Cytotoxicity across OECD methods is not a single endpoint: context-dependent roles, variability and implications for decision-making

The regulatory meaning of cytotoxicity depends on its role within the test method, which may be as a classification endpoint, predictive surrogate, validity criterion, or concentration constraint.

“Their regulatory meaning depends on assay role, where they may function as classification endpoints, predictive surrogates, validity criteria, or constraints for concentration selection.”

• Also stated in: § Introduction · hedged

  “Depending on the assay context, it may represent tissue damage, define the biologically interpretable concentration range, constrain dose selection, or serve as a validity filter for mechanistic endpoints (OECD, 2012; Kolle et al., 2021; Gabbert et al., 2022) and serve various endpoints, including irritation, sensitization, endocrine activity, mutagenicity and acute toxicity estimation.”

• cites OECD, 2012Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites Kolle et al., 2021 sourceNot checked — no full text of the source
• cites Gabbert et al., 2022 sourcePartly supported

  Table I (Empirical Application, Selection of Testing Methods; page unavailable) uses relative cell viability as a classification threshold for skin irritation and corrosion, while the h-CLAT entry requires at least 50% viability for a positive result based on cell-surface markers. These examples support the point that viability has different roles across assays, but the copy does not establish the full four-role account of cytotoxicity: it does not identify cytotoxicity as a predictive surrogate or a constraint on concentration selection. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

In Fischer et al.'s comparison, an apparent positive response in the MicroFlow assay was not confirmed by microscopic evaluation when cytotoxicity/cytostasis was assessed using count-based metrics recommended for micronucleus tests without cytochalasin B.

“In their comparison of flow-cytometric evaluation using the MicroFlow kit (Litron Laboratories) and microscopic evaluation, an apparent positive response observed in the MicroFlow assay was not confirmed by microscopic evaluation when cytotoxicity/cytostasis was assessed using count-based metrics recommended for micronucleus tests without cytochalasin B.”

• cites Fischer et al., 2026 sourceSupported

Assay variability near decision points may be large enough to affect classification, particularly where the threshold is close to the steep part of a concentration-response curve.

“Ring-trial and validation data indicate that assay variability near decision points may be large enough to affect classification, particularly where the threshold is close to the steep part of a concentration-response curve (Kolle et al., 2021; Gabbert et al., 2022; Leontaridou, 2017).”

• cites Kolle et al., 2021 sourceNot checked — no full text of the source
• cites Gabbert et al., 2022 sourcePartly supported

  The Conclusions state that technical and biological variability can cause results near a classification threshold to switch between positive and negative on retesting. The paper does not establish the added condition that this is particularly so when the threshold lies near the steep part of a concentration-response curve. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites Leontaridou, 2017 sourcePartly supported

  The Abstract says biological and technical variability can make results near classification thresholds inconclusive and can affect the overall assessment. It does not link that effect specifically to thresholds on the steep part of a concentration-response curve or identify ring-trial data as its basis. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

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).”

• cites Judson et al., 2016 sourceSupported
• cites Escher et al., 2020 sourceSupported

The concept of borderline ranges is well established for several defined approaches and in vitro skin sensitization methods.

“The concept is well established for several defined approaches and in vitro skin sensitization methods (Kolle et al., 2021; Gabbert et al., 2022; Leontaridou, 2017; OECD, 2025e).”

• cites Kolle et al., 2021 sourceNot checked — no full text of the source
• cites Gabbert et al., 2022 sourcePartly supported

  The Introduction reports borderline-range analyses for three nonanimal skin-sensitization methods and one defined approach, the “2-out-of-3” approach. But it says borderline ranges had been explored for only a few methods and that evidence remained limited; it does not support calling the concept well established for several defined approaches. Page numbers were not available in our copy. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites Leontaridou, 2017 sourcePartly supported

  The Abstract reports quantified borderline ranges for three non-animal skin-sensitization methods and examines their effect on one integrated testing strategy. It does not establish that borderline ranges were already well established across several defined approaches; instead, it proposes adding a borderline alert to standard classifications. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

The width of borderline ranges depends on the precision of the test method.

