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Cited by 123Animal testing and alternativesCarcinogens and Genotoxicity AssessmentEnvironmental Toxicology and Ecotoxicology

Cytotoxicity Burst? Differentiating Specific from Nonspecific Effects in Tox21 in Vitro Reporter Gene Assays

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Escher, Beate I., Henneberger, Luise, König, Maria, Schlichting, Rita, Fischer, Fabian C.·2020·Environmental Health Perspectives
DOI 10.1289/ehp6664PMC PMC7377237
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Abstract

BACKGROUND: High-throughput screening of chemicals with in vitro reporter gene assays in Tox21 has produced a large database on cytotoxicity and specific modes of action.However, the validity of some of the reported activities is questionable due to the "cytotoxicity burst," which refers to the supposition that many stress responses are activated in a nonspecific way at concentrations close to cell death.OBJECTIVES: We propose a pragmatic method to identify whether reporter gene activation is specific or cytotoxicity-triggered by comparing the measured effects with baseline toxicity.METHODS: Baseline toxicity, also termed narcosis, is the minimal toxicity any chemical causes.Quantitative structure-activity relationships (QSARs) developed for baseline toxicity in mammalian reporter gene cell lines served as anchors to define the chemical-specific threshold for the cytotoxicity burst and to evaluate the degree of specificity of the reporter gene activation.Measured 10% effect concentrations were related to measured or QSARpredicted 10% cytotoxicity concentrations yielding specificity ratios (SR).We applied this approach to our own experimental data and to ∼ 8,000 chemicals that were tested in six of the high-throughput Tox21 reporter gene assays.RESULTS: Confirmed baseline toxicants activated reporter gene activity around cytotoxic concentrations triggered by the cytotoxicity burst.In six Tox21 assays, 37%-87% of the active hits were presumably caused by the cytotoxicity burst (SR < 1) and only 2%-14% were specific with SR ≥ 10 against experimental cytotoxicity but 75%-97% were specific against baseline toxicity.This difference was caused by a large fraction of chemicals showing excess cytotoxicity.CONCLUSIONS: The specificity analysis for measured in vitro effects identified whether a cytotoxicity burst had likely occurred.The SR-analysis not only prevented false positives, but it may also serve as measure for relative effect potency and can be used for quantitative in vitro-in vivo extrapolation and risk assessment of chemicals.

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  • In the region near cytotoxic concentrations, oxidative stress, mitochondrial dysfunction, heat-shock responses, unfolded-protein signaling and loss of membrane integrity may occur in parallel, so that reporter gene or receptor-transactivation signals can reflect non-specific stress rather than a discrete molecular initiating event.

    “In this region, oxidative stress, mitochondrial dysfunction, heat-shock responses, unfolded-protein signaling and loss of membrane integrity may occur in parallel, so that reporter gene or receptor-transactivation signals can reflect non-specific stress rather than a discrete molecular initiating event (Judson et al., 2016; Escher et al., 2020).”

    From Cytotoxicity across OECD methods is not a single endpoint: context-dependent roles, variability and implications for decision-making (2026) · cites this paper as Escher et al., 2020

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  • Analyses of ToxCast and Tox21 data showed that chemicals may activate multiple, unrelated assay endpoints close to cytotoxic concentrations, indicating a transition from specific bioactivity to generalized cellular stress.

    “Analyses of ToxCast and Tox21 data showed that chemicals may activate multiple, unrelated assay endpoints close to cytotoxic concentrations, indicating a transition from specific bioactivity to generalized cellular stress (Judson et al., 2016; Escher et al., 2020).”

    From Cytotoxicity across OECD methods is not a single endpoint: context-dependent roles, variability and implications for decision-making (2026) · cites this paper as Escher et al., 2020

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Cited by

Sorted by citations

Authors
Escher, B. I., Henneberger, L., König, M., Schlichting, R., & Fischer, F. C.
Affiliations
Helmholtz Centre for Environmental Research (DE)
Corresponding author
Escher, Beate I.
ORCID
Venue
Environmental Health Perspectives
Publisher
American Chemical Society (ACS)
Published
2020-07
Volume
128
Issue
7
Pages
77007-77007
ISSN
  • 0091-6765 (ISSN-L)
  • 1552-9924
DOI
10.1289/ehp6664
PMC
PMC7377237
OpenAlex
W3044039596
Cited by
123
References
2
Citations by year
2026: 33 · 2025: 17 · 2024: 25 · 2023: 13 · 2022: 16 · 2021: 17 · 2020: 2
FWCI
11.12
Type
journal article
Language
en
Open access
Licence
public-domain
Locations
  • Environmental Health Perspectives (publishedVersion, public-domain)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
Funders
  • European Chemical Industry Council (ECO36)
  • Danmarks Tekniske Universitet
Topics
  • Health Sciences › Veterinary › Small Animals › Animal testing and alternatives
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Carcinogens and Genotoxicity Assessment
  • Physical Sciences › Environmental Science › Health, Toxicology and Mutagenesis › Environmental Toxicology and Ecotoxicology
Keywords
baseline toxicity, narcosis, Tox21, reporter gene assay, QSAR, false positives, in vitro-in vivo extrapolation, risk assessment, high-throughput screening
MeSH terms
Biological Assay, Humans, Genes, Reporter, Toxicity Tests, Quantitative Structure-Activity Relationship

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