Toxicological Data Research Articles

Category-Based Toxicokinetic Evaluations of Data-Poor Per- and Polyfluoroalkyl Substances (PFAS) using Gas Chromatography Coupled with Mass Spectrometry.

Concern over per- and polyfluoroalkyl substances (PFAS) has increased as more is learned about their environmental presence, persistence, and bioaccumulative potential. The limited monitoring, toxicokinetic (TK), and toxicologic data available are inadequate to inform risk across this diverse domain. Here, 73 PFAS were selected for in vitro TK evaluation to expand knowledge across lesser-studied PFAS alcohols, amides, and acrylates. Targeted methods developed using gas chromatography-tandem mass spectrometry (GC-MS/MS) were used to measure human plasma protein binding and hepatocyte clearance. Forty-three PFAS were successfully evaluated in plasma, with fraction unbound (fup) values ranging from 0.004 to 1. With a median fup of 0.09 (i.e., 91% bound), these PFAS are highly bound but exhibit 10-fold lower binding than legacy perfluoroalkyl acids recently evaluated. Thirty PFAS evaluated in the hepatocyte clearance assay showed abiotic loss, with many exceeding 60% loss within 60 min. Metabolic clearance was noted for 11 of the 13 that were successfully evaluated, with rates up to 49.9 μL/(min × million cells). The chemical transformation simulator revealed potential (bio)transformation products to consider. This effort provides critical information to evaluate PFAS for which volatility, metabolism, and other routes of transformation are likely to modulate their environmental fates.

Ecological safety evaluation of chlorpyrifos on agricultural soil: Effects on soil microbes

The health of farmland soils is directly related to the proper application of pesticides. Chlorpyrifos is a widely used broad-spectrum organophosphorus insecticide, and its ecological security risk to farmland soils requires evaluation. A trial was conducted to study the effects of chlorpyrifos on soil microbes in a wheat–maize rotation field. A 45 % chlorpyrifos emulsion was sprayed at the recommended and dual doses, after which the chlorpyrifos residue, soil microbial content, bacterial community structure, functional microbial abundance, and enzyme activities were measured. The results showed that chlorpyrifos had a half-life of 8.94–9.88 and 2.17–2.18 days in soil for wheat and maize, respectively. Although the final chlorpyrifos residues in the crop grains were lower than the maximum residue limit recommended by the Chinese government, soil fungal abundance was significantly inhibited, and the structure and function of soil bacterial communities were disturbed. Furthermore, the quantities of nitrification-related genera (Acidobacteria GP4 and Acidobacteria GP6) and functional genes (AOB-amoA and AOA-amoA) decreased, whereas those of denitrification-related functional genes (nirS and nirK) and urease activity increased. Gemmatimonas and alkaline phosphatase involved in phosphorus cycling were activated, and the abundance of cbbLR and cbbLG genes involved in carbon fixation decreased. The integrated biomarker response index showed that nirK and β-glucosidase were the most sensitive indicators in wheat and maize stages. The present study provides toxicological data and theoretical guidance for the ecological safety evaluation and rational application of chlorpyrifos in soil.

Derivation of Sustainable Reference Chemical Levels for the Protection of Italian Freshwater Ecosystems

The current environmental quality standards (EQSs) for freshwater ecosystems have been established in relation to the priority substances covered by Directive 2013/39/EU. The procedure for deriving EQSs that rely on the selection of the most sensitive toxicological data, with the application of arbitrary safety factors, is probably unrealistic for the Italian freshwater ecosystem. In this work, a procedure for the evaluation of specific sensitivity of 13 taxonomic groups from bacteria to amphibians and the derivation of protective chemical reference values specifically for the Italian aquatic communities was developed. Toxicological raw data of species belonging to the same taxonomic group spending at least one phase of their life cycle in Italian freshwater ecosystems were downloaded from EnviroTox and USEPA ECOTOX databases, aggregated, and then used as input for the model called Species Sensitivity Distribution in order to estimate the predicted no effect concentrations (PNECs). The comparison of relative sensitivity factors (RFSs) made it possible to identify the amphibians as the most sensitive group toward metals, trace elements, and pesticides, whereas crustacean were identified as the most sensitive group toward towards polycyclic aromatic hydrocarbons (PAHs). PNECs were estimated to cover 62 substances, of which 37 identified by Directive 2013/39/EU, and in most of the cases, the values were higher than EQSs. The PNECs reported in this work should be considered more realistic and tailored for Italian freshwater ecosystems, having significant repercussions in the classification of water bodies and the estimation of environmental impact assessment.

