Nonclinical Safety Research Articles

Importance of tailored non-clinical safety testing of novel antimalarial drugs: Industry best-practice

Malaria is an acute, debilitating parasitic illness. There were 249 million cases of malaria in 2022, resulting in 608,000 deaths globally, 76% of which were children ≤5 years. The unique nature of this disease (recurrences leading to re-treatments and numerous organ systems affected), specific clinical treatment regimens, poor compliance, and diversity of affected populations (predominantly pediatrics, women of childbearing potential, pregnant and lactating women), often makes standard testing approaches inadequate, and tailor-made safety assessments are more appropriate.We provide best practice recommendations based on company experience for the non-clinical safety assessment of antimalarial drugs, with a focus on small molecules since they represent the majority of drug candidates for this illness. We focus on specific testing considerations for repeat dose toxicity studies, including combination toxicity assessments, since new drug treatment regimens typically foresee short treatment durations to improve compliance (i.e., 1 day) with combinations of compounds to improve efficacy and limit potential resistance. Due to the target population, the timing of reproductive, developmental, and juvenile toxicity studies may be earlier than general testing roadmaps for other small molecule drugs.In conclusion, key recommendations presented should enable a more effective and efficient development path whilst protecting clinical trial participants and patients.

Are Repeat-Dose Toxicity Studies Informative for Safety Assessment of Vaccine Candidates? A Survey of Vaccine Developers.

A BioSafe-sponsored survey investigated how vaccine companies (n = 12) perceive the value of the repeat-dose toxicity studies for safety assessment of vaccine candidates. As all major vaccine developers were part of the survey, it was considered representative for the industry practices up to 2022. Vaccine developers indicated that they see scientific value in performing repeat-dose toxicity studies with vaccines, especially when novel components (e.g., adjuvant) or technology is being used. However, a few (3/12) also indicated that repeat-dose toxicity studies could be replaced by a pharmacology study with additional toxicity parameters. For the majority of companies (9/12), findings from the repeat-dose toxicity studies never prevented or postponed a first-in-human (FIH) trial. In the remaining 3 companies, a total of 4 occurrences of postponement or prevention of clinical development occurred and in only 2 of these cases was the finding considered related to the vaccine. A platform approach has been successfully implemented for influenza vaccines already in 2016, and an outline of the regulatory requirements for a platform approach has been recently documented in the latest infectious disease mRNA-LNP vaccine guideline, as well as in the guidance on the development and licensure of COVID-19 vaccines presented by the FDA. Vaccine developers are seeking to extend this platform approach to the development of new vaccines, building on established technologies and using well-defined manufacturing processes. This approach could support reduction of animal use (a principle of 3Rs) while still providing reassurance of the nonclinical safety of these products.

Intra-abdominal Abscesses in Two Göttingen Minipigs.

Minipigs are valued alternatives to dogs and non-human primates in non-clinical safety and toxicity studies, and Göttingen minipigs are bred specifically for experimental purposes. They are bred under barrier conditions and monitored regularly for many pathogens and opportunistic agents, and spontaneous disease is rare when compared to what is seen in production pigs. Knowledge of spontaneous background lesions is important when toxicological pathologists evaluate microscopic findings in pre-clinical toxicity studies to avoid interference with study data interpretation. In this brief communication, intra-abdominal granulomas/abscesses were seen in Göttingen minipigs. The minipigs did not show any clinical signs, but nodules were present in the abdominal peritoneum at necropsy. Microscopic evaluation revealed chronic inflammation, with abscess or granuloma formation. Areas of inflammation, occasionally associated with the presence of the Splendore-Hoeppli material, were surrounded by a fibrotic capsule. Special stains were applied to investigate for the presence of microorganisms.

