The U.S. Food and Drug Administration (FDA), Center for Drug Evaluation and Research (CDER), and Office of New Drugs (OND) has continuously encouraged the submission of nonclinical tests utilizing new approach methodologies (NAMs) and sponsor engagement with regulators to optimize NAM utility in supporting the safety and efficacy of new drugs. Previously, we published an FDA/CDER perspective on nonclinical testing strategies, discussed the opportunities and challenges of using NAMs to replace, reduce, and refine animal testing in drug development, and reported gaps and challenges underserved by existing nonclinical testing approaches that CDER Pharmacology/Toxicology reviewers face. Here, we demonstrate how FDA/CDER has historically incorporated NAMs into standard nonclinical assessments, describing how specific tests became validated and internationally accepted alternatives to animal testing for regulatory decision-making. We also provide a CDER/OND Pharmacology/Toxicology reviewer perspective on NAMs submitted to support new drug development, in an effort to provide insight into our experience with NAMs submitted for CDER-regulated products. Furthermore, we provide a CDER/OND Pharmacology/Toxicology reviewer perspective on the future of NAM incorporation into nonclinical development programs for new drugs as scientific technology continues to evolve. Ultimately, we hope that by sharing the FDA/CDER/OND experience with NAMs thus far and providing considerations for refining NAM submissions, we will (1) illustrate our scientific approach to evaluating NAM submissions, (2) reiterate FDA/CDER's steadfast commitment to the 3Rs, and (3) foster confidence in our continued efforts to encourage nonclinical test NAM submissions for regulatory decision-making, while maintaining our mission to protect public health and patients from unintended harm.

Exposure to diesel exhaust air pollutants is a key environmental threat for pulmonary and cardiovascular diseases. Oxidative stress and Transient Receptor Potential Vanilloid-1 (TRPV1) receptors exhibit pivotal contributions in mediating lung injury induced by environmental pollutants. This study investigates the histological changes in lung tissue induced by short-term particulate-free filtered diesel exhaust (FDE) exposure, with a focus on TRPV1 receptor expression and oxidative stress pathways. Male rats were allocated to four groups randomly: Non-exposed (NE), clean air exposed (CAE), FDE-exposed, and NAC pre-treated FDE-exposed groups. FDE exposure lasted 5hours per day for five days, with histological examination and TRPV1 expression analysis conducted on day six. The NAC pre-treated group received NAC (200mg/kg) for five days prior to each exposure. Lung tissue samples were analyzed using hematoxylin and eosin staining, and immunofluorescence for TRPV1 expression. FDE exposure caused significant histological alterations, including alveolar septal thickening, interstitial inflammation, capillary congestion, perivascular inflammation, bronchial epithelial necrosis, endothelial discontinuity, and interstitial fibrosis, as classified by a semi-quantitative INHAND (International Harmonization of Nomenclature and Diagnostic) scoring criteria for rats, visualized on a heat map. TRPV1 expression was upregulated in FDE-exposed lung tissues, particularly around congested vessels and thickened septa. NAC pre-treatment significantly reduced both histological damage and TRPV1 expression. Our study highlights a potential mechanistic relationship between TRPV1 receptor expression and oxidative stress pathways in the lung damage induced by FDE exposure, underscoring the therapeutic potential of NAC in countering these effects.

Although Bisphenol A (BPA) is still used in consumer products, concerns about its toxicity led to the adoption of structurally related replacement products such as Bisphenol F (BPF) and Bisphenol S (BPS). Unfortunately, comparing the biological responses to BPA and BPA substitutes in vivo can be challenging, as the available information is often derived from different studies using various animal strains and experimental protocols. To address this issue, we directly compared the impacts of BPA, BPF, and BPS in the same in vivo exposure study. Briefly, 8-week-old male Fischer rats were exposed to BPA, BPF, or BPS (at five different doses) and to 17α-ethinylestradiol (positive control for estrogenicity) by gavage for 28 consecutive days. Rat health, dietary intakes, and weight gains were monitored, 24-hour urine samples were collected, and blood and tissues were harvested at the terminal necropsy. The impacts of BPA, BPF, and BPS on rat weight gains, organ weights and histology, liver enzymatic activities, hematology, clinical chemistry, and serum hormone levels were relatively modest and mostly limited to the highest doses administered. However, bisphenol cross-contamination observed in urine samples from the vehicle control group may have interfered with the evaluation of their effects at lower doses. Although BPA, BPF, and BPS exposures all shared similarities with the 17α-ethinylestradiol positive control group, their impacts on serum hormone levels and endocrine-responsive tissues also presented noticeable differences. This suggests that BPA, BPF, and BPS may interfere with endocrine functions through slightly different molecular mechanisms.