20 An Approach Towards in Vitro-Based Human Toxicity Effect Factors for the Life Cycle Impact Assessment of Inhaled Low-Solubility Particles.

Abstract Life Cycle Assessment (LCA) calculations of products containing nanomaterials are limited in their comprehensiveness by the lack of characterization factors linking nanomaterial emissions to their impacts on human health. This is mainly due to the scarcity of animal toxicological data compared to the number of existing nanomaterials, a constraint that could be lifted if in vivo data could be substituted by in vitro data to calculate nanomaterials' effect factors (EF). To support a future animal-independent LCA methodology, we present a step-by-step procedure to calculate in vitro-to-in vivo extrapolation factors to estimate human Benchmark Doses and subsequently in vitro-based EFs for inhaled non-soluble nanomaterials. Titanium dioxide, amorphous silica, cerium oxide and crystalline silica are presented as case studies. Based on mouse data, the in vitro-based EF of TiO2 is between 2.76*10-4 and 1.10*10-3 cases/(m2/g *kg intake), depending on the aerodynamic size of the particle, which is in good agreement with in vivo-based EFs (1.51*10-4 - 5.6*10-2 cases/(m2/g *kg intake)). The EF for amorphous silica is in a similar range as for TiO2, but the result is less robust due to only few in vivo data available. The discrepancy between in vivo and animal in vitro data in terms of availability and quality limits the coverage of further nanomaterials. While the work focused on LCA, the procedure can be easily adapted for risk assessment as well, thus increasing the relevance of our findings.

94 Metalsafety – In Vitro, ex Vivo and in Vivo Investigations on Particulate and Nanofibrous Metal Compounds

Abstract Metals and metal compounds are essential in daily life. Besides particulate materials nanofibrous material (nanowires) gain importance in innovative processes like optoelectronics. Toxicological data to assess the hazard is often available only for granular metals while data on nanowires is sparse. The project MetalSafety funded by the German BMBF aims at the development of an in vitro model to allow the grouping of different metal compounds with different bioavailability according to their toxicological hazard. Ex vivo and in vivo investigations are used to analyse the predictivity of the in vitro results and the underlying mode-of-action and dose-response relationship. Within this contribution the study concept is introduced. Silver and copper, both in particulate and nanofibrous form, are used across all examinations in vitro, ex vivo and in vivo. To allow an interspecies comparison cell lines derived from human and rat lung are used. Exposure to the materials can be investigated either submerse or with air-liquid-interface (ALI) conditions. The correlation between both species can also be examined ex vivo using precision-cut lung slices (PCLS) derived from human and rat lung tissue. Application in vivo by intratracheal instillation in rats is conducted to enable a validation of the in vitro and ex vivo screening models in a best case scenario under physiological conditions. Comparable endpoints in vitro, ex vivo as well as in vivo are measured during the study. This includes cytotoxicity endpoints and cytokine measurements as well as gene expression analyses. The lung tissue is also subjected to a histopathological examination.

The Impact of Linoleic Acid on Infant Health in the Absence or Presence of DHA in Infant Formulas

Both linoleic acid (LA) and α-linolenic acid (ALA) are essential dietary fatty acids, and a balanced dietary supply of these is of the utmost importance for health. In many countries across the globe, the LA level and LA/ALA ratio in breast milk (BM) are high. For infant formula (IF), the maximum LA level set by authorities (e.g., Codex or China) is 1400 mg LA/100 kcal ≈ 28% of total fatty acid (FA) ≈ 12.6% of energy. The aims of this study are: (1) to provide an overview of polyunsaturated fatty acid (PUFA) levels in BM across the world, and (2) to determine the health impact of different LA levels and LA/ALA ratios in IF by reviewing the published literature in the context of the current regulatory framework. The lipid composition of BM from mothers living in 31 different countries was determined based on a literature review. This review also includes data from infant studies (intervention/cohort) on nutritional needs regarding LA and ALA, safety, and biological effects. The impact of various LA/ALA ratios in IF on DHA status was assessed within the context of the current worldwide regulatory framework including China and the EU. Country averages of LA and ALA in BM range from 8.5–26.9% FA