Preclinical immunogenicity and safety of hemagglutinin-encoding modRNA influenza vaccines

Seasonal epidemics of influenza viruses are responsible for a significant global public health burden. Vaccination remains the most effective way to prevent infection; however, due to the persistence of antigenic drift, vaccines must be updated annually. The selection of vaccine strains occurs months in advance of the influenza season to allow adequate time for production in eggs. RNA vaccines offer the potential to accelerate production and improve efficacy of influenza vaccines. We leveraged the nucleoside-modified RNA (modRNA) platform technology and lipid nanoparticle formulation process of the COVID-19 mRNA vaccine (BNT162b2; Comirnaty®) to create modRNA vaccines encoding hemagglutinin (HA) (modRNA-HA) for seasonal human influenza strains and evaluated their preclinical immunogenicity and toxicity. In mice, a monovalent modRNA vaccine encoding an H1 HA demonstrated robust antibody responses, HA-specific Th1-type CD4+ T cell responses, and HA-specific CD8+ T cell responses. In rhesus and cynomolgus macaques, the vaccine exhibited durable functional antibody responses and HA-specific IFN-γ+ CD4+ T cell responses. Immunization of mice with monovalent, trivalent, and quadrivalent modRNA-HA vaccines generated functional antibody responses targeting the seasonal influenza virus(es) encoded in the vaccines that were greater than, or similar to, those of a licensed quadrivalent influenza vaccine. Monovalent and quadrivalent modRNA-HA vaccines were well-tolerated by Wistar Han rats, with no evidence of systemic toxicity. These nonclinical immunogenicity and safety data support further evaluation of the modRNA-HA vaccines in clinical studies.

Non-clinical safety evaluation of Yongdamsagan-tang water extract: A 13-week subchronic oral toxicity study in Sprague Dawley rats.

Despite the continued clinical application of traditional herbal medicines, scientific evidence such as toxicological safety profile of Yongdamsagan-tang water extract (YSTW) has not yet been established. The purpose of this study was to investigate the potential toxicity profile of YSTW following a 13-week repeated oral administration at various concentrations to Sprague Dawley rats. YSTW was administered orally to rats once daily at doses of 0, 1000, 2000, and 5000 mg/kg bw/day for 13 weeks. During the experimental period, there were no significant toxicological changes related to YSTW treatment. These results indicate that the administration of YSTW for 13 weeks in the rat resulted in no signs of toxicity at up to 5000 mg/kg bw/day, which was considered the no-observed-adverse-effect level.

Rethinking the necessity of long-term toxicity studies for biotherapeutics using weight of evidence assessment

The registration of biotherapeutics for chronic indications requires 6-month toxicity studies. However, extensive experience has shown that the non-clinical safety profiles of biotherapeutics are generally predictable. This suggests that conducting multiple studies, especially a 6-month study may not be necessary. In a meta-analysis of biologics developed for non-oncology indications over last 25 years at Pfizer, we compared organ system findings between short-term (1–3 month) and long-term (6-month) animal studies. Our goal was to determine if there were differences in the safety profiles between the two study durations and their relevance to human risk assessment. Our analysis revealed that most clinically relevant toxicities could be detected in shorter-term studies (87%; 26/30 programs). This suggests either an undifferentiated safety profile between short-and long-term studies, or anticipated toxicities based on the modality, such as immunogenicity or exaggerated pharmacology. However, for 4 out of 30 programs (13%), long-term studies did identify either potential new toxicities or more severe manifestation of exaggerated pharmacology, leading to modifications in clinical trial designs and human risk assessment. Our experience suggests that 3-month toxicity studies may be sufficient to support late-stage clinical development for a majority of standard biotherapeutic programs. This pragmatic and science-based approach aligns with the goal of advancing 3R's initiatives in nonclinical safety assessment.

An industry perspective on the FDA modernization act 2.0/3.0: Potential next steps for sponsors to reduce animal use in drug development

Abstract Pharmaceutical developers are encouraged to adopt the best practices of being purposefully thoughtful about the use of animals, seeking alternatives wherever possible. They should engage with health authorities to increase their familiarity with the methods, study designs, data outputs and the context of use for new approach methodologies (NAMs). Although current state of technology does not yet provide adequate models to fully replace in vivo studies, many models are sufficiently good for an augmented approach that will enhance our understanding of in vitro to in vivo correlations and advance the long-term goal of reducing animal use through innovative NAMs. The goal of future nonclinical safety packages is to advance the utilization of such enabling technologies towards appropriate human risk characterization. Establishing confidence in NAMs is a critical first step. For example, sponsors may include both “traditional” and NAM-based nonclinical safety data in regulatory submissions to establish confidence with health authorities. In addition, regulators should create a “safe-harbor” for hybrid nonclinical data packages to facilitate iterative learning, refinement, and implementation of NAM-based safety assessment strategies. Sponsors are urged to contribute to NAMs evolution through consortia participation, peer-reviewed publications, and documenting animal reduction in studies/programs, accelerating the eventual elimination of animal use in pharmaceutical development, as envisioned in the FDA Modernization Act 3.0.

