Toxicology Certification: The Need, the Value, and an Evolving Landscape.

Certification in toxicology remains a subject of interest to those in the field, as evidenced by the number of presentations at major meetings and publications in the past decade. In 2009, Brock and colleagues summarized the certifications available in the field of toxicology and provided an international perspective of the pros and cons of gaining certification. Though that article has been viewed thousands of times, the certification processes have evolved over the past decade. Therefore, the purpose of the article is to build upon the prior work and present current information for toxicologists seeking certification with the American Board of Toxicology (DABT), Academy of Toxicological Sciences (ATS), as a European Registered Toxicologist (ERT), as a Diplomate, Japanese Society of Toxicology (DJSOT), and as a Diplomate of the Chinese Society of Toxicology (DCST). In addition, other certifications are presented, but available details are limited.

Opportunities for use of one species for longer-term toxicology testing during drug development: A cross-industry evaluation.

An international expert working group representing 37 organisations (pharmaceutical/biotechnology companies, contract research organisations, academic institutions and regulatory bodies) collaborated in a data sharing exercise to evaluate the utility of two species within regulatory general toxicology studies. Anonymised data on 172 drug candidates (92 small molecules, 46 monoclonal antibodies, 15 recombinant proteins, 13 synthetic peptides and 6 antibody-drug conjugates) were submitted by 18 organisations. The use of one or two species across molecule types, the frequency for reduction to a single species within the package of general toxicology studies, and a comparison of target organ toxicities identified in each species in both short and longer-term studies were determined. Reduction to a single species for longer-term toxicity studies, as used for the development of biologicals (ICHS6(R1) guideline) was only applied for 8/133 drug candidates, but might have been possible for more, regardless of drug modality, as similar target organ toxicity profiles were identified in the short-term studies. However, definition and harmonisation around the criteria for similarity of toxicity profiles is needed to enable wider consideration of these principles. Analysis of a more robust dataset would be required to provide clear, evidence-based recommendations for expansion of these principles to small molecules or other modalities where two species toxicity testing is currently recommended.

Intermittent oral coadministration of a gamma secretase inhibitor with dexamethasone mitigates intestinal goblet cell hyperplasia in rats.

Dexamethasone was given in 2 oral dosing regimens with repeat dose oral administration of the gamma secretase inhibitor (GSI), PF-03084014, in Sprague-Dawley (SD) rats in order to evaluate the effects of coadministration of dexamethasone on GSI-induced goblet cell hyperplasia (GCH) in the intestinal tract. Safety end points were evaluated in 1 week and 1 month studies. The dosing regimens tested in the 1-month studies included a 1-week pretreatment with 1.0 mg/kg dexamethasone followed by a 3-week repeat dose treatment with 100 mg/kg GSI or concurrent intermittent treatment with 1.0 mg/kg dexamethasone on weeks 1 and 3 and repeat dose treatment with 100 mg/kg GSI for 4 weeks. Pretreatment with dexamethasone for 1 week transiently mitigated the severity of intestinal GCH for up to 1 week. Intermittent coadministration of dexamethasone on weeks 1 and 3 with GSI repeat dosing for 4 weeks mitigated intestinal GCH for up to 4 weeks post treatment. Treatment-related morbidity and mortality occurred on day 7 with 150 mg/kg GSI and 5 mg/kg dexamethasone coadministration, and on days 13, 14, and 23 with 100 mg/kg GSI and 1 mg/kg dexamethasone coadministration.

Recommendations from a global cross-company data sharing initiative on the incorporation of recovery phase animals in safety assessment studies to support first-in-human clinical trials.

An international expert group which includes 30 organisations (pharmaceutical companies, contract research organisations, academic institutions and regulatory bodies) has shared data on the use of recovery animals in the assessment of pharmaceutical safety for early development. These data have been used as an evidence-base to make recommendations on the inclusion of recovery animals in toxicology studies to achieve scientific objectives, while reducing animal use. Recovery animals are used in pharmaceutical development to provide information on the potential for a toxic effect to translate into long-term human risk. They are included on toxicology studies to assess whether effects observed during dosing persist or reverse once treatment ends. The group devised a questionnaire to collect information on the use of recovery animals in general regulatory toxicology studies to support first-in-human studies. Questions focused on study design, the rationale behind inclusion or exclusion and the impact this had on internal and regulatory decisions. Data on 137 compounds (including 53 biologicals and 78 small molecules) from 259 studies showed wide variation in where, when and why recovery animals were included. An analysis of individual study and programme design shows that there are opportunities to reduce the use of recovery animals without impacting drug development.

