Using in silico methods as alternatives to using protected animals within toxicology research

Introduction

Exposure to potentially harmful chemicals is always a possibility throughout varying areas of everyday life. They can be found as pesticides sprayed onto food, compounds for a new potential pharmaceutical or even as a new formula for a popular cosmetic product. Toxicology can be defined as the field of science dedicated to the investigation of the harmful effects caused by chemical substances on living organisms (Soto, 2018). Measuring the adverse effects that a specific chemical may have on a body, whether that be animal or human, is paramount for assessing the safety of a product. Measuring the dosage of chemicals is also extremely important when assessing safety. When assessing safety of chemicals, it is also imperative to evaluate how the body reacts to different dosages. The dose response curve refers to the relationship between the size of the dose given and its affect the body. In the right dosages, these affects could be very positive, but too much and the chemical could then become toxic and extremely harmful (Tsatsakis et al., 2018). Within the field of toxicology in vivo experiments involving animals have been widely used. Shukla et al., (2010) discusses how animal models have been used to investigate specific toxicological end points such as; immunotoxicity, genotoxicity and carcinogenicity. Although the outcomes of testing on animal models have provided useful information, they aren’t always consistently predictive of human biology, and they come at a large expensive. Ethical debates arise around the subject of in vivo experimentation. Where possible, the replacement of animals within research is essential. In silico is a method of testing which involves computer analysis and simulation (Raunio, 2011). Raies and Bajic, (2016) describe how in silico testing is widely used throughout toxicology to estimate the toxicity of chemicals. The aim of in silico testing is to complement existing toxicology tests by; minimising late-stage failures in drug development, prioritise chemicals and guide toxicity tests. This report will discuss how protected animals are used within research, the use of in silico technology methods as an alternative to using protected animals within toxicology research and discussions will be made into the ethics surrounding the use of research animals and why is it important to focus on the development of alternative testing methods.

The use of protected animals within research

The use of animals for scientific research has been a heavily debated subject for years. Animals have been considered good model systems for humans and human disease, therefore in vivo testing methods have been widely used within toxicology and other related research. Human obligation to consider animal ethics within research has been paramount for the development of ethical and legal guidelines that must be considered and followed by researchers (Cheluvappa et al., 2017). In 1959, William Russell and Rex Burch introduced the concept of the “3 Rs”; replacement, reduction and refinement (Ferdowsian and Beck, 2011). Miziara et al., (2012) goes on to explain how the 3 Rs were a tool designed to rationalise the use of animals in research and humanise the care of animals. Russel and Burch further argued that alternative methods such as in vitro testing and computer modelling should always be considered to replace and reduce the number of animals used where possible. Where animals must be used, techniques should be refined so as to reduce any pain or suffering caused to research animals. Such techniques included care with analgesia and postoperative antisepsis treatments. Within the UK, legislation was introduced with the purpose of protecting laboratory animals as much as possible. This legislation is known as the Animals (Scientific Procedures) Act, 1986 (ASPA). In accordance with ASPA, any living vertebrate (other than man) and any living cephalopod is deemed as “protected” once it has reached two thirds of its gestation or incubation period or is able to feed independently (‘Animals (Scientific Prodecures) Act 1986, Section 1.,’ 2019). The principles of the 3Rs and the protection of laboratory animals should also extend to the breeding, accommodation and care of any protected animals used for research (Guidance on the Operation of the Animals (Scientific Procedures) Act 1986., 2014).

