Statement on the forced swim test in rodent models

Date published: 13 December 2023 (updated 24 January 2024 to include definitions)

Background

NHMRC is aware of reported ethical, welfare and scientific concerns about the forced swim test in rodents. This procedure has also been the subject of growing public concern within Australia and internationally.

In November 2022, the NSW Animal Research Review Panel published Guideline 30: The forced swim test in rats and mice.¹ Concerns about the forced swim test in rodents were also raised during the 2022 inquiry on the use of primates and other animals in medical research in New South Wales.²

The forced swim test has been decommissioned by several global pharmaceutical companies (for example, AbbVie, Pfizer).^3,4,5,6 Use of data from the forced swim test is not a regulatory requirement as part of the pharmacodynamic data submitted to demonstrate efficacy of medicines for depression and anxiety in humans prior to clinical trials.^7,8,9 The use of the forced swim test has also been discontinued at several universities in Australia and internationally.²

About the procedure

The forced-swim test was developed in 1977 to assess antidepressant treatments in rodents¹⁰, and involves placing a mouse or rat into a tank of water too deep for the animal to stand in and from which escape is impossible. The animal tries to swim or escape by attempting to climb the walls of the cylinder, and eventually becomes immobile and floats. The swimming is classed as a forced procedure because it is not a voluntary behaviour of the animal. The test measures the time taken for the animal to stop swimming and become immobile. It is typically halted after six minutes for mice and after up to 15 minutes for rats, or earlier if the animal begins sinking. Traditionally, researchers have associated immobility in the water with a lack of drive, equivalent to the symptoms of human depression. It has also been used in neurobiology research and in drug studies. Procedures involving swimming of rodents that may be used in other fields of research (for example, the induction of cardiac hypertrophy) are not considered in this Statement.

The concerns

The transition from swimming to immobility in the rodent forced swim test may assess individual coping with inescapable stressors.¹¹ However, there are reported concerns about the scientific validity of this procedure in animal models. For example, the forced swim test in rodents is no longer considered a suitable model for depression in humans, to study depression-like behaviour (including in the phenotyping of genetically altered mice), or for studies of anxiety disorders and their treatment.^{8,11,12,13,14,15,16,17} In addition, evidence indicates that its use for studying the neurobiology of stress and its accuracy as a screen for new antidepressant drugs is problematic.^{1,8,16,18,19,20}

The potential adverse impacts of the forced swim test on animal wellbeing include the combined effects of stress or distress from being placed in an inescapable situation, social isolation during and after the procedure, and handling before and after the procedure. Other potential adverse impacts on the animal include fatigue, hypothermia and aspiration of water during the procedure.^1,21 There are innate differences between rats and mice in their responses to water and the potential responses of an animal to the forced swim test may be influenced by the strain and genotype.^22,23

At the time of publication, there are no validated non-animal alternatives to the forced swim test. It may be possible to replace the forced swim test with other procedures that have less impact on animal wellbeing.^1,8

Requirements in the Code

The Australian code for the care and use of animals for scientific purposes (the Code) provides guidance for investigators, animal carers, institutions and animal ethics committees (AECs) about the ethical, humane and responsible care and use of animals for scientific purposes.²⁴ As the Code is incorporated into legislation in all Australian states and territories, its requirements are therefore embedded in legislation.

The governing principles in the Code that are particularly relevant to the use of the forced swim test in rodents are outlined in Appendix A. These principles include the necessity for methods used to be scientifically valid and for the use of animals to be ethically justified, balancing whether the potential effects on the wellbeing of the animals involved are justified by the potential benefits of the research.

NHMRC’s requirements

NHMRC requires NHMRC-funded research involving animals to comply with relevant legislation, the Code and other relevant NHMRC guidelines and policies. These requirements are a condition of NHMRC’s funding agreement.²⁵

Relevant NHMRC guidelines include NHMRC’s Best practice methodology in the use of animals for scientific purposes, which provides guidance for the conduct of high-quality animal-based studies that are rigorous, transparent and reproducible and lead to useful outcomes.²⁶ This Guideline emphasises the necessity for systematic review of animal-based studies.

It also outlines that the validity and relevance of a proposed animal model must be assessed, and that if there is insufficient evidence to support the validity of an animal model, its use must be rejected.