Parkinson's disease (PD) is characterized by the abnormal aggregation of α-synuclein, which can originate in the gut and propagate to the brain. Recent evidence suggests a correlation between metabolic disorders, particularly diabetes, and PD pathogenesis through the gut-brain axis. Methylglyoxal (MGO), a glucose-derived metabolite produced by gut bacteria such as Proteus mirabilis, is implicated in protein misfolding and glycation. This study investigated whether MGO induced α-synuclein aggregation in intestinal enteroendocrine cells and explored the underlying mechanisms. Mouse enteroendocrine STC-1 cells were treated with MGO (0.01-1mM) for 36h, and changes in α-synuclein aggregation, neuronal markers, and relevant signaling pathways were assessed. MGO at 1mM significantly reduced cell viability and neuronal marker expression, and concentrations of 0.1 and 1mM increased α-synuclein aggregation. MGO also inhibited SIRT1 expression, leading to increased Hif-1α transcription and reduced expression of autophagy-related proteins Beclin1 and LC3B. These changes were accompanied by mitochondrial dysfunction, as evidenced by decreased Bcl2, increased cytochrome C expression, and reduced levels of the antioxidant factor HO-1. Our findings provide the first evidence that MGO directly induces α-synuclein aggregation in enteroendocrine cells via the SIRT1-Hif-1α-autophagy pathway dysregulation, establishing a potential mechanistic link between gut microbiome-derived metabolites and PD pathogenesis. These results suggest that intestinal glycation may be a critical target for preventing α-synuclein pathology originating in the gut.

The Expert Panel for Cosmetic Ingredient Safety (Panel) reviewed the safety of Polyquaternium-6 as used in cosmetic formulations. This ingredient is reported to function as an antimicrobial agent, antistatic agent, film former, and hair fixative in cosmetics. The Panel cautions that products containing Polyquaternium-6 should be formulated to avoid the formation of nitrosamines. The Panel reviewed data relevant to the safety of this ingredient in cosmetic formulations, and concluded that Polyquaternium-6 is safe in cosmetics in the present practices of use and concentration described in this safety assessment.

Platelet hyperactivation represents a significant risk factor for atherosclerotic cardiovascular diseases. This study investigated the expression and functional roles of integrin αvβ3 and (Multimerin 1) MMRN1 in platelets from atherosclerotic conditions and evaluated the therapeutic potential of integrin αvβ3 antagonism in atherosclerotic progression. We examined the expression patterns of αvβ3 and MMRN1 in platelets from healthy controls, patients with coronary heart disease (CHD), and patients with acute myocardial infarction (AMI) using qRT-PCR and ELISA techniques. The correlation between αvβ3 and MMRN1 expression levels with platelet counts and aggregation was analyzed. Additionally, we established a mouse model of atherosclerosis to investigate the effects of an αvβ3 antagonist (SB273005) and MMRN1 knockdown on platelet activation and atherosclerosis progression. Our findings revealed positive correlations between MMRN1 and αvβ3 expression levels with both platelet counts and aggregation. Notably, elevated platelet counts and aggregation observed in CHD and AMI patient samples were effectively reduced by the αvβ3 antagonist treatment. Furthermore, both αvβ3 antagonist treatment and MMRN1 knockdown significantly ameliorated disease severity in the mouse atherosclerosis model. These results demonstrate that upregulation of αvβ3 integrin and MMRN1 contributes to platelet hyperactivation in atherosclerotic vascular diseases, and targeting the αvβ3/MMRN1 axis may serve as a promising intervention strategy for the clinical management of atherosclerotic vascular diseases.

