Integrating toxicokinetics into toxicology studies and the human health risk assessment process for chemicals: Reduced uncertainty, better health protection.

The database of practical examples where toxicokinetic (TK) data has benefitted all stages of the human health risk assessment process are increasingly being published and accepted. This review aimed to highlight and summarise notable examples and to describe the "state of the art" in this field. The overall recommendation is that for any in vivo animal study conducted, measurements of TK should be very carefully considered for inclusion as the numerous benefits this brings continues to grow, particularly during the current march towards animal free toxicology testing and ambitions to eventually conduct human health risk assessments entirely based upon non-animal methods.

Harmonized 3Rs-based non-mutagenic impurity qualification study designs developed using the results of an IQ consortium survey.

As per the ICH Q3A(R2) and Q3B(R2) regulatory guidelines, safety studies may be needed when an impurity in new drug substances or products is above the qualification threshold, and such qualification studies should be conducted in one nonclinical species for a duration of 14-90 days. However, the guidelines do not specify details about species selection, recommended study design, and the exact study duration that would support clinical use of a specific duration. This lack of guidance leads to ambiguity and sponsors have used various study designs to qualify impurities. In 2018, the European Medicines Agency provided a draft reflection paper encouraging the incorporation of 3Rs (Replacement, Reduction, and Refinement) principles for animal use into impurity qualification. As a response, the IQ DruSafe Impurity Working Group (WG) surveyed the IQ member companies to capture the current practices for impurity qualification, and evaluate study designs for a potential reduction in animal testing. This article summarizes the results and learnings from the survey. Additionally, the WG leveraged the survey learnings and provided harmonized study design considerations aimed towards achieving the study objectives, while supporting the 3Rs initiative in reducing the total number of animals used (up to 90%) for impurity qualification.

Considering aspects of the 3Rs principles within experimental animal biology.

The 3Rs - Replacement, Reduction and Refinement - are embedded into the legislation and guidelines governing the ethics of animal use in experiments. Here, we consider the advantages of adopting key aspects of the 3Rs into experimental biology, represented mainly by the fields of animal behaviour, neurobiology, physiology, toxicology and biomechanics. Replacing protected animals with less sentient forms or species, cells, tissues or computer modelling approaches has been broadly successful. However, many studies investigate specific models that exhibit a particular adaptation, or a species that is a target for conservation, such that their replacement is inappropriate. Regardless of the species used, refining procedures to ensure the health and well-being of animals prior to and during experiments is crucial for the integrity of the results and legitimacy of the science. Although the concepts of health and welfare are developed for model organisms, relatively little is known regarding non-traditional species that may be more ecologically relevant. Studies should reduce the number of experimental animals by employing the minimum suitable sample size. This is often calculated using power analyses, which is associated with making statistical inferences based on the P-value, yet P-values often leave scientists on shaky ground. We endorse focusing on effect sizes accompanied by confidence intervals as a more appropriate means of interpreting data; in turn, sample size could be calculated based on effect size precision. Ultimately, the appropriate employment of the 3Rs principles in experimental biology empowers scientists in justifying their research, and results in higher-quality science.

AAPS Perspective on the EURL Recommendation on the use of Non-Animal-Derived Antibodies.

In May 2020, the EU Reference Laboratory for alternatives to animal testing (EURL ECVAM) published a recommendation report entitled "Recommendation on nonanimal-derived antibodies". In this report, the EURL ECVAM specifically states: "Therefore, taking into consideration the ESAC Opinion on the scientific validity of replacements for animal-derived antibodies, EURL ECVAM recommends that animals should no longer be used for the development and production of antibodies for research, regulatory, diagnostic and therapeutic applications. The provisions of Directive 2010/63/EU should be respected, and EU countries should no longer authorise the development and production of antibodies through animal immunisation, where robust, legitimate scientific justification is lacking." (1). Here, we are providing the American Association of Pharmaceutical Scientists (AAPS) opinion on the EURL ECVAM recommendation report. In brief, there has been a clear and strong progress in reduction of animal use in the drug discovery and development process, including significant reduction of animal use in production of antibody reagents. Yet, it is proposed that more data need to be generated, shared and discussed within the scientific community before a decision to implement the change to non-animal derived antibodies is made.

3D cell culture models: Drug pharmacokinetics, safety assessment, and regulatory consideration.