“Gabbert et al. provided the formal statistical basis for this approach by defining borderline ranges as expressions of precision uncertainty around classification thresholds and showed that their width depends on the precision of the test method (Gabbert et al., 2022).”

• cites Gabbert et al., 2022 sourceSupported

Fischer et al. showed in V79 cells that methodological handling of cytotoxicity and cytostasis can determine whether an apparent micronucleus response is accepted as relevant or rejected as toxicity-associated and non-scorable.

“Fischer et al. showed in V79 cells that the methodological handling of cytotoxicity and cytostasis can determine whether an apparent micronucleus response is accepted as relevant or rejected as toxicity-associated and non-scorable.”

• cites Fischer et al., 2026 sourceSupported

Interlaboratory studies show substantial variability in IC50 and related cytotoxicity metrics, frequently in the range of approximately 20% to >40%, depending on system and endpoint.

“Interlaboratory studies further show substantial variability in IC50 and related cytotoxicity metrics, frequently in the range of approximately 20% to >40%, depending on system and endpoint (Tani et al., 1999; Gruber and Nickel, 2023).”

• cites Tani et al., 1999 sourceNot checked — no full text of the source
• cites Gruber and Nickel, 2023 sourcePartly supported

  The Results section (‘Outcome of the interlaboratory comparison: highly variable cytotoxicity test results for PVC’; page unavailable) reports substantial variation: only 62% of laboratories classified PVC as cytotoxic, and 42% did not classify at least one material as expected. It does not report interlaboratory variability in IC50 or establish the claimed approximately 20% to >40% range for IC50 and related metrics across systems and endpoints; its percentages describe classification outcomes. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

Cytotoxicity and cytostasis metrics are not auxiliary measurements in the in vitro micronucleus assay, but assay-defining constraints for valid concentration selection and interpretation.

“This supports the view that cytotoxicity and cytostasis metrics are not auxiliary measurements in the in vitro micronucleus assay, but assay-defining constraints for valid concentration selection and interpretation (Fischer et al., 2026).”

• cites Fischer et al., 2026 sourceSupported

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).”

• cites Judson et al., 2016 sourcePartly supported

  Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  Experimental — accuracy not yet measured
• cites Escher et al., 2020 sourcePartly supported

  Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  Experimental — accuracy not yet measured

In vitro assays often test nominal concentrations that may not reflect freely available, intracellular or in vivo-relevant concentrations.

“In vitro assays often test nominal concentrations that may not reflect freely available, intracellular or in vivo-relevant concentrations (Dimitrijevic et al., 2022; Kramer et al., 2012).”

• cites Dimitrijevic et al., 2022 sourceSupported
• cites Kramer et al., 2012 sourcePartly supported

  The indexed Abstract (no page given) reports that phenanthrene’s free concentration is reduced in a typical in vitro setup by binding to serum protein and well-plate plastic, and that free concentrations can differ between in vitro and in vivo systems. This supports the concern about nominal versus freely available and in vivo-relevant concentrations, but the passage does not establish a mismatch with intracellular concentrations. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

Measured exposure concentrations are particularly valuable because nominal concentrations can misrepresent the actual exposure of cells or tissues.

“Measured exposure concentrations are particularly valuable because nominal concentrations can misrepresent the actual exposure of cells or tissues (Henneberger et al., 2025).”

• cites Henneberger et al., 2025 sourceSupported

OECD TG 439 requires a mandatory check for direct chemical reduction of MTT by the test substance to avoid artificially inflated viability scores and false-negative irritancy classifications.

“To ensure the accuracy of results, OECD TG 439 (in vitro skin irritation) requires a mandatory check for direct chemical reduction of MTT by the test substance, as such interactions can artificially inflate viability scores and lead to false-negative irritancy classifications (OECD, 2025b).”

• cites OECD, 2025bNot matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Variability levels frequently overlap with commonly applied decision thresholds such as ∼50% viability or ∼55 ± 5% cytostasis.

