Why are two techniques that can help reduce our reliance on research animals so underutilized?

Two important safety assessment tools that are also helpful in meeting 3Rs objective are microsampling and the extended one-generation reproductive toxicity study. One helps to reduce the number of animals needed for screening, the other both reduces and refines our use of animals. Yet they remain underutilized by laboratories. Alan Hoberman, Executive Director, Global Development, Reproductive and Juvenile Toxicology at Charles River Laboratories, discusses why.

How are newer techniques, like the extended one-generation reproductive toxicity study and microsampling, helping us to meet our 3Rs objectives?

AH: While the the ultimate goal of 3Rs is replacement, reduction and refinement are the interim steps that reduce animal usage while improving quality of the information we obtain from our animal model. The extended one-generation reproductive toxicity study (EOGRTS) is designed to reduce the number of animals required for screening the toxicity of a chemical, utilize the animals more effectively and reduce the time required to develop our hazard assessment. The EOGRTS is a refinement on the multigenerational study and reduces the number of animals, while completely eliminating the need (reduction) for a separate developmental neurtoxicity and immunotoxicity study. The incentive to conducting the EORGTS for the manufacturer of chemical is a reduction in time, money in addition to animal welfare.

Microsampling, is defined as a blood sample less than 60 uL as opposed to a typical blood sample of 500 uL (0.5 mL) to allow for analysis of exposure (amount of drug or metabolite in the blood) or evaluation of a biomarker in the blood.  Similar to the EOGRTS, the addition of microsampling to a study focuses on reduction and refinement. In small animals it can allow for a complete toxicokinetic profile (exposure overtime) from a single animal (reduction) as opposed to a single animal being needed for each time point. It is a refinement for blood collection in large animals and even humans when a pin prick can be used to obtain a blood sample as opposed to a needle stick needed to collect the blood sample. For both small and large animals, microsampling allows exposure or a biomarker to be evaluated in the same animal that is being used to evaluate the toxicity of test material. This is better science while eliminating the need for large numbers of satellite animals for blood collection.

The EOGRTS has been slow to take hold. Why is this so?

AH: Because EOGRTS do not replace animals and while comprehensive it is also very expensive to do. Although it is very comprehensive, it is only conducted when required by regulatory authorities. In fact, under the European Union (EU) regulations known as REACH (Registration, Evaluation, Authorisation and Restriction of Chemicals), animal testing cannot be conducted until required or mandated by the EU. So when appropriate the study will reduce animal usage, but not having to conduct the study because other studies do not indicate a need is still less expensive for a manufacturer.

How widely used is microsampling?

AH: The drug development process has been divided into various phases including discovery, non-clinical testing and clinical testing. Microsampling has been used in the discovery phase for many years and was always considered very appropriate for proof-of-concept studies, metabolic profiling and subsequent refinement of a molecule for candidate selection to move into the clinic. But clinical candidates (potential new drugs) require validated methods of analysis for both non-clinical (animal studies) and human studies. Until about 15 years ago, the validated method was always for a large blood volume (0.5mL) as humans have plenty of blood for analysis and the 3Rs were not a major concern to most pharmaceutical companies. More recently the advantages of microsampling have been realized in terms of 3Rs and reduction in costs for the amount of test material required and the possibility of human subjects taking their own blood samples instead of coming into a clinic. Several large pharmaceutical companies have gone completely toward microsampling. Charles River has been promoting the technique with medium and small pharma and just last year the International Conference of Harmonization (ICH) issued a guidance that promoted microsampling. This should speed the acceptance of the technique. 

Are the 3Rs still an important guideline for animal research today? Why?

AH: The transition to in silico and human cells for testing will involve the continued use of animals in research.  Therefore the 3Rs will remain important in our thinking for several reasons including the importance of understanding how a chemical/drug interacts with gene, cells, tissues and organs in the fewest number of animals, with the best techniques that will provide the data to lead to replacement. It has been and will remain very important to conduct our work in the best models that give us the best data and this requires animals that are not stressed and do not suffer from our procedures.

How do you think the 3Rs has changed animal research in the last 50 years?

AH: Since 1959 when Russell and Burch proposed the concept of 3Rs, the realization that these principles are improving our testing methods and the quality of data has become fundamental to the design of our experiments.  The incorporation 3R principles into the regulations that govern our directed research has helped to speed products to the market while ensuring the safe use of these products. The 3Rs have forced us to use the best models available and search for better models more appropriate for the development of drugs. It has always been realized that animals are not the best models but they have been the appropriate model whose real value is to direct us to better models.