Student or educator? Sign up with your school email and get 6 months of Pro free.
CoChat
Sign Up
Cited by 1

The DOI matches its Crossref record; 28 of the 28 references it cites were found in a registry or an index.

Development of a tool for measuring uncertainty of acute cytotoxicity results using the methodology of OECD Guidance Document 129

Neves, Lorena de O., Garrido, Bruno C., Granjeiro, José Mauro, Balottin, Luciene B.L.·2025·NAM Journal
DOI 10.1016/j.namjnl.2025.100021PMC PMC13396913
Open this paper in CoChat — free account

Abstract

Despite the use of strict acceptance criteria in validated methods for in vitro toxicology safety non-animal methods, the evaluation of statistical uncertainties in the results and their impact on the reproducibility and reliability of these methods is limited. Technical and operational variations in intra- and inter-laboratory settings can interfere with the results, and measurement uncertainty estimations are crucial for a better understanding of the potential sources of errors and ensuring that the assays are under control. The measurement uncertainty characterizes the dispersion of values, which can be attributed to the measurement results. This study applied the Monte Carlo statistical model to calculate the measurement uncertainty for the half-maximal inhibitory concentration (IC50) of sodium dodecyl sulfate (SDS) using OECD Guidance Document 129. We focused on acute oral cytotoxicity in vitro and developed a specific tool for this purpose. The Monte Carlo method enabled the identification of the contributions to the measurement uncertainty of IC50 and their confidence intervals for the in vitro cytotoxicity assay recommended by OECD GD 129. Furthermore, the statistical tool developed in this study, although specifically applied to OECD GD 129, has the potential to be adapted and extended to other in vitro toxicological assays based on dose-response curves, thus supporting broader applications in New Approach Methodologies (NAMs) by enhancing reproducibility and confidence in alternative methods validation processes. In conclusion, this study demonstrates the effectiveness of the Monte Carlo statistical model in quantifying the measurement uncertainty of sodium dodecyl sulfate (SDS) IC50 values, with a comprehensive analysis yielding a combined IC50 of 34.6 μg/mL with a combined uncertainty of 1.21 μg/mL. These findings highlight the importance of incorporating metrological evaluations to increase the reliability of in vitro methods. By enabling more informed decision-making, this study can significantly contribute to the advancement of toxicological research and NAMs.

Overview

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.1016/j.namjnl.2025.100021

Cites

28 of 28 references verified0 citation errors · 0 not checkable

Statistics

Figures

Fig 1 Micrographs of the wells representing the Vehicle Control. BALB/c 3T3 clone A31 incubated at 37 °C ± 1 °C, 90 % ± 10 % humidity, 5.0 % ± 1.0 % CO2/air. A – After plating 2.5 x 103 cells in 100 μL. B – After incubation of 24 h ± 2 h. C – Left Vehicle Control (VC1) after exposure for 46 h. D – Right Vehicle Control (VC2) after exposure for 46 h.
Fig 1 Micrographs of the wells representing the Vehicle Control. BALB/c 3T3 clone A31 incubated at 37 °C ± 1 °C, 90 % ± 10 % humidity, 5.0 % ± 1.0 % CO2/air. A – After plating 2.5 x 103 cells in 100 μL. B – After incubation of 24 h ± 2 h. C – Left Vehicle Control (VC1) after exposure for 46 h. D – Right Vehicle Control (VC2) after exposure for 46 h.CC BY · PubMed Central
Fig 2 Historical series in NRU 3T3 assays. The historical series comprises the results obtained in the laboratory, seven retrospective results, and the results of the present study. Mean absorbance values of negative controls (cells on vehicle) across multiple experimental replicates (A). This control chart monitors the stability of the negative control responses, ensuring consistency across assays. The data represent the average absorbance values with standard deviations, subsequent to blank correction. IC50 values for sodium dodecyl sulfate (SDS) obtained from multiple independent rounds of the NRU 3T3 assay (B). IC50 equivalent to (1) 36.02 µg/mL; (2) 34.51 µg/mL; (3) 35.95 µg/mL; (4) 34.39 µg/mL; (5) 31.16 µg/mL; (6) 28.58 µg/mL; (7) 38.61 µg/mL; (8) 25.96 µg/mL; (9) 33.32 µg/mL; (10) 38.58 µg/mL. Limits: mean of 33.71 µg/mL; Standard deviation (SD) of 4.49 µg/mL and 2.5 SD with a value of 11.23 µg/mL.
Fig 2 Historical series in NRU 3T3 assays. The historical series comprises the results obtained in the laboratory, seven retrospective results, and the results of the present study. Mean absorbance values of negative controls (cells on vehicle) across multiple experimental replicates (A). This control chart monitors the stability of the negative control responses, ensuring consistency across assays. The data represent the average absorbance values with standard deviations, subsequent to blank correction. IC50 values for sodium dodecyl sulfate (SDS) obtained from multiple independent rounds of the NRU 3T3 assay (B). IC50 equivalent to (1) 36.02 µg/mL; (2) 34.51 µg/mL; (3) 35.95 µg/mL; (4) 34.39 µg/mL; (5) 31.16 µg/mL; (6) 28.58 µg/mL; (7) 38.61 µg/mL; (8) 25.96 µg/mL; (9) 33.32 µg/mL; (10) 38.58 µg/mL. Limits: mean of 33.71 µg/mL; Standard deviation (SD) of 4.49 µg/mL and 2.5 SD with a value of 11.23 µg/mL.CC BY · PubMed Central
Fig 3 Cause-and-effect Ishikawa diagram in the Neutral Red Uptake (NRU) test of OECD GD 129. The sources of uncertainty linked to the test were identified to estimate the measurement uncertainty associated with the traditional protocol.
Fig 3 Cause-and-effect Ishikawa diagram in the Neutral Red Uptake (NRU) test of OECD GD 129. The sources of uncertainty linked to the test were identified to estimate the measurement uncertainty associated with the traditional protocol.CC BY · PubMed Central
Fig 4 Contribution of the standard uncertainties of the volume measured using the Multichannel Micropipette and Graduated Pipette. These are standard uncertainty values ​​on the x-axis. To Multichannel Micropipette the expanded air density uncertainty and standard deviation of the measurements are the significant contributors. To Graduated Pipette the expanded air density uncertainty and standard deviation of measurements are the significant contributors.
Fig 4 Contribution of the standard uncertainties of the volume measured using the Multichannel Micropipette and Graduated Pipette. These are standard uncertainty values ​​on the x-axis. To Multichannel Micropipette the expanded air density uncertainty and standard deviation of the measurements are the significant contributors. To Graduated Pipette the expanded air density uncertainty and standard deviation of measurements are the significant contributors.CC BY · PubMed Central
Fig 5 Graphical results of the cytotoxicity analyses. Dose-response curve for sodium dodecyl sulfate (SDS) in the NRU 3T3 assay. The dots show the mean relative viability ( %) of the six replicates produced at each of the eight chemical concentrations.
Fig 5 Graphical results of the cytotoxicity analyses. Dose-response curve for sodium dodecyl sulfate (SDS) in the NRU 3T3 assay. The dots show the mean relative viability ( %) of the six replicates produced at each of the eight chemical concentrations.CC BY · PubMed Central
Fig 6 Unilateral degrees of equivalence on Design of Experiments (DoE) based on Bayesian procedures. The purple points indicate the Bayesian posterior means for each evaluated sample, and the blue error bars reflect the corresponding uncertainty (95 % credible intervals). These intervals represent the range within which the true value of the degree of equivalence is expected to fall with 95 % probability. Any points significantly deviating from zero suggest a systematic bias, while values close to zero indicate agreement with the consensus value within the uncertainty limits.
Fig 6 Unilateral degrees of equivalence on Design of Experiments (DoE) based on Bayesian procedures. The purple points indicate the Bayesian posterior means for each evaluated sample, and the blue error bars reflect the corresponding uncertainty (95 % credible intervals). These intervals represent the range within which the true value of the degree of equivalence is expected to fall with 95 % probability. Any points significantly deviating from zero suggest a systematic bias, while values close to zero indicate agreement with the consensus value within the uncertainty limits.CC BY · PubMed Central

