Associations between clinical signs and pathological findings in toxicity testing

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Antero V. Silva
Ulf Norinder
Elin Liiv
Björn Platzack
Mattias Öberg
Elin Törnqvist


Animal testing for toxicity assessment of chemicals and pharmaceuticals must take the 3R principles into consideration. During toxicity testing in vivo, clinical signs are used to monitor animal welfare and to inform about potential toxicity. This study investigated possible associations between clinical signs, body weight change and histopathological findings observed after necropsy. We hypothesized that clinical signs and body weight loss observed during experiments could be used as early markers of organ toxicity. This represents a potential for refinement in terms of improved study man­agement and decreasing of pain and distress experienced during animal experiments. Data from three sequential toxicity studies of an anti-cancer drug candidate in rats were analyzed using the multivariate partial least squares (PLS) regression method. Associations with a predictive value over 80% were found between the occurrence of mild to severe clinical signs and histopathological findings in the thymus, testes, epididymides and bone marrow. Piloerection, eyes half shut and slightly decreased motor activity were most strongly associated with the pathological findings. A 5% body weight loss was found to be a strong empirical predictor of pathological findings but could also be predicted accurately by clinical signs. Thus, we suggest using mild clinical signs and a 5% body weight loss as toxicity markers and as a non-invasive surveillance tool to monitor research animal welfare in toxicity testing. These clinical signs may also enable reduction of animal use due to their informative potential to support scientific decisions regarding drug candidate selection, dose setting, study design, and toxicity assessment.

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How to Cite
Silva, A. V., Norinder, U., Liiv, E., Platzack, B., Öberg, M. and Törnqvist, E. (2021) “Associations between clinical signs and pathological findings in toxicity testing”, ALTEX - Alternatives to animal experimentation, 38(2), pp. 198-214. doi: 10.14573/altex.2003311.

Chapman, K., Sewell, F., Allais, L. et al. (2013). A global pharmaceutical company initiative: An evidence-based approach to define the upper limit of body weight loss in short term toxicity studies. Regul Toxicol Pharmacol 67, 27-38. doi:10.1016/j.yrtph.2013.04.003

EC – European Commission (2006). Regulation (EC) No 1907/2006 of the European Parliament and of the Council of 18 December 2006 concerning the Registration, Evaluation, Authorisation and Restriction of chemicals (REACH), establishing a European Chemicals Agency, amending Directive 1999/45/EC and repealing Council Regulation (EEC) No 793/93 and Commission Regulation (EC) No 1488/94 as well as Council Directive 76/769/EEC and Commission Directives 91/155/EEC, 93/67/EEC, 93/105/EC and 2000/21/EC. OJ L 396, 1-849. (accessed 15.11.2019)

EC (2012). Working document on a Severity Assessment Framework. Brussels, 11-12 July 2012. (accessed 15.02.2020)

EC (2019). 2019 report on the statistics on the use of animals for scientific purposes in the Member States of the European Union in 2015-2017. COM (2020) 16 final. (accessed 15.02.2020)

Ekins, S., M. Clark, A. M., Perryman, A. L. et al. (2018). Accessible machine learning approaches for toxicology. In S. Ekins (ed.), Computational Toxicology: Risk Assessment for Chemicals (1-29). Wiley. doi:10.1002/9781119282594.ch1

EMA – European Medicines Agency (2010). Guideline on repeated dose toxicity. CPMP/SWP/1042/99 Rev 1. (accessed 25.10.2019)

Eriksson, L., Johansson, E. and Lundstedt, T. (2005). Regression- and projection-based approaches in predictive toxicology. In C. Helma (ed.), Predictive Toxicology (177-222). Taylor and Francis. doi:10.1201/9780849350351

Eriksson, L., Byrne, T., Johansson, E. et al. (2013). Multi-and Megavariate Data Analysis Basic Principles and Applications. 3rd edition. Umetrics Academy. ISBN 9197373052.