Developing a pragmatic consensus procedure supporting the ICH S1B(R1) weight of evidence carcinogenicity assessment.

The ICH S1B carcinogenicity global testing guideline has been recently revised with a novel addendum that describes a comprehensive integrated Weight of Evidence (WoE) approach to determine the need for a 2-year rat carcinogenicity study. In the present work, experts from different organizations have joined efforts to standardize as much as possible a procedural framework for the integration of evidence associated with the different ICH S1B(R1) WoE criteria. The framework uses a pragmatic consensus procedure for carcinogenicity hazard assessment to facilitate transparent, consistent, and documented decision-making and it discusses best-practices both for the organization of studies and presentation of data in a format suitable for regulatory review. First, it is acknowledged that the six WoE factors described in the addendum form an integrated network of evidence within a holistic assessment framework that is used synergistically to analyze and explain safety signals. Second, the proposed standardized procedure builds upon different considerations related to the primary sources of evidence, mechanistic analysis, alternative methodologies and novel investigative approaches, metabolites, and reliability of the data and other acquired information. Each of the six WoE factors is described highlighting how they can contribute evidence for the overall WoE assessment. A suggested reporting format to summarize the cross-integration of evidence from the different WoE factors is also presented. This work also notes that even if a 2-year rat study is ultimately required, creating a WoE assessment is valuable in understanding the specific factors and levels of human carcinogenic risk better than have been identified previously with the 2-year rat bioassay alone.

Workshop report: A study roadmap to evaluate the safety of recombinant human lactoferrin expressed in Komagataella phaffii intended as an ingredient in conventional foods - Recommendations of a scientific expert panel.

Human milk lactoferrin (hmLF) is a glycoprotein with well-known effects on immune function. Helaina Inc. has used a glycoengineered yeast, Komatagaella phaffii, to produce recombinant human lactoferrin (Helaina rhLF, Effera™) that is structurally similar to hmLF with intended uses as a food ingredient. However, earlier FDA reviews of rhLF were withdrawn due to insufficient safety data and unanswered safety questions the experts and FDA raised about the immunogenicity/immunotoxicity risks of orally ingested rhLF. Helaina organized a panel of leading scientists to build and vet a safety study roadmap containing the studies and safety endpoints needed to address these questions. Panelists participated in a one-day virtual workshop in June 2023 and ensuing discussions through July 2023. Relevant workshop topics included physicochemical properties of LF, regulatory history of bovine LF and rhLF as food ingredients in the FDA's generally recognized as safe (GRAS) program, and synopses of publicly available studies on the immunogenicity/alloimmunization, immunotoxicology, iron homeostasis, and absorption, distribution, metabolism, and excretion of rhLF. Panelists concluded that the safety study roadmap addresses the unanswered safety questions and the intended safe use of rhLF as a food ingredient for adults and agreed on broad applications of the roadmap to assess the safety and support GRAS of other recombinant milk proteins with immunomodulatory functions.

In silico approaches in carcinogenicity hazard assessment: case study of pregabalin, a nongenotoxic mouse carcinogen.

