Characteristics to consider when selecting a positive control material for an in vitro assay

Main Article Content

Elijah J. Petersen , Andrew Nguyen, Jeffrey Brown, John T. Elliott, Amy J. Clippinger, John Gordon, Nicole Kleinstreuer, Matthias Roesslein
[show affiliations]


The use of in vitro assays to inform decision-making requires robust and reproducible results across studies, laboratories, and time. Experiments using positive control materials are an integral component of an assay procedure to demonstrate the extent to which the measurement system is performing as expected. This paper reviews ten characteristics that should be considered when selecting a positive control material for an in vitro assay: 1) the biological mechanism of action, 2) ease of preparation, 3) chemical purity, 4) verifiable physical properties, 5) stability, 6) ability to generate responses spanning the dynamic range of the assay, 7) technical or biological interference, 8) commercial availability, 9) user toxicity, and 10) disposability. Examples and a case study of the monocyte activation test are provided to demonstrate the application of these characteristics for identification and selection of potential positive control materials. Because specific positive control materials are often written into testing standards for in vitro assays, selection of the positive control material based on these characteristics can aid in ensuring the long-term relevance and usability of these standards.

Article Details

How to Cite
Petersen, E. J. (2021) “Characteristics to consider when selecting a positive control material for an in vitro assay”, ALTEX - Alternatives to animal experimentation, 38(2), pp. 365–376. doi: 10.14573/altex.2102111.

Alexopoulou, L., Holt, A. C., Medzhitov, R. et al. (2001). Recognition of double-stranded RNA and activation of NF-κB by toll-like receptor 3. Nature 413, 732-738. doi:10.1038/35099560

Aliprantis, A. O., Yang, R. B., Mark, M. R. et al. (1999). Cell activation and apoptosis by bacterial lipoproteins through toll-like receptor-2. Science 285, 736-739. doi:10.1126/science.285.5428.736

Avila, A. M., Bebenek, I., Bonzo, J. A. et al. (2020). An FDA/CDER perspective on nonclinical testing strategies: Classical toxicology approaches and new approach methodologies (NAMs). Regul Toxicol Pharmacol 114, 104662. doi:10.1016/j.yrtph.2020.104662

Barosova, H., Maione, A. G., Septiadi, D. et al. (2020). Use of EpiAlveolar lung model to predict fibrotic potential of multiwalled carbon nanotubes. ACS Nano 14, 3941-3956. doi:10.1021/acsnano.9b06860

Basketter, D., Azam, P., Casati, S. et al. (2019). Applying non-animal strategies for assessing skin sensitisation report from an EPAA/cefic-LRI/IFRA Europe cross sector workshop, ECHA, February 7th and 8th 2019. Regul Toxicol Pharmacol 109, 104477. doi:10.1016/j.yrtph.2019.104477

Borrel, A., Huang, R., Sakamuru, S. et al. (2020a). High-throughput screening to predict chemical-assay interference. Sci Rep 10, 3986. doi:10.1038/s41598-020-60747-3

Borrel, A., Mansouri, K., Nolte, S. et al. (2020b). InterPred: A webtool to predict chemical autofluorescence and luminescence interference. Nucleic Acids Res 48, W586-W590. doi:10.1093/nar/gkaa378

Borton, L. K. and Coleman, K. P. (2018). Material-mediated pyrogens in medical devices: Applicability of the in vitro monocyte activation test. ALTEX 35, 453-463. doi:10.14573/altex.1709221

Brown, J., Clippinger, A. J., Fritz Briglia, C. et al. (2021). Using the monocyte activation test as a stand-alone release test for medical devices. ALTEX 38, 151-156. doi:10.14573/altex.2012021

Browne, P., Judson, R. S., Casey, W. M. et al. (2015). Screening chemicals for estrogen receptor bioactivity using a computational model. Environ Sci Technol 49, 8804-8814. doi:10.1021/acs.est.5b02641

Chipinda, I., Ajibola, R. O., Morakinyo, M. K. et al. (2010). Rapid and simple kinetics screening assay for electrophilic dermal sensitizers using nitrobenzenethiol. Chem Res Toxicol 23, 918-925. doi:10.1021/tx100003w