EU – European Union (2001). Directive 2001/83/EC of the European Parliament and of the Council of 6 November 2001 on the Community code relating to medicinal products for human use. OJ L 311, 67-128. (accessed 25.10.2019)

EU (2010). Directive 2010/63/eu of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes. Volume 53 ed. OJ L 276, 33-79. doi:10.3000/17252555.L_2010.276.eng (accessed 25.10.2019)

Gould, S. and Scott, R. C. (2005). 2-hydroxypropyl-β-cyclodextrin (HP-β-CD): A toxicology review. Food Chem Toxicol 43, 1451-1459. doi:10.1016/j.fct.2005.03.007

Hornberg, J. J., Laursen, M., Brenden, N. et al. (2014). Exploratory toxicology as an integrated part of drug discovery. Part I: Why and how. Drug Discov Today 19, 1131-1136. doi:10.1016/j.drudis.2013.12.008

Hubert, M. and Branden, K. V. (2003). Robust methods for partial least squares regression. J Chemometr 17, 537-549. doi:10.1002/cem.822

ICH – International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (2009). Guidance on nonclinical safety studies for the conduct of human clinical trials and marketing authorization for pharmaceuticals M3 (R2). International conference on harmonisation of technical requirements for registration of pharmaceuticals for human use. (accessed 05.11.2019)

JECFA – Joint FAO/WHO Expert Committee on Food Additives (2016). Evaluation of certain food additives and contaminants. 80th report of the Joint FAO/WHO Expert Committee on Food Additives. World Health Organization. IBSN 9789241209953. (accessed 15.03.2020)

Jonsson, O., Villar, R. P., Nilsson, L. B. et al. (2012). Capillary microsampling of 25 µl blood for the determination of toxicokinetic parameters in regulatory studies in animals. Bioanalysis 4, 661-674. doi:10.4155/bio.12.25

Kalantari, F., Ringblom, J., Sand, S. et al. (2017). Influence of distribution of animals between dose groups on estimated benchmark dose and animal distress for quantal responses. Risk Anal 37, 1716-1728. doi:10.1111/risa.12741

Kilkenny, C., Browne, W. J., Cuthill, I. C. et al. (2010). Improving bioscience research reporting: The ARRIVE guidelines for reporting animal research. PLoS Biol 8, e1000412. doi:10.1371/journal.pbio.1000412

Langford, D. J., Bailey, A. L., Chanda, M. L. et al. (2010). Coding of facial expressions of pain in the laboratory mouse. Nat Methods 7, 447-449. doi:10.1038/nmeth.1455

Lazraq, A., Cléroux, R. and Gauchi, J.-P. (2003). Selecting both latent and explanatory variables in the PLS1 regression model. Chemometr Intell Lab Syst 66, 117-126. doi:10.1016/S0169-7439(03)00027-3

Morton, D. B. and Griffiths, P. H. (1985). Guidelines on the recognition of pain, distress and discomfort in experimental animals and an hypothesis for assessment. Vet Rec 116, 431-436. doi:10.1136/vr.116.16.431

Morton, D. B. (1997). A scheme for the recognition and assessment of adverse effects in animals. Animal Alternatives, Welfare, and Ethics 27, 235-240. ISBN:0-444-82424-3

NAC – National Advisory Committee for Acute Exposure Guideline Levels for Hazardous Substances (2001). Standing Operating Procedures for Developing Acute Exposure Guideline Levels for Hazardous Chemicals. National Research Council, Committee on Toxicology, Subcommittee on Acute Exposure Guideline Levels (AEGL). Washington, DC, USA: National Academy Press. Online ISBN 9780309570114. (accessed 21.02.2020)

NC3Rs – National Centre for the Replacement, Refinement and Reduction of Animals in Research (2009). Guidance on dose level selection for regulatory general toxicology studies for pharmaceuticals. London: NC3Rs/LASA. (accessed 05.11.2019)

NC3Rs (2010). ARRIVE Guidelines. (accessed 05.11.2019)

OECD – Organisation for Economic Co-operation and Development (2000). Guidance Document on the Recognition, Assessment, and Use of Clinical Signs as Humane Endpoints for Experimental Animals Used in Safety Evaluation. Series on Testing and Assessment, No. 19. OECD Publishing, Paris. doi:10.1787/9789264078376-en

