Beyond chemicals: Opportunities and challenges of integrating non-chemical stressors in adverse outcome pathways

Main Article Content

Laure-Alix Clerbaux , Julija Filipovska, Penny Nymark, Vinita Chauhan, Katherina Sewald, Miriam Alb, Madgalini Sachana, Anna Beronius, Maria-Joao Amorim, Clemens Wittwehr
[show affiliations]


Adverse outcome pathways (AOPs) were developed to accelerate evidence-based chemical risk assessment by leveraging data from new approach methodologies. Thanks to their stressor-agnostic approach, AOPs were seen as instrumental in other fields. Here, we present AOPs that report non-chemical stressors along with the challenges encountered for their development. Challenges regarding AOPs linked to nanomaterials include non-specific molecular initiating events, limited understanding of nanomaterial biodistribution, and needs for adaptations of in silico modeling and testing systems. Development of AOPs for radiation faces challenges in how to incorporate ionizing events type, dose rate, energy deposition, and how to account for targeting multiple macromolecules. AOPs for COVID-19 required the inclusion of SARS-CoV-2-specific replicative steps to capture the essential events driving the disease. Developing AOPs to evaluate efficacy and toxicity of cell therapies necessitates addressing the cellular nature and the therapeutic function of the stressor. Finally, addressing toxicity of emerging biological stressors like microbial pesticides can learn from COVID-19 AOPs. We further discuss that the adaptations needed to expand AOP appli­cability beyond chemicals are mainly at the molecular and cellular levels, while downstream key events at tissue or organ level, such as inflammation, are shared by many AOPs initiated by various stressors. In conclusion, although it is challenging to integrate non-chemical stressors within AOPs, this expands opportunities to account for real-world scenarios, to identify vulnerable individuals, and to bridge knowledge on mechanisms of adversity.

Plain language summary
The adverse outcome pathway (AOP) framework was developed to help predict whether chemicals have toxic effects on humans. Structuring available information in an accessible database can reduce animal testing. AOPs usually capture the path from the interaction of a stressor, usually a chemical, with the human body to an adverse outcome, e.g., a disease symptom. The concept of AOPs has now been expanded to include non-chemical stressors such as nanomaterials, radiation, viruses, cells used to treat patients, and microorganisms employed as pesticides. We discuss how these stressors need to be accommodated within the framework and point out that pathways initiated by these stressors share downstream events like inflammation with chemical stressors. By integrating non-chemical stressors into the framework, real-world scenarios where people may be exposed to different stressor types can be considered, vulnerable individuals can be identified, and knowledge on toxic effects can be compounded.

Article Details

How to Cite
Clerbaux, L.-A. (2024) “Beyond chemicals: Opportunities and challenges of integrating non-chemical stressors in adverse outcome pathways”, ALTEX - Alternatives to animal experimentation, 41(2), pp. 233–247. doi: 10.14573/altex.2307061.

Abraham, K., Mielke, H., Fromme, H. et al. (2020). Internal exposure to perfluoroalkyl substances (PFASs) and biological markers in 101 healthy 1-year-old children: Associations between levels of perfluorooctanoic acid (PFOA) and vaccine response. Arch Toxicol 94, 2131-2147. doi:10.1007/s00204-020-02715-4

Andersen, M. E. and Krewski, D. (2010). The vision of toxicity testing in the 21st century: Moving from discussion to action. Toxicol Sci 117, 17-24. doi:10.1093/toxsci/kfq188

Ankley, G. T., Bencic, D. C., Breen, M. S. et al. (2009). Endocrine disrupting chemicals in fish: Developing exposure indicators and predictive models of effects based on mechanism of action. Aquat Toxicol 92, 168-178. doi:10.1016/j.aquatox.2009.01.013

Ankley, G. T., Bennett, R. S., Erickson, R. J. et al. (2010). Adverse outcome pathways: A conceptual framework to support ecotoxicology research and risk assessment. Environ Toxicol Chem 29, 730-741. doi:10.1002/etc.34

Azimzadeh, O., Moertl, S., Ramadan, R. et al. (2022). Application of radiation omics in the development of adverse outcome pathway networks: An example of radiation-induced cardiovascular disease. Int J Radiat Biol 98, 1722-1751. doi:10.1080/09553002.2022.2110325

Beronius, A., Zilliacus, J., Hanberg, A. et al. (2020). Methodology for health risk assessment of combined exposures to multiple chemicals. Food Chem Toxicol 143, 111520. doi:10.1016/j.fct.2020.111520