Application of this Statement

This Statement applies to institutions, investigators and AECs involved with, or considering, the use of the forced swim test in rodents in NHMRC-funded research. However, NHMRC anticipates that this Statement will provide guidance for those involved with, or considering, the use of the forced swim test in rodents in research not funded by NHMRC.

Implementation of this Statement

Those involved with NHMRC-funded research (see definitions) must implement the recommendations in this Statement from the date of its publication.

Appendix A – Requirements in the Code

The Australian code for the care and use of animals for scientific purposes (the Code) provides guidance for investigators, animal carers, institutions and animal ethics committees (AECs) about the ethical, humane and responsible care and use of animals for scientific purposes.²⁴ As the Code is incorporated into legislation in all Australian states and territories, its requirements are therefore embedded in legislation.

The governing principles in the Code that are particularly relevant to the use of the forced swim test in rodents include:

• A judgement as to whether a proposed use of animals is ethically acceptable must be based on information that demonstrates the principles in Clause 1.1 (of the Code) and must balance whether the potential effects on the wellbeing of the animals involved is justified by the potential benefits. (Clause 1.3)
• Steps must be taken at all times to safeguard the wellbeing of animals by avoiding or minimizing harm, including pain and distress to the animals. (Clause 1.11)
• Regardless of the potential benefits of a project, the methods used must be scientifically valid, feasible, well designed and carefully conducted so that there is a reasonable expectation that the aims of the project will be achieved. Projects that are not scientifically valid must not be performed, no matter how mild the impact on the wellbeing of the animals. (Clause 1.15)
• Investigators must use methods that accord with current best practice that:
  • take into consideration relevant aspects of species-specific biology, physiology and behaviour
  • are based on the best available scientific evidence, which include the potential adverse impact of conditions and procedures on the wellbeing of animals
  • include strategies to minimise adverse impacts. (Clause 1.16)
• Methods that replace or partially replace the use of animals must be investigated, considered and, where applicable, implemented. (Clause 1.18)
• All possible steps must be taken to reduce factors that are not part of the experimental design of the project and are known to contribute to variability of experimental results. (Clause 1.25)
• Steps must be taken at all times to support and safeguard animal wellbeing. The effectiveness of strategies for supporting and safeguarding animal wellbeing must be kept under review during the lifetime of activities, including projects. Where relevant and applicable, the outcome of this review must be implemented in current activities and taken into account in planning future activities, including projects. (Clause 1.28)

The governing principles in the Code underpin the requirements in the entire Code including the responsibilities of investigators, animal carers, institutions and AECs. They are also reflected in the requirements about animal wellbeing (Section 3) and those related to specific procedures (Clause 3.3.1).

The Code applies at all stages of animal care and use including during the lifetime of a project – when investigators are planning a project (Clauses 2.4.6–2.4.9), conducting the project and reviewing the project (Clauses 2.4.13–2.4.29, 2.4.34).

Definitions 

For the purposes of this Statement, the following definitions apply. Note that these definitions apply to NHMRC research grants and are independent of when applications are submitted to, or approved by, an Animal Ethics Committee.

New project: Any application submitted under NHMRC Grant Opportunity Guidelines issued on or after 1 January 2024.
An application submitted under NHMRC Grant Opportunity Guidelines issued prior to 1 January 2024 will not be regarded as a ‘new project’. However, if such an application is successful, NHMRC will regard the project as a ‘current project’ for the purposes of the Statement.

Current project: Any NHMRC-funded project that is active after the date of publication of the Statement and was submitted under NHMRC Grant Opportunity Guidelines issued prior to 1 January 2024.