Verification

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Citations

Cites

Show all 28 references

Research works printed but not linked: 6

  1. 2010, Directive 2010/63 on the protection of animals used for scientific purposes, Official J. European Union, 47
  2. 2006, ICCVAM-recommended test method protocol BALB/c 3T3 NRU cytotoxicity test method, ICCVAM Test Method Eval Rep Vitr Cytotox Test Methods Estim Start Doses Acute Oral Syst Toxic Tests
  3. 2012, International vocabulary of metrology – Basic and general concepts and associated terms (VIM), Tetrahedron Lett.
  4. JCGM/WG. Evaluation of measurement data. Vol. 50, GUM - guide to the expression of uncertainty in measurement. 2008.
  5. 2014, Series on Testing and Assessment No. 129. GUIDANCE DOCUMENT ON USING CYTOTOXICITY TESTS TO ESTIMATE STARTING DOSES FOR ACUTE ORAL SYSTEMIC TOXICITY TESTS, ENV/JM/MONO(2010), 20, 54
  6. Valadares, 2006, Avaliação de toxicidade Aguda: estratégias após a “era do Teste dl 50, Rev Eletrônica Farmácia, 3, 93

Cited by

See all 1

Metadata

Authors
Neves, L. D. O., Garrido, B. C., Granjeiro, J. M., & Balottin, L. B. L.
ORCID
Venue
NAM Journal
Publisher
Elsevier BV
Published
2025
Volume
1
Pages
100021
ISSN
3050-6204
DOI
10.1016/j.namjnl.2025.100021
PMC
PMC13396913
Cited by
1
References
28
Language
en
Licence
Funders
  • Coordination of Higher Education Personnel Improvement
  • National Council for Scientific and Technological Development (465656/2014-5, 400030/2018-7)
  • Instituto Nacional de Ciência e Tecnologia em Medicina Regenerativa
  • Ministry of Science Technology and Innovations (404149/2020-0)
  • Carlos Chagas Filho Foundation for Research Support of Rio de Janeiro State (E-26/10.000981/2019)

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review

How to cite

Cite CoChat for the checks on this page, and the paper for its own findings.

This page
CoChat. (2026). Citation check: Development of a tool for measuring uncertainty of acute cytotoxicity results using the methodology of OECD Guidance Document 129. https://app.cochat.ai/papers/10.1016/j.namjnl.2025.100021
The paper
Neves, L. D. O., Garrido, B. C., Granjeiro, J. M., & Balottin, L. B. L. (2025). Development of a tool for measuring uncertainty of acute cytotoxicity results using the methodology of OECD Guidance Document 129. NAM Journal. https://doi.org/10.1016/j.namjnl.2025.100021

Data sources

Bibliographic data: Semantic Scholar, OpenAlex, Crossref.

Contains information from the Semantic Scholar Academic Graph Datasets, made available under the ODC Attribution License (ODC-BY 1.0).

Semantic Scholar’s data is described in: Kinney, R., et al. (2023). The Semantic Scholar Open Data Platform. arXiv:2301.10140.