Advances in Animal Models and Cutting-Edge Research in Alternatives: Proceedings of the Third International Conference on 3Rs Research and Progress, Vishakhapatnam, 2022.

Animal experimentation has been integral to drug discovery and development and safety assessment for many years, since it provides insights into the mechanisms of drug efficacy and toxicity (e.g. pharmacology, pharmacokinetics and pharmacodynamics). However, due to species differences in physiology, metabolism and sensitivity to drugs, the animal models can often fail to replicate the effects of drugs and chemicals in human patients, workers and consumers. Researchers across the globe are increasingly applying the Three Rs principles by employing innovative methods in research and testing. The Three Rs concept focuses on: the replacement of animal models (e.g. with in vitro and in silico models or human studies), on the reduction of the number of animals required to achieve research objectives, and on the refinement of existing experimental practices (e.g. eliminating distress and enhancing animal wellbeing). For the last two years, Oncoseek Bio-Acasta Health, a 3-D cell culture-based cutting-edge translational biotechnology company, has organised an annual International Conference on 3Rs Research and Progress. This series of global conferences aims to bring together researchers with diverse expertise and interests, and provides a platform where they can share and discuss their research to promote practices according to the Three Rs principles. In November 2022, the 3rd international conference, Advances in Animal Models and Cutting-Edge Research in Alternatives, took place at the GITAM University in Vishakhapatnam (AP, India) in a hybrid format (i.e. online and in-person). These conference proceedings provide details of the presentations, which were categorised under five different topic sessions. It also describes a special interactive session on in silico strategies for preclinical research in oncology, which was held at the end of the first day.

Support for Regulatory Assessment of Percutaneous Absorption of Retronecine-type Pyrrolizidine Alkaloids through Human Skin.

1,2-unsaturated pyrrolizidine alkaloids are found naturally in Symphytum officinale, well known as comfrey, which has a longstanding use for the topical treatment of painful muscle and joint complaints. Pyrrolizidine alkaloids (PA) are a relevant concern for the safety assessment due to their liver genotoxicity profile, and close attention is paid during manufacturing to minimizing their levels. Current regulatory risk assessment approaches include setting limits that derive from toxicity data coming from the oral route of exposure. This study investigated to what extent pyrrolizidine alkaloids are bioavailable following topical exposure, assessing penetration of retronecine-type PAs in an in vitro human skin model. A single comfrey root formulation was spiked with 3 different congeners (a 7R-monoester, an open-chained 7R-diester, and a cyclic diester) and percutaneous absorption measured per OECD guidelines and good laboratory practices. The measured penetration for all 3 PAs was low and compared favourably with existing in vitro data. Although consideration of different regulatory guidance influences the determination of dermally absorbed dose, these data facilitate the understanding of absorption differences following topical exposure, which in turn can be taken into account in the risk assessment.

Evaluation of the toxicity of sodium dodecyl sulphate (SDS) in the MucilAir™ human airway model in vitro.

The aim of the study was to use multiple in vitro assays to assess the effects of a model irritant, sodium dodecyl sulphate (SDS) (≤10 mM (0.29 %, w/v)), on an in vitro model of the airway, MucilAir™. The use of MucilAir™ in recovery studies was also explored. A 24 h exposure increased IL-8 release at an SDS concentration ≥0.63 mM (0.018 %, w/v). Mucin secretion increased and transepithelial electrical resistance (TEER) decreased at SDS concentrations ≥1.25 mM (0.04 %, w/v). Cytotoxicity (lactate dehydrogenase (LDH) release into basolateral chamber) was observed at SDS concentrations of ≥2.5 mM (0.07 %, w/v). The sensitivity of the assays was IL-8 release > TEER = mucin secretion > LDH release. After 7 days, full or partial recovery was observed for intermediate concentrations of SDS using all assays but not at 5 and 10 mM SDS. Morphologically, erosion and cell loss were observed at these concentrations. Resazurin metabolism at 7 days tended to decrease in a dose-dependent manner at SDS concentrations above 2.5 mM (0.07 %, w/v). Together, these data support a No Observable Effect Level of 0.31 mM (0.009 % w/v) SDS and the use of MucilAir™ as a relevant model for airway toxicity studies.