In silico toxicology protocols are meant to support computationally-based assessments using principles that ensure that results can be generated, recorded, communicated, archived, and then evaluated in a uniform, consistent, and reproducible manner. We investigated the availability of in silico models to predict the carcinogenic potential of pregabalin using the ten key characteristics of carcinogens as a framework for organizing mechanistic studies. Pregabalin is a single-species carcinogen producing only one type of tumor, hemangiosarcomas in mice via a nongenotoxic mechanism. The overall goal of this exercise is to test the ability of in silico models to predict nongenotoxic carcinogenicity with pregabalin as a case study. The established mode of action (MOA) of pregabalin is triggered by tissue hypoxia, leading to oxidative stress (KC5), chronic inflammation (KC6), and increased cell proliferation (KC10) of endothelial cells. Of these KCs, in silico models are available only for selected endpoints in KC5, limiting the usefulness of computational tools in prediction of pregabalin carcinogenicity. KC1 (electrophilicity), KC2 (genotoxicity), and KC8 (receptor-mediated effects), for which predictive in silico models exist, do not play a role in this mode of action. Confidence in the overall assessments is considered to be medium to high for KCs 1, 2, 5, 6, 7 (immune system effects), 8, and 10 (cell proliferation), largely due to the high-quality experimental data. In order to move away from dependence on animal data, development of reliable in silico models for prediction of oxidative stress, chronic inflammation, immunosuppression, and cell proliferation will be critical for the ability to predict nongenotoxic compound carcinogenicity.

Neurotoxicity of an Hepatitis B Virus (HBV) Transcript Inhibitor in 13-Week Rat and Monkey Studies.

The nonclinical safety profile of GS-8873, a hepatitis B virus RNA transcript inhibitor was evaluated in rat and monkey 13-week toxicity studies with 8-week recovery phases. Vehicle or GS-8873 was dosed orally for 13 weeks at 2, 6, 20, and 60 mg/kg/day to Wistar Han rats and at 0.5, 1.5, 3, and 6 mg/kg/day to cynomolgus monkeys. In vitro and in vivo screening results from an analog discovered prior to GS-8873 informed the 13-week toxicology study designs. Neuroelectrophysiology and neurobehavioral evaluations were included at weeks 4 and 13 of the dosing and recovery phases for GS-8873. No adverse neurobehavioral effects were observed. Significant nerve conduction velocity (NCV) decreases and latency increases occurred at the high doses after 4 weeks of dosing. By week 13, dose-responsive NCV reductions and latency increases worsened across all dose groups compared with controls. Some reversal occurred 8 weeks after the last dose administered, but not to vehicle control levels. A minimal, axonal degeneration was observed in rat spinal and peripheral nerves across dose groups compared with controls. No monkey nervous system microscopic findings were observed. No-observed-adverse-effect-levels could not be determined for either species due to the neuroelectrophysiology findings and development was halted in the interest of safety. A retrospective risk assessment approach utilizing benchmark dose (BMD) modeling contributed 13-week NCV BMDL estimates (lower limits of the 95% confidence interval) in lieu of no-observed-adverse-effect-levels. The best-fitted models extrapolated NCV BMDLs for the rat caudal and monkey sural nerve at 0.3 and 0.1 mg/kg/day, respectively.

Impaired reproduction in adult male, but not female, rats following juvenile treatment with the aromatase inhibitor, exemestane.

BACKGROUND: Exemestane is an irreversible steroidal inhibitor of cytochrome-P450 aromatase required for estrogen synthesis. The safety of the drug in the pediatric population, particularly in males, has not previously been evaluated. Given the increased interest in treating children with aromatase inhibitors, we undertook a study in rats to assess the potential for exemestane to alter reproductive development and function when administered to juveniles. METHODS: Male and female rats were treated with exemestane at doses anticipated to produce exposures approximately 2- and 35-fold the expected clinical plasma exposure in young adult males during the period of reproductive maturation. After maturation, treated rats were mated to evaluate the potential impact on reproductive function. RESULTS AND CONCLUSION: There were no effects on sexual maturation in either sex or on female reproductive function. Treatment of juvenile male rats caused increased cohabitation time and decreased copulation rates; pregnancy rates and litter size were not affected in rats that mated. Decreased testis (10-15%) and epididymis (20-30%) weights, and decreased Sertoli cell numbers were noted at all doses. This indicates that exemestane can reduce Sertoli cell proliferation during maturation. The sensitive window for this effect is expected to be limited to the period of Sertoli cell proliferation, which is completed by around postnatal day 15 in rats and before puberty in humans. Treatment beginning at a later time relative to the window for Sertoli cell proliferation or for a longer duration is not expected to have additional adverse effect as the effect was not shown to be degenerative.