Coleman, K. P., Grailer, T. P., McNamara, L. R. et al. (2018). Preparation of irritant polymer samples for an in vitro round robin study. Toxicol In Vitro 50, 401-406. doi:10.1016/j.tiv.2018.01.018

Dahlin, J. L., Nelson, K. M., Strasser, J. M. et al. (2017). Assay interference and off-target liabilities of reported histone acetyltransferase inhibitors. Nat Commun 8, 1527. doi:10.1038/s41467-017-01657-3

de Oliviera Nascimento, L., Massari, P. and Wetzler, L. (2012). The role of TLR2 in infection and immunity. Front Immunol 3, 79. doi:10.3389/fimmu.2012.00079

Deininger, S., Stadelmaier, A., von Aulock, S. et al. (2003). Definition of structural prerequisites for lipoteichoic acid-inducible cytokine induction by synthetic derivatives. J Immunol 170, 4134-4138. doi:10.4049/jimmunol.170.8.4134

Deininger, S., Figueroa-Perez, I., Sigel, S. et al. (2007). Use of synthetic derivatives to determine the minimal active structure of cytokine-inducing lipoteichoic acid. Clin Vaccine Immunol 14, 1629-1633. doi:10.1128/CVI.00007-07

EC – European Commission (2020). 2019 Report on the Statistics on the Use of Animals for Scientific Purposes in the Member States of the European Union in 2015-2017.

EDQM – European Directorate for the Quality of Medicines and HealthCare (2020). Monocyte Activation Test Chapter 2.6.30 European Pharmacopoeia (Version 07/2017), 10th edition.

Elliott, J. T., Rosslein, M., Song, N. W. et al. (2017). Toward achieving harmonization in a nanocytotoxicity assay measurement through an interlaboratory comparison study. ALTEX 34, 201-218. doi:10.14573/altex.1605021

EPA – Environmental Protection Agency (2014). SW-846 Test Method 4435: Screening for Dioxin-Like Chemical Activity in Soils and Sediments Using the Calux Bioassay and Toxic Equivalents (TEQs) Determinations.

Figueiredo, R. T., Carneiro, L. A. M. and Bozza, M. T. (2011). Fungal surface and innate immune recognition of filamentous fungi. Front Microbiol 2, 248. doi:10.3389/fmicb.2011.00248

Groff, K., Allen, D., Casey, W. et al. (2020). Increasing the use of animal-free recombinant antibodies. ALTEX 37, 309-311. doi:10.14573/altex.2001071

Guadagnini, R., Kenzaoui, B. H., Walker, L. et al. (2015). Toxicity screenings of nanomaterials: Challenges due to interference with assay processes and components of classic in vitro tests. Nanotoxicology 9, 13-24. doi:10.3109/17435390.2013.829590

Hanna, S. K., Montoro Bustos, A. R., Peterson, A. W. et al. (2018). Agglomeration of Escherichia coli with positively charged nanoparticles can lead to artifacts in a standard Caenorhabditis elegans toxicity assay. Environ Sci Technol 52, 5968-5978. doi:10.1021/acs.est.7b06099

Hartmann, G. and Krieg, A. M. (1999). CpG DNA and LPS induce distinct patterns of activation in human monocytes. Gene Ther 6, 893-903. doi:10.1038/

Hartung, T., Balls, M., Bardouille, C. et al. (2002). Good cell culture practice. ECVAM good cell culture practice task force report 1. Altern Lab Anim 30, 407-414. doi:10.1177/026119290203000404

Hartung, T. (2015). The human whole blood pyrogen test – Lessons learned in twenty years. ALTEX 32, 79-100. doi:10.14573/altex.1503241

Hartung, T. (2021). Pyrogen testing revisited on occasion of the 25th anniversary of the whole blood monocyte activation test. ALTEX 38, 3-19. doi:10.14573/altex.2101051

Hasiwa, N., Daneshian, M., Bruegger, P. et al. (2013). Evidence for the detection of non-endotoxin pyrogens by the whole blood monocyte activation test. ALTEX 30, 169-208. doi:10.14573/altex.2013.2.169

Hayashi, F., Smith, K. D., Ozinsky, A. et al. (2001). The innate immune response to bacterial flagellin is mediated by toll-like receptor 5. Nature 410, 1099-1103. doi:10.1038/35074106