Towards More Predictive, Physiological and Animal-free In Vitro Models: Advances in Cell and Tissue Culture 2020 Conference Proceedings.

Experimental systems that faithfully replicate human physiology at cellular, tissue and organ level are crucial to the development of efficacious and safe therapies with high success rates and low cost. The development of such systems is challenging and requires skills, expertise and inputs from a diverse range of experts, such as biologists, physicists, engineers, clinicians and regulatory bodies. Kirkstall Limited, a biotechnology company based in York, UK, organised the annual conference, Advances in Cell and Tissue Culture (ACTC), which brought together people having a variety of expertise and interests, to present and discuss the latest developments in the field of cell and tissue culture and in vitro modelling. The conference has also been influential in engaging animal welfare organisations in the promotion of research, collaborative projects and funding opportunities. This report describes the proceedings of the latest ACTC conference, which was held virtually on 30th September and 1st October 2020, and included sessions on in vitro models in the following areas: advanced skin and respiratory models, neurological disease, cancer research, advanced models including 3-D, fluid flow and co-cultures, diabetes and other age-related disorders, and animal-free research. The roundtable session on the second day was very interactive and drew huge interest, with intriguing discussion taking place among all participants on the theme of replacement of animal models of disease.

Application of in vitro skin penetration measurements to confirm and refine the quantitative skin sensitization risk assessment of methylisothiazolinone.

Use of quantitative risk assessment (QRA) for assessing the skin sensitization potential of chemicals present in consumer products requires an understanding of hazard and product exposure. In the absence of data, consumer exposure is based on relevant habits and practices and assumes 100% skin uptake of the applied dose. To confirm and refine the exposure, a novel design for in vitro skin exposure measurements was conducted with the preservative, methylisothiazolinone (MI), in beauty care (BC) and household care (HHC) products using realistic consumer exposure conditions. A difference between measured exposure levels (MELs) for MI in leave-on versus rinse-off BC products, and lower MELs for MI in HHC rinse-off compared to BC products was demonstrated. For repeated product applications, the measured exposure was lower than estimations based on summation of applied amounts. Compared to rinse-off products, leave-on applications resulted in higher MELs, correlating with the higher incidences of allergic contact dermatitis associated with those product types. Lower MELs for MI in rinse-off products indicate a lower likelihood to induce skin sensitization, also after multiple daily applications. These in vitro skin exposure measurements indicate conservatism of default exposure estimates applied in skin sensitization QRA and might be helpful in future risk assessments.

Use of an in vitro human skin permeation assay to assess bioequivalence of two topical cream formulations containing butenafine hydrochloride (1%, w/w).

The primary objective of this work was to investigate, using an in vitro human skin permeation study, whether changes in the excipients of butenafine hydrochloride cream would have any effect on bioperformance of the formulation. Such in vitro data would be a surrogate for any requirement of a bioequivalence (BE) study to demonstrate formulation similarity. A LC-MS/MS method for quantitation of butenafine in various matrices was developed and validated. A pilot study was performed to validate the in vitro skin permeation methodology using three cream formulations containing butenafine hydrochloride at concentrations of 0.5, 1.0 and 1.5% (w/w). Finally, a definitive in vitro human skin permeation study was conducted, comparing the extent of butenafine hydrochloride permeation from the new formulation to that from the current formulation. The results of the study comparing the two formulations showed that there was no statistically significant difference in the extent of butenafine permeation into human skin. In conclusion, these in vitro data demonstrated that the formulation change is likely to have no significant impact on the bioperformance of 1% (w/w) butenafine hydrochloride cream.

Assessing the safety of cosmetic chemicals: Consideration of a flux decision tree to predict dermally delivered systemic dose for comparison with oral TTC (Threshold of Toxicological Concern).

