In December 2021, the United States Food and Drug Administration (FDA) issued the final guidance for industry titled Pathology Peer Review in Nonclinical Toxicology Studies: Questions and Answers. The stated purpose of the FDA guidance is to provide information to sponsors, applicants, and nonclinical laboratory personnel regarding the management and conduct of histopathology peer review as part of nonclinical toxicology studies conducted in compliance with good laboratory practice (GLP) regulations. On behalf of and in collaboration with global societies of toxicologic pathology and the Society of Quality Assurance, the Scientific and Regulatory Policy Committee (SRPC) of the Society of Toxicologic Pathology (STP) initiated a review of this FDA guidance. The STP has previously published multiple papers related to the scientific conduct of a pathology peer review of nonclinical toxicology studies and appropriate documentation practices. The objectives of this review are to provide an in-depth analysis and summary interpretation of the FDA recommendations and share considerations for the conduct of pathology peer review in nonclinical toxicology studies that claim compliance to GLP regulations. In general, this working group is in agreement with the recommendations from the FDA guidance that has added clear expectations for pathology peer review preparation, conduct, and documentation.

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Background: siRNA is a promising therapeutic modality highlighted by several US FDA approvals since 2018, with many more oligonucleotide assets in clinical development. To support siRNA discovery and development, robust and sensitive quantitative platforms for bioanalysis must be established to assess pharmacokinetic/pharmacodynamic relationships and toxicology. Droplet digital PCR offers improved sensitivity and throughput, as well as reduced susceptibility to matrix effects, compared with other analytical platforms. Methodology: The authors developed a stem-loop reverse transcription droplet digital PCR method to measure siRNA in mouse plasma and liver extract using bioanalytical method qualification guidelines. Conclusion: This newly developed assay has been demonstrated to be a superior alternative to other platforms, with the added benefit of greater sensitivity, with dynamic range from 390 to 400,000 copies/reaction and readiness for FDA investigational new drug-enabling applications.

We describe a roadmap for a fully digital artificial intelligence (AI)-augmented nonclinical pathology laboratory across three continents. Underpinning the design are Good Laboratory Practice (GLP)-validated laboratory information management systems (LIMS), whole slide-scanners (WSS), image management systems (IMS), and a digital microscope intended for use by the nonclinical pathologist. Digital diagnostics are supported by tools that include AI-based virtual staining and deep learning-based decision support. Implemented during the COVID-19 pandemic, the initial digitized workflow largely mitigated disruption of pivotal nonclinical studies required to support pharmaceutical clinical testing. We believe that this digital transformation of our nonclinical pathology laboratories will promote efficiency and innovation in the future and enhance the quality and speed of drug development decision making.

Biodistribution assays are integral to gene therapy commercialization and have traditionally used real-time qPCR. Droplet digital PCR (ddPCR), however, has distinct advantages including higher sensitivity and absolute quantification but is underused because of lacking regulatory guidance and meaningful examples in the literature. We report a fit-for-purpose model process to validate a good laboratory practice (GLP)-compliant ddPCR assay for AVGN7, a Smad7 gene therapeutic for muscle wasting. Duplexed primer/probe sets for Smad7 and mouse TATA-box binding protein were optimized using gBlock DNA over a dynamic range of 10-80,000 copies/reaction in 250 ng mouse gDNA. Linearized plasmid and mouse gDNA were used for validation, which determined precision, accuracy, ruggedness/robustness, selectivity, recovery, specificity, dilution linearity, and stability. Inter-run precision and accuracy met previously established criteria with bias between -5% and 15%, coefficient of variation (CV) less than 19%, and total error within 8%-35%. The limit of detection was 2.5 copies/reaction, linearity was confirmed at 40-80,000 copies/reaction, specificity was demonstrated by single droplet populations and assay stability was demonstrated for benchtop, refrigerated storage, and repeated freeze-thaw cycles. The procedural road map provided exceeds recently established standards. It is also relevant to many IND-enabling processes, as validated ddPCR assays can be used in biodistribution studies and with vector titering and manufacturing quality control.

Bovine tuberculosis (bTB) is a global disease of livestock that has damaging economic, animal health and public health consequences. Conventional bTB disease control strategies, based around the testing and slaughter of cattle infected with bTB, are typically used to help limit or reduce the transmission of this disease but in many low- and middle-income countries such strategies may often be economically unviable, culturally unacceptable or logistically impracticable. The use of vaccination to protect cattle against bTB could provide a potentially more affordable, ethically acceptable and practical additional disease control measure. The protective efficacy of the commercially produced and readily available human vaccine against tuberculosis (Mycobacterium bovis Bacille Calmette-Guérin; BCG) in cattle has been demonstrated in many experimental laboratory and field studies. However, Good Laboratory Practice (GLP) studies assessing the safety of BCG vaccination in cattle have not previously been reported. We describe here the results of two GLP safety studies in which calves and lactating cows were vaccinated with BCG (Danish 1331 strain). From an animal health and welfare perspective, the results of these studies indicate that BCG vaccine is well tolerated in these categories of cattle with only transient and minor local or systemic reactions. Furthermore, there was no evidence that BCG was shed in raw milk, saliva or faeces collected from vaccinates and vaccination did not have a detrimental effect on milk yields in lactating cattle. These data, underpinned by GLP principles, further support the existing data on the safety of BCG vaccine in cattle and complement the abundant available cattle efficacy data for this potential cattle bTB vaccine.