Considerations regarding nonhuman primate use in safety assessment of biopharmaceuticals.

Selection of a pharmacologically responsive species can represent a major challenge in designing nonclinical safety assessment programs for many biopharmaceuticals (eg, monoclonal antibodies (mAbs)). Frequently, the only relevant species for nonclinical testing of mAbs is the non-human primate (NHP). This situation, coupled with a rapidly increasing number of mAb drugs in development, has resulted in a significant increase in the number of NHPs used in nonclinical safety assessment. Apart from ethical considerations related to responsible animal use, there is a clear need for more efficient and innovative approaches to drug discovery and development; these factors drive the need to investigate alternative approaches and strategies for the safety assessment. This review summarizes important scientific and regulatory perspectives derived from presentations and audience discussions in an educational forum at the 2010 annual American College of Toxicology meeting regarding opportunities for employing alternative approaches to minimize NHP use in mAb drug development.

Species-Specific Urothelial Toxicity With an Anti-HIV Noncatalytic Site Integrase Inhibitor (NCINI) Is Related to Unusual pH-Dependent Physicochemical Changes.

GS-9695 and GS-9822 are next-generation noncatalytic site integrase inhibitors (NCINIs) with significantly improved potency against human immunodeficiency virus compared with previous drugs such as BI-224436. Development stopped due to vacuolation of the bladder urothelium seen in cynomolgus monkey but not in rat; this lesion was absent in equivalent preclinical studies with BI-224436 (tested in dog and rat). Lesions were unlikely to be attributable to target because NCINIs specifically target viral integrase protein and no mammalian homologue is known. Secondary pharmacology studies, mitochondrial toxicity studies, immunophenotyping, and analysis of proteins implicated in cell-cell interactions and/or bladder integrity (E-cadherin, pan-cytokeratin, uroplakins) failed to offer any plausible explanation for the species specificity of the lesion. Because it was characterized by inflammation and disruption of urothelial morphology, we investigated physicochemical changes in the bladder of cynomolgus monkey (urinary pH 5.5-7.4) that might not occur in the bladder of rats (urinary pH 7.3-8.5). In measurements of surface activity, GS-9822 showed an unusual transition from a monolayer to a bilayer at the air/water interface with decreasing pH, attributed to the strong association between drug molecules in adjacent bilayer leaflets and expected to be highly disruptive to the urothelium. Structural analysis of GS-9822 and GS-9695 showed zwitterionic characteristics over the range of pH expected in cynomolgus monkey but not rat urine. This exotic surface behavior is unlikely with BI-224436 since it would transition from neutral to cationic (never zwitterionic) with decreasing pH. These data provide useful insights to guide discovery and development of NCINIs, related compounds, and zwitterions.

Lack of an association between clinical INSTI-related body weight gain and direct interference with MC4 receptor (MC4R), a key central regulator of body weight.

An increasing prevalence of overweight and obesity in people living with HIV has been associated with initiation of antiretroviral therapy with integrase strand transfer inhibitors (INSTIs). An off-target inhibition of the endogenous ligand binding to the human melanocortin 4 receptor (MC4R) has been suggested as a potential mechanism for clinical body weight gain following initiation of dolutegravir, an INSTI. In this study, we interrogated several INSTIs for their capacity for antagonism or agonism of MC4R in an in vitro cell-based assays including at concentrations far exceeding plasma concentrations reached at the recommended dosages. Our results indicate that while INSTIs do exhibit the capacity to antagonize MC4R, this occurs at concentrations well above predicted clinical exposure and is thus an implausible explanation for INSTI-associated weight gain.

The West coast regional safety pharmacology society meeting update: Filling translational gaps in safety assessment.

The Safety Pharmacology Society (SPS) held a West Coast Regional Meeting in Foster City, CA on November 14, 2018 at the Gilead Sciences Inc. site. The meeting was attended by scientists from the pharmaceutical and biotechnology industry, contract research organizations (CROs) and academia. A variety of scientific topics were presented by speakers, covering a broad variety of topics in the fields of safety risk assessment; from pro-arrhythmia and contractility risk evaluation, to models of heart failure and seizure in-a-dish; and discovery sciences; from stem cells and precision medicine, to models of inherited cardiomyopathy and precision cut tissue slices. The present review summarizes the highlights of the presentations and provides an overview of the high level of innovation currently underlying many frontiers in safety pharmacology.

