Statement on smoke inhalation procedures 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 smoke inhalation procedures in rodents. These procedures have also been the subject of growing public concern within Australia and internationally. 

In July 2022, the NSW Animal Research Review Panel published the guideline: Smoke inhalation procedures in rodents.¹ Concerns about smoke inhalation procedures in rodents – and the nose-only exposure procedure in particular – were also raised by the research and the general community during the 2022 inquiry on the use of primates and other animals in medical research in New South Wales.² A report from the UK Animals in Science Committee on harm-benefit analysis in the use of animals in research identified research related to controversial purposes and associated with societal concerns, which included ‘exposing animals to tobacco smoke (as a pharmacological tool to induce diseases such as chronic obstructive pulmonary disease)’.³

About the procedures

Smoke inhalation procedures are respiratory inhalation models used to study respiratory conditions such as chronic obstructive pulmonary disease, emphysema and lung cancer, as well as the relationship between cigarette smoking and other conditions such as gastrointestinal disease and cardiovascular disease.^1,4 They involve the exposure of mice or rats to inhaled substances (such as tobacco smoke) by either nose-only exposure or whole-body exposure. The nose-only exposure procedure mimics direct first-hand smoking by tightly restraining mice or rats in individual chambers and delivering smoke directly to their nose or head. The whole-body exposure procedure delivers smoke to unrestrained animals in a chamber. 

The concerns

Both the nose-only exposure procedure and the whole body exposure procedure cause significant harm to the animals as a result of the acute and cumulative effects of exposure to smoke.¹ The effects on the animal include irritation to the eyes and respiratory tract, nicotine withdrawal (for research involving tobacco smoke since animals are typically exposed only five out of seven days during a week), and the debilitating effects of the chronic lung disease that the procedures are designed to model.¹ During the nose-only exposure procedure, the animals are at a heightened risk of injury or mortality, they suffer stress as a result of being restrained and kept in isolation, and there is the potential for hypothermia and weight loss.^1,5 The whole-body exposure procedure reduces the impact of stress caused by restraint and isolation.^6,7

The scientific validity and reproducibility of both the nose-only exposure procedure and whole-body exposure procedure have been questioned because of the lack of experimental standardisation in dosing, length and method of exposure; the uncontrolled experimental variable resulting from the pain and distress caused in the animals; and the variability in response between strains of rodents.^1,8,9,10,11 The potential exposure of the animal to smoke uptake via routes other than inhalation (for example, dermal, gastro-intestinal following ingestion of residue during grooming) can prevent accurate measurement of smoke absorption via inhalation, resulting in the procedure being less controlled and less reproducible.^7,8 Tight physical restraint necessary for the nose-only exposure procedure and acute pain seen with both types of procedures have no counterpart in typical smoke exposure in humans. These factors, combined with the inherent limitation of animal models, mean that the features of human disease mimicked in these procedures could be limited considerably and could exacerbate any issues relating to translation of results to the human condition.^12,13

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.¹⁴ 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 smoke inhalation procedures 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.¹⁵

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.¹⁶ 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 smoke inhalation procedures in rodents in NHMRC-funded research. However, NHMRC anticipates that this Statement will provide guidance for those involved with, or considering, the use of smoke inhalation procedures in rodents in research not funded by NHMRC. This Statement may also provide guidance for those involved with, or considering, the use of inhalational procedures in rodents that do not involve smoke (for example, inhalation of substances included in e-cigarette or vaping products), which may have similar adverse impacts on animal wellbeing due to restraint and forced inhalation. 

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.¹⁴ 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 smoke inhalation procedures 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 are 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 28: Smoke Inhalation Procedures in Rodents. NSW Department of Primary Industries, July 2022. Retrieved 6 January 2023. This provides a useful checklist of matters that should be considered as part of a review of a project by investigators and for consideration of the outcome of this review by AECs
2. Use of primates and other animals in medical research in New South Wales: Report (21 October 2022) and NSW Government response (23 January 2023)
3. UK Government, Animals in Science Committee. Review of harm-benefit analysis in the use of animals in research (November 2017). Retrieved 24 May 2023
4. Kogel U, Wong ET, Szostak J, Tan WT, Lucci F, Leroy P, et al. 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. 2021;41(10):1598-619. doi
5. van Eijl S, van Oorschot R, Olivier B, Nijkamp FP, Bloksma N. Stress and hypothermia in mice in a nose-only cigarette smoke exposure system. Inhal Toxicol. 2006 Oct;18(11):911-8. doi: 10.1080/08958370600822672. PMID: 16864409
6. Shu J, Li D, Ouyang H, Huang J, Long Z, Liang Z, et al. Comparison and evaluation of two different methods to establish the cigarette smoke exposure mouse model of COPD. Scientific Reports. 2017;7(1):15454. doi: 10.1038/s41598-017-15685-y
7. OECD 2018. Guidance Document on Acute Inhalation Toxicity Testing. Series on Testing and Assessment No 39. Second ed. Paris, France: Organisation for Economic Co-operation and Development. Retrieved 23 May 2023
8. Ghorani V, Boskabady MH, Khazdair MR, Kianmeher M. Experimental animal models for COPD: a methodological review. Tob Induc Dis. 2017;15:25. Epub 2017/05/05. doi: 10.1186/s12971-017-0130-2. PubMed PMID: 28469539; PubMed Central PMCID: PMCPMC5414171
9. Jones B, Donovan C, Liu G, Gomez HM, Chimankar V, Harrison CL, et al. Animal models of COPD: What do they tell us? Respirology. 2017;22(1):21-32. doi
10. T Tanner L, Single AB. Animal Models Reflecting Chronic Obstructive Pulmonary Disease and Related Respiratory Disorders: Translating Pre-Clinical Data into Clinical Relevance. J Innate Immun. 2020;12(3):203-25. Epub 2019/09/19. doi: 10.1159/000502489. PubMed PMID: 31527372; PubMed Central PMCID: PMCPMC7265725
11. Fröhlich E. Replacement Strategies for Animal Studies in Inhalation Testing. Sci. 2021;3(4):45. PubMed PMID: doi:10.3390/sci3040045
12. Bonniaud P, Fabre A, Frossard N, Guignabert C, Inman M, Kuebler WM, et al. Optimising experimental research in respiratory diseases: an ERS statement. European Respiratory Journal. 2018;51(5):1702133. doi: 10.1183/13993003.02133-2017
13. Barnes PJ, Bonini S, Seeger W, Belvisi MG, Ward B, Holmes A. Barriers to new drug development in respiratory disease. European Respiratory Journal. 2015;45(5):1197. doi: 10.1183/09031936.00007915.
14. National Health and Medical Research Council. Australian code for the care and use of animals for scientific purposes. 8th ed (2013, updated 2021)
15. National Health and Medical Research Council. Funding Agreement.
16. National Health and Medical Research Council. Best practice methodology in the use of animals for scientific purposes (2017, updated July 2018)