the subsequent analysis, EPA’s Office of Pesticide Programs finalized its Guidance for Waiving Acute Dermal Toxicity Tests for Pesticide Formulations November 29, 2016. This action supports EPA’s commitment to only require data that informs regulatory decision-making and to avoid unnecessary use of time and resources, data generation costs, and animal testing. The agency estimates that this action will save 2,500 or more laboratory animals every year.

A critical component to achieving OPP’s vision of a more informative and efficient testing approach is its “Strategic Vision for Adopting 21st Century Science Methodologies.” It describes how OPP will use an IATA to promote a hypothesis based, systematic, integrative use of exposure and hazard information. The plan is to develop a progressive, tiered-testing approach using existing exposure and toxicity information combined with computer modeling and ‘new’ diagnostic non-animal assays to target toxicity testing to the specific data needed for human health and ecological risk assessments. Development and use of Adverse Outcome Pathways (AOPs) are an integral component of the strategy.

AOPs and IATA are terms developed by the OECD. They provide a means to link alternative test methods into batteries that approximate events leading to toxicity in animals in a manner that is acceptable to the condition of use for which they are applied. OECD has issued guidance on how to prepare an AOP, a process to evaluate them, and it has established a database, AOPWiki, to store those AOPs judged to be appropriate for use.

As defined by OECD, “an AOP is an analytical construct that describes a sequential chain of causally linked events at different levels of biological organisation that lead to an adverse health or ecotoxicological effect.” It requires knowledge of an initial molecular initiating event (MIE) and a well-defined adverse outcome that can result from the MIE. It also requires the identification of key events linking the MIE to the adverse outcome. A complete mechanistic understanding of the disease process is not required.

AOPs provide an essential means to understand what portion of the adverse outcome pathway each alternative test method approximates. Unlike whole animal tests that integrate all events from external exposure to adverse outcome in a living organism, alternative methods mimic only a subset of these events. AOPs provide a means to clearly define which part of the overall process leading to toxicity is mimicked by each alternative test and this provides a basis to combine a suite of tests in such a way that the results from all the tests combined covers the full range of events from exposure triggering an MIE to an adverse outcome.

An IATA involves the collection of all information available about a substance, and organizing that information with respect to a specific hypothesis(es) about toxicity (or lack thereof) for one or more AOP(s). OECD defines IATAs as “pragmatic, science-based approaches for chemical hazard characterisation that rely on an integrated analysis of existing information coupled with the generation of new information using testing strategies.” In essence this is a process to gather all that is known about the toxicity of a chemical, organize that information in a weight of evidence manner to arrive at conclusions about whether the substance causes toxicity or not. It also involves evaluation of how well the tests were conducted with respect to the degree to which the results are useful to inform decisions, and it defines what follow up studies are needed. IATA is a “learn as you go,” self-correcting process.

To better define how IATAs and AOPs apply to acute systemic toxicity, a workshop was held in September 2015, “Alternative Approaches for Identifying Acute Systemic Toxicity: Moving from Research to Regulatory Testing.” The workshop report offers recommendations to facilitate the adoption of alternative approaches for systemic acute toxicity testing, including overcoming the limitations of whole animal reference data by developing validated non-animal comparators, by conducting research to better understand the mechanism of toxicity, by improving the means to evaluate absorption, distribution, metabolism, and excretion in alternative test systems, by developing integrated approaches to testing and assessment, by education and training to help overcome the barriers to change, and by understanding regulatory requirements to identify the needs of regulatory agencies, how data are used, and what is the best path to regulatory acceptance.

Workshop participants noted that a major challenge to developing AOPs for acute systemic toxicity tests is a lack of knowledge of the MIE. Systemic toxicity is an organism-wide effect and death can be caused by failure of a variety of biologic pathways and processes. It may result from non-specific events rather than disruption of one or more specific biological pathways. As a way to define whether or not this is the case they recommend the use of “a suite of high-throughput screens (as this) may be most useful for covering nonspecific mechanisms as some substances created responses in many assays, and are therefore acting against multiple pathways. High throughput assays are also suitable for bringing together common key events based on downstream specific mechanisms, for example, altered signal transduction pathways or ion homeostasis that cover a wide array of MIEs.”

Another limitation of current in vitro methods is a lack of biokinetics and metabolism, and the difficulty in testing for volatile compounds. EPA and others are working to overcome these limitations. These efforts will not be discussed further here, because the focus of this section is on acute systemic toxicity tests not dosimetry and metabolism. Those interested in learning more may wish to explore EPA’s “Chemical Exposure and Dose Research” webpage.