Astodrimer sodium nasal spray forms a barrier to SARS-CoV-2 in vitro and preserves normal mucociliary function in human nasal epithelium

COVID-19 remains a severe condition for many including immunocompromised individuals. There remains a need for effective measures against this and other respiratory infections, which transmit via virus-laden droplets that reach the nasal or oral mucosae. Nasal sprays offer potential protection against viruses. Such formulations should preserve normal nasal mucociliary function. The antiviral barrier efficacy and effects on mucociliary function of astodrimer sodium nasal spray (AS-NS) were evaluated and compared with other available nasal sprays—low pH hydroxypropyl methylcellulose (HPMC-NS), iota-carrageenan (Carr-NS), nitric oxide (NO-NS), and povidone iodine (PI-NS). Assays simulated clinical conditions. Antiviral barrier function and cell viability were assessed in airway cell monolayers, while a model of fully differentiated human nasal epithelium (MucilAir™) was utilized to evaluate tissue integrity, cytotoxicity, cilia beating frequency, and mucociliary clearance. AS-NS reduced infectious virus in cell monolayers and demonstrated a benign cytotoxicity profile. In human nasal epithelium ex vivo, AS-NS had no impact on mucociliary function (cilia beating nor mucociliary clearance). Carr-NS, HPMC-NS, NO-NS and PI-NS demonstrated limited antiviral effects, while HPMC-NS caused inhibition of mucociliary function. Astodrimer sodium nasal spray demonstrates an acceptable nonclinical efficacy and safety profile as a barrier nasal spray against respiratory viral infection in the nasal cavity.

P19-77 Comparing non-clinical safety studies to pharmacovigilance evaluation for assessment of safety: the case of anti-COVID-19 vaccines in France

P19-77 Comparing non-clinical safety studies to pharmacovigilance evaluation for assessment of safety: the case of anti-COVID-19 vaccines in France

Inclusion of Histopathology in Dose Range-Finding Nonclinical Studies for Inhaled Drug Products.

Drug development is a lengthy process that promotes and protects the health and safety of future patients. Nonclinical safety studies follow essentially similar designs that fulfill regulatory requirements but are amended based on factors including the mechanism of action, class of molecule, and route of administration. Clinical observations, clinical pathology, and macroscopic pathology in dose range-finding (DRF) studies generally provide sufficient information to select doses for pivotal studies by most delivery routes. Inhaled drug candidates are recognized for producing adverse effects on the respiratory system at the microscopic level that may otherwise be unpredictable; therefore, unlike other routes of administration, inhalation DRF studies typically include histopathology of the respiratory tract. Histopathology evaluations can add several weeks to the Investigational New Drug (IND) application timeline along with additional costs but have been considered necessary to support accurate dose selection for adequate safety margins, thereby potentially avoiding additional studies and animal usage by ensuring achievement of a NOAEL in the pivotal studies. Therefore, DRF inhalation studies initiated from 2018 to 2021 at Labcorp were reviewed to determine whether inclusion of histopathology on preliminary inhalation studies was necessary for subsequent dose selection. Histopathology findings in the DRF impacted dose selection in pivotal inhalation studies for approximately 45% of rat and dog studies. This review identified histopathology findings in rat and dog that support continued inclusion of respiratory tract histopathology in DRF studies. Future investigations will evaluate potential surrogate endpoints for these findings, which could reduce nonclinical drug development timelines by several weeks.

Diterpenoid DGT alleviates atopic dermatitis–like responses in vitro and in vivo via targeting IL-4Rα