Diabetes and hypertension often coexist and need complex pharmacological management. Sitagliptin, a dipeptidyl peptidase-4 inhibitor, is widely used for glycemic control, and telmisartan, an angiotensin II receptor blocker, is the preferred treatment for hypertension. Thus, we have conducted a 1-month exploratory combination safety and toxicity study in male Wistar Han rats. Animals were administered 100mg/kg/day of sitagliptin or telmisartan individually, or in combination by oral gavage. We have evaluated effects on body weight, feed intake, clinical chemistry, and hematology parameters, and microscopic examination of liver, kidney, adrenal glands, heart, lungs, lymphatic organs, and gastrointestinal tract. The combination showed a decrease in body weight gain, feed intake, hematocrit, reticulocytes, WBCs, serum triglycerides, AST, total protein, globulin, and heart weight, and increased serum levels of urea, phosphorous, magnesium, potassium, and kidney weight. Co-administration showed an additive effect on decrease in WBC counts, potentiating the effects on decreased serum triglycerides and increased phosphorous levels and kidney weights, and a synergistic effect on serum AST levels. Sitagliptin attenuated the changes caused by telmisartan on reticulocyte counts, serum creatinine levels, and liver and thymus weights. Administration of the individual compounds caused a reduction in spleen weight, though the combination had no effect. Sitagliptin, telmisartan, or combination dosing showed no significant organ toxicity upon histopathological examination. It appears that co-administration of telmisartan and sitagliptin is well tolerated in rats. Three-month toxicity studies, in both genders, with full toxicokinetic profiling and thorough tissue examination, will be necessary to support the combination's toxicity profile.

The Expert Panel for Cosmetic Ingredient Safety (Panel) assessed the safety of 8 glycerin ethoxylates, as used in cosmetic formulations. All of these ingredients are reported to function in cosmetics as skin-conditioning agents, and most are also reported to function as viscosity-decreasing agents. The Panel reviewed relevant data relating to the safety of these ingredients. The Panel concluded that these ingredients are safe in cosmetics in the present practices of use and concentration as described in this safety assessment when formulated to be non-irritating.

The Expert Panel for Cosmetic Ingredient Safety (Panel) reviewed the safety of 4 Scutellaria baicalensis-derived ingredients in cosmetic products; these ingredients are reported to have the following functions in cosmetics: antimicrobial agent, skin conditioning agent, abrasives, fragrance ingredients, skin protectants, and antioxidants. The Panel reviewed relevant data relating to the safety of these ingredients in cosmetic formulations, and concluded that Scutellaria Baicalensis Root Extract and Scutellaria Baicalensis Root Powder are safe in cosmetics in the present practices of use and concentration described in this safety assessment. The Panel also concluded that the available data are insufficient to make a determination of safety for Scutellaria Baicalensis Extract and Scutellaria Baicalensis Sprout Extract under the reported conditions of use in cosmetic formulations.

2-Deoxy-D-Glucose (2-DG) has anticonvulsant and antiseizure effects in rodent models and is in the development phase for novel antiseizure treatment. To evaluate potential toxicity, Beagle dogs (five/sex/group) were orally gavaged with either vehicle (deionized water) or 2-DG (5, 30, and 90mg/kg BID) for 28days followed by a 14-day recovery period. The safety endpoints evaluated were mortality, clinical observations, body temperature, respiratory assessment, body weights, food consumption, ophthalmic examinations, electrocardiograph (ECG), blood pressure, cardiac biomarker (NT-proBNP), and pathology. Toxicokinetic analysis was conducted after the first dose on days 1 and 28. The dose formulation analysis confirmed that 2-DG concentrations were within 93%-107% of the target concentrations. There were no 2-DG associated effects observed in mortality, clinical signs, body temperature, respiratory parameters, body weights, food consumption, ophthalmic examination, ECG, blood pressure, and NT-proBNP. There was an increase (∼1.7X) in aspartate transaminase on day 29, while histopathological evaluation revealed hepatic cytoplasmic alterations in 2 of 6 dogs on day 29 and only in 1 of 4 dogs on day 43 at 90mg/kg BID. These changes were considered non-adverse because of minimal severity, reversibility trend after recovery period and no correlative increase in alanine transaminase. Toxicokinetic evaluation revealed dose dependent increases in Cmax of ∼7.8, 39.5, and 114μg/mL, and AUCs of 12.2, 70.8, and 202 h*μg/mL at 5, 30, and 90mg/kg, respectively with Tmax of ∼0.5-0.9h and T1/2 of ∼3.8-5.4h. In conclusion, 90mg/kg BID of 2-DG was considered as the No Observed Adverse Effect Level following 28-day administration in dogs.