Threshold of Toxicological Concern (TTC) aids assessment of human health risks from exposure to low levels of chemicals when toxicity data are limited. The objective here was to explore the potential refinement of exposure for applying the oral TTC to chemicals found in cosmetic products, for which there are limited dermal absorption data. A decision tree was constructed to estimate the dermally absorbed amount of chemical, based on typical skin exposure scenarios. Dermal absorption was calculated using an established predictive algorithm to derive the maximum skin flux adjusted to the actual 'dose' applied. The predicted systemic availability (assuming no local metabolism), can then be ranked against the oral TTC for the relevant structural class. The predictive approach has been evaluated by deriving the experimental/prediction ratio for systemic availability for 22 cosmetic chemical exposure scenarios. These emphasise that estimation of skin penetration may be challenging for penetration enhancing formulations, short application times with incomplete rinse-off, or significant metabolism. While there were a few exceptions, the experiment-to-prediction ratios mostly fell within a factor of 10 of the ideal value of 1. It can be concluded therefore, that the approach is fit-for-purpose when used as a screening and prioritisation tool.

Skin barrier function for regulatory skin absorption tests and effects on testosterone and sucrose absorption.

In vitro absorption through human skin is a critical component in the safety assessment of chemicals, crop protection products, consumer healthcare products and cosmetics. A barrier integrity assay is used to identify skin samples which are potentially damaged. A retrospective analysis of 9978 electrical resistance (ER) measurements generated in a single laboratory (DTL) over a 15-year period was performed. Skin absorption experiments were performed using two model penetrants, testosterone and sucrose, utilising no ER acceptance criteria, and the results assessed. Using a barrier integrity test, to remove potentially damaged samples, was offset against one that can be used to remove intact skin samples with a poorer barrier function (i.e. false positives). The previously identified barrier integrity limit (10 kΩ for a 2.54 cm2 diffusion cell; Davies et al., 2004) was demonstrated to identify half of all samples tested, many of which would be false positive samples. This retrospective analysis identified 5.0 kΩ (17.5th percentile) as an acceptance criterion for a 2.54 cm2 diffusion cell, whilst not considerably changing results generated in skin absorption studies. This was confirmed from the cumulative absorption of the model penetrants tested. Using this limit would, therefore, provide suitable skin samples for regulatory skin absorption studies.

A comparative study of the in vitro dermal absorption of radiolabeled benzophenone through human skin.

Octocrylene is a common sun filter ingredient used to protect the skin from damaging UV rays. Benzophenone is an impurity found in formulations containing octocrylene. [14C]-Benzophenone was spiked (0.1 g/L) into 2 commercial sunscreen formulations; Neutrogena® Beach Defense Sunscreen Spray Broad Spectrum SPF 70 Aerosol, Neutrogena® Ultra Sheer Body Mist Sunscreen Broad Spectrum SPF 30 Aerosol, and an acetone vehicle. The formulations were applied (ca 2 µL/cm2) to dermatomed human skin mounted in static diffusion cells in vitro. Receptor fluid was collected up to 24 h post dose. All samples were analyzed by liquid scintillation counting. The dermal delivery of [14C]-Benzophenone was 10.02, 9.04 and 5.19% for the 3 formulations. However, the [14C]-Benzophenone mass balances were low; 81.5, 85.3 and 8.02%, respectively. A volatility test was performed replacing skin with aluminum foil for the sunscreen formulations only. The [14C]-Benzophenone mass balance at dosing was 99% but fell to 56.9 and 60.6% at 24 h post dose, confirming the losses were due to [14C]-Benzophenone volatility. A conservative dermal absorption value of 12.42% was proposed to cover [14C]-Benzophenone containing formulations.

New approach methodologies (NAMs): identifying and overcoming hurdles to accelerated adoption.