Boobis, A. R., Cohen, S. M., Dellarco, V. et al. (2006). IPCS framework for analyzing the relevance of a cancer mode of action for humans. Crit Rev Toxicol 36, 781-792. doi:10.1080/10408440600977677

Boobis, A. R., Doe, J. E., Heinrich-Hirsch, B. et al. (2008). IPCS framework for analyzing the relevance of a noncancer mode of action for humans. Crit Rev Toxicol 38, 87-96. doi:10.1080/10408440701749421

Braakhuis, H. M., Gosens, I., Heringa, M. B. et al. (2021). Mechanism of action of TiO2: Recommendations to reduce uncertainties related to carcinogenic potential. Annu Rev Pharmacol Toxicol 61, 203-223. doi:10.1146/annurev-pharmtox-101419-100049

Brosseau, L. M., Escandón, K., Ulrich, A. K. et al. (2022). Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) dose, infection, and disease outcomes for coronavirus disease 2019 (COVID-19): A review. Clin Infect Dis 75, E1195-E1201. doi:10.1093/cid/ciab903

Bulka, C. M., Enggasser, A. E., Fry, R. C. et al. (2022). Epigenetics at the intersection of COVID-19 risk and environmental chemical exposures. Curr Environ Health Rep 9, 477-489. doi:10.1007/s40572-022-00353-9

Burtt, J. J., Leblanc, J., Randhawa, K. et al. (2022). Radiation adverse outcome pathways (AOPs) are on the horizon: Advancing radiation protection through an international horizon-style exercise. Int J Radiat Biol 98, 1763-1776. doi:10.1080/09553002.2022.2121439

Carnesecchi, E., Langezaal, I., Browne, P. et al. (2023). OECD harmonised template 201: Structuring and reporting mechanistic information to foster the integration of new approach methodologies for hazard and risk assessment of chemicals. Regul Toxicol Pharmacol 142, 105426. doi:10.1016/j.yrtph.2023.105426

Carusi, A., Filipovska, J., Wittwehr, C. et al. (2023). CIAO: A living experiment in interdisciplinary large-scale collaboration facilitated by the adverse outcome pathway framework. Front Publ Health 11, 1212544. doi:10.3389/fpubh.2023.1212544

Chatterjee, N. and Walker, G. C. (2017). Mechanisms of DNA damage, repair, and mutagenesis. Environ Mol Mutagen 58, 235-263. doi:10.1002/em.22087

Chauhan, V., Said, Z., Daka, J. et al. (2019). Is there a role for the adverse outcome pathway framework to support radiation protection? Int J Radiat Biol 95, 225-232. doi:10.1080/09553002.2019.1532617

Chauhan, V., Hamada, N., Monceau, V. et al. (2021a). Expert consultation is vital for adverse outcome pathway development: A case example of cardiovascular effects of ionizing radiation. Int J Radiat Biol 97, 1516-1525. doi:10.1080/09553002.2021.1969466

Chauhan, V., Sherman, S., Said, Z. et al. (2021b). A case example of a radiation-relevant adverse outcome pathway to lung cancer. Int J Radiat Biol 97, 68-84. doi:10.1080/09553002.2019.1704913

Chauhan, V., Stricklin, D., Cool, D. et al. (2021c). The integration of the adverse outcome pathway framework to radiation risk assessment. Int J Radiat Biol 97, 60-67. doi:10.1080/09553002.2020.1761570

Chauhan, V., Villeneuve, D., Cool, D. et al. (2021d). Collaborative efforts are needed among the scientific community to advance the adverse outcome pathway concept in areas of radiation risk assessment. Int J Radiat Biol 97, 815-823. doi:10.1080/09553002.2020.1857456

Chauhan, V., Wilkins, R. C., Beaton, D. et al. (2021e). Bringing together scientific disciplines for collaborative undertakings: A vision for advancing the adverse outcome pathway framework. Int J Radiat Biol 97, 431-441. doi:10.1080/09553002.2021.1884314

Chauhan, V., Hamada, N., Garnier-Laplace, J. et al. (2022a). Establishing a communication and engagement strategy to facilitate the adoption of the adverse outcome pathways in radiation research and regulation. Int J Radiat Biol 98, 1714-1721. doi:10.1080/09553002.2022.2086716

Chauhan, V., Hamada, N., Wilkins, R. et al. (2022b). A high-level overview of the OECD AOP development programme. Int J Radiat Biol 98, 1704-1713. doi:10.1080/09553002.2022.2110311

Clerbaux, L.-A. (2022). COVID-19 through adverse outcome pathways: Building networks to better understand the disease – 3rd CIAO AOP design workshop. ALTEX 39, 322-335. doi:10.14573/altex.2112161

