Forced Swim Test and Forced Smoke Inhalation Experiments

Purpose

To advocate for cessation of forced swim tests and nose-only forced smoke inhalation experiments, and for a phase-out of whole body smoke inhalation experiments as soon as possible.

Policy

1. The AVA does not support the use of forced swim tests on animals in medical research.
2. The AVA does not support the use of forced smoke inhalation experiments on animals in medical research. 

Background

Forced Swim Test (FST)

The Posolt forced swim test (FST) is a research procedure involving animals that has been used since the 1970s for studies relating to depression and other mental health conditions and for screening potential anti-depressants and other medications.

This test involves placing an individual animal into a container of water where there is no means of escape, and they are unable to touch the bottom. Researchers record the amount of time between when the animal is placed in the water and when they cease to swim. Historically, it has been thought that the interval is reduced in animal models of depression and may be reversed when animals are treated with certain anti-depressants. Since the animal has no means of escape, the test is aversive due to animals experiencing fear and anxiety as they frantically swim to avoid drowning.

While the FST may have previously played a role in screening anti-depressants, there is strong evidence that it is not reliable in screening compounds with novel mechanisms of action. A comprehensive review recommended that it not be used as a model of depression or to study depression-like behaviour, nor for studies of anxiety disorders and their treatment (ASC 2023; Commons et al 2017). Armario (2021) also suggests that cessation of swimming may be an energy conserving strategy rather than indicating a mental state such as despair. Animals also may very quickly learn to associate cessation of swimming with removal from the water apparatus resulting in potentially confounding research outcomes with this test (Parra et al 1999). In recent years more robust alternatives have been developed to test depression and anti-depressant and other medications in rodents.

There is no regulatory requirement to submit FST-related efficacy data prior to human clinical trials, and some global pharmaceutical companies no longer use the test.

While there are no specific equivalent non-animal alternatives to the test, it has been argued that there are much less aversive behavioural tests which can be substituted, for example the sucrose preference test. Further, moving from reliance on behavioural to quantifiable neurochemical outcomes in animal studies has been advocated, such as by measuring translationally relevant and predictive biomarkers, for example Brain Derived Neurotropic Factor (B-DNF) (Sewell et al 2021).

Finally, non-animal alternatives are also being considered as alternative methods to study various mental health conditions, for example, use of pluripotent human stem cells (Wang et al 2019).

Current status in Australia

In December 2023, the National Health and Medical Research Council (NHMRC) published a statement announcing that “The forced swim test in rodents must not be used in any new projects for any other purpose unless there is robust evidence to support the scientific validity of its use and compelling justification that use of alternatives to the forced swim test will not achieve the scientific aims of the proposed research”[1]. Whilst this is likely to limit the use of the FST, it falls short of a complete ban on such use.

In February 2024, NSW became the first Australian jurisdiction to prohibit the use of the forced swim test through the passing of the Animal Research Amendment (Prohibition of Forced Swim Tests and Forced Smoke Inhalation Experiments) Bill 2023.

Prior to these events, several Australian universities had already announced that they would no longer conduct the FST (NSW Parliament 2022).

Forced Smoke Inhalation Experiments (FSIE)

FSIEs are used in mice to study chronic obstructive pulmonary (lung) disease (COPD) and smoking/vaping induced effects in humans (Wright et al 2008; Ghorani et al 2017). The two different methods used for forced smoke inhalation experiments involve laboratory animals being exposed to either:

1.     smoke through a nose cone (known as nose only exposure) or
2.   over their whole body (known as whole body exposure)

Nose only smoke exposure is considered more aversive as the smoke is forced directly into the face and respiratory tract for up to an hour at a time, with consecutive days of exposure (usually five days per week) for up to six months.

Animals subjected to nose only smoke exposure have been reported to show signs indicating compromised welfare including abnormal grooming, face-rubbing, reduced activity, incoordination, muscle tremor, abnormal position of the tail, and eye squinting which can be indicative of pain (Beckett et al 2013). In addition, fear and anxiety are likely to be experienced due to repeated and extended periods of restraint in a cylinder. The aim of the experiment is to cause damage to the lungs, which, in addition to irritation of the lining of the respiratory tract at the time of smoke exposure, is detrimental to welfare. Mice exposed to nose only cigarette smoke have also been reported to show tremors and breathing difficulty (Kogel et al 2021). Mice used in nose only and sham exposure have showed lower body weights than whole body exposure suggesting that the significant restraint may cause stress (van Eijl et al 2006; Kogel et al 2021).

The use of this procedure as a model to study COPD in humans has been criticised due to limitations in modelling the complexity of the disease progression and pathophysiology (Williams & Roman 2016; Bonniaud et al 2018; Tanner et al 2020). There is concurrently an increasing focus on non-animal alternatives for studying COPD and other respiratory diseases (Fröhlich 2021; Hynes et al 2020; Movia et al 2020). Ongoing research in Australia using in vitro lung models that mimic pathophysiology is showing some potential to replace and reduce the use of smoke inhalation tests for drug testing, screening and validation (Phan et al 2023).

Current status in Australia

The use of forced smoke inhalation experiments (including nose-only) occurs at a number of universities across Australia (NSW Parliament 2022).

In December 2023, the National Health and Medical Research Council (NHMRC) released a policy statement that nose-only smoke inhalation research will no longer be funded, and that additional restrictions will be placed on whole-body exposure. This falls short of NHMRC imposing a complete ban on the use of these techniques.

In February 2024, NSW became the first Australian jurisdiction to prohibit the use of the nose only forced smoke inhalation experiments through the passing of the Animal Research Amendment (Prohibition of Forced Swim Tests and Forced Smoke Inhalation Experiments) Bill 2023.