BACKGROUND: Broflanilide is a newly discovered insecticide with a novel mode of action targeting insect γ-aminobutyric acid receptors. The efficacy of VECTRON™ T500, a wettable powder formulation of broflanilide, was assessed for IRS against wild pyrethroid-resistant malaria vectors in experimental huts in Benin.
METHODS: VECTRON™ T500 was evaluated at 100 mg/m2 in mud and cement-walled experimental huts against wild pyrethroid-resistant Anopheles gambiae sensu lato (s.l.) in Covè, southern Benin, over 18 months. A direct comparison was made with Actellic® 300CS, a WHO-recommended micro-encapsulated formulation of pirimiphos-methyl, applied at 1000 mg/m2. The vector population at Covè was investigated for susceptibility to broflanilide and other classes of insecticides used for vector control. Monthly wall cone bioassays were performed to assess the residual efficacy of VECTRON™ T500 using insecticide susceptible An. gambiae Kisumu and pyrethroid-resistant An. gambiae s.l. Covè strains. The study complied with OECD principles of good laboratory practice.
RESULTS: The vector population at Covè was resistant to pyrethroids and organochlorines but susceptible to broflanilide and pirimiphos-methyl. A total of 23,171 free-flying wild pyrethroid-resistant female An. gambiae s.l. were collected in the experimental huts over 12 months. VECTRON™ T500 induced 56%-60% mortality in wild vector mosquitoes in both cement and mud-walled huts. Mortality with VECTRON™ T500 was 62%-73% in the first three months and remained > 50% for 9 months on both substrate-types. By comparison, mortality with Actellic® 300CS was very high in the first three months (72%-95%) but declined sharply to < 40% after 4 months. Using a non-inferiority margin defined by the World Health Organization, overall mortality achieved with VECTRON™ T500 was non-inferior to that observed in huts treated with Actellic® 300CS with both cement and mud wall substrates. Monthly in situ wall cone bioassay mortality with VECTRON™ T500 also remained over 80% for 18 months but dropped below 80% with Actellic® 300CS at 6-7 months post spraying.
CONCLUSIONS: VECTRON™ T500 shows potential to provide substantial and prolonged control of malaria transmitted by pyrethroid-resistant mosquito vectors when applied for IRS. Its addition to the current list of WHO-approved IRS insecticides will provide a suitable option to facilitate rotation of IRS products with different modes of action.