Nonclinical testing has served as a foundation for evaluating potential risks and effectiveness of investigational new drugs in humans. However, the current two-dimensional (2D) in vitro cell culture systems cannot accurately depict and simulate the rich environment and complex processes observed in vivo, whereas animal studies present significant drawbacks with inherited species-specific differences and low throughput for increased demands. To improve the nonclinical prediction of drug safety and efficacy, researchers continue to develop novel models to evaluate and promote the use of improved cell- and organ-based assays for more accurate representation of human susceptibility to drug response. Among others, the three-dimensional (3D) cell culture models present physiologically relevant cellular microenvironment and offer great promise for assessing drug disposition and pharmacokinetics (PKs) that influence drug safety and efficacy from an early stage of drug development. Currently, there are numerous different types of 3D culture systems, from simple spheroids to more complicated organoids and organs-on-chips, and from single-cell type static 3D models to cell co-culture 3D models equipped with microfluidic flow control as well as hybrid 3D systems that combine 2D culture with biomedical microelectromechanical systems. This article reviews the current application and challenges of 3D culture systems in drug PKs, safety, and efficacy assessment, and provides a focused discussion and regulatory perspectives on the liver-, intestine-, kidney-, and neuron-based 3D cellular models.

Possibility of Immediate Introduction of a Single-Dose Antibody Induction Test as a Refinement of the NIH Test for Inactivated Rabies Vaccine Potency Determination.

Antibody induction test (AIT) is a promising candidate as a refinement of the troublesome National institutes of Health (NIH) test in the sense of animal welfare 3R approach for determination of potency of inactivated rabies vaccines for veterinary and human use. In this study, we initially try to develop AIT as a suitable alternative to NIH test, to achieve a reduction of test duration and diminish animal suffering by omitting intracerebral CVS infection and measuring humoral immunity upon vaccination. Designs of both multi-dose and single-dose AIT were examined. Biological reference preparation, batch 5 with assigned titer of 10 IU/vial, was taken as both standard and test vaccine. Six consecutive AITs were performed and eight pools of sera in each AIT were tested in triplicate by rapid fluorescent focus inhibition test. We estimated the upper detection limit and calculated test variability for individual dilutions. For multi-dose AIT, we estimated the dose-response function and performed calculations of final test results and statistical validity parameters for both linear and sigmoidal model using CombiStats program. Sigmoidal 4-parameter dose-response model was found optimal. Presented design of multi-dose AIT showed a satisfactory detection limit for testing of inactivated rabies vaccines for both veterinary and human use. However, due to nonconformity of obtained results with statistical validity criteria, we concluded that the presented model of multi-dose AIT was unsuitable for introduction in routine practice. However, we concluded that there was a realistic option for introduction of two versions of single-dose AIT. The first version would be with two standard vaccine controls and could be introduced immediately, while the second version would include testing of the sample only and rely on comparison of the induced rabies antibody level with absolute cut-off limits set in advance.

Gonadal sex and animal experimentation: Perfection vs. 3R principle?

Replicability of experimental results and optimal use of experimental animals are everybody's concern. Current efforts towards increased replicability include guidelines and checklists as tools for experimenters, referees, editors and publishers. Guidelines are also provided for appropriate use of animals. To ensure the quality of experimental results, the number of animals must be adequate, that is, sufficiently large, for the purpose of the given experiment. To comply with current ethical recommendations, the use of animals should be reduced as much as possible. Therefore, determination of the number of animals for a given scientific objective includes contrasting considerations. Current guidelines for animal experimentation, notably from the National Institute of Health, mandate (with very few exceptions) inclusion of animals of both sexes in experimental designs statistically powered to address the difference between the two groups. Notably, absence of evidence for sex differences between the organ or system functions under study does not qualify as an exception. Mandatory, equal representation of both sexes raises several questions including ethical ones. Other guidelines, by public regulators and major publishers, do not seem to have a similar selective focus on sex differences. In summary, current concerns about replicability of scientific results are justified. Concomitantly, the knowledge of sex differences also between non-reproductive, non-endocrine organ functions is increasing. In principle, sex matters in any experimental context. However, an indiscriminate demand for inclusion of both sexes in all experimental protocols seems a waste of animals, money and time, violating traditional principles of animal experimentation, particularly that of reduction.

More is less: reducing animal use by raising awareness of the principles of efficient study design and analysis.

Good experimental design and the appropriate use of statistical tests form the corner stone of high-quality scientific research. This is especially important when the experiments involve the use of laboratory animals, to ensure that their use is appropriate and that the minimum number of animals will be used that will provide data which are sufficiently statistically-sound to meet the objectives of the study. One way to raise awareness of the importance of efficient study design and analysis is to provide training courses. This paper reports the views of participants at two such training schools, with reference to why they felt that attendance was necessary and how effective they felt the experience had been. The implications of the responses are discussed, and considerations for future training events are noted.

Safety testing of acellular pertussis vaccines: Use of animals and 3Rs alternatives.