BackgroundAtopic dermatitis is a common chronic inflammatory skin disease characterized by relapsing eczema and intense itch. DGT is a novel synthetic heterocyclic diterpenoid derived from plants. Its therapeutic potential and mechanism(s) of action are poorly understood. ObjectivesWe investigated the potent therapeutic effect of DGT on atopic dermatitis, exploring the underlying mechanisms and determining whether DGT is a safe and well-tolerated topical treatment. MethodsWe observed anti-inflammatory effects of DGT on tumor necrosis factor-α/interferon-γ–treated human keratinocytes, and anti-allergic effects on immunoglobulin E–sensitized bone marrow–derived mast cells. In vivo, DGT was topically applied to two experimental mouse models of atopic dermatitis: oxazolone-induced sensitization and topically applied calcipotriol. Then the therapeutic effects of DGT were evaluated physiologically and morphologically. Moreover, we performed nonclinical toxicology and safety pharmacology research, including general toxicity, pharmacokinetics, and safety pharmacology on the cardiovascular, respiratory, and central nervous systems. ResultsIn keratinocytes, DGT reduced the expression of inflammatory factors, promoting the expression of barrier functional proteins and tight junctions and maintaining the steady state of barrier function. DGT also inhibited the activation and degranulation of mast cells induced by immunoglobulin E. Moreover, we found that interleukin-4 receptor-α was the possible target of DGT. Meanwhile, DGT had therapeutic effects on oxazolone/calcipotriol-treated mice. Notably, our pharmacology results demonstrated that DGT was safe and nontoxic in our studies. ConclusionDGT’s potent anti-inflammatory effects and good safety profile suggest that it is a potential candidate for the treatment of atopic dermatitis.

Weight of Evidence: Is an Animal Study Warranted? Assessments for Carcinogenicity, Drug Abuse Liability, and Pediatric Safety.

Nonclinical safety studies are typically conducted to establish a toxicity profile of a new pharmaceutical in clinical development. Such a profile may encompass multiple differing types of animal studies, or not! Some types of animal studies may not be warranted for a specific program or may only require a limited evaluation if scientifically justified. The goal of this course was to provide a practical perspective on regulatory writing of a dossier(s) using the weight of evidence (WOE) approach for carcinogenicity, drug abuse liability and pediatric safety assessments. These assessments are typically done after some clinical data are available and are highly bespoke to the pharmaceutical being developed. This manuscript will discuss key data elements to consider and strategy options with some case studies and examples. Additionally, US FDA experience with dossier(s) including WOE arguments is discussed.

Nonclinical safety and immunogenicity assessment of a combined DTacP vaccine in animal models.

The (diphtheria, tetanus, and pertussis [acellular, component] [DTacP]) vaccine is a combined vaccine designed to prevent three potentially fatal diseases including pertussis, tetanus, and diphtheria in both children and adults. We utilized advanced technology to develop a novel DTacP vaccine that was previously unavailable in China. The nonclinical studies were performed to evaluate the immunogenicity, potential toxicity, and local tolerance of the vaccine in animal models. In the immunogenicity study, three batches of the vaccine were intraperitoneally administered to National Institutes of Health (NIH)mice, resulting in 100% seropositivity for all three batches. Additionally, antibody levels notably increased as the immunization dosage increased. In acute toxicity study, no mortality was observed among the animals during the 14-day observation period, and no abnormalities in clinical signs were reported. Active systemic anaphylaxis assessment in guinea pigs showed no evidence of serious allergic reactions in the vaccine groups. In the repeat-dose toxicity study, where five intramuscular doses were administered every 2weeks, gross autopsy and histopathological examination revealed no vaccine-related systemic pathological changes in rats, with dose site irritant reactions mostly recovered at the end of recovery period. In conclusion, the vaccine demonstrated good local and systemic tolerance, supporting its clinical development.

Safety Implications of Modulating Nuclear Receptors: A Comprehensive Analysis from Non-Clinical and Clinical Perspectives.

The unintended modulation of nuclear receptor (NR) activity by drugs can lead to toxicities amongst the endocrine, gastrointestinal, hepatic cardiovascular, and central nervous systems. While secondary pharmacology screening assays include NRs, safety risks due to unintended interactions of small molecule drugs with NRs remain poorly understood. To identify potential nonclinical and clinical safety effects resulting from functional interactions with 44 of the 48 human-expressed NRs, we conducted a systematic narrative review of the scientific literature, tissue expression data, and used curated databases (OFF-X™) (Off-X, Clarivate) to organize reported toxicities linked to the functional modulation of NRs in a tabular and machine-readable format. The top five NRs associated with the highest number of safety alerts from peer-reviewed journals, regulatory agency communications, congresses/conferences, clinical trial registries, and company communications were the Glucocorticoid Receptor (GR, 18,328), Androgen Receptor (AR, 18,219), Estrogen Receptor (ER, 12,028), Retinoic acid receptors (RAR, 10,450), and Pregnane X receptor (PXR, 8044). Toxicities associated with NR modulation include hepatotoxicity, cardiotoxicity, endocrine disruption, carcinogenicity, metabolic disorders, and neurotoxicity. These toxicities often arise from the dysregulation of receptors like Peroxisome proliferator-activated receptors (PPARα, PPARγ), the ER, PXR, AR, and GR. This dysregulation leads to various health issues, including liver enlargement, hepatocellular carcinoma, heart-related problems, hormonal imbalances, tumor growth, metabolic syndromes, and brain function impairment. Gene expression analysis using heatmaps for human and rat tissues complemented the functional modulation of NRs associated with the reported toxicities. Interestingly, certain NRs showed ubiquitous expression in tissues not previously linked to toxicities, suggesting the potential utilization of organ-specific NR interactions for therapeutic purposes.

