Why in vitro methods are the future of risk assessments for inhaled substances 

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Inhaled substances are primarily tested on rats for toxicity, but key differences between rat and human lungs suggest it’s time to look towards alternative methods.

We breathe in all kinds of substances, from medicines to perfumes to smoke. To protect our health, it’s important to check whether the things we inhale are likely to cause us harm. The most common method of assessing inhaled compounds for safety is to test them on rats. However, substances that are harmful to human lungs can be safe for rats, and vice versa. 

According to regulatory guidelines, inhaled substances should only be tested for acute toxicity – severe health effects resulting from a single exposure – on rats or mice. Rats have long served as a suitable species in many areas of biological research thanks to their small size, ease of maintenance and anatomical similarity to humans. But although rats may be more like us than many other animals, their respiratory systems still significantly differ from our own. 

Physiological differences 

The differences between rat and human respiratory tracts start right at the nose. Compared to most other mammals, humans and other primates have rather simple nasal anatomy. The noses of animals like rats, on the other hand, have complex, scroll-like internal structures. Additionally, rats are unable to breathe through their mouths. Due to these differences, inhaled particles larger than a few micrometres are less likely to enter the airways of rats than humans. 

In rats, the transition from the largest branches of the lungs to the air sacs, or alveoli, is far more abrupt. Their lungs lack respiratory bronchioles, the deepest and narrowest airways of the lungs. The presence of respiratory bronchioles in humans makes it more difficult for us to clear insoluble particles from our alveoli. Since rats can rid their lungs of harmful particles faster than humans, some substances may pose greater risks to our health than theirs. 

The opposite problem can also arise. Substances that are safe for us to inhale may be harmful to rats. Although rat airways are larger than ours relative to body size, they are still smaller overall. Insoluble aerosols can lead to obstruction of rat lungs and cause death, even if they are non-toxic to humans. 

A review of 52 studies concluded that inhalation toxicity testing in rats was not relevant for humans. Current tests struggle to predict the human condition, and this has major consequences. From 2011-2012, 33% of respiratory drugs failed during clinical trials due to toxicological reasons, despite many of these drugs having performed well in animal models. 

An alternative path 

If we can’t use rat studies to accurately assess the safety of inhaled substances in humans, what is the alternative? The obvious solution may be to look for a different animal model, but all have limitations. Other small mammals such as mice, rabbits and guinea pigs are most commonly used in place of rats. Using mice leads to many of the same problems as using rats, and since mice are smaller, it can be even more difficult to extrapolate findings to humans. Guinea pig lungs are divided into seven lobes instead of our five, and their pulmonary blood vessels behave differently to our own. As for rabbit lungs, while they exhibit more human-like branching patterns than rat lungs do, rabbits have different immune system tissues and lack a cough reflex.  

Lung studies are sometimes performed on larger animals, usually pigs. Although pigs have the respiratory bronchioles that are absent in many smaller mammals, the ratio between the trachea and body mass differs from that of humans, and white blood cell counts are also much higher. Large animals are also far more expensive to use in research. 

The future is in vitro 

The best option may be to expand beyond animal studies. In recent years, there has been a surge in the number of companies working to develop alternative approaches to safety assessment. There is a particular focus on in vitro tests, i.e. tests conducted outside a living organism. 

In vitro methods include “lung-on-a-chip” systems, micro-engineered devices that replicate the physiological functions of the lung, and cell cultures such as ImmuONE’s ImmuLUNGTM and ImmuPHAGETM models. In vitro cell culture technology allows researchers to create and manipulate cells in a controlled environment outside the body, providing a more cost-effective and efficient alternative to animal testing.  

Researchers can test substances for toxicity by adding them to human cells that resemble those found in the lung and checking for any changes. In vitro approaches have demonstrated the ability to measure immune function, cell health, protein interactions and other aspects of lung health.  

To meet the increasing demand for personalised medicine and the need for more reliable pre-clinical testing methods, organisations like the National Centre for the Replacement, Refinement & Reduction of Animals in Research (NC3Rs) in the UK and the National Institutes of Health (NIH) in the US are funding new research into in vitro technologies. For example, the NC3Rs has launched the Animal-free in vitro CRACK-IT Challenge, a collaborative research project in which ImmuONE and partners are adapting safety testing approaches to be more relevant to humans. The accuracy of animal-free methods is improving all the time, and the range of biological information they can be used to obtain is ever-expanding. Expertise and the number of people who are skilled in using in vitro approaches is also on the rise.  

Regulatory push 

Animal-free approaches are the future for human safety testing. They are necessary not only to increase the relevance of assessments to humans, but to keep pace with legislative and ethical developments. Due to animal welfare concerns, authorities around the world are phasing out animal testing. The US Environmental Protection Agency plans to end funding for mammal studies by 2035, and the EU has already banned animal testing for cosmetic purposes. By developing alternative testing methods, we can ensure that risk assessments only continue to become more effective as these regulations come into place. 

Right now, the UK still heavily relies on animal studies. According to a report published by the UK Home Office, the number of tests carried out on animals increased by 6% from 2020 to 2021. Between 2015 and 2017, the UK was found to be the biggest user of animals for experimental purposes in Europe. Regulatory bodies often require animal studies to establish product safety and provide little information regarding alternatives. It can be challenging for companies to move towards in vitro methods, even if such methods offer better insight into how substances affect the human body. However, innovative in vitro specialists like ImmuONE are using their comprehensive knowledge of cutting-edge human focused solutions to guide companies through the complex regulatory landscape. 

The burgeoning development of in vitro solutions represents a promising trend towards more ethical and human-relevant safety testing methods. Although animal testing cannot yet be fully replaced, we should aim to reduce and refine animal studies wherever possible while supporting the uptake of human-relevant alternatives. 

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