New approach methodologies (NAMs) can deliver improved chemical safety assessment through the provision of more protective and/or relevant models that have a reduced reliance on animals. Despite the widely acknowledged benefits offered by NAMs, there continue to be barriers that prevent or limit their application for decision-making in chemical safety assessment. These include barriers related to real and perceived scientific, technical, legislative and economic issues, as well as cultural and societal obstacles that may relate to inertia, familiarity, and comfort with established methods, and perceptions around regulatory expectations and acceptance. This article focuses on chemical safety science, exposure, hazard, and risk assessment, and explores the nature of these barriers and how they can be overcome to drive the wider exploitation and acceptance of NAMs. Short-, mid- and longer-term goals are outlined that embrace the opportunities provided by NAMs to deliver improved protection of human health and environmental security as part of a new paradigm that incorporates exposure science and a culture that promotes the use of protective toxicological risk assessments.

Evaluation of in vitro rat and human airway epithelial models for acute inhalation toxicity testing.

In vivo models (mostly rodents) are currently accepted by regulatory authorities for assessing acute inhalation toxicity. Considerable efforts have been made in recent years to evaluate in vitro human airway epithelial models (HAEM) as replacements for in vivo testing. In the current work, an organotypic in vitro rat airway epithelial model (RAEM), rat EpiAirway, was developed and characterized to allow a direct comparison with the available HAEM, human EpiAirway, in order to address potential interspecies variability in responses to harmful agents. The rat and human models were evaluated in 2 independent laboratories with 14 reference chemicals, selected to cover a broad range of chemical structures and reactive groups, as well as known acute animal and human toxicity responses, in 3 replicate rounds of experiments. Toxicity endpoints included changes in tissue viability (MTT assay), epithelial barrier integrity (TEER, transepithelial electrical resistance), and tissue morphology (histopathology). The newly developed rat EpiAirway model produced reproducible results across all replicate experiments in both testing laboratories. Furthermore, a high level of concordance was observed between the RAEM and HAEM toxicity responses (determined by IC25) in both laboratories, with R2=0.78 and 0.88 when analyzed by TEER; and R2=0.92 for both when analyzed by MTT. These results indicate that rat and human airway epithelial tissues respond similarly to acute exposures to chemicals. The new in vitro RAEM will help extrapolate to in vivo rat toxicity responses and support screening as part of a 3Rs program.

Incorporating new approach methodologies into regulatory nonclinical pharmaceutical safety assessment.

New approach methodologies (NAMs) based on human biology enable the assessment of adverse biological effects of pharmaceuticals and other chemicals. Currently, however, it is unclear how NAMs should be used during drug development to improve human safety evaluation. A series of 5 workshops with 13 international experts (regulators, preclinical scientists, and NAMs developers) was conducted to identify feasible NAMs and to discuss how to exploit them in specific safety assessment contexts. Participants generated four "maps" of how NAMs can be exploited in the safety assessment of the liver, respiratory, cardiovascular, and central nervous systems. Each map shows relevant endpoints measured and tools used (e.g., cells, assays, platforms), and highlights gaps where further development and validation of NAMs remains necessary. Each map addresses the fundamental scientific requirements for the safety assessment of that organ system, providing users with guidance on the selection of appropriate NAMs. In addition to generating the maps, participants offered suggestions for encouraging greater NAM adoption within drug development and their inclusion in regulatory guidelines. A specific recommendation was that pharmaceutical companies should be more transparent about how they use NAMs in-house. As well as giving guidance for the four organ systems, the maps provide a template that could be used for additional organ safety testing contexts. Moreover, their conversion to an interactive format would enable users to drill down to the detail necessary to answer specific scientific and regulatory questions.

Visualisation of drug distribution in skin using correlative optical spectroscopy and mass spectrometry imaging.