“Together, these factors contribute to variability levels that frequently overlap with commonly applied decision thresholds, such as ∼50% viability or ∼55 ± 5% cytostasis, as summarized in the supplementary information, Table S2.”

OECD guidelines for mechanistic assays define viability limits or concentration constraints, commonly requiring viability to remain at or above approximately 70-80%, depending on the assay.

“For this reason, the guidelines define viability limits or concentration constraints, commonly requiring viability to remain at or above approximately 70–80%, depending on the assay (OECD, 2024a,b,2021,2025d).”

• cites OECD, 2024a,b,2021,2025dNot matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Substantial and assay-specific variability of cytotoxicity and cytostasis endpoints is consistently observed across interlaboratory datasets.

“Substantial and assay-specific variability of cytotoxicity and cytostasis endpoints is consistently observed across interlaboratory datasets (supplementary information, Table S2).”

In mutagenicity testing (OECD TG 473, 476, 487, 490), cytotoxicity and cytostasis are not the primary endpoints but define the concentration range within which chromosomal aberrations, mutations or micronuclei can be interpreted reliably.

“In mutagenicity testing, cytotoxicity and cytostasis have a distinct regulatory function. In OECD TG 473, TG 476, TG 487 and TG 490, they are not the primary mutagenicity endpoints. Instead, they define the concentration range within which chromosomal aberrations, mutations or micronuclei can be interpreted reliably (OECD, 2012,2016a,b,c,2023).”

• cites OECD, 2012,2016a,b,c,2023Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

OECD GD 114 showed that RPD, RICC and CBPI differ in sensitivity, dynamic range and variability for the in vitro micronucleus test.

“OECD GD 114 analyzed cytotoxic and cytostatic measures for the in vitro micronucleus test and showed that RPD, relative increase in cell counts (RICC) and CBPI differ in sensitivity, dynamic range and variability (OECD, 2012).”

• cites OECD, 2012Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

In androgen receptor agonist testing based on LDH release in U2OS cells, different laboratories identified divergent cytotoxic concentrations and selected different top-dose limits despite using the same test system and endpoint.

“This variability has direct practical consequences, as illustrated in Table 3 for androgen receptor agonist testing based on LDH release in U2OS cells, where different laboratories identified divergent cytotoxic concentrations and selected different top-dose limits despite using the same test system and endpoint.”

Schürmeyer et al. (2025) illustrated variability of cytotoxicity analyses of valproic acid in six independent experiments (HepG2 cells, CellTiter Blue assay) with 50 concentrations each, demonstrating that even within one cytotoxicity assay framework, concentration-response estimates are uncertain.

“This is supported by data from Schürmeyer et al. (2025) who have illustrated the variability of cytotoxicity analyses of the test compound valproic acid in six independent experiments (HepG2 cells, CellTiter Blue assay) with 50 concentrations each (Fig. 3), demonstrating that even within one cytotoxicity assay framework, concentration-response estimates are uncertain.”

• cites Schürmeyer et al., 2025 sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

Uncertainty in in vitro IC50 estimates propagates into uncertainty in predicted acute toxicity values in vivo.

“Independent evaluations and modelling approaches have shown that uncertainty in IC50 estimates in vitro cytotoxicity tests propagates into uncertainty in predicted acute toxicity values in vivo (Schrage et al., 2011; Neves L de et al., 2025).”

• cites Schrage et al., 2011 sourceNot checked — no full text of the source
• cites Neves L de et al., 2025Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Cytotoxicity and proliferation measurements used in OECD in vitro test guidelines do not represent a uniform endpoint.

“Cytotoxicity and proliferation measurements are widely used in OECD in vitro test guidelines but do not represent a uniform endpoint.”

Excessive cytotoxicity can compromise interpretability through non-specific stress and assay interference.

“Excessive cytotoxicity may further compromise interpretability through non-specific stress and assay interference.”

