In the US and EU, proposals are advanced to reduce the use of animals for acute systemic toxicity testing

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In the US and EU, proposals are advanced to reduce the use of animals for acute systemic toxicity testing

by Marilyn Matevia, Humane Society of the United States
Acute systemic toxicity refers to the adverse reactions that occur within 24 hours of oral or dermal exposure to a substance, or within 4 hours of inhalation exposure. In traditional toxicity testing procedures it is measured by the median lethal dose (LD50 for oral or dermal routes of exposure, or LC50 – median lethal concentration – for inhalation routes), or the dose of the substance that kills 50% of the animals tested, within 14 days. As noted by Gennari, et al. (2004), “A major use of acute systemic toxicity data is for the classification and labelling of chemicals in relation to their manufacture, transport and use, but the data are also used for defining modes of action, setting dose levels for other tests, and providing medical advice in cases of poisoning (based on the toxic effects observed).”

Procedures to establish median lethal dose/concentration have changed little since the measure was introduced in 1927, and they are not uncontroversial. While refinements have been developed to reduce the number of animals required for each test (Spielmann et al., 1999, and Halle, 2003), the methods for administering doses inflict considerable pain and distress on test animals. At the same time, the scientific value of LD50 measures is the subject of longstanding debate (Ekwall et al., 1998; Robinson et al., 2008; Seidle et al., 2011). An analysis of preclinical and clinical data on 74 pharmaceutical compounds by Robinson, et al. (2008) demonstrated that “acute toxicity data was not used to (i) terminate drugs from development, (ii) support dose selection for repeat dose studies in animals, or (iii) to set doses in the first clinical trials in humans.” (According to the Globally Harmonised System of Classification and Labelling of Chemicals (GHS), rats are the “preferred species” for oral and inhalation tests; rats or rabbits are preferred for dermal exposure tests.)

Despite the dubious scientific value and clear animal welfare concerns, acute toxicity data are required or recommended for a wide range of chemical substances by regulatory agencies around the world (Gennari et al., 2004; Seidle et al., 2010). In the United States, for example, the Environmental Protection Agency (EPA) and Department of Transportation (DOT) require acute toxicity test data for hazard classifications of certain industrial and agricultural chemicals. In the European Union, REACH regulations specify that registration applications for new chemicals being produced or imported in volumes greater than 10 tonnes (22,046 pounds, or just over 11 US tons) include acute toxicity data. Although some regulatory agencies have explored strategies to reduce, refine, and replace animal tests for acute toxicity (for example, Gennari et al., 2004), progress toward approved non-animal alternatives has been slow. The Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) began to assess the performance of methods to reduce animal use in acute toxicity testing, designed to reduce animal use by defining a narrower range of starting doses in the animal test, in the early 2000’s. Since that time, several reduction methods for determining starting doses for the animal studies have been endorsed by ICCVAM, the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM), and the Japanese Center for the Validation of Alternative Methods (JaCVAM). Currently, there are several internationally-accepted in vitro and in vivo reduction methods used to establish the starting doses for required animal tests: two cell-based methods(normal human keratinocyte neutral red uptake [NHK NRU] assay, and Balb/c 3T3 neutral red uptake assay; OECD GD 129), and three animal-based procedures (OECD TG 420, 423, 425). There are no non-animal acute toxicity replacement methods/approaches accepted by regulatory agencies at this time.

This sluggish progress could soon change. In the late part of 2014 and early 2015, agencies in the EU and US released proposals that – if implemented – will increase the use of alternative methods and greatly reduce the numbers of animals used for acute toxicity testing.

In December 2014, the European Commission released the EURL ECVAM strategy to replace, reduce and refine the use of animals in the assessment of acute mammalian systemic toxicity. The report outlines two “strategic aims:” (1) to reduce and replace the use of animals in acute toxicity studies, and (2) to refine those animal studies that are still required.