"Omics" Technologies and the Immune System (a) , (b).

Recent advances in genomics-based identification of gene families and gene polymorphisms associated with immune system dysfunction have answered basic questions in immunology and have begun to move forward our understanding of immune-related disease processes. In toxicology, "omic" technologies have the potential to replace or supplement current immunotoxicological screening procedures, to provide insight into potential mode or mechanisms of action, and to provide data suitable for risk assessment. The application of omic technologies to the study of the immune system also has great potential to appreciably impact the diagnosis and treatment of immune-related diseases. This review focuses on the use of omic technologies in immunopharmacology and immunotoxicology, specifically considering the potential for these technologies to impact chemical hazard identification, risk characterization and risk assessment, and the development and application of novel therapeutics. The state of the science of omics technologies and the immune system is addressed in terms of a continuum of understanding of how omics technologies can and cannot yet be applied in the various aspects of immunopharmacology and immunotoxicology. Additionally, information gaps are identified that, once addressed, will move each area further down the continuum of understanding.

Developmental toxicology evaluations--issues with including neurotoxicology and immunotoxicology assessments in reproductive toxicology studies.

Developmental and reproductive toxicology (DART) has routinely been a part of safety assessment. Attention is now focused on the effects of chemicals on the developing nervous and immune systems. This focus on developmental neurotoxicology (DNT) and developmental immunotoxicology (DIT) is based on the premise that children differ from adults in some aspects of their biology and, thus, may also differ in their responses to chemicals. This session's objective was to discuss issues common to DNT and DIT as they relate to DART protocols, including high dose selection and maternal toxicity, adequacy of pup exposure during lactation, use of a different dosing paradigm for DART versus DNT or DIT studies, and whether DIT and DNT endpoints can be incorporated into a single DART study for hazard identification purposes. Consensus was achieved on all topics except the adequacy for risk assessment purposes of the use of a limited number of endpoints for DIT and DNT, with the DNT endpoints being the primary focus of disagreement. Panelists indicated that a combination study design for hazard identification was feasible, though flexibility to meet the scientific needs of the project was emphasized. The adequacy of existing triggers for additional developmental studies was also questioned. Panelists iterated the importance of understanding pup exposure during the various life stages and the use of toxicokinetic data in designing these studies. The group agreed to consider the HESI ACSA Life Stages Task Force recommendations as a next step to address some of the issues and challenges raised during this session.

A proposed testing framework for developmental immunotoxicology (DIT).

A group of thirty immunotoxicology experts from the U.S. and E.U. representing government, industry, and academia met in May 2003, in Washington, D.C., to reach consensus regarding the most appropriate methods to assess developmental immunotoxicology (DIT) for hazard identification, including under what conditions such testing might be required. The following points represent the major conclusions from this roundtable discussion: (1) the rat is the preferred model; (2) any DIT protocol should be based on immune assays already validated; (3) DIT methods should be incorporated into standard developmental and reproductive toxicity protocols to the extent possible rather than a "stand-alone" protocol; (4) the approach to address DIT potential should be similar for chemicals and drugs, but the experimental design should be flexible and should reflect the specific questions to be answered; (5) it is possible to utilize a study design that assesses all critical windows in one protocol, with the results leading to further study of specific effects, as warranted; (6) animals should be exposed throughout the treatment protocol; (7) the triggers for DIT may include structure-activity-relationships, results from other toxicity studies, the intended use of a drug/chemical and/or its anticipated exposure of neonates and/or juveniles.

Immunotoxicity evaluation of nelfinavir in rats.