• Also stated in: § Cytotoxicity as a validity criterion in mechanistic assays · plain

  “Excessive cytotoxicity can distort transcriptional activity, receptor signaling, membrane integrity, metabolic capacity and protein expression, thereby generating false-negative or false-positive pathway responses.”

Cytotoxicity and cytostasis are not biologically or operationally equivalent.

“In this review, cytotoxicity refers primarily to loss of cellular viability, integrity or function, whereas cytostasis refers to reduced proliferation or cell-cycle progression; both may affect assay interpretation, but they are not biologically or operationally equivalent.”

• Also stated in: § Cytotoxicity and cytostasis as dose-setting in mutagenicity assays · plain

  “This distinction is particularly important because cytotoxicity, cytostasis and cell-cycle delay are not equivalent.”

The same numerical result (e.g., 70% viability) can have different regulatory meanings depending on the method context.

“The same numerical result can therefore have different regulatory meanings.”

• Also stated in: § Introduction · hedged

  “Thus, a value such as 70% viability may indicate an acceptable concentration, an exclusion criterion, an uninterpretable condition or no classification-relevant effect, depending on the method context.”

A result just above or just below a cutoff may fall within the empirical variability of the assay.

“A result just above or just below a cutoff may fall within the empirical variability of the assay.”

In mechanistic assays, cytotoxic stress may distort pathway-specific signals.

“In mechanistic assays, cytotoxic stress may distort pathway-specific signals.”

The interpretation of cytotoxicity depends not only on numerical effect levels but on how the measurement is generated and used within a test method.

“The interpretation of cytotoxicity depends not only on numerical effect levels but on how the measurement is generated and used within a test method.”

Test substances may interfere chemically with endpoint detection through color, fluorescence, reduction potential or binding to assay reagents.

“Test substances may also interfere chemically with endpoint detection, for example through color, fluorescence, reduction potential or binding to assay reagents.”

In the 3T3 NRU assay, basal cytotoxicity shows inter-laboratory dispersion with a CV of ∼47%.

“For example, basal cytotoxicity in the 3T3 NRU assay shows inter-laboratory dispersion with a CV of ∼47%, and the assay results with the historical positive control (sodium lauryl sulfate, SLS) exhibit an SD of 41.5 ± 4.8 µg/mL, providing a quantitative estimate of assay precision (OECD GD 129 (OECD, 2010)).”

• cites OECD, 2010Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

The concentration range described as the cytotoxicity burst provides a mechanistic explanation for many cytotoxicity-related artefacts in pathway-based assays.

“The concentration range described as the cytotoxicity burst provides a mechanistic explanation for many cytotoxicity-related artefacts in pathway-based assays.”

Mechanistic assay responses are interpretable only when cellular function is sufficiently preserved; excessive cytotoxicity can distort responses and generate false-negative or false-positive pathway responses.

“These responses are interpretable only when cellular function is sufficiently preserved. Excessive cytotoxicity can distort transcriptional activity, receptor signaling, membrane integrity, metabolic capacity and protein expression, thereby generating false-negative or false-positive pathway responses.”

RPD, RICC and CBPI are not interchangeable.

“It shows that RPD, RICC and CBPI are not interchangeable and that their variability can influence concentration selection.”

• cites OECD, 2012Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Benchmark concentration approaches can provide an additional quantitative basis for defining cytotoxicity-related decision points and their uncertainty where sufficiently resolved concentration-response data are available.

“Where sufficiently resolved concentration-response data are available, benchmark concentration approaches can provide an additional quantitative basis for defining cytotoxicity-related decision points and their uncertainty (Krebs, 2019).”

• cites Krebs, 2019Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Variability of RPD, RICC and CBPI can influence concentration selection.

“It shows that RPD, RICC and CBPI are not interchangeable and that their variability can influence concentration selection.”

• cites OECD, 2012Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Cytotoxicity-based decisions carry a non-negligible probability of misclassification, particularly near common thresholds such as 70-80% viability or 55 ± 5% cytostasis.