Characterization of an Uncommon Finding in the Basal Nuclei in Beagle Dogs: A Novel Potential Background Lesion in the Canine Brain.

Degenerative lesions specific to the basal nuclei have not been described as a background finding in Beagle dogs. This report comprises a documentation of seven cases. In the context of a nonclinical safety studies, the authors suggest documenting the lesion descriptively as degeneration neuropil, basal nuclei, bilateral as it is characterized by (1) vacuolation, neuropil; (2) gliosis (astro- and/or microgliosis); and (3) demyelination. This novel lesion is considered a potential new background change for several reasons: (1) It occurred in animals from test item-treated and also vehicle-treated groups; (2) no dose dependency was observed; (3) in one of six affected test item-treated dogs, the given compound was shown not to penetrate the blood-brain barrier; and (4) statistical comparison between the proportions of affected dogs in the treatment and control groups did not yield a statistically significant difference. The etiology remains unknown and is subject to further investigations.

Maximizing insights from nonclinical safety studies in the context of rising costs and changing regulations

The traditional paradigm of non-rodent safety assessment studies, primarily reliant on non-human primates (NHPs) and dogs, is undergoing a transformation. During the 2023 Safety Pharmacology Society Annual Meeting, scientists from leading nonclinical contract organizations discussed how traditional IND-enabling studies can benefit from employing underutilized alternative non-rodent models, such as the swine. Swine offer a cost-effective approach to drug development and share many anatomical and physiological similarities with humans. The inclusion of non-traditional species in safety assessments, coupled with advanced measurement techniques, aids in de-risking compounds early on and adapting projects to the evolving cost landscape.

Thyrogrit, supplemented with a sub-optimal dose of levothyroxine, restores thyroid function in rat model of propylthiouracil-induced hypothyroidism

BackgroundHypothyroidism is a common endocrine ailment, whose current standard of care is hormonal replacement therapy with levothyroxine (LT4). There is a medical need for alternative and safer therapies as LT4 is associated with special treatment considerations and adverse effects. Thyrogrit (THY) is a polyherbal formulation indicated for the treatment of hypothyroidism. The present study, describes the characterization of the phytocompounds present in THY and its in-vivo efficacy in rat model of hypothyroidism, in combination with a sub-optimal dose of LT4.MethodsUltra High Performance Liquid chromatography was employed for the identification of the phytocompounds present in THY. For the evaluation of its in-vivo efficacy, female Wistar rats were administered THY orally, 15-days prior to disease induction, and continued throughout the experiment. Subsequently, hypothyroidism was induced by oral administration of propylthiouracil (PTU). From day 45 onwards, animals were administered orally with a sub-optimal dose of LT4 (2 μg/kg) till the end of the study. On day 79, animals were euthanized, blood was collected for measurement of thyroid hormones and other clinical chemistry parameters. Weights of liver, kidney and thyroid were recorded. Finally, the thyroid was subjected to histopathological evaluation through hematoxylin and eosin (H&E staining), immunohistochemistry as well as immunofluorescence.ResultsThe principal phyto-components detected in THY by Ultra High Performance Liquid Chromatography included gallic acid, protocatechuic acid, corilagin, ellagic acid, piperine, guggulsterone E and Z, which are documented to exerted beneficial effects on thyroid function. In the in-vivo study, THY when supplemented with a low dose of levothyroxine restored the PTU-induced reduction in the serum levels of T3 and T4 and improved PTU-induced renal impairment. THY treatment ameliorated the hallmark histopathological changes associated with hypothyroidism and C-cell hyperplasia. Further, co-administration of THY and LT4 did not show any major non-clinical safety concerns even after the administration for more than twelve weeks.ConclusionThis study has demonstrated that co-administration of THY and LT4 improves the PTU-evoked alterations in the thyroid ultrastructure and function, abrogates hypothyroidism-associated renal impairment and exhibits an acceptable basic safety profile.Graphical abstract