Different countries have diverse approaches to safety evaluation with the result that there are variations in what information is acceptable to meet the test and regulatory authority requirements. Even within a country there are differences in the regulatory requirements, such as between those developed to ensure worker safety compared to those developed to ensure environmental protection. Over the years, a variety of approaches have been taken to develop rigorous approaches to “validate” the alternative methods developed for toxicity testing, including the establishment of a variety of authoritative validation bodies. These include the Interagency Coordinating Committee for the Validation of Alternative Methods (ICCVAM) in the US, the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM), the Japanese Center for the Validation of Alternative Methods (JaCVAM), the Korean Center for the Validation of Alternative Methods (KoCVAM), and the Environmental Health Science and Research Bureau within Health Canada. They work together through the International Cooperation on Alternative Test Methods (ICATM) to conduct test method validation studies, to conduct independent peer review of the validation studies, and to develop formal recommendations.

Prior to publication of the NAS “Toxicity Testing in the 21st Century Report” the approach to alternative method test validation typically involved running the test system in several different laboratories each using the same reference set of coded chemicals of known toxicological effect. The results are then compared to those obtained in another test system that has been accepted by competent regulatory authorities to represent “the truth.” The process is described in OECD GD 34 “Guidance Document On the Validation and International Acceptance of New or Updated Test Methods for Hazard Assessment”. The approach has been used successfully, but only to a limited extent, because the process is inefficient, costly, and time consuming. Furthermore, it freezes technology, and is not suited to “validating” the large numbers of newly developed Tox21 approaches to testing and assessment that are rapidly being developed.

Thus, new “fit for purpose” evaluations, including bioinformatics approaches, are being developed as a means to “validate” alternative test systems to provide certainty about the utility of the results and to meet the requirement in the enabling legislation for some agencies that the test systems be “validated” (e.g., the endocrine disruption screening requirements in FIFRA and SDWA). They generally involve developing a set of performance metrics to assess the ability of a test method to evaluate hazard properly for a clearly defined set of conditions. Once such performance standards are defined and accepted any alternative test approach that can correctly meet the performance metrics would be considered “validated” and fit for that defined purpose. Chapter 6 “Model and Assay Validation and Acceptance” of National Academy of Sciences report “Using 21st Century Science to Improve Risk-Related Evaluations” discusses validation approaches being considered for evaluating and “validating” Tox21 alternative test approaches.

Some alternative test approaches “validated” using the traditional pre-Tox21 validation approaches are summarized in Table 4.

Table 4. Non-animal alternative methods for assessing acute toxicity for regulatory testing purposes.



Test Purpose

Validation Authority*

International Acceptance*

Acute mammalian toxicity (oral)Normal human keratinocyte neutral red uptake (NHK NRU) assayIn vitro basal cytotoxicity assay: Neutral red uptake (NRU) test with normal human epidermal keratinocytes (NHK). Adjunct to in vivo acute oral toxicity tests for determining starting doses 3ICCVAM (2008)
JaCVAM (2011)
OECD GD 129 (2010)
Balb/c 3T3 neutral red uptake (NRU) assayIn vitro basal cytotoxicity assay: NRU test with mouse 3T3 fibroblasts. Adjunct to in vivo acute oral toxicity tests for determining starting doses 3ICCVAM (2008)
JaCVAM (2011)
OECD GD 129 (2010)
3T3 NRU Assay supporting identification of substances not requiring classification for acute oral toxicityIn vitro 3T3 NRU test method should be considered as an initial screening tool together with complimentary information to possibly reduce or avoid animal testingEURL ECVAM (2013)
Acute mammalian toxicity (hematotoxicity)Colony Forming Unit-Granulocyte/ Macrophage Assay for acute neutropenia in humansIn vitro haematotoxicity test for acute neutropeniaEURL ECVAM Science Advisory Committee ESAC (2006)

*Links for accessing documents from organizations mentioned in the table:

Further information on the use and/or development of in vitro methods being developed for Acute Systemic Toxicity testing can be found at:


AltTox Editorial Board reviewer(s):

The information provided here is intended only as an overview, and is neither guidance nor a comprehensive review of the laws and regulations on acute systemic toxicity testing. Individual countries/ regions and their regulatory authorities usually provide specific guidance on hazard/toxicity testing requirements.