Holland-Letz, T. and Kopp-Schneider, A. (2015). Optimal experimental designs for dose-response studies with continuous endpoints. Arch Toxicol 89, 2059-2068. doi:10.1007/s00204-014-1335-2

Holland-Letz, T. (2017). On the combination of c- and D-optimal designs: General approaches and applications in dose-response studies. Biometrics 73, 206-213. doi:10.1111/biom.12545

Huth, J. R., Mendoza, R., Olejniczak, E. T. et al. (2005). ALARM NMR: A rapid and robust experimental method to detect reactive false positives in biochemical screens. J Am Chem Soc 127, 217-224. doi:10.1021/ja0455547

ICCVAM – Interagency Coordinating Committee on the Validation of Alternative Methods (2008). ICCVAM Test Method Evaluation Report: Validation Status of Five In Vitro Test Methods Proposed for Assessing Potential Pyrogenicity of Pharmaceuticals and Other Products.

ISO– International Organization for Standardization (2010). 10993-10:2010: Biological Evaluation of Medical Devices – Part 10: Tests for Irritation and Skin Sensitization. Geneva, Switzerland: International Organization for Standardization.

ISO (2012). 10993-12:2012: Biological Evaluation of Medical Devices – Part 12: Sample Preparation and Reference Materials. Geneva, Switzerland: International Organization for Standardization.

ISO (2015). Guide 30: 2015: Reference Materials – Selected Terms and Definitions. Geneva, Switzerland: International Organization for Standardization.

Keene, A. M., Bancos, S. and Tyner, K. M. (2014). Considerations for in vitro nanotoxicity testing. In S. C. Sahu and D. A. Casciano (eds.), Handbook of Nanotoxicology, Nanomedicine and Stem Cell Use in Toxicology. Chapter 2. John Wiley and Sons. doi:10.1002/9781118856017.ch2

Kleinstreuer, N. C., Ceger, P., Watt, E. D. et al. (2017). Development and validation of a computational model for androgen receptor activity. Chem Res Toxicol 30, 946-964. doi:10.1021/acs.chemrestox.6b00347

Kleinstreuer, N. C., Karmaus, A., Mansouri, K. et al. (2018). Predictive models for acute oral systemic toxicity: A workshop to bridge the gap from research to regulation. Comput Toxicol 8, 21-24. doi:10.1016/j.comtox.2018.08.002

Kolle, S. N., Hil, E., Raabe, H. et al. (2019). Regarding the references for reference chemicals of alternative methods. Toxicol In Vitro 57, 48-53. doi:10.1016/j.tiv.2019.02.007

Leibrock, L., Jungnickel, H., Tentschert, J. et al. (2020). Parametric optimization of an air-liquid interface system for flow-through inhalation exposure to nanoparticles: Assessing dosimetry and intracellular uptake of CeO2 nanoparticles. Nanomaterials 10, 2369. doi:10.3390/nano10122369

Molenaar-de Backer, M. W. A., Gitz, E., Dieker, M. et al. (2021). Performance of monocyte activation test supplemented with human serum compared to fetal bovine serum. ALTEX 38, 307-315. doi:10.14573/altex.2008261

Morath, S., Stadelmaier, A., Geyer, A. et al. (2002). Synthetic lipoteichoic acid from Staphylococcus aureus is a potent stimulus of cytokine release. J Exp Med 195, 1635-1640. doi:10.1084/jem.20020322

Morath, S., von Aulock, S. and Hartung, T. (2005). Structure/function relationships of lipoteichoic acids. J Endotoxin Res 11, 348-356. doi:10.1179/096805105X67328

Nelson, B. C., Petersen, E. J., Marquis, B. J. et al. (2013). NIST gold nanoparticle reference materials do not induce oxidative DNA damage. Nanotoxicology 7, 21-29. doi:10.3109/17435390.2011.626537

Netea, M. G., Kullberg, B. J. and Van der Meer, J. W. M. (2000). Circulating cytokines as mediators of fever. Clin Infect Dis 31, Suppl 5, S178-S184. doi:10.1086/317513

Nomura, Y., Lee, M., Fukui, C. et al. (2018). Proof of concept testing of a positive reference material for in vivo and in vitro skin irritation testing. J Biomed Mater Res B Appl Biomater 106, 2807-2814. doi:10.1002/jbm.b.34061

OECD – Organization for Economic Cooperation and Development (2005). Guidance Document on the Validation and International Acceptance of New or Updated Test Methods for Hazard Assessment. Series on Testing and Assessment, No. 34. OECD Publishing, Paris.