There are many different factors surrounding the husbandry of laboratory animals. If husbandry is not correct, dependent on the species, it can have detrimental effects on the animal’s health and welfare. Animals can sense the seasons even when housed in windowless rooms. Reports have indicated that seasonal variation can cause immunosuppression induced by chronic stress. Other research has also shown that mice living with a 12:12-hour photoperiod were at higher risk of death from peritonitis in the summer or autumn compared with other season, highlighting the importance of understanding seasonal variation in regards to animal health (Nevalainen, 2014). A similar study conducted by (Hawkins and Golledge, 2018) describes how it is common practice to carry out scientific procedures on rats and mice during the day time. Because rodents are nocturnal, this tends to interrupt their inactive period. Testing on rodents during their inactive periods can cause cognitive defects and induced stress. Species behaviour and species enrichment are further examples as to why species specific husbandry is so significant. Alho et al., (2016) explains how a lack of feline enrichment for indoor housed cats can lead to disorders such as; anxiety, stress, obesity and feline idiopathic cystitis. As laboratory cats must be housed in doors due to health, hygiene and other relating factors that could affect research, it is paramount for laboratory workers to ensure that there is always available enrichment to enable laboratory cats to be able to exhibit their natural behaviours.

The law states that a procedure that is undertaken on a protected animal becomes regulated if that procedure has the potential of causing pain, suffering, distress or lasting harm equivalent to, or higher than, pain induced by the insertion of a hypodermic needle according to good veterinary practice. Such procedures must only be carried out if they have a scientific or educational purpose (Guidance on the Operation of the Animals (Scientific Procedures) Act 1986., 2014). The breeding of genetically modified animals also falls under the umbrella of regulated procedure. A home office report from 2017 indicates that approximately 0.28m (15%) genetically altered animals had experienced some form of potentially harmful effect due to the genetic alteration procedure (Great Britain and Home Office, 2018). Li et al., (2015) further explains how pig genomes are manipulated to increase the compatibility with human biology. This raises the question as to how reliable animal models can be if it is needed to manipulate their genome to make them more representative of human biology. Alternative methods may be a more feasible and ethical alternative. Further regulated procedures within toxicology include the assessment of multiple different toxicological endpoints including; acute oral toxicity and skin sensitisation. Oral toxicity studies involve the determination of the median lethal dose of a substance, otherwise known as LD50 testing (Zakari and Kubmarawa, 2016). This test determines the dose responsible for killing 50% of the participant animals within 24 hours. LD50 testing is a high debated method of endpoint testing. It requires a large number of animals in order to gain enough statistical data and is deemed as cruel and an inappropriate use of animals due to its questionable translatability to humans (Buesen et al., 2016). Skins sensitisation is another major toxicology endpoint that has to be assessed particularly in the development of dermatology products. Guinea pigs are often used as test models for skin sensitisation, but again the translatability of the results for human skin has been argued. Skin sensitisation also involve the application of an adjuvant that causes pain and distress to treated animals, highlighting the desirability of a replacement method (Basketter and Kimber, 2018).

 

The use of in silico methods as an alternative to using protected animals within research

Before a research project can begin it is required that a comprehensive project evaluation is conducted. This evaluation should consider ethical deliberations surrounding the use of animals for said project. The purpose of this ethical evaluation is to ensure that principles of the 3R’s (replacement, reduction and refinement) have been implemented within the study design (Guidance on the Operation of the Animals (Scientific Procedures) Act 1986., 2014). The 3Rs provide a useful strategy to rationally consider how animals are used in a project, without compromising the quality of the research being undertaken (Kandárová and Letašiová, 2011). Replacement is defined as a substitution of protected animals by non-sentient material or methods. Replacement does not necessarily mean that no animals are used at all. Alternative methods can be used to partially replace animals for example; replacing the use of animals for certain kind of test substances or for a particular range of toxicological hazard. Alternative methods implement the principles of reduction by decreasing the number of animals used within a project. Finally, refinement is defined as any decrease in the incidence or severity of procedures conducted for research. For example, in silico analysis may be able to predict the severity or lethality of a substance before it is tested on an animal. This allows researchers to refine their testing strategies, evidently reducing the amount of pain or suffering experienced by the animal (Mushtaq et al., 2018). In silico toxicology (IST) methods use computer resources to analyse, simulate, visualise or predict toxicity of chemicals and substances  (Myatt et al., 2018). IST methodologies aim to complement existing toxicology tests to potentially minimise the need for animal testing and improve toxicity prediction and safety assessment (Raies and Bajic, 2016). IST is a very fast expanding example of alternatives methods to animal testing used within the field of toxicology.

