Scientific and Regulatory Policy Committee Points to Consider: Review of the United States Food and Drug Administration (FDA) Guidance on Pathology Peer Review in Nonclinical Toxicology Studies.

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.

Tracking Charge Migration with Frequency-Matched Strobo-Spectroscopy.

We present frequency-matched strobo-spectroscopy (FMSS) of charge migration (CM) in bromobutadiyne, simulated with time-dependent density functional theory. CM + FMSS is a pump-probe scheme that uses a frequency-matched high harmonic generation (HHG)-driving laser as an independent probe step, following the creation of a localized hole on the bromine atom that induces CM dynamics. We show that the delay-dependent harmonic yield tracks the phase of the CM dynamics through its sensitivity to the amount of electron density on the bromine end of the molecule. FMSS takes advantage of the intrinsic attosecond time resolution of the HHG process in which different harmonics are emitted at different times and thus probe different locations of the electron hole. Finally, we show that the CM-induced modulation of the HHG signal is dominated by the recombination step of the HHG process, with a negligible contribution from the ionization step.

Attochemistry Regulation of Charge Migration.

Charge migration (CM) is a coherent attosecond process that involves the movement of localized holes across a molecule. To determine the relationship between a molecule's structure and the CM dynamics it exhibits, we perform systematic studies of para-functionalized bromobenzene molecules (X-C6H4-R) using real-time time-dependent density functional theory. We initiate valence-electron dynamics by emulating rapid strong-field ionization leading to a localized hole on the bromine atom. The resulting CM, which takes on the order of 1 fs, occurs via an X localized → C6H4 delocalized → R localized mechanism. Interestingly, the hole contrast on the acceptor functional group increases with increasing electron-donating strength. This trend is well-described by the Hammett σ value of the group, which is a commonly used metric for quantifying the effect of functionalization on the chemical reactivity of benzene derivatives. These results suggest that simple attochemistry principles and a density-based picture can be used to predict and understand CM.

Investigation of Interferences in Carbon Dioxide through Multidimensional Molecular-Frame High-Harmonic Spectroscopy.

We present molecular-frame high-harmonic spectroscopic measurements of the spectral intensity and group delay of carbon dioxide. Using four different driving wavelengths and a range of intensities at each wavelength for high-harmonic generation, we observe a well-characterized minimum in the harmonic emission that exhibits both a wavelength and intensity dependence. Using the intensity dependence at each driving wavelength, we classify the minimum as due to either a structural two-center interference or dynamic multichannel interference, consistent with previous literature. By additionally measuring the group delay at each driving wavelength and intensity, we find that the sign of the group delay excursion across the interference is an acute probe of the interference mechanism. The experimental results are confirmed by ab initio time-dependent density functional theory calculations of both the spectral intensity and the phase of the harmonic emission.

Scientific and Regulatory Policy Committee Points to Consider: Fixation, Trimming, and Sectioning of Nonrodent Eyes and Ocular Tissues for Examination in Ocular and General Toxicity Studies.

A Working Group of the Society of Toxicologic Pathology's Scientific and Regulatory Policy Committee conducted a technical and scientific review of current practices relating to the fixation, trimming, and sectioning of the nonrodent eye to identify key points and species-specific anatomical landmarks to consider when preparing and evaluating eyes of rabbits, dogs, minipigs, and nonhuman primates from ocular and general toxicity studies. The topics addressed in this Points to Consider article include determination of situations when more comprehensive evaluation of the globe and/or associated extraocular tissues should be implemented (expanded ocular sampling), and what constitutes expanded ocular sampling. In addition, this manuscript highlights the practical aspects of fixing, trimming, and sectioning the eye to ensure adequate histopathological evaluation of all major ocular structures, including the cone-dense areas (visual streak/macula/fovea) of the retina for rabbits, dogs, minipigs, and nonhuman primates, which is a current regulatory expectation for ocular toxicity studies.[Box: see text].