Re: Re: Laboratory safety evaluation of bedinvetmab, a canine anti-nerve growth factor monoclonal antibody, in dogs

In their letter to the editor, Farrell et al., (2024) presented questions related to canine joint health after treatment with the anti-Nerve Growth Factor (NGF) monoclonal antibody (mAb) bedinvetmab, which was presented as a component of a non-clinical laboratory safety assessment published in Krautmann et al., (2021). Their questions appear to have stemmed from an anti-NGF mAb developed for the treatment of osteoarthritis in humans (tanezumab; FDA, 2021) which in 2021 failed to achieve marketing approval due to an unfavorable benefit: risk profile, primarily due to a syndrome called Rapidly Progressive Osteoarthritis (RPOA) which occurred more commonly in treatment groups when compared to controls. Farrell et. al. (2024) have posed questions on radiographic and histopathologic bone findings from studies included in Krautmann, et al., (2021) and communicated in the FDA’s Freedom of Information summary for Librela (FDA, 2023). These findings have previously been determined to be incidental and not bedinvetmab-associated. To address the questions posed, it is important to briefly define RPOA and summarize the syndrome in humans, review why the bone/joint findings in bedinvetmab safety studies in dogs are not indicative of RPOA or an RPOA-like condition, provide an update on joint health after use of bedinvetmab since market approval (>3 years in some markets), and summarize why Zoetis, the manufacturer of Librela, has confidence in joint safety after use of bedinvetmab in dogs.

A Nonclinical Safety Evaluation of Cold Atmospheric Plasma for Medical Applications: The Role of Genotoxicity and Mutagenicity Studies.

Atmospheric nonthermal plasma (ANTP) has rapidly evolved as an innovative tool in biomedicine with various applications, especially in treating skin diseases. In particular, the formation of reactive oxygen species (ROS) and nitrogen species (RNS), which are generated by ANTP, plays an important role in the biological signaling pathways of human cells. Unfortunately, excessive amounts of these reactive species significantly result in cellular damage and cell death induction. To ensure the safe application of ANTP, preclinical in vitro studies must be conducted before proceeding to in vivo or clinical trials involving humans. Our study aimed to investigate adverse effects on genetic substances in murine fibroblast cells exposed to ANTP. Cell viability and proliferation were markedly reduced after exposing the cells with plasma. Both extracellular and intracellular reactive species, especially RNS, were significantly increased upon plasma exposure in the culture medium and the cells. Notably, significant DNA damage in the cells was observed in the cells exposed to plasma. However, plasma was not classified as a mutagen in the Ames test. This suggested that plasma led to the generation of both extracellular and intracellular reactive species, particularly nitrogen species, which affect cell proliferation and are also known to induce genetic damage in fibroblast cells. These results highlight the genotoxic and mutagenic effects of ANTP, emphasizing the need for the cautious selection of plasma intensity in specific applications to avoid adverse side effects resulting from reactive species production.

A Minimal Approach to Demonstrate Concordance of Digital and Conventional Microscopy in Toxicologic Pathology.

Digitalization of pathology workflows has undergone a rapid evolution and has been widely established in the diagnostic field but remains a challenge in the nonclinical safety context due to lack of regulatory guidance and validation experience for good laboratory practice (GLP) use. One means to demonstrate that digital slides are fit for purpose, that is, provide sufficient quality for pathologists to reach a diagnosis, is conduction of comparison studies, which have been published both, for veterinary and human diagnostic pathology, but not for toxicologic pathology. Here, we present an approach that uses study material from nonclinical safety studies and that allows for the statistical comparison of concordance rates for glass and digital slide evaluation while minimizing time and effort for the involved personnel. Using a benchmark study design, we demonstrate that evaluation of digital slides fits the purpose of nonclinical safety evaluation. These results add to reports of successful workflow validations and support the full adaptation of digital pathology in the regulatory field.