“Because many regulatory acceptance criteria coincide with empirical variability bands, cytotoxicity-based decisions carry a non-negligible probability of misclassification, particularly near common thresholds such as 70–80% viability or 55 ± 5% cytostasis.”

• AssumptionRests on an assumption: Many regulatory acceptance criteria coincide with empirical variability bands.
• AssumptionRests on an assumption: Common thresholds such as 70–80% viability or 55 ± 5% cytostasis are used in regulatory decisions.

Stress pathway activation may increase reporter activity or surface marker expression and contribute to false-positive results.

“Conversely, stress pathway activation may increase reporter activity or surface marker expression and contribute to false-positive results.”

The commonly used target range of approximately 55 ± 5% cytostasis is an operational compromise to ensure adequate test concentration while preserving a sufficiently proliferating and scorable cell population.

“The commonly used target range of approximately 55 ± 5% cytostasis is therefore an operational compromise: it aims to ensure adequate test concentration while preserving a sufficiently proliferating and scorable cell population.”

Excessive cellular stress may produce secondary responses that mimic the endpoint of interest.

“Excessive cellular stress may produce secondary responses that mimic the endpoint of interest as illustrated in Fig. 4.”

Cytotoxicity and proliferation endpoints show substantial variability from sources including cell type, tissue model, donor/cell-line characteristics, passage number, metabolic competence, culture conditions, medium composition, serum content, exposure duration, endpoint chemistry, operator effects and laboratory-specific procedures.

“Cytotoxicity and proliferation endpoints show substantial variability. Sources include cell type, tissue model, donor or cell-line characteristics, passage number, metabolic competence, culture conditions, medium composition, serum content, exposure duration, endpoint chemistry, operator effects and laboratory-specific procedures.”

OECD GD 114 explicitly addresses differences between cytostasis measures in the in vitro micronucleus test.

“OECD GD 114 is important because it explicitly addresses differences between cytostasis measures in the in vitro micronucleus test (OECD, 2012).”

• cites OECD, 2012Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Membrane damage can affect dye uptake, fluorescence, antibody staining or enzyme release.

“Membrane damage can affect dye uptake, fluorescence, antibody staining or enzyme release.”

The 3T3 NRU assay results with the historical positive control sodium lauryl sulfate exhibit an SD of 41.5 ± 4.8 μg/mL.

“For example, basal cytotoxicity in the 3T3 NRU assay shows inter-laboratory dispersion with a CV of ∼47%, and the assay results with the historical positive control (sodium lauryl sulfate, SLS) exhibit an SD of 41.5 ± 4.8 µg/mL, providing a quantitative estimate of assay precision (OECD GD 129 (OECD, 2010)).”

• cites OECD, 2010Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

In TG 487, cytotoxicity determines whether the scored cell population has undergone sufficient and interpretable cell-cycle progression; excessive cytotoxicity or cytostasis can lead to micronucleus frequencies that no longer reflect primary genotoxic activity.

“In TG 487, cytotoxicity is not merely a tolerability criterion; it determines whether the scored cell population has undergone sufficient and interpretable cell-cycle progression. If excessive cytotoxicity or cytostasis removes damaged cells, prevents mitosis, delays cell-cycle progression or leaves only a selected survivor population, micronucleus frequencies may no longer reflect primary gen”

Mitochondrial impairment can alter tetrazolium or resazurin readouts without proportional cell death.

“Mitochondrial impairment can alter tetrazolium or resazurin readouts without proportional cell death.”

Cytotoxicity is time-dependent; exposure duration, recovery period and timing of endpoint measurement strongly influence the observed result.

“Cytotoxicity is time-dependent. The exposure duration, recovery period and timing of endpoint measurement strongly influence the observed result.”

Chemical interference (e.g., colored, fluorescent, reducing, oxidizing, precipitating, volatile, protein-binding or particle-forming substances) can distort optical, fluorometric or luminescent readouts without causing proportional cellular injury.

“Chemical interference is a specific and often under-recognized source of cytotoxicity misclassification. Colored, fluorescent, reducing, oxidizing, precipitating, volatile, protein-binding or particle-forming substances may distort optical, fluorometric or luminescent readouts without causing proportional cellular injury.”