International Harmonization of Nomenclature and Diagnostic Criteria (INHAND): Nonproliferative and Proliferative Lesions of the Rabbit.

The INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions Project (www.toxpath.org/inhand.asp) is a joint initiative of the Societies of Toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP) and North America (STP) to develop an internationally accepted nomenclature for proliferative and non-proliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature for classifying microscopic lesions observed in most tissues and organs from the laboratory rabbit used in nonclinical safety studies. Some of the lesions are illustrated by color photomicrographs. The standardized nomenclature presented in this document is also available electronically on the internet (http://www.goreni.org/). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous lesions as well as lesions induced by exposure to test materials. Relevant infectious and parasitic lesions are included as well. A widely accepted and utilized international harmonization of nomenclature for lesions in laboratory animals will provide a common language among regulatory and scientific research organizations in different countries and increase and enrich international exchanges of information among toxicologists and pathologists.

Molecular Modes of Attosecond Charge Migration.

First-principles calculations are employed to elucidate the modes of attosecond charge migration (CM) in halogenated hydrocarbon chains. We use constrained density functional theory (DFT) to emulate the creation of a localized hole on the halogen and follow the subsequent dynamics via time-dependent DFT. We find low-frequency CM modes (∼1 eV) that propagate across the molecule and study their dependence on length, bond order, and halogenation. We observe that the CM speed (∼4 Å/fs) is largely independent of molecule length, but is lower for triple-bonded versus double-bonded molecules. Additionally, as the halogen mass increases, the hole travels in a more particlelike manner as it moves across the molecule. These heuristics will be useful in identifying molecules and optimal CM detection methods for future experiments, especially for halogenated hydrocarbons which are promising targets for ionization-triggered CM.

Investigation of the use of microwave ablation with and without cooling urethral perfusion for thermal ablation of the prostate gland in canine cadavers.

OBJECTIVE: To investigate the use of microwave ablation (MWA) with cooling urethral perfusion and with no perfusion (MWA-UP and MWA-NP, respectively) for prostate gland ablation in canine cadavers. ANIMALS: Cadavers of 18 sexually intact male dogs. PROCEDURES: After technique refinement in 2 cadavers, laparotomy with ultrasound-guided MWA-UP (n = 8) or MWA-NP (8) of the prostate gland was performed in 16 cadavers. Normograde cystourethroscopy was performed before and after treatment; recorded images were reviewed in a blinded manner for scoring of urethral mucosal discoloration and loss of integrity. Difficulty with cystoscope insertion was recorded if present. Excised prostate glands were fixed for serial sectioning, gross measurements, and calculation of percentage ablation. Percentages of prostate tissue necrosis from MWA, denuded urethral mucosa, and depth of epithelial surface loss in an adjacent section of the colon were estimated histologically. Variables of interest were statistically analyzed. RESULTS: Difficulty with cystoscope insertion after treatment was significantly more common and scores for urethral mucosal discoloration and loss of integrity were significantly higher (indicating more severe lesions) for the MWA-NP group than for the MWA-UP group. The histologically assessed percentage of denuded urethral mucosa was also greater for the MWA-NP group. Overall median percentage prostate gland ablation was 73%; this result was not associated with prostate gland volume or chronological order of treatment. CONCLUSIONS AND CLINICAL RELEVANCE: MWA-UP induced subtotal thermal necrosis of prostate glands in canine cadavers while limiting urethral mucosal injury. Further study is required to optimize the technique and evaluate its safety and efficacy in vivo as a future curative-intent treatment for prostatic tumors in dogs.

Introduction to Special Issue on Ocular Pathology and Drug Development.