Clerbaux, L. A., Albertini, M. C., Amigó, N. et al. (2022a). Factors modulating COVID-19: A mechanistic understanding based on the adverse outcome pathway framework. J Clin Med 11, 4464. doi:10.3390/jcm11154464

Clerbaux, L. A., Fillipovska, J., Muñoz, A. et al. (2022b). Mechanisms leading to gut dysbiosis in COVID-19: Current evidence and uncertainties based on adverse outcome pathways. J Clin Med 11, 5400. doi:10.3390/jcm11185400

Clerbaux, L. A., Mayasich, S. A., Muñoz, A. et al. (2022c). Gut as an alternative entry route for SARS-CoV-2: Current evidence and uncertainties of productive enteric infection in COVID-19. J Clin Med 11, 5691. doi:10.3390/jcm11195691

del Giudice, G., Serra, A., Saarimäki, L. A. et al. (2023). An ancestral molecular response to nanomaterial particulates. Nat Nanotechnol 18, 957-966. doi:10.1038/s41565-023-01393-4

Diamond, M. S. and Kanneganti, T. D. (2022). Innate immunity: The first line of defense against SARS-CoV-2. Nat Immunol 23, 165-176. doi:10.1038/s41590-021-01091-0

Ding, X., Pu, Y., Tang, M. et al. (2023). Pulmonary hazard identifications of graphene family nanomaterials: Adverse outcome pathways framework based on toxicity mechanisms. Sci Total Environ 857, 159329. doi:10.1016/j.scitotenv.2022.159329

Drasler, B., Sayre, P., Steinhäuser, K. G. et al. (2017). In vitro approaches to assess the hazard of nanomaterials. NanoImpact 8, 99-116. doi:10.1016/j.impact.2017.08.002

Fadeel, B. (2022). Nanomaterial characterization: Understanding nano-bio interactions. Biochem Biophys Res Commun 633, 45-51. doi:10.1016/j.bbrc.2022.08.095

Garcia-Reyero, N. (2015). Are adverse outcome pathways here to stay? Environ Sci Technol 49, 3-9. doi:10.1021/es504976d

Gerloff, K., Landesmann, B., Worth, A. et al. (2017). The adverse outcome pathway approach in nanotoxicology. Comput Toxicol 1, 3-11. doi:10.1016/j.comtox.2016.07.001

Halappanavar, S., Van Den Brule, S., Nymark, P. et al. (2020). Adverse outcome pathways as a tool for the design of testing strategies to support the safety assessment of emerging advanced materials at the nanoscale. Part Fibre Toxicol 17, 16. doi:10.1186/s12989-020-00344-4

Halappanavar, S., Ede, J. D., Mahapatra, I. et al. (2021a). A methodology for developing key events to advance nanomaterial-relevant adverse outcome pathways to inform risk assessment. Nanotoxicology 15, 289-310. doi:10.1080/17435390.2020.1851419

Halappanavar, S., Nymark, P., Krug, H. F. et al. (2021b). Non-animal strategies for toxicity assessment of nanoscale materials: Role of adverse outcome pathways in the selection of endpoints. Small 17, 2007628. doi:10.1002/smll.202007628

Hoffmann, M., Kleine-Weber, H., Schroeder, S. et al. (2020). SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Cell 181, 271-280.e8. doi:10.1016/j.cell.2020.02.052

Hogberg, H. T., Lam, A., Ohayon, E. et al. (2022). The adverse outcome pathway framework applied to neurological symptoms of COVID-19. Cells 11, 3411. doi:10.3390/cells11213411

Jaylet, T., Coustillet, T., Jornod, F. et al. (2023). AOP-helpFinder 2.0: Integration of an event-event searches module. Environ Int 177, 108017. doi:10.1016/j.envint.2023.108017

Kim, Y., Park, C., Lim, S. et al. (2021). Advanced adverse outcome pathways potentially bridging pathogenesis of COVID-19. Preprints 2021, 2021010065. doi:10.20944/preprints202101.0065.v1

Klokov, D., Applegate, K., Badie, C. et al. (2022). International expert group collaboration for developing an adverse outcome pathway for radiation induced leukemia. Int J Radiat Biol 98, 1802-1815. doi:10.1080/09553002.2022.2117873

Knapen, D., Angrish, M. M., Fortin, M. C. et al. (2018). Adverse outcome pathway networks I: Development and applications. Environ Toxicol Chem 37, 1723-1733. doi:10.1002/etc.4125