OBJECTIVE: Three years after the start of the ESHRE ART Centre Certification (ARTCC) programme, what is the current state of the system, in terms of the interest expressed in it and experiences during the assessment of ART services?
CONCLUSIONS: As of 1 December 2021, 25 European ART centres have been involved in the various stages of certification and the most common recommendations from inspectors were the need for documented training, verification of competencies for all staff members, verification of laboratory and clinical performance indicators, implementation of a quality management system and avoidance of overusing ICSI and add-ons.
BACKGROUND: European Union (EU) legislation has included ART activities in the EU Tissue and Cells Directives (EUTCDs). Following inspections by national EUTCD authorities, many details regarding documentation, laboratory environment, handling of reproductive cells and tissues, traceability, coding and patient testing have become standardized. However, the EUTCDs do not cover all ART-specific aspects. For this reason, the ARTCC was established to focus on peculiar areas, including relevant staff qualifications, training, continuing professional development, workload, equipment suitability, (non)-evidence-based laboratory and clinical methods used, treatment approaches according to ESHRE guidelines, recommendations and laboratory and clinical key performance indicators.
UNASSIGNED: The article reviews the state-of-the-art of the ESHRE certification of ART centres for good clinical and laboratory practice over an initial 3-year period of operation, including the number of ART centres involved in the different stages of certification and the most common recommendations by inspectors.
METHODS: In 2016, the ARTCC working group began to establish a new ESHRE ARTCC programme. Since then, the working group has organized 4 preparatory courses and appointed 37 inspectors (19 clinicians, 17 embryologists and one paramedical). A tool to verify compliance with ESHRE recommendations for good laboratory and clinical practice was developed. The ARTCC has been open for applications since September 2018. In Step 1, the applicant enters basic information about the ART centre, staff and ART activities into the application platform. After review and approval, the applicant is given the opportunity to enter Step 2 and provide detailed online checklists on general, laboratory, clinical services and clinical outcomes. Two inspectors (one clinician and one embryologist) independently evaluate the submitted checklists. The condition to proceed to evaluation is a positive mean score (at least 66%) from each of the four checklists. In Step 3, a live site visit (or virtual owing to the coronavirus disease 2019 (COVID-19) pandemic) is organized and the inspectors prepare a final report with appropriate recommendations. The application may be rejected at any time if the criteria required to advance to the next stage are not met. The ARTCC programme is currently available for European countries listed in ESHRE internal rules, available on the ESHRE website. The certificate is valid for 3 years, after which an application for renewal can be submitted.
RESULTS: Over a 3-year period (until 1 December 2021), 63 ART centres from 25 countries started applying through an online platform. So far, 38 applications did not progress owing to lack of completion of the initial application within a 1-year period or because applications came from non-European countries. Of the remaining 25 applications, 8 centres have been inspected and 7 centres have been certified. The most common recommendations given by inspectors to assessed centres were the need for documented training, verification of competencies, skills and continuing professional development for all staff members, verification of laboratory and clinical performance indicators and implementation of a quality management system. The inspectors identified some recurring areas of medically assisted reproduction that deviate from good practice: the overuse of ICSI, preimplantation genetic testing for aneuploidies, freeze-all and other add-ons. They often reported that the clinical outcomes could not be objectively assessed because of non-inclusion of the started cycles or the frequent use of freeze-all cycles.
CONCLUSIONS: No major modifications have been made to the application platform and checklists since the early stages of the certification programme. However, in this short time, quite a few changes in clinical practice have occurred, especially concerning the more frequent use of the \'freeze-all\' strategy. As a result, problems arose in the evaluation of clinical outcomes. In addition, because of the COVID-19 pandemic, site visits were substituted by the implementation of virtual visits. While this enabled the certification programme to continue, it is possible that certain critical details that would have been noticed during a traditional site visit may have been overlooked.
CONCLUSIONS: Regular monitoring of the observations of ARTCC inspectors and analysis of their reports is certainly useful to harmonize inspectors\' criteria in the assessment process and to identify chronic deficiencies in clinical and laboratory practice. Non-conformities can be addressed by ESHRE through guidelines and recommendations, as well as through discussion with EU institutions and competent authorities.
BACKGROUND: The ARTCC programme was developed and funded by ESHRE, covering expenses associated with the meetings. The Steering Committee members who are the authors of this article did not receive payments for the completion of this study. The inspectors were remunerated for their work with an honorarium. The authors have no conflicts of interest to declare.

Recent advances in human pluripotent stem cell (hPSC)-derived cell therapies and genome editing technologies such as CRISPR/Cas9 make regenerative medicines promising for curing diseases previously thought to be incurable. However, the possibility of off-target effects during genome editing and the nature of hPSCs, which can differentiate into any cell type and infinitely proliferate, inevitably raises concerns about tumorigenicity. Tumorigenicity acts as a major obstacle to the application of hPSC-derived and gene therapy products in clinical practice. Thus, regulatory authorities demand mandatory tumorigenicity testing as a key pre-clinical safety step for the products. In the tumorigenicity testing, regulatory guidelines request to include human cancer cell line injected positive control group (PC) animals, which must form tumors. As the validity of the whole test is determined by the tumor-forming rates (typically above 90%) of PC animals, establishing the stable tumorigenic condition of PC animals is critical for successful testing. We conducted several studies to establish the proper positive control conditions, including dose, administration routes, and the selection of cell lines, in compliance with Good Laboratory Practice (GLP) regulations and/or guidelines, which are essential for pre-clinical safety tests of therapeutic materials. We expect that our findings provide insights and practical information to create a successful tumorigenicity test and its guidelines.

(1) Background: the use of Mesenchymal Stromal Cells (MSC) in emerging therapies for spinal cord injury (SCI) hold the potential to improve functional recovery. However, the development of cell-based medicines is challenging and preclinical studies addressing quality, safety and efficacy must be conducted prior to clinical testing; (2) Methods: herein we present (i) the characterization of the quality attributes of MSC from the Wharton\'s jelly (WJ) of the umbilical cord, (ii) safety of intrathecal infusion in a 3-month subchronic toxicity assessment study, and (iii) efficacy in a rat SCI model by controlled impaction (100 kdynes) after single (day 7 post-injury) and repeated dose of 1 × 106 MSC,WJ (days 7 and 14 post-injury) with 70-day monitoring by electrophysiological testing, motor function assessment and histology evaluation; (3) Results: no toxicity associated to MSC,WJ infusion was observed. Regarding efficacy, recovery of locomotion was promoted at early time points. Persistence of MSC,WJ was detected early after administration (day 2 post-injection) but not at days 14 and 63 post-injection. (4) Conclusions: the safety profile and signs of efficacy substantiate the suitability of the presented data for inclusion in the Investigational Medicinal Product Dossier for further consideration by the competent Regulatory Authority to proceed with clinical trials.