Dysfunction of the visual system remains a leading cause of human disability worldwide. Preclinical studies are a key component of efforts to develop drugs and devices to ameliorate visual impairment. Although new opportunities for the delivery of targeted ocular therapeutics have been created, clinical success has been confounded by unique challenges of drug development for the eye. This Special Issue brings together a broad range of articles that augment our current understanding of the visual system and highlight methods for assessing ocular toxicity and some of the current challenges in ocular drug development. Topics addressed include the anatomy, developmental anatomy, and/or immunobiology of the visual system and associated lymphoid tissues; animal models; methods for assessing ocular toxicity; spontaneous background and procedure-related microscopic findings and common artifacts in histologic sections of ocular tissues; and novel ocular drug delivery systems.

Opinion on Performing Pathology Peer-Review During the Global Pandemic: Challenges and Opportunities.

The coronavirus disease 2019 pandemic has affected business on numerous fronts in unprecedented and abrupt ways. From site closures and local "stay-at-home orders" to travel advisories and restrictions, the day-to-day practice of toxicologic pathology has been impacted dramatically and rapidly. A critical function of Toxicologic Pathologists is performing pathology peer review for nonclinical studies. Traditionally, corroborating the findings of histological assessment could be achieved through shipment of histopathological slides to the peer review pathologist, or by the peer review pathologist traveling to the location of the slides (eg, the test facility). Since early 2020, many pathologists have been unable to perform the latter due to local, regional, national, test facility, company, and/or personal restrictions. The disruption for some has been minimal, while others are working from home for the first time. We recommend that contingency plans for all peer review procedures and personnel should be in-place to accommodate sudden and unexpected workflow transitions. Now, more than ever, approaching peer reviews with enhanced adaptability will help ensure success.

Attosecond Time-Domain Measurement of Core-Level-Exciton Decay in Magnesium Oxide.

Excitation of ionic solids with extreme ultraviolet pulses creates localized core-level excitons, which in some cases couple strongly to the lattice. Here, core-level-exciton states of magnesium oxide are studied in the time domain at the Mg L_{2,3} edge with attosecond transient reflectivity spectroscopy. Attosecond pulses trigger the excitation of these short-lived quasiparticles, whose decay is perturbed by time-delayed near-infrared pulses. Combined with a few-state theoretical model, this reveals that the infrared pulse shifts the energy of bright (dipole-allowed) core-level-exciton states as well as induces features arising from dark core-level excitons. We report coherence lifetimes for the two lowest core-level excitons of 2.3±0.2 and 1.6±0.5 fs and show that these are primarily a consequence of strong exciton-phonon coupling, disclosing the drastic influence of structural effects in this ultrafast relaxation process.

Attosecond pulse shaping using a seeded free-electron laser.

Attosecond pulses are central to the investigation of valence- and core-electron dynamics on their natural timescales1-3. The reproducible generation and characterization of attosecond waveforms has been demonstrated so far only through the process of high-order harmonic generation4-7. Several methods for shaping attosecond waveforms have been proposed, including the use of metallic filters8,9, multilayer mirrors10 and manipulation of the driving field11. However, none of these approaches allows the flexible manipulation of the temporal characteristics of the attosecond waveforms, and they suffer from the low conversion efficiency of the high-order harmonic generation process. Free-electron lasers, by contrast, deliver femtosecond, extreme-ultraviolet and X-ray pulses with energies ranging from tens of microjoules to a few millijoules12,13. Recent experiments have shown that they can generate subfemtosecond spikes, but with temporal characteristics that change shot-to-shot14-16. Here we report reproducible generation of high-energy (microjoule level) attosecond waveforms using a seeded free-electron laser17. We demonstrate amplitude and phase manipulation of the harmonic components of an attosecond pulse train in combination with an approach for its temporal reconstruction. The results presented here open the way to performing attosecond time-resolved experiments with free-electron lasers.

High-harmonic spectroscopy of transient two-center interference calculated with time-dependent density-functional theory.