Kozbenko, T., Adam, N., Lai, V. et al. (2022). Deploying elements of scoping review methods for adverse outcome pathway development: A space travel case example. Int J Radiat Biol 98, 1777-1788. doi:10.1080/09553002.2022.2110306

Krewski, D., Acosta, D. J., Andersen, M. et al. (2010). Toxicity testing in the 21st century: A vision and a strategy. J Toxicol Environ Health B Crit Rev 13, 51-138. doi:10.1080/10937404.2010.483176

Landsiedel, R., Fabian, E., Ma-Hock, L. et al. (2012). Toxico-/biokinetics of nanomaterials. Arch Toxicol 86, 1021-1060. doi:10.1007/s00204-012-0858-7

Laurier, D., Rühm, W., Paquet, F. et al. (2021). Areas of research to support the system of radiological protection. Radiat Environ Biophys 60, 519-530. doi:10.1007/s00411-021-00947-1

Mazein, A., Shoaib, M., Alb, M. et al. (2023). Using interactive platforms to encode, manage and explore immune-related adverse outcome pathways. bioRxiv. doi:10.1101/2023.03.21.533620

Murugadoss, S., Vinković Vrček, I., Schaffert, A. et al. (2024). Linking nanomaterial-induced mitochondrial dysfunction to existing adverse outcome pathways for chemicals. ALTEX 41, 76-90. doi:10.14573/altex.2305011

NCRP – National Council on Radiation Protection and Measurements (2020). Approaches for Integrating Information from Radiation Biology and Epidemiology to Enhance Low-Dose Health Risk Assessment. Report No. 186.

Nel, A., Xia, T., Meng, H. et al. (2013). Nanomaterial toxicity testing in the 21st century: Use of a predictive toxicological approach and high-throughput screening. Acc Chem Res 46, 607-621. doi:10.1021/ar300022h

NRC (2007). Toxicity Testing in the 21st Century: A Vision and a Strategy. Washington, DC, USA: National Academies Press.

Nymark, P., Bakker, M., Dekkers, S. et al. (2020). Toward rigorous materials production: New approach methodologies have extensive potential to improve current safety assessment practices. Small 16, 1904749. doi:10.1002/smll.201904749

Nymark, P., Karlsson, H. L., Halappanavar, S. et al. (2021a). Adverse outcome pathway development for assessment of lung carcinogenicity by nanoparticles. Front Toxicol 3, 653386. doi:10.3389/ftox.2021.653386

Nymark, P., Sachana, M., Leite, S. B. et al. (2021b). Systematic organization of COVID-19 data supported by the adverse outcome pathway framework. Front Public Health 9, 638605. doi:10.3389/fpubh.2021.638605

OECD (2002). Consensus Document on Information Used in the Assessment of Environmental Applications Involving Baculoviruses. Series on Harmonization of Regulatory Oversight in Biotechnology, No. 20.

OECD (2011). Report of the Workshop on Using Mechanistic Information in Forming Chemical Categories. Series on Testing and Assessment, No. 138.

OECD (2012a). The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins. Part 2: Use of the AOP to Develop Chemical Categories and Integrated Assessment and Testing Approaches. Series on Testing and Assessment, No. 168.

OECD (2012b). The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins. Part 1: Scientific Evidence. Series on Testing and Assessment, No. 168.

OECD (2018). User’s Handbook Supplement to the Guidance Document for Developing and Assessing Adverse Outcome Pathways. OECD Environ Health Saf Publ. doi:10.1787/5jlv1m9d1g32-en

OECD (2020). Case study on the use of integrated approaches to testing and assessment for identification and characterisation of parkinsonian hazard liability of deguelin by an AOP-based testing and read across approach. Series on Testing and Assessment, No. 326.

OECD (2022). Test No. 442D: In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method. OECD Guidelines for the Testing of Chemicals, Section 4. OECD Publishing, Paris. doi:10.1787/9789264229822-en

OECD (2023a). 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

OECD (2023b). Test No. 442E: In Vitro Skin Sensitisation: In Vitro Skin Sensitisation assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation. OECD Guidelines for the Testing of Chemicals, Section 4. OECD Publishing, Paris. doi:10.1787/9789264264359-en

OECD (2023c). Guideline No. 497: Defined Approaches on Skin Sensitisation. OECD Guidelines for the Testing of Chemicals, Section 4. OECD Publishing, Paris. doi:10.1787/b92879a4-en

Pollesch, N. L., Villeneuve, D. L., O’Brien, J. M. et al. (2019). Extracting and benchmarking emerging adverse outcome pathway knowledge. Toxicol Sci 168, 349-364. doi:10.1093/toxsci/kfz006