A correlative methodology for label-free chemical imaging of soft tissue has been developed, combining non-linear optical spectroscopies and mass spectrometry to achieve sub-micron spatial resolution and critically improved drug detection sensitivity. The approach was applied to visualise the kinetics of drug reservoir formation within human skin following in vitro topical treatment with a commercial diclofenac gel. Non-destructive optical spectroscopic techniques, namely stimulated Raman scattering, second harmonic generation and two photon fluorescence microscopies, were used to provide chemical and structural contrast. The same tissue sections were subsequently analysed by secondary ion mass spectrometry, which offered higher sensitivity for diclofenac detection throughout the epidermis and dermis. A method was developed to combine the optical and mass spectrometric datasets using image registration techniques. The label-free, high-resolution visualisation of tissue structure coupled with sensitive chemical detection offers a powerful method for drug biodistribution studies in the skin that impact directly on topical pharmaceutical product development.

Optimization of topical formulations using a combination of in vitro methods to quantify the transdermal passive diffusion of drugs.

This paper describes a new approach to the early-stage optimization of topical products and selection of lead formulation candidates. It demonstrates the application of open flow microperfusion in vitro in conjunction with the Franz diffusion cell to compare time-resolved, 24-hour profiles of diclofenac passive diffusion through all skin layers (including the skin barrier, dermis, and subcutis) resulting from nine topical formulations of different composition. The technique was successfully validated for in vitro sampling of diclofenac in interstitial fluid. A multi-compartmental model integrating the two datasets was analyzed and revealed that the passive diffusion of diclofenac through the dermis and subcutis does not correlate with its diffusion through the skin barrier and cannot be predicted using Franz diffusion cell data alone. The combined application of the two techniques provides a new, convenient tool for product development and selection enabling the comparison of topical formulation candidates and their impact on drug delivery through all skin layers. This approach can also generate the experimental data required to improve the robustness of mechanistic PBPK models, and when combined with clinical sampling via open flow microperfusion - for the development of better in vivo-in vitro correlative models.

Inter- and intra-individual variability in human skin barrier function: a large scale retrospective study.

In vitro transepidermal tritiated water flux measurements are frequently used to evaluate skin barrier integrity for quality control purposes. However, research in this area to date has been largely based upon small-scale studies, each involving relatively few skin permeation measurements. In order to enhance our understanding in this area, we have conducted a much larger scale retrospective statistical analysis of tritiated water kp values. These values reflected the permeability of 2400 skin samples that were derived from 112 female volunteers over a 4 year period. It was found that the population of tritiated water kp values constituted a positively skewed, non-Normal distribution. Mean kp was 2.04 x 10(-3)cm/h while the 95th percentile was 4.50 x 10(-3)cm/h. Both values are higher than those reported in previous smaller studies. Hence, our study indicates that previously suggested upper limits for tritiated water flux are too low and that they be revised upwards to a value of 4.5 x 10(-3)cm/h. Analysis was also performed on smaller data subsets allowing inter-individual and intra-individual comparisons. For intra-individual kp variability, site-related differences yielded a non-Normal, positively skewed pattern in most individuals. Inter-individual variability was Normally-distributed and showed scatter that was much smaller in magnitude.

Development of a modified in vitro skin absorption method to study the epidermal/dermal disposition of a contact allergen in human skin.

In vitro skin absorption methods exist in Organisation for Economic Co-operation and Development (OECD) guideline form (No. 428) and are used to estimate the degree of systemic penetration of chemicals through skin. More detailed kinetics of permeation through skin compartments are not described well by existing methods. This study was designed to assess the practical feasibility of generating compartmental (stratum corneum/epidermal/dermal) disposition and kinetic data of topically applied chemicals. For chemically induced effects initiated in the skin (e.g., skin allergy), the delivery of tissue concentrations of chemical will impact the incidence and severity of biological effect. Explicit data on the kinetics of chemical disposition in skin have not traditionally been needed for skin allergy risk assessment: current in vivo assays embody delivery implicitly. Under the 7th Amendment to the European Cosmetics Directive, in vivo assays (such as the local lymph node assay for skin sensitization) will not be permitted to assess cosmetic ingredients. New in vitro and in silico alternative approaches and ways of predicting risk of adverse effects in humans need to be developed, and new methods such as that described here provide a way of estimating delivered concentrations and the effect of formulation changes on that delivery. As we continue to deconstruct the contributing factors of skin allergy in humans, it will be useful to have methods available that can measure skin tissue compartment exposure levels delivered from different exposure use scenarios. Here we provide such a method. The method could also be used to generate useful data for developing in silico kinetic models of compartmental skin delivery and for refining data for skin delivery in relation to the evaluation of systemic toxicity.