We demonstrate high-harmonic spectroscopy in many-electron molecules using time-dependent density-functional theory. We show that a weak attosecond-pulse-train ionization seed that is properly synchronized with the strong driving mid-infrared laser field can produce experimentally relevant high-harmonic generation (HHG) signals, from which we extract both the spectral amplitude and the target-specific phase (group delay). We also show that further processing of the HHG signal can be used to achieve molecular-frame resolution, i.e., to resolve the contributions from rescattering on different sides of an oriented molecule. In this framework, we investigate transient two-center interference in CO2 and OCS, and how subcycle polarization effects shape the oriented/aligned angle-resolved spectra.

Nonlinear XUV signal generation probed by transient grating spectroscopy with attosecond pulses.

Nonlinear spectroscopies are utilized extensively for selective measurements of chemical dynamics in the optical, infrared, and radio-frequency regimes. The development of these techniques for extreme ultraviolet (XUV) light sources facilitates measurements of electronic dynamics on attosecond timescales. Here, we elucidate the temporal dynamics of nonlinear signal generation by utilizing a transient grating scheme with a subfemtosecond XUV pulse train and two few-cycle near-infrared pulses in atomic helium. Simultaneous detection of multiple diffraction orders reveals delays of ≥1.5 fs in higher-order XUV signal generation, which are reproduced theoretically by solving the coupled Maxwell-Schrödinger equations and with a phase grating model. The delays result in measurable order-dependent differences in the energies of transient light induced states. As nonlinear methods are extended into the attosecond regime, the observed higher-order signal generation delays will significantly impact and aid temporal and spectral measurements of dynamic processes.

Good Laboratory Practice in the Academic Setting: Fundamental Principles for Nonclinical Safety Assessment and GLP-Compliant Pathology Support When Developing Innovative Biomedical Products.

Development of new biomedical products necessitates nonclinical safety assessment in animals as a means of assessing potential risk to human patients. Pivotal nonclinical safety studies that support human clinical trials are performed according to Good Laboratory Practice (GLP) guidelines, which are designed to ensure that the study was conducted under carefully controlled conditions using standardized and validated procedures that will yield a reliable, reproducible, and traceable data set. The GLP guidelines established by different regulatory agencies address organizational structure, personnel responsibilities, personnel training practices, quality assurance (ensuring compliance), facilities, equipment, standard operating procedures, study documentation (record keeping), and record and sample retention. Academic institutions engaging in nonclinical safety assessment on-site have multiple options for implementing a GLP quality system. This article outlines the rationale supporting the use of a GLP-compliant or GLP-like quality system in academia and reviews key concepts needed to efficiently and effectively implement GLP in the academic setting. Emphasis is given to provision of GLP-compliant pathology support as (1) pathology data are an essential component of GLP nonclinical safety testing, (2) familiarity with pathology-related GLP procedures typically is gained first outside the academic setting, and (3) microscopic pathology diagnoses and interpretations require special accommodations to ensure that they are undertaken in a GLP-compliant fashion.

Historical Data: Histopathology Lesions Observed in the Eyes of Control Rabbits in Topical Ocular Administration and Contact Lens Studies.

Information on background changes in the ocular tissues of rabbits ( Oryctolagus cuniculus), a common species employed in ophthalmic toxicology studies, is sparse. This complicates interpretation of changes in light of small sample sizes on any single study. The purpose of this publication is to document the interstudy incidence of spontaneous or iatrogenic changes occurring in eyes of control rabbits. Photomicrographs of select lesions are provided. The data set was derived from a total of 54 studies conducted over an eleven-year period at Alcon Research Ltd., a Novartis Division, which featured topical ocular and contact lens routes of administration. It includes a total of 1,222 pigmented and albino New Zealand rabbits and a total of 2,084 eyes which were either untreated or treated with innocuous control articles. There were no noteworthy differences across routes of administration. Changes in anterior segment ocular and adnexal tissues were more common than in posterior segment ocular tissues. Overall, mononuclear cell infiltration was the most common finding. The retina was the posterior tissue most commonly observed with spontaneous changes, with folds and rosettes being the most common retinal finding. Retinal changes were more common in albino as compared to pigmented rabbits. Understanding the incidence and characteristics of spontaneous ocular lesions facilitates accurate and consistent diagnosis and data interpretation.