Rolo, D., Assunção, R., Ventura, C. et al. (2022). Adverse outcome pathways associated with the ingestion of titanium dioxide nanoparticles – A systematic review. Nanomaterials 12, 3275. doi:10.3390/nano12193275

Shahbaz, M. A., De Bernardi, F., Alatalo, A. et al. (2022). Mechanistic understanding of the olfactory neuroepithelium involvement leading to short-term anosmia in COVID-19 using the adverse outcome pathway framework. Cells 11, 3027. doi:10.3390/cells11193027

Stainforth, R., Schuemann, J., McNamara, A. L. et al. (2021). Challenges in the quantification approach to a radiation relevant adverse outcome pathway for lung cancer. Int J Radiat Biol 97, 85-101. doi:10.1080/09553002.2020.1820096

Tanabe, S., Beaton, D., Chauhan, V. et al. (2022a). Report of the 1st and 2nd mystery of reactive oxygen species conferences. ALTEX 39, 336-338. doi:10.14573/altex.2203011

Tanabe, S., O’Brien, J., Tollefsen, K. E. et al. (2022b). Reactive oxygen species in the adverse outcome pathway framework: Toward creation of harmonized consensus key events. Front Toxicol 4, 887135. doi:10.3389/ftox.2022.887135

Tanabe, S., Beaton, D., Chauhan, V. et al. (2023). Report of the 3rd and 4th mystery of reactive oxygen species conference. ALTEX 40, 689-693. doi:10.14573/altex.2307041

Tanonaka, K. and Marunouchi, T. (2016). Angiotensin-converting enzyme 2. Folia Pharmacol Jpn 147, 120-121. doi:10.1254/fpj.147.120

Tollefsen, K. E., Alonzo, F., Beresford, N. A. et al. (2022). Adverse outcome pathways (AOPs) for radiation-induced reproductive effects in environmental species: State of science and identification of a consensus AOP network. Int J Radiat Biol 98, 1816-1831. doi:10.1080/09553002.2022.2110317

Villeneuve, D. L., Crump, D., Garcia-Reyero, N. et al. (2014). Adverse outcome pathway (AOP) development I: Strategies and principles. Toxicol Sci 142, 312-320. doi:10.1093/toxsci/kfu199

Villeneuve, D. L., Angrish, M. M., Fortin, M. C. et al. (2018). Adverse outcome pathway networks II: Network analytics. Environ Toxicol Chem 37, 1734-1748. doi:10.1002/etc.4124

Villeneuve, D. L., Landesmann, B., Allavena, P. et al. (2019). Representing the process of inflammation as key events in adverse outcome pathways. Tox Sci 163, 346-352. doi:10.1093/toxsci/kfy047

Vinken, M. (2021). COVID-19 and the liver: An adverse outcome pathway perspective. Toxicology 455, 152765. doi:10.1016/j.tox.2021.152765

Volz, D. C., Belanger, S., Embry, M. et al. (2011). Adverse outcome pathways during early fish development: A conceptual framework for identification of chemical screening and prioritization strategies. Toxicol Sci 123, 349-358. doi:10.1093/toxsci/kfr185

Watanabe, K. H., Andersen, M. E., Basu, N. et al. (2011). Defining and modeling known adverse outcome pathways: Domoic acid and neuronal signaling as a case study. Environ Toxicol Chem 30, 9-21. doi:10.1002/etc.373

Whelan, M. and Andersen, M. (2013). Toxicity pathways – From concepts to application in chemical safety assessment. Joint Research Centre of the European Commission, Publications Office. doi:10.2788/49626

Wittwehr, C., Amorim, M. J., Clerbaux, L.-A. et al. (2021). Understanding COVID-19 through adverse outcome pathways – 2nd CIAO AOP design workshop. ALTEX 38, 351-357. doi:10.14573/altex.2102221

Wyrzykowska, E., Mikolajczyk, A., Lynch, I. et al. (2022). Representing and describing nanomaterials in predictive nanoinformatics. Nat Nanotechnol 17, 924-932. doi:10.1038/s41565-022-01173-6

Yu, J., Tu, W., Payne, A. et al. (2022). Adverse outcome pathways and linkages to transcriptomic effects relevant to ionizing radiation injury. Int J Radiat Biol 98, 1789-1801. doi:10.1080/09553002.2022.2110313

Zhou, P., Yang, X. Lou, Wang, X. G. et al. (2020). A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579, 270-273. doi:10.1038/s41586-020-2012-7

Most read articles by the same author(s)