OECD (2018). Guidance Document on Good In Vitro Method Practices (GIVIMP). OECD Series on Testing and Assessment, No. 286. OECD Publishing, Paris. doi:10.1787/9789264304796-en

OECD (2019). Test No. 442C: In Chemico Skin Sensitisation: Assays addressing the Adverse Outcome Pathway key event on covalent binding to proteins. OECD Guidelines for the Testing of Chemicals, Section 4. OECD Publishing, Paris. doi:10.1787/9789264229709-en

Park, B. S. and Lee, J. O. (2013). Recognition of lipopolysaccharide pattern by TLR4 complexes. Exp Mol Med 45, e66. doi:10.1038/emm.2013.97

Petersen, E. J., Henry, T. B., Zhao, J. et al. (2014). Identification and avoidance of potential artifacts and misinterpretations in nanomaterial ecotoxicity measurements. Environ Sci Technol 48, 4226-4246. doi:10.1021/es4052999

Petersen, E. J., Hirsch, C., Elliott, J. T. et al. (2020). Cause-and-effect analysis as a tool to improve the reproducibility of nanobioassays: Four case studies. Chem Res Toxicol 33, 1039-1054. doi:10.1021/acs.chemrestox.9b00165

Plant, A. L., Locascio, L. E., May, W. E. et al. (2014). Improved reproducibility by assuring confidence in measurements in biomedical research. Nat Methods 11, 895-898. doi:10.1038/nmeth.3076

Poole, S., Desai, T., Findlay, L. et al. (2012). WHO international standard for endotoxin: Report of an international collaborative study to evaluate three preparations of endotoxin for their suitability to serve as the third international standard for bacterial endotoxin. World Health Organization, 1-40.

Roesslein, M., Hirsch, C., Kaiser, J. P. et al. (2013). Comparability of in vitro tests for bioactive nanoparticles: A common assay to detect reactive oxygen species as an example. Int J Mol Sci 14, 24320-24337. doi:10.3390/ijms141224320

Rösslein, M., Elliott, J. T., Salit, M. et al. (2015). Use of cause-and-effect analysis to design a high-quality nanocytotoxicology assay. Chem Res Toxicol 28, 21-30. doi:10.1021/tx500327y

Rudbach, J. A., Akiya, F. I., Elin, R. J. et al. (1976). Preparation and properties of a national reference endotoxin. J Clin Microbiol 3, 21-25.

Schwandner, R., Dziarski, R., Wesche, H. et al. (1999). Peptidoglycan- and lipoteichoic acid-induced cell activation is mediated by toll-like receptor 2. J Biol Chem 274, 17406-17409. doi:10.1074/jbc.274.25.17406

Stadelmaier, A., Morath, S., Hartung, T. et al. (2003). Synthesis of the first fully active lipoteichoic acid. Angew Chem Int Edit 42, 916-920. doi:10.1002/anie.200390243

Urbisch, D., Mehling, A., Guth, K. et al. (2015). Assessing skin sensitization hazard in mice and men using non-animal test methods. Regul Toxicol Pharmacol 71, 337-351. doi:10.1016/j.yrtph.2014.12.008

United States (1973). Federal Hazardous Substances Act. Title 16. Code of Federal Regulations.

Part 1500: United States Code.

US FDA – United States Food and Drug Administration (2012). Guidance for Industry. Pyrogen and Endotoxins Testing: Questions and Answers.

US FDA (2016). Use of International Standard ISO 10993-1, “Biological evaluation of medical devices – Part 1: Evaluation and testing within a risk management process”. Department of Health and Human Services Food and Drug Administration.

US FDA (2018). Bioanalytical Method Validation Guidance for Industry.

US Pharmacopeia (2012). Bacterial Endotoxins Test – Chapter 85. United States Pharmacopeia 37, 1-5.

Most read articles by the same author(s)

1 2 > >>