The story of the possible replacement of traditional toxicology approaches with more integrated and modern methods started many years ago with the declaration of the 3Rs Principle.
In 1959 two Academics, Rex Burch and William Russell, Members of the University Federation of Animal Welfare (USA), published a book that will become, in the following years, a milestone in the animal welfare policy, “The Principles of Human Experimental Technique”. In such a book the two scientists presented a road map addressed to practice an animal experimentation that will take into account both the data quality and the welfare of animals used in testing. Their approach is still known as the 3R principles where 3Rs means:
Replacement: “any scientific method employing non sentient material which may, in the history of experimentation, replace methods which use conscious living vertebrates;
Reduction: “reduction in the number of animals used to obtain information of a given amount and precision”;
Refinement: “simply to reduce to an absolute minimum the amount of stress imposed on those animals that are still used”.
Continues advancements in toxicological science, bioinformatics and biology have provided a number of tools to commute the traditional toxicology (based mainly on “in vivo” animal experimental studies) into a “predictive science”.
Traditional animal models are approximate models for human toxic responses: using their knowledge about physical, chemical and biological processes, the actual view adopt a hypothesis-driven, multi-disciplinary approach integrating “in vitro” methods with computational based methodologies (“in silico”) together with ‘omics’ approaches (e.g. proteomics, genomics and metabolomics, promising approaches to study impact of toxicants on expression of genes, proteins and cellular metabolites). This is aimed at identifying potential toxicants on the basis on an understanding of their mechanisms of biological action. These methods, sometimes referred to as “non-testing methods”, can be used to reduce our reliance on experimental testing, and in particular animal testing. In practice, these methods are used in Integrated Testing Strategies (ITS), along with experimental data generated by alternative (non-animal) tests, such as in vitro tests and high throughput screening.
Along the last four decades, many efforts were done by the scientific community to apply the 3Rs principles and, at the end, reduce the “in vivo” animal use in the experimental science.
A good number of “in vitro” studies are now available in GLP testing laboratories. Most of them cover the local effects end-point such as corrosion, irritation (skin and eye) as well as sensitization potential and cytotoxicity. The “in vitro” packages are considered by EU regulations an indispensable step of the safety research before coming to the “in vivo” ones. We’d like to remind that suitable “in vitro” studies need to be validated by EURL ECVAM (EU Reference Laboratory for Alternatives to Animal testing) before getting the status of recognized alternative method/protocol to standard “in vivo” test. An update on validated studies can be found in its web site (1)
On the other hand, important regulations include such principles in their approaches.
The Directive on the protection of animals used for scientific purposes (2010/63), the Regulation on cosmetic products (1223/2009), REACH (2007/2006) and Classification, Labeling and Packaging (CLP) (1272/2008) are examples of EU legislation that require, or strongly encourage, the replacement of animal testing and , of course, the REACH Regulation (EC 1907/2006).The EU Regulation on chemicals and their safe use – REACH (Registration, Evaluation, Authorization and Restriction of Chemical substances) – which came into force in 2007 has implications for the testing methods used for evaluating the effects of chemical substances. To minimize the number of animal tests required in implementing the REACH Regulation, provision has been made to provide a number of possibilities for adapting the testing requirements, and to use existing data and alternative assessment approaches instead. Even more… REACH Art. 117(3) claim that “Every three years the Agency, in accordance with the objective of promoting non-animal testing methods, shall submit to the Commission a report on the status of implementation and use of non-animal test methods and testing strategies used to generate information on intrinsic properties and for risk assessment to meet the requirements of this Regulation”
The use of alternatives to animal testing strictly depends on the specific toxicological end-points and reflects the availability of suitable methods (e.g. QSAR) to predict such end-point. It has to be noted that an important part of end-points was covered by Read Across and Weight of Evidence (WoE) approaches; smaller part by QSAR. Many were omitted probable on scientific waiving criteria application or not reported.
What are the numbers in EU
Based on EU Commission information (European Commission, C (2023) 5041, final July 25, 2023) the number of animals used in the experimental research or for regulatory purposes is 7.9 millions (M) in 2020; a still high number even though lower than in the previous years (8.9M in 2019, 8.8M in 2018). 72% (6.37M) of these are used for research purposes; 41% (2.60M) for basic research and 31% (1.96M) for applied and translational research. The animals used for regulatory purposes represents the 17% of the total, i.e. 1.4 M animals. Of these, 54% for human medicinal development; 22.8% for veterinary drug development and 8.7% for chemical safety studies. Much less are the percentages values for other applications: 2.8% for feed products; 4,8% for Plant Protection Products; 3.6% for Medical Devices; 0.3% for biocidal products, 3% for other purposes. Species more used are mouses (49%) and fishes (27%) and others normally used in experimental toxicology studies.
