Tracking progress: They say, knowledge is power

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Tracking progress: They say, knowledge is power

by Sherry Ward, AltTox Contributing Editor
Posted: May 31, 2016

One of our content updates this month is an updated version of AltTox’s Table of Validated & Accepted Alternative Methods (“Table”). This Table, which provides a comprehensive list of alternative methods endorsed as scientifically valid by ICCVAM (United States), EURL ECVAM (European Union), and/or JaCVAM (Japan), has become one of the most popular resources on AltTox. Related OECD Test Guidelines, which indicate some degree of international acceptance, as well as some regionally accepted regulatory approaches, are also identified in the Table.

What could be considered the original “Table” was created in 2004 as a means to inform and update colleagues on the status of validated alternative methods in the US and EU, as well as to illustrate the success of ICCVAM considering their limited resources relative to their much larger EU counterpart, ECVAM. At that time, a similar summary of methods validated by ECVAM and ICCVAM was not available elsewhere, and JaCVAM was not yet established.

The “Table” was first published with the launch of AltTox in December 2007, and is now updated several times per year to reflect the current status of alternative methods formally accepted for regulatory use. The methods listed in the Table include those addressing the 3R’s – methods intended to Replace, Reduce, and/or Refine the use of animals in toxicity and biologics testing for the purpose of fulfilling regulatory testing obligations.

AltTox’s Table is still possibly the only resource where information on methods endorsed by the three major validation authorities is integrated into one table, but there are many other useful summary sources of information on 3R methods; these are cited in the Table and elsewhere in AltTox content. EURL ECVAM provides extensive information on methods they have reviewed on their website, and developed one of my favorite resources, the DB-ALM database, which provides information on many in vitro methods, including those considered to be “validated.” Since ICCVAM reorganized their website in mid-2008, they provide an extremely comprehensive table of ICCVAM-endorsed methods along with each method’s international acceptance status. With the launch of their English-version website in 2010, JaCVAM maintains an up-to-date table of methods they have under review or have endorsed, and a second table with links to the validation and regulatory review documents for each method. Health Canada also provides useful tables on 3Rs methods on their 3Rs Microsite. And, the PETA International Science Consortium recently added a table of endorsed methods to its website. Each of these organizations developed a novel format that they found useful for presenting the information.

Current Table updates

There are many exciting updates in the latest version of the Table, plus the usual corrections for broken links and other edits. Current updates include the following:

  • Fixed concentration procedure for acute inhalation toxicity (Draft OECD Test Guideline (TG) 433, 2015): OECD TG 433 will be a new guideline that uses a series of fixed concentrations to determination acute inhalation toxicity in only one sex of animal. This refinement approach uses only moderately toxic concentrations of the test substance, avoids using death as the exclusive or intended endpoint, and may reduce the number of animals used.
  • European Pharmacopoeia’s changes to the general monograph Vaccines for Veterinary Use (0062) and to about 40 vaccine-specific monographs (adopted November 2015; to be effective in 2017) will result in a considerable reduction in animal use.
  • Alternative testing framework for classification of eye irritation potential for EPA pesticide products (EPA OPP, 2015): The applicability of the Bovine Corneal Opacity and Permeability (BCOP) assay was expanded so that it can be used for identifying toxicity category I, II, and III eye irritants for antimicrobial cleaning products (AMCPs). The EPA’s alternative approach for AMCPs utilizes a decision tree approach involving three in vitro/ex vivo assays (BCOP, EpiOcular, and Cytosensor Microphysiometer) to differentiate among EPA’s four eye irritation hazard categories. “For other classes of pesticides and pesticide products…the agency will consider alternative tests conducted and submitted on a case-by-case basis.”
  • Stably transfected human Androgen Receptor (AR) Transcriptional Activation (TA) assay for detection of androgenic agonist and antagonist activity of chemicals (AR STTA) (Draft OECD TG, 2015): The AR STTA is an in vitro assay “that evaluates transcriptional activation and inhibition of AR-mediated responses…considered to be one of the key mechanisms of possible endocrine disruption related health hazards.”
  • Reconsideration of the ICE [isolated chicken eye] Decision Criteria for identification of UN GHS No Category test chemicals for eye hazard (Updated OECD guidance, 2015): “Since the revisions from 2013, the criteria for acceptance of test methods to identify UN GHS No Category chemicals have changed…it was agreed to revise the Decision Criteria of the ICE test method for the identification of UN GHS No Category chemicals based on the current acceptance standards.” This position paper explains the revised ICE Decision Criteria for UN GHS classification.
  • Human cytochrome P450 (CYP) n-fold induction in vitro test method (OECD draft proposal for a new Performance Based Test Guideline, 2014): The draft guideline was published in 2014, but this is the first time it is listed in the Table. The guideline describes two in vitro methods using human hepatocytes (primary hepatocytes and the HepRG cell line) used “to assess the potential of test chemicals to induce three cytochrome P450 isoforms CYP1A2, CYP2B6, and CYP3A4….”
  • OECD adoption of the human Cell Line Activation Test (h-CLAT) TG for skin sensitization testing (approved by the OECD Working Group in April 2016): The h-CLAT is the third validated non-animal method for skin sensitization assessment, and is to be used in combination with the Direct Peptide Reactivity Assay and the ARE-Nrf2 Luciferase Test Method in an integrated approach. “The h-CLAT method is proposed to address the third key event (dendritic cell [DC] activation) of the skin sensitisation [adverse outcome pathway] (AOP) by quantifying changes in the expression of cell surface markers associated with the process of activation of DC…following exposure to sensitisers.”
  • Revision of regulatory requirements for skin sensitization under REACH: On April 20, 2016, the REACH Committee “adopted a revision to Annex VII of the REACH regulation which means that validated and accepted non-animal tests will become the default information requirement for assessing whether chemicals have the potential to cause skin sensitisation.”

A comment made at last week’s ICCVAM Workshop in Bethesda, Maryland, indicated there were many favorable responses to EPA’s recent request for public comment that waivers be granted for acute dermal toxicity studies for pesticide formulations, and that this policy is likely to be adopted. We look forward to adding this information to the Table after it is formally announced.

Saved by big data – yes, but not yet

In looking over the Table and other lists of alternative methods endorsed by the validation authorities, it appears there are around 100 methods available for refining, reducing, and/or replacing the use of animals in testing. However, the use of these validated and accepted methods remains limited, and, additionally, the available alternatives are not sufficient to meet many testing needs.

A major initiative undertaken in the US and EU is the big data computational toxicology approach to developing models that can predict human toxicity. High-throughput screening (HTS) approaches, like Tox21, are being developed to prioritize the large numbers of chemicals in the marketplace for traditional toxicology testing, but may not reduce overall animal use. The benefits of these and other new technologies lies in their future potential to replace animal use, and to even more accurately predict potentially toxic substances. In the near-term, however, laboratory-based toxicity test methods, like many of those listed in the Table, are the practical types of methods needed now by industry and testing labs to effectively reduce animal use over the next five years.


We always welcome your comments, and especially your contributions to improving AltTox content. If you find anything we overlooked in the Table, please send it to