OECD (2008). Test No. 407: Repeated Dose 28-day Oral Toxicity Study in Rodents. OECD Guidelines for the Testing of Chemicals, Section, 4. OECD Publishing, Paris. doi:10.1787/9789264070684-en

OECD (2018). 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 (2019). Test No. 442C: In Chemico Skin Sensitisation. OECD Guidelines for the Testing of Chemicals, Section, 4. OECD Publishing, Paris. doi:10.1787/9789264229709-en

Olson, H., Betton, G., Robinson, D. et al. (2000). Concordance of the toxicity of pharmaceuticals in humans and in animals. Regul Toxicol Pharmacol 32, 56-67. doi:10.1006/rtph.2000.1399

Remesh, A. (2012). Toxicities of anticancer drugs and its management. Int J Basic Clin Pharmacol 1, 2-12. doi:10.5455/2319-2003.ijbcp000812

Ringblom, J., Kalantari, F., Johanson, G. et al. (2017a). Influence of distribution of animals between dose groups on estimated benchmark dose and animal welfare for continuous effects. Risk Anal 38, 1143-1153. doi:10.1111/risa.12929

Ringblom, J., Törnqvist, E., Hansson, S. O. et al. (2017b). Assigning ethical weights to clinical signs observed during toxicity testing. ALTEX 34, 148-156. doi:10.14573/altex.1512211

Sewell, F., Chapman, K., Baldrick, P. et al. (2014). Recommendations from a global cross-company data sharing initiative on the incorporation of recovery phase animals in safety assessment studies to support first-in-human clinical trials. Regul Toxicol Pharmacol 70, 413-429. doi:10.1016/j.yrtph.2014.07.018

Sewell, F., Ragan, I., Marczylo, al. (2015). A global initiative to refine acute inhalation studies through the use of ‘evident toxicity’ as an endpoint: Towards adoption of the fixed concentration procedure. Regul Toxicol Pharmacol 73, 770-779. doi:10.1016/j.yrtph.2015.10.018

SFS – Svensk författningssamling (1988). L 1 Djurskyddslag. 1988:534 [Article in Swedish]. (accessed 25.10.2019)

SJVFS – Statens Jordbruksverks Föreskrifter (2012). L 150 Statens Jordbruksverks Föreskrifter och allmänna råd om försöksdjur. 2012:26. ISSN 1102-0970 [Article in Swedish]. (accessed 25.10.2019)

SJVFS (2015). L 150 Statens Jordbruksverks Föreskrifter och allmänna råd om försöksdjur SJVFS 2015:24. ISSN 1102-0970 [Article in Swedish]. (accessed 25.10.2019)

Sparrow, S. S., Robinson, S., Bolam, S. et al. (2011). Opportunities to minimise animal use in pharmaceutical regulatory general toxicology: A cross-company review. Regul Toxicol Pharmacol 61, 222-229. doi:10.1016/j.yrtph.2011.08.001

Törnqvist, E., Annas, A., Granath, B. et al. (2014). Strategic focus on 3R principles reveals major reductions in the use of animals in pharmaceutical toxicity testing. PLoS One 9, e101638. doi:10.1371/journal.pone.0101638

USDA – United States Department of Agriculture (1966). The Animal Welfare Act – Public Law 89-544. (accessed 15.03.2020)

US FDA – Food and Drug Administration (2010). Guidance for industry – M3 (R2) nonclinical safety studies for the conduct of human clinical trials and marketing authorization for pharmaceuticals. Rev 1. (accessed 15.08.2019)

WHO – World Health Organization (2009). Principles and methods for the risk assessment of chemicals in food. EHC 240.

Wold, H. (1975). Soft modelling by latent variables: The non-linear iterative partial least squares (NIPALS) approach. J Appl Probab 12, 117-142. doi:10.1017/S0021900200047604

Zidar, J., Weber, E. M., Ewaldsson, B. et al. (2019). Group and single housing of male mice: Collected experiences from research facilities in Sweden. Animals 9, 1010. doi:10.3390/ani9121010