Pathway-based predictive approaches for non-animal assessment of acute inhalation toxicity.

New approaches are needed to assess the effects of inhaled substances on human health. These approaches will be based on mechanisms of toxicity, an understanding of dosimetry, and the use of in silico modeling and in vitro test methods. In order to accelerate wider implementation of such approaches, development of adverse outcome pathways (AOPs) can help identify and address gaps in our understanding of relevant parameters for model input and mechanisms, and optimize non-animal approaches that can be used to investigate key events of toxicity. This paper describes the AOPs and the toolbox of in vitro and in silico models that can be used to assess the key events leading to toxicity following inhalation exposure. Because the optimal testing strategy will vary depending on the substance of interest, here we present a decision tree approach to identify an appropriate non-animal integrated testing strategy that incorporates consideration of a substance's physicochemical properties, relevant mechanisms of toxicity, and available in silico models and in vitro test methods. This decision tree can facilitate standardization of the testing approaches. Case study examples are presented to provide a basis for proof-of-concept testing to illustrate the utility of non-animal approaches to inform hazard identification and risk assessment of humans exposed to inhaled substances.

Determining epidermal disposition kinetics for use in an integrated nonanimal approach to skin sensitization risk assessment.

Development of risk assessment methods for skin sensitization in the absence of toxicological data generated in animals represents a major scientific and technical challenge. The first step in human skin sensitization induction is the transport of sensitizer from the applied dose on the skin surface to the epidermis, where innate immune activation occurs. Building on the previous development of a time course in vitro human skin permeation assay, new kinetic data for 10 sensitizers and 2 nonsensitizers are reported. Multicompartmental modeling has been applied to analyze the data and determine candidate dose parameters for use in integrated risk assessment methods: the area under the curve (AUC) and maximum concentration (C(max)) in the epidermis. A model with two skin compartments, representing the stratum corneum and viable skin (epidermis and dermis), was chosen following a formal model selection process. Estimates of the uncertainty, as well as average values of the epidermal disposition kinetics parameters, were made by fitting to the time course skin permeation data from individual skin donors. A potential reduced time course method is proposed based on two time points at 4 and 24 h, which gives results close to those from the full time course for the current data sets. The time course data presented in this work have been provided as a resource for development of predictive in silico skin permeation models.

On the correlation between single-frequency impedance measurements and human skin permeability to water.

The objective of this study was to quantitatively compare measurements of tritiated water permeability with impedance determined at either 100 or 1000 Hz using an LCR databridge on the same pieces of skin. A previously published expression based on a simple circuit of a parallel resistor and constant phase element (CPE) was used to relate (RPARA) measured at different frequencies to the DC resistance (RskinA) and the steady-state skin permeability of tritiated water (kp). Using this analysis, kp and (RPARA) data from three laboratories were shown to be consistent with each other, and kp and (RskinA) estimated from (RPARA) were linearly correlated. Compared with urea and mannitol, which are known to permeate skin through a polar pathway, the value of kp for water was found to be about two times larger than expected for transport through only the polar pathway, suggesting an approximately equal contribution from the lipophilic pathway. Equations relating kp to (RPARA) and (RskinA) were used to compare on a consistent basis proposed tests for identifying and excluding damaged skin from chemical absorption studies. The criterion of 20 kΩ cm2 for (RskinA) corresponds to a tritiated water permeability of 3.2×10(-3) cm/h, which should exclude damaged skin without screening undamaged but higher permeability skin samples from study.