Recommendations

1. Animal Ethics Committees and Institutions:

• Should not approve any future use of any Forced Swim Test; and  
• Should not approve ongoing use of any Forced Swim Test in renewal ethics applications for current projects

2. Forced Swim Tests should be banned under legislation in all Australian jurisdictions.

3. Animal Ethics Committees and Institutions:

• Should end research using nose-only Forced Smoke Inhalation Tests;
• Should not approve use of this technique in renewal applications for current projects 

4. Whole body smoke inhalation is not recommended, and alternatives should be sought. 

5. Where whole body smoke inhalation experiments are used, institutions are urged to:

• ensure the use of more humane endpoints
• ensure less intense exposure regimes are used; and 
• adopt more humane alternatives to smoke inhalation test to facilitate phasing out the use of this test as soon as possible

References

Armario, A. (2021). The forced swim test: Historical, conceptual and methodological considerations and its relationship with individual behavioral traits. Neuroscience & Biobehavioral Reviews, 128, 74–86. https://doi.org/10.1016/j.neubiorev.2021.06.014

Animals in Science Committee. (2023). Forced swim test report. https://www.gov.uk/government/publications/forced-swim-test-report

Beckett, E. L., et al. (2013). A new short-term mouse model of chronic obstructive pulmonary disease identifies a role for mast cell tryptase in pathogenesis. The Journal of Allergy and Clinical Immunology, 131(3), 752–762. https://doi.org/10.1016/j.jaci.2012.11.053

Bonniaud, P., et al. (2018). Optimising experimental research in respiratory diseases: An ERS statement. European Respiratory Journal, 51(5), 1702133. https://doi.org/10.1183/13993003.02133-2017

Commons, K. G., Cholanians, A. B., Babb, J. A., & Ehlinger, D. G. (2017). The rodent forced swim test measures stress-coping strategy, not depression-like behavior. ACS Chemical Neuroscience, 8(5), 955–960. https://doi.org/10.1021/acschemneuro.7b00042

Fröhlich, E. (2021). Replacement strategies for animal studies in inhalation testing. Science, 3, 45. https://doi.org/10.3390/sci3040045

Ghorani, V., Boskabady, M. H., Khazdair, M. R., & Kianmeher, M. (2017). Experimental animal models for COPD: A methodological review. Tobacco Induced Diseases. https://doi.org/10.1186/s12971-017-0130-2

Hynes, J., Marshall, L., Adcock, I., Novotny, T., Nic, M., Dibusz, K., & Gribaldo, L. (2020). Advanced non-animal models in biomedical research: Respiratory tract diseases (EUR 30334 EN). Publications Office of the European Union. https://doi.org/10.2760/725821

Kogel, U., Wong, E. T., Szostak, J., et al. (2021). Impact of whole-body versus nose-only inhalation exposure systems on systemic, respiratory, and cardiovascular endpoints in a 2-month cigarette smoke exposure study in the ApoE−/− mouse model. Journal of Applied Toxicology, 41(10), 1598–1619.

Movia, D., Bruni-Favier, S., & Prina-Mello, A. (2020). In vitro alternatives to acute inhalation toxicity studies in animal models – A perspective. Frontiers in Bioengineering and Biotechnology, 8, 549. https://doi.org/10.3389/fbioe.2020.00549

National Health and Medical Research Council. (2023). Statement on the forced swim test in rodent models. https://www.nhmrc.gov.au/about-us/news-centre/statement-forced-swim-test-rodent-models

NSW Animal Research Review Panel. (2022). Guideline 30: The forced swim test in rats and mice. NSW Department of Primary Industries.

NSW Parliament. (2022). Use of primates and other animals in medical research in New South Wales (Report No. 59). Legislative Council, Portfolio Committee No. 2 - Health.

Parra, A. (1999). Learned immobility is also involved in the forced swimming test in mice. Psicothema, 11(2), 239–246.

Phan, T. H., Shi, H., Denes, C., et al. (2023). Advanced pathophysiology mimicking lung models for accelerated drug discovery. Biomaterials Research, 27, 35.

Pharma. (2019). European Pharmaceutical Manufacturer: Pharma Manufacturing News.

Reardon, S. (2019). Depression researchers rethink popular mouse swim tests. Nature, 571(7766), 456–457. https://doi.org/10.1038/d41586-019-02133-2

Sewell, F., Waterson, I., Jones, D., Tricklebank, M. D., & Ragan, I. (2021). Preclinical screening for antidepressant activity– Shifting focus away from the forced swim test to the use of translational biomarkers. Regulatory Toxicology and Pharmacology, 125, 105002.

Tanner, L., & Single, A. B. (2020). Animal models reflecting chronic obstructive pulmonary disease and related respiratory disorders: Translating pre-clinical data into clinical relevance. Journal of Innate Immunity, 12(3), 203–225. https://doi.org/10.1159/000502489

Trunnell, E. R., & Carvalho, C. (2021). The forced swim test has poor accuracy for identifying novel antidepressants. Drug Discovery Today, 26(12), 2898–2904.

Van Eijl, S., van Oorschot, R., Olivier, B., et al. (2006). Stress and hypothermia in a nose-only cigarette smoke exposure system. Inhalation Toxicology, 18(11), 911–918.

Wang, M., Zhang, L., & Gage, F. H. (2019). Modelling neuropsychiatric disorders using human induced pluripotent stem cells. Protein & Cell, 11(1), 45–59.

Williams, K., & Roman, J. (2016). Studying human respiratory disease in animals—Role of induced and naturally occurring models. The Journal of Pathology, 238(2), 220–232. https://doi.org/10.1002/path.4658

Wright, J. L., Cosio, M., & Churg, A. (2008). Animal model of chronic obstructive pulmonary disease. American Journal of Physiology. https://doi.org/10.1152/ajplung.90200.2008