Additional information

1. NSW Animal Research Review Panel. Guideline 30: The forced swim test in rats and mice. NSW Department of Primary Industries, November 2022. Accessed 18 August 2022
2. Use of primates and other animals in medical research in New South Wales: Report (21 October 2022)
3. AbbVie. Responsible Use of Animals in Research – AbbVie’s Commitment. Retrieved 11 July 2023
4. Pfizer, Letter to the U.S. Securities and Exchange Commission (19 December 2019). Retrieved 18 May 2023
5. Reardon S. Depression researchers rethink popular mouse swim tests. Nature. 2019 Jul;571(7766):456-457. doi: 10.1038/d41586-019-02133-2. PMID: 31337906
6. European Pharmaceutical Manufacturer, Pharma Manufacturing News (1 November 2019). Retrieved 12 July 2023
7. Sewell F, Waterson I, Jones D, Tricklebank MD, Ragan I. Preclinical screening for antidepressant activity - shifting focus away from the Forced Swim Test to the use of translational biomarkers. Regul Toxicol Pharmacol. 2021;125:105002. Epub 2021/07/11. doi: 10.1016/j.yrtph.2021.105002. PubMed PMID: 34245825
8. UK Government. Animals in Science Committee. Advice on the use of the forced swim test (5 July 2023). Retrieved 13 July 2023
9. Therapeutic Goods Administration, Australia. Pers comm (28 July 2023). While TGA does not mandate that particular nonclinical studies must be undertaken in support of prescription medicines, TGA expects that either nonclinical data or scientific justification for the absence of nonclinical data must be provided. Additional information retrieved 28 July 2023
10. Porsolt RD, Le Pichon M, Jalfre M. Depression: a new animal model sensitive to antidepressant treatments. Nature. 1977;266(5604):730-2. Epub 1977/04/21. doi: 10.1038/266730a0. PubMed PMID: 559941
11. Commons KG, Cholanians AB, Babb JA, Ehlinger DG. The Rodent Forced Swim Test Measures Stress-Coping Strategy, Not Depression-like Behavior. ACS Chemical Neuroscience. 2017;8(5):955-60. doi: 10.1021/acschemneuro.7b00042
12. Armario A. The forced swim test: Historical, conceptual and methodological considerations and its relationship with individual behavioral traits. Neurosci Biobehav Rev. 2021;128:74-86. Epub 2021/06/13. doi: 10.1016/j.neubiorev.2021.06.014. PubMed PMID: 34118295
13. Borsini F, Volterra G, Meli A. Does the behavioral “despair” test measure “despair”? Physiology & Behavior. 1986;38(3):385-6. doi
14. Molendijk ML, de Kloet ER. Immobility in the forced swim test is adaptive and does not reflect depression. Psychoneuroendocrinology. 2015;62:389-91. Epub 2015/09/21. doi: 10.1016/j.psyneuen.2015.08.028. PubMed PMID: 26386543
15. Molendijk ML, de Kloet ER. Coping with the forced swim stressor: Current state-of-the-art. Behavioural Brain Research. 2019;364:1-10. doi
16. Ferreira MF, Castanheira L, Sebastião AM, Telles-Correia D. Depression Assessment in Clinical Trials and Pre-clinical Tests: A Critical Review. Curr Top Med Chem. 2018;18(19):1677-1703. doi: 10.2174/1568026618666181115095920. PMID: 30430942
17. British Association for Psychopharmacology: Fact sheet on the forced swim test. (2020). Retrieved 15 February 2023
18. Slattery DA, Cryan JF. Using the rat forced swim test to assess antidepressant-like activity in rodents. Nature Protocols. 2012;7(6):1009-14. doi: 10.1038/nprot.2012.044
19. Kokras N, Antoniou K, Mikail HG, Kafetzopoulos V, Papadopoulou-Daifoti Z, Dalla C. Forced swim test: What about females? Neuropharmacology. 2015;99:408-21. doi
20. Trunnell ER, Carvalho C. The forced swim test has poor accuracy for identifying novel antidepressants. Drug Discovery Today. 2021;26(12):2898-904. doi
21. Beausoleil, N. 2021. Use of the Five Domains Model to assess the potential animal welfare impacts for a cost-benefit analysis of the test as a research tool. Panel discussion – Porsolt Forced Swim Test. Proceedings. Australian and New Zealand Council for the Care of Animals in Research and Teaching. 2021 Conference Proceedings
22. Sluyter F, Van Oortmerssen GA. A Mouse is Not Just a Mouse. Animal Welfare. 2000;9(2):193-205. Epub 2023/01/11. doi: 10.1017/S0962728600022521
23. Jacobson LH, Cryan JF. Feeling strained? Influence of genetic background on depression-related behavior in mice: a review. Behav Genet. 2007;37(1):171-213. Epub 2006/10/10. doi: 10.1007/s10519-006-9106-3. PubMed PMID: 17029009
24. National Health and Medical Research Council. Australian code for the care and use of animals for scientific purposes. 8th ed (2013, updated 2021)
25. National Health and Medical Research Council. Funding Agreement
26. National Health and Medical Research Council. Best practice methodology in the use of animals for scientific purposes (2017, updated July 2018).