In mutagenicity testing, excessive cytotoxicity or cytostasis may cause misleading positives through oxidative stress, nuclease activation, impaired DNA repair, disturbed cell-cycle progression, chromosomal mis-segregation or selective survival of damaged cells.

“In mutagenicity testing, excessive cytotoxicity or cytostasis may cause misleading positives through oxidative stress, nuclease activation, impaired DNA repair, disturbed cell-cycle progression, chromosomal mis-segregation or selective survival of damaged cells (OECD, 2012,2016,2023; Fowler et al., 2012; Azqueta et al., 2022).”

• cites OECD, 2012Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites Fowler et al., 2012 sourceNot checked — no full text of the source
• cites Azqueta et al., 2022 sourceNot checked — no full text of the source

Cytotoxicity readouts are not interchangeable; a membrane-integrity assay, a metabolic assay and a proliferation endpoint may respond differently to the same substance even when expressed on a common percentage scale.

“This requirement recognizes that cytotoxicity readouts are not interchangeable. A membrane-integrity assay, a metabolic assay and a proliferation endpoint may respond differently to the same substance, even when expressed on a common percentage scale.”

Metabolic impairment can reduce reporter gene expression and cause false-negative results.

“For example, metabolic impairment can reduce reporter gene expression and cause false-negative results.”

Variability is an operational determinant of interpretation.

“Consequently, variability is not merely a technical characteristic but an operational determinant of interpretation, requiring uncertainty-aware handling of results near threshold values and, where available, predefined borderline ranges.”

• AssumptionRests on an assumption: Variability in cytotoxicity and cytostasis endpoints can be driven by methodological and biological factors including cell system characteristics, experimental conditions, technical execution, and endpoint-specific properties and assay interference.

Cytotoxicity measurements in OECD in vitro methods are not generic indicators of toxicity but acquire regulatory meaning only through their defined role within a given method.

“Cytotoxicity measurements in OECD in vitro methods are not generic indicators of toxicity but acquire regulatory meaning only through their defined role within a given method.”

The cytotoxicity burst is a boundary of assay interpretability.

“The cytotoxicity burst should therefore be regarded as a boundary of assay interpretability.”

Toxicokinetic information can improve concentration selection and interpretation.

“Toxicokinetic information can improve concentration selection and interpretation (Landsiedel et al., 2024).”

• cites Landsiedel et al., 2024 sourceNot checked — no full text of the source

Reported cytotoxicity or cytostasis values should not be interpreted as interchangeable across methods as they may lead to different regulatory conclusions, depending on assay design, endpoint biology, and decision criteria.

“Reported cytotoxicity or cytostasis values should not be interpreted as interchangeable across methods as they may lead to different regulatory conclusions, depending on assay design, endpoint biology, and decision criteria.”

Cytotoxicity observed at high nominal concentrations may have limited relevance if those concentrations are not kinetically plausible.

“Consequently, cytotoxicity observed at high nominal concentrations may have limited relevance if those concentrations are not kinetically plausible.”

• AssumptionRests on an assumption: Solubility, volatility, adsorption to plastic, binding to serum proteins, degradation, metabolism and accumulation can all alter effective exposure.

Incorporating measured concentrations can reveal instability, poor bioavailability, loss to the test system or unexpected accumulation.

“Incorporating measured concentrations can reveal instability, poor bioavailability, loss to the test system or unexpected accumulation.”

As a result, fixed cytotoxicity thresholds are applied as operational decision criteria without consistent characterization of the variability of the underlying biological measurements.

“As a result, fixed cytotoxicity thresholds are applied as operational decision criteria without consistent characterization of the variability of the underlying biological measurements.”

Depending on context, cytotoxicity measurements may serve as classification-driving endpoints, predictive surrogates, validity criteria, or constraints for concentration selection.

“Depending on context, they may serve as classification-driving endpoints, predictive surrogates, validity criteria, or constraints for concentration selection.”