Toxicology testing is a necessary step in the process of all new product developments containing potentially hazardous chemicals or substances. For example; pharmaceutical drug design, food production, pesticide development and the making of cosmetic products. The developmental stage requires an analysis of different data sets to predict the toxicity of chemicals for potential new products. Data sets tend to fall into two main types of classification; single-label and multi-label classification (Yang et al., 2018). Single-label classification is used when each sample in a data set falls into only one class, or if the data set is binary and can be split into two classes. For example; carcinogenic or noncarcinogenic compounds. If a data set can be split into more than two classes, it then becomes a multi-class classification. An example of this would be classifying a compound on the degree of skin sensitisation (e.g. low, moderate, high) (Raies and Bajic, 2018). A study conducted by Zhang et al., (2015) describes the use of in silico methods used to predict chemical toxicity of pesticides within avian species. More than 663 diverse chemicals were assessed, and all chemicals were classified into three categories; highly toxic, slightly toxic and non-toxic. Although this study still used protected animals to obtain data, the prediction models significantly reduced the number of animals needed within the project. The high accuracy of the prediction models that were developed meant that pain and suffering was kept to a minimum as the highly toxic substances had already been predicted. The data that was recorded for the study can be used again for further avian toxicology research, reducing the number of animals needed in the future.

Although the development of in silico methods for toxicology testing is a seemingly popular and expanding field of research, there are some areas of limitation. There is a continuing need to ensure that new methods which are developed for use within toxicology are validated correctly. Clarity is needed about what to validate new methods against; is it already available test methods or accredited knowledge of adverse health effects of chemicals. A limitation to validating new test methods is the cost of potential time delays for the industry. For example, development of new in silico methods in parallel with traditional methods can add an extensive amount of time to a research project (Prior et al., 2019). Further limitations can include the need for whole organism interactions. Planchart et al., (2016) explains how zebra fish are still commonly used within toxicology as fish and humans share similar developmental and physiological responses to chemical exposures. Furthermore, although alternative models such as in silico are excellent for providing insight into reactions within specific areas of the body, there needs to be further development into in silico predictions regarding adverse effects to the whole organism. Although limitations are clearly apparent, in silico toxicology still has many benefits. Toxicology accounts for around 50% of failures in preclinical drug development, indicating the need for reliable prediction strategies to minimise these failures (Ford, 2016). A study conducted by Tambunan et al., (2019) explains how including in silico methods into their study design greatly reduced resource requirements in comparison to conventional methods. This is beneficial to the project as it saves time and expenses but also works in accordance with the 3Rs. Additionally, a differing study led by Passini et al., (2017) describes how in silico drug trials institute influential methodology to predict clinical risk of arrhythmias in human cardiotoxicity. The benefits shown by the methodology allow it to be integrated into existing cardiotoxicity assessment schemes, thus contributing to the reduction of animals used for experimentation.

Conclusion

Animal contributions to the field of toxicology research have provided extremely advantageous information and knowledge and have shaped the development of many life-saving drugs, safe and effective pesticides and many other advances in toxicology safety assessments. The use of in silico technology as an alternative to using animal models has been demonstrated as extremely effective and beneficial for both the research it has been involved with and to minimising the number of protected animals used within toxicology research, working in accordance with ASPA and respecting the 3Rs. Although in silico testing methods still must be used alongside animal models, the toxic predictability allows researchers to rapidly refine their studies. Predicting the toxicity of chemicals before they are tested on protected animals means that animals will experience less pain and suffering as researchers can already gauge how the animal may react to a certain chemical. Further research needs to be carried out to development more in silico models and testing methods to further increase the benefits in silico testing in the future, but needless to say in silico is an extremely positive alternative to using protected animals in research will continue to improve welfare and minimise the use of protected animals with research.

 

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