The current test of acellular Bordetella pertussis (aP) vaccines for residual pertussis toxin (PTx) is the Histamine Sensitization test (HIST), based on the empirical finding that PTx sensitizes mice to histamine. Although HIST has ensured the safety of aP vaccines for years, it is criticized for the limited understanding of how it works, its technical difficulty, and for animal welfare reasons. To estimate the number of mice used worldwide for HIST, we surveyed major aP manufacturers and organizations performing, requiring, or recommending the test. The survey revealed marked regional differences in regulatory guidelines, including the number of animals used for a single test. Based on information provided by the parties surveyed, we estimated the worldwide number of mice used for testing to be 65,000 per year: ∼48,000 by manufacturers and ∼17,000 by national control laboratories, although the latter number is more affected by uncertainty, due to confidentiality policies. These animals covered the release of approximately 850 final lots and 250 in-process lots of aP vaccines yearly. Although there are several approaches for HIST refinement and reduction, we discuss why the efforts needed for validation and implementation of these interim alternatives may not be worthwhile, when there are several in vitro alternatives in various stages of development, some already fairly advanced. Upon implementation, one or more of these replacement alternatives can substantially reduce the number of animals currently used for the HIST, although careful evaluation of each alternative's mechanism and its suitable validation will be necessary in the path to implementation.

Anticoccidial efficacy testing: In vitro Eimeria tenella assays as replacement for animal experiments.

SCOPE: Availability of an accurate in vitro assay is a crucial demand to determine sensitivity of Eimeria spp. field strains toward anticoccidials routinely. In this study we tested in vitro models of Eimeria tenella using various polyether ionophores (monensin, salinomycin, maduramicin, and lasalocid) and toltrazuril. Minimum inhibitory concentrations (MIC95, MIC50/95) for the tested anticoccidials were defined based on a susceptible reference (Houghton strain), Ref-1. In vitro sporozoite invasion inhibition assay (SIA) and reproduction inhibition assay (RIA) were applied on sensitive laboratory (Ref-1 and Ref-2) and field (FS-1, FS-2, and FS-3) strains to calculate percent of inhibition under exposure of these strains to the various anticoccidials (%ISIA and%IRIA, respectively). The in vitro data were related to oocyst excretion, lesion scores, performance, and global resistance indices (GI) assessed in experimentally infected chickens. RESULTS: Polyether ionophores applied in the RIA were highly effective at MIC95 against Ref-1 and Ref-2 (%IRIA≥95%). In contrast, all tested field strains displayed reduced to low efficacy (%IRIA<95%).%IRIA values significantly correlated with oocyst excretion determined in the animal model (p<0.01) for polyether ionophores. However, this relationship could not be demonstrated for toltrazuril due to unexpected lack of in vitro sensitivity in Ref-2 (%IRIA=56.1%). In infected chickens, toltrazuril was generally effective (GI>89%) against all strains used in this study. However, adjusted GI (GIadj) for toltrazuril-treated groups exhibited differences between reference and field strains which might indicate varying sensitivity. CONCLUSION: RIA is a suitable in vitro tool to detect sensitivity of E. tenella towards polyether ionophores, and may thus help to reduce, replace, or refine use of animal experimentation for in vivo sensitivity assays.

Revision of the ICH guideline on detection of toxicity to reproduction for medicinal products: SWOT analysis.

SWOT analysis was used to gain insights and perspectives into the revision of the ICH S5(R2) guideline on detection of toxicity to reproduction for medicinal products. The current ICH guideline was rapidly adopted worldwide and has an excellent safety record for more than 20 years. The revised guideline should aim to further improve reproductive and developmental (DART) safety testing for new drugs. Alternative methods to animal experiments should be used whenever possible. Modern technology should be used to obtain high quality data from fewer animals. Additions to the guideline should include considerations on the following: limit dose setting, maternal toxicity, biopharmaceuticals, vaccines, testing strategies by indication, developmental immunotoxicity, and male-mediated developmental toxicity. Emerging issues, such as epigenetics and the microbiome, will most likely pose challenges to DART testing in the future. It is hoped that the new guideline will be adopted even outside the ICH regions.

The need for a formal invalidation process for animal and non-animal tests.

A plethora of regulations require that many chemicals and chemical products are tested for efficacy and/or toxicity. When permitted to operate effectively and without bias, the ECVAM/ICCVAM/OECD validation process can be used to independently establish that new animal and non-animal test procedures are sufficiently relevant and reliable for their stated purposes and should be considered for regulatory use. However, the validation process is under threat because of vested interests of various kinds, and it is clear that many currently-accepted animal tests and candidate animal and non-animal tests do not, and could never, meet the agreed criteria for necessity, test development, prevalidation, validation and acceptance. We therefore need an invalidation process to parallel and protect the validation process, so that such methods could be independently reviewed and declared irrelevant and/or unreliable for their claimed purposes. An additional advantage of such a process would be that valuable resources would no longer be wasted in attempts to secure the acceptance of inherently inadequate tests.

The '3Rs' model and the concept of alternatives in animal research: a questionnaire survey.

It has been 45 years since Russell and Burch first proposed the concept of the '3Rs', yet it remains unclear how those individuals involved in animal research view and implement these concepts. The authors used a questionnaire survey to determine how well-known experts judged issues related to the 3Rs.