A recent European Citizens Initiative (ECI) has driven attention again on the need to improve animal experimentation alternatives saying that despite the huge public investment made by EU Commission in the last 20 years (more than 1 Billion Euro) the progresses aimed to replace live animal experimentation are modest and the road to reach this target is still long and difficult.
The EU Commission answered on July 25, 2023 (see previous reference) saying that while sharing the overall target to avoid any live animal in the regulatory research, such target must be pursued in the long term and by consecutive steps carefully evaluating, on a scientific basis, the risk posed by non-animal testing. EU Commission believes that animal experimentation is still an acceptable and good tool in the short mid-term to evaluate the safety of chemical substances for the human health and the environment. The REACH regulation revision, expected for 2024, will be a good occasion to start a new process of replacement. Furthermore, a strong cooperation between the major EU agencies (ECHA, EFSA and EMA) will be crucial to get the utmost results. In any case EU Commission has announced the preparation of a new Road Map to reach to an animal-free regulatory system on a long-term perspective.
Furthermore, EU Commission claimed that among the investment for the replace of “in vivo” animal studies, new methods for the evaluation of the hazard/risk of chemical substances have been recently developed or are under development. This attitude is in line with the principles of the EU Green Deal project and the Chemical Strategy for Sustainability (CSS). These new approaches include the NAM (New Alternative Methodologies) and Next Generation Risk Assessment (NGRA). Both these approaches will encourage much more the use of alternative methods which include “in vitro” testing and, in particular “in silico” approaches. These last are following the implementation of the safe and sustainable by design approach.
EURL ECVAM (EU Reference Laboratory of the European Center for validation of Alternative Methods) worked very hard in the last 25 years to propose and validate new testing approaches. The areas primarily involved are the cytotoxicity, the skin sensitization and genotoxicity which are still under development. A key area is the Endocrine disrupting properties evaluation; work is in progress for methods assessing the impact of the thyroid endocrine system, an endpoint which will be crucial for the evaluation of chemical substance safety in the next future.
Conclusion
Even though there are strong and totally shared ethical reasons to replace and definitively abandon the animal use in the chemical safety assessment approaches and, in general, in the regulatory research, some critical points still remain and need to be underlined:
– a strong need to assess the quality of the alternative method used. It’s well known that some read across approaches (particularly of the beginning of the Reach registration process) were very weak and not sufficiently scientific robust. ECHA insisted on this point issuing a detailed and specific guideline some time ago;
- it’s known that some waiving criteria were not scientifically supported. The lack of data/studies on a specific key end-point (e.g. reproductive toxicity) led to lack of classification with huge impact on human health and/or environment safety;
- there is a difficulty in assessing the Integrated Testing Strategy as very complex. Furthermore experts are strongly needed;
- for some end-points, the toxicological approaches need to be integrated with the MoA (Mechanism of Action) evaluation (e.g EDS). Again, this is a scientific multidisciplinary approach that needs scientific and economic resources;
- the costs of alternative methods are not comparable to the “old” toxicological studies; therefore, there is a sort of “motivation” to adopt such methods instead of the traditional ones even if the final results are not so reliable (see point one in conclusion);
- computational methods are under a great rate of update and scientific evolution: hence there is a constant need to be up to date with the latest possibilities and integrate them into our evaluation;
- NAMs and NGRA will represent valid tools to face the risk assessment on chemical but probably they need a further development to be definitively adopted. They have been evaluated by PARERE (Preliminary Assessment of Regulatory Relevance) in the light to update the REACH Regulation introducing such new approaches. During 2024 news are expected in this extend.
- last but not least, we all need to be sure that new alternative methods can be utmost reliable to assure the proper results. At the end, based on these methods we are evaluating impacts on our health and the safety of environment.
In conclusion, we can say that a lot of progresses from a scientific and regulatory point of view are expected in the 2024-2025 period regarding the introduction of new alternative methods. Obviously, all methods must be validated to give reliable results leading to a proper classification and risk management of chemical substances. On the other way, Authorities and EU Agency should help to further implement the data sharing procedure between companies (which is already working with some regulation _ REACH and BIOCIDES mainly). An efficient data sharing procedure will reduce the animal use avoiding duplications. Last but not least, it must be said that all EU policies on the safety for chemical substances (including medicines) will be probably affected and determined by political directions of the new EU Commission which will be appointed following the political election of June 2024. Hence, an update will be needed at the end of 2024.
REFERENCES AND NOTES
- https://ec.europa.eu/jrc/en/eurl/ecvam/alternative-methods-toxicity-testing/validated-test-methods