The objective of these investigations was to determine whether exposure to the HIV-1 protease inhibitor nelfinavir compromises immune function in Sprague Dawley rats. Animals (20/sex per group) were exposed orally for 1 or 6 months to nelfinavir at doses of 0 (1% carboxymethylcellulose vehicle), 100, 300 or 1000 mg/kg per day. Animals were observed daily for morbidity/mortality and for clinical signs of toxicity. Body weights were recorded weekly (weeks 1-14) and then monthly thereafter and at study termination. At termination (1 month or 6 months; 10/sex per group), serum was collected and retained for toxicokinetic analysis. The spleen, thymus and liver were removed from each animal and weighed; thymuses and liver were discarded after weighing. Spleens were prepared and immunophenotyping, natural killer (NK) cell activity, and proliferative responses to mitogenic stimuli (e.g., concanavalin A, Salmonella typhimurium) were evaluated. There were no treatment-related effects on immune cell populations (absolute or percent values) or in proliferative responses. At the 1-month interval, a decrease in NK cell activity (0.45-fold control) was noted in male rats at 100 and 1000 mg/kg per day but not at the middle dose of 300 mg/kg per day. Female rats at 1 month were noted for an increase in NK cell activity (1.4-fold control) at 100 mg/kg per day, but there was no difference in the NK response between vehicle-treated animals and those exposed to higher doses of nelfinavir. No effects on NK activity were noted in female animals after 6 months of nelfinavir treatment. Assay difficulties prevented evaluation of male rats at the 6-month interval. Taken together, the absence of a dose-response effect for NK activity in male rats treated for 1 month, the lack of suppressive effects in females treated for either 1 or 6 months, and the unchanged splenic NK cell numbers in nelfinavir-treated animals at both 1 and 6 months suggest that the decreased NK activity noted in male rats at 1 month is not biologically relevant. It was therefore concluded that, under the experimental conditions used, oral treatment with nelfinavir for 1 or 6 months at doses up to 1000 mg/ kg per day is not immunosuppressive in rats. C8hr values following nelfinavir treatment at 1000 mg/kg per day for 6 months were between 1- and 2.7-fold the reported Cmax values in humans.

Cardiovascular differentiation of imatinib and bosutinib in the rat.

Imatinib and bosutinib were administered to rats for up to 6 months at clinically relevant exposures to investigate the effects on the cardiovascular system. Imatinib treatment resulted in increased volume, wall thickness and mass suggesting a hypertrophic heart in male and female rats at one and fivefold clinical exposures, respectively. Bosutinib treatment resulted in milder cardiac hypertrophy in female rats only at fivefold clinical exposures. Analysis of excised hearts and cultured myocytes demonstrated increased expression of hypertrophic genes with imatinib or analogs, but not bosutinib or c-Abl RNAi treatment. The current dataset suggests that cardiovascular liability of imatinib and bosutinib are differentiated preclinically and c-Abl independent.

Developmental immunotoxicity (DIT) testing of pharmaceuticals: current practices, state of the science, knowledge gaps, and recommendations.

The development and regulatory approval of immunomodulatory pharmaceuticals to treat many human diseases has increased significantly over the last two decades. As discussed by FDA and ICH guidelines, all human pharmaceuticals in development should be evaluated for potential adverse effects on the immune system. Developmental immunotoxicology (DIT) focuses on the concern that early-life (during pre-/post-natal development) exposure to agents which target the immune system may result in enhanced susceptibility to immune-related disease (e.g., infection, autoimmunity, and cancer, particularly leukemia) compared to adults, unique effects not observed in adults, or more persistent effects in comparison to those following adult exposure. This article provides a substantive review of the literature and presents detailed considerations for DIT testing strategies with a specific focus on pharmaceuticals and biopharmaceuticals. In this regard, differences between small molecule and large molecule therapeutics will be considered, along with recommendations for best practices in the assessment of DIT during drug development. In addition, gaps in the DIT knowledge base and current testing strategies are identified. Finally, a summary of an ILSI-HESI-ITC sponsored Workshop conducted in 2010, entitled 'Developmental Immunotoxicity Testing of Pharmaceuticals' will be presented. This Workshop consisted of participants from the pharmaceutical, biotechnology, academic, and regulatory sectors, where many of the issues relating to DIT outlined in this review were discussed, key points of consensus reached, and current gaps in the science identified.