To reduce and replace animal studies, the report authors recommend a combination of tactics, including:

  • making more extensive use of existing in vitro and chemoinformatic methods
  • making better use of existing mechanistic and repeated dose data
  • developing approaches for in vitro-to-in vivo and inter-species extrapolation
  • developing scientifically-based arguments for waiving certain animal tests in acute toxicity studies

These objectives will be facilitated by an Integrated Approach to Testing and Assessment (IATA) – a framework that combines and evaluates the strength of evidence from existing in vitro and in vivo test data, computational models, Adverse Outcome Pathways (AOPs), and other sources. The report authors note that while “the development of IATA in this area is hampered by the lack of sufficient mechanistic understanding of the numerous toxicity pathways and/or modes-of-action that lead to acute systemic toxicity” (p. 10), a number of programs or projects – e.g. the OECD’s AOP development programs, and the US EPA’s ToxCast – will advance IATA development.

Recommendations for refinement include:

  • avoiding the use of lethal endpoints wherever possible, and reducing the number of animals required for assessments, and
  • refining acute dermal toxicity test methods, in particular

The authors note that refinement and reduction approaches exist for the assessment of acute oral and inhalation toxicity, but for acute dermal toxicity “the only guideline currently available is the classic dermal LD50 study… that uses lethality as the primary endpoint and requires an average number of animals between 10 (limit test) and 30.” However, the OECD Working Group of National Coordinators for Test Guidelines (WNT) “recently approved a project proposal…to either revise or replace the OECD TG402 (acute dermal toxicity testing) in line with the 3Rs principles.”

Not long after EURL ECVAM published its strategy document, the US EPA’s Office of Pesticide Programs released its own draft guidance document, “Process for Establishing & Implementing Alternative Approaches to Traditional In Vivo Acute Toxicity Studies.” It outlines “a transparent, stepwise process for evaluating and implementing alternative methods of testing for acute oral, dermal, inhalation toxicity, along with skin and eye irritation and skin sensitization,” the so-called “six pack studies” required by the EPA for registering pesticides.

The OPP program has three components: evaluation, transition, and implementation. The evaluation phase will determine whether a particular alternative method can be used for regulatory purposes. This process considers whether the method has been validated by other agencies; the types of chemicals to which it applies; and whether it is appropriate for single chemicals, mixtures, or both. It also considers whether a method can be used alone as a replacement for an in vivo test, or if it must be used in combination with other methods in an IATA; specifics about the reliability, ease of use, and cost; and whether the information it yields is suitable for labeling and regulatory decisions.

During the transition phase, OPP will accept data generated from the alternative method, along with data from the in vivo counterpart (or reference to previously submitted in vivo data). “This would provide the broader registrant community with the opportunity to practice conducting the method(s) and the agency additional experience in assessing the data” (p. 6). The agency will invite public comment on draft guidance during this period, as well.

In the implementation phase, the OPP – having evaluated comments and procedures – will issue a final guidance document, and the agency will accept data generated by the alternative method in place of its in vivo counterpart.

The authors of the EURL ECVAM strategy hope their recommendations are in place in time for the 2018 REACH registration deadline, but note that “the timely achievement of the objectives and aims presented…will depend on the proactive and coordinated engagement of multiple stakeholders” (p. 17). The EPA invited and accepted comments on its proposal until March 10, 2015; a final guidance has yet to be issued.

Regulatory acceptance of alternative approaches for identifying acute systemic toxicity will be discussed at an upcoming two-day workshop sponsored by the National Toxicology Program’s Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM), the PETA International Science Consortium Ltd., and the Physicians Committee for Responsible Medicine. The limited-attendance workshop, September 24-25 in Bethesda, Maryland, is designed to “explore and discuss the state of the science of approaches to identifying acute systemic toxicity that replace, reduce, and refine animal use and to explore ways to facilitate their implementation.” Registration information is available here.

Posted: September 15, 2015