Has Toxicity Testing Moved into the 21st Century? A Survey and Analysis of Perceptions in the Field of Toxicology

Background: Ten years ago, leaders in the field of toxicology called for a transformation of the discipline and a shift from primarily relying on traditional animal testing to incorporating advances in biotechnology and predictive methodologies into alternative testing strategies (ATS). Governmental agencies and academic and industry partners initiated programs to support such a transformation, but a decade later, the outcomes of these efforts are not well understood. Objectives: We aimed to assess the use of ATS and the perceived barriers and drivers to their adoption by toxicologists and by others working in, or closely linked with, the field of toxicology. Methods: We surveyed 1,381 toxicologists and experts in associated fields regarding the viability and use of ATS and the perceived barriers and drivers of ATS for a range of applications. We performed ranking, hierarchical clustering, and correlation analyses of the survey data. Results: Many respondents indicated that they were already using ATS, or believed that ATS were already viable approaches, for toxicological assessment of one or more end points in their primary area of interest or concern (26–86%, depending on the specific ATS/application pair). However, the proportions of respondents reporting use of ATS in the previous 12 mo were smaller (4.5–41%). Concern about regulatory acceptance was the most commonly cited factor inhibiting the adoption of ATS, and a variety of technical concerns were also cited as significant barriers to ATS viability. The factors most often cited as playing a significant role (currently or in the future) in driving the adoption of ATS were the need for expedited toxicology information, the need for reduced toxicity testing costs, demand by regulatory agencies, and ethical or moral concerns. Conclusions: Our findings indicate that the transformation of the field of toxicology is partly implemented, but significant barriers to acceptance and adoption remain. https://doi.org/10.1289/EHP1435

. ATS Technology Categories Table S2. Use categories of ATS Table S3. Sampling frame characterization of SOT respondents at time of the survey. Table S4. Perception of current viability as a function of familiarity. Percent respondents reporting ATS as currently viable as a function of familiarity with specific techniques. P values are based on independent Chi-square tests of independence. Table S5. Respondent characteristics by 4 categories of familiarity. P values refer to chisquare tests of independence for categorical outcomes, and F tests for continuous outcomes. Figure S1. Survey respondents clustered by patterns of viability. Darker features indicate higher perceived viability. The two clusters are separated at the red line. Two classes of respondents emerge as, one group identifying most technologies as being currently viable for ATS, and a second group exhibiting varying degrees of skepticism towards the viability of some applications. The percentage of subjects identified as perceiving a large number of viable technologies is 27.81.

Additional Files
Data and Code ZIP File PDF Documents -Survey Questionnaire and Responses Table S1 ATS Technology Categories

Technology
Description Mechanistically based in vitro assays Examines the effect of a chemical on a cultured bacterial or mammalian cell, or biological molecule such as a protein. Chemicals of interest are introduced into the testing medium, and observations are made regarding changes in biologic processes that may lead to toxicity. Depending upon the particular in vitro assay, mechanisms of injury will vary, including such phenomena as enzyme inhibition, cell membrane injury, and oxidative stress. Mechanistically based in vivo assays Focuses upon biological pathways for complex endpoints such as reproductive toxicity and developmental toxicity in small lower animals such as the vertebrate zebrafish and invertebrate Caenorhabditis elegans as surrogates for higher animals. Unlike mechanistic in vitro assays, this ATS takes into account the activity of the chemical and its metabolites at the cellular, organ and organism level 1 .

High throughput in vitro assays
Using advanced robotics and automation of in vitro testing to assess hundreds or even thousands of materials at once across a range of concentrations for a variety of parameters. The cells or molecules of interest are placed in small wells on plates containing hundreds or thousands of wells. The materials to be tested are added to the wells at varying concentrations, and readings are automatically made at specified intervals 2 . High throughput in vivo assays Using a variant of HTS in which the readout of the assay captures more complex data than in an HTS screen, such as a microscopic image from which quantitative information may be drawn regarding observable physical or biochemical characteristics of the cell or organism. For example, HCS of the effects of material on zebrafish embryos would generate quantitative data regarding hatching, developmental abnormalities and mortality using high content imaging software 2 . Quantitative Structure Activity Relationship (QSAR) Modeling the likely toxicity of a compound using the relationship between chemical structures and biological activities. Uses mathematical modeling to predict the likely toxicity for one substance based upon existing toxicity data for a large set of other relevant chemicals (the "training set.") The predictions are derived from analysis of the relationship between the training set's physicochemical properties or other descriptors and observed toxicity 3 . Biomarkers Assessing an in vitro-derived measure that provides mechanisticallybased, quantitative information predictive of an adverse effect in vivo.
Biomarkers of toxicity take into account adaptive responses of the organism that may prevent or mitigate the apical outcome associated with exposure 4 .

Table S2
Use categories of ATS Use Description Screening for prioritization for further testing Prioritizing chemicals that ATS assays identify as inducing changes in biological pathways that are associated with in vivo end points for more in-depth testing 5 . Screening for prioritization for other action Prioritizing chemicals that ATS assays identify as inducing changes in biological pathways that are associated with in vivo end points for risk assessment or risk management purposes. Comparative assessment of chemicals to alternative Comparing a chemical of concern to possible alternatives across a range of endpoint, as in alternatives analysis 6 . Weight of evidence in quantitative risk assessment Using ATS to determine how much weight to give specific lines of traditional toxicological of evidence in quantitative risk assessment Qualitative risk assessment Assigning a qualitative measurement of risk (e.g. high, medium, or low) based on ATS results Setting doses for in vivo testing Extrapolating doses to use in whole animal testing from ATS results Quantitative risk assessment Using ATS results in establishing a point of departure such as a no observed adverse effect levels