Practical Strategies for Navigating Toxicologic Pathology in One's Early Career and Beyond!

The toxicologic pathologist plays a vital role in the scientific community, using their unique blend of diagnostic and investigative skills to advance biomedical research, public health, drug discovery, or regulatory practices. But what exactly do toxicologic pathologists contribute? Where do these specialized professionals work? How can toxicologic pathologists maximize their efficiency and potential? To enlighten students and trainees, as well as early- or mid-career toxicologic pathologists, or even those approaching retirement, the Career Development and Outreach Committee of the Society of Toxicologic Pathology (STP) sponsored a career development workshop entitled "Practical Strategies for Navigating Toxicologic Pathology in One's Early Career…and Beyond!" in conjunction with the STP 37th annual symposium. The workshop featured toxicologic pathologists from contract research organizations and the pharmaceutical industry, who provided their perspectives on career preparation, evolving veterinary pathologist roles within various sectors of toxicologic pathology, the fundamentals of safety assessment, logistics of projects involving good laboratory practices, tools for effective interpretation and communication of anatomic and clinical pathology results, and a recap of scientific resources available to support the toxicologic pathologist in his or her journey. This article provides brief summaries of the talks presented during this career development workshop.

Controlling attosecond transient absorption with tunable, non-commensurate light fields.

We demonstrate a transient absorption scheme that uses a fixed-spectrum attosecond pulse train in conjunction with a tunable probe laser to access a wide range of nonlinear light-atom interactions. We exhibit control over the time-dependent Autler-Townes splitting of the 1s4p absorption line in helium, and study its evolution from a resonant doublet to a light-induced sideband with changing probe wavelength. The non-commensurate probe also allows for the background-free study of two-infrared-photon emission processes in a collinear geometry. Using this capability, we observe two different emission pathways with non-trivial delay dependencies, one prompt and the other delayed. We identify the nonlinear processes underlying these emissions by comparing the experimental results to calculations based on the time-dependent Schrödinger equation.

A safety and immunogenicity study of immunization with hVEGF26-104/RFASE in cynomolgus monkeys.

INTRODUCTION: Vascular endothelial growth factor (VEGF) is pivotal in tumor angiogenesis and therapies targeting the VEGF axis are widely used in the clinic for the treatment of cancer. We have developed a therapeutic vaccine targeting human (h)VEGF165. hVEGF26-104/RFASE is based on the truncated protein hVEGF26-104 as antigen formulated in an oil-in-water emulsion containing the sulpholipopolysaccharide RFASE as adjuvant. Here we describe the toxicity and immunogenicity of this therapeutic vaccine in cynomolgus monkeys. METHODS: In total 54 cynomolgus monkeys were used and divided in 7 groups. Groups 1-3 were control groups, either receiving PBS alone (group 1), RFASE alone (group 2) or hVEGF26-104 alone (group 3). Animals allocated to groups 4-7 received hVEGF26-104 together with RFASE, but with varying doses of the antigen or the adjuvant. All animals were immunized four times with 2-week intervals and safety and immunogenicity were monitored until 3 days after the final immunization. RESULTS: Immunization induced an RFASE adjuvant dependent acute phase response. High titers of antibodies against hVEGF26-104 and cross-reactive with hVEGF165, were found in monkey sera, 28 days after primer immunization. These antibodies were able to inhibit the binding of the monoclonal antibody bevacizumab with hVEGF165 in a competition ELISA. Moreover, the biological activity of hVEGF165 could be inhibited by the addition of immunized monkey serum in a VEGF specific bioassay. Importantly, no adverse events commonly observed with VEGF neutralization were observed throughout the study. CONCLUSION: These data show that hVEGF26-104/RFASE can be safely administered in cynomolgus monkeys, induces the desired immune response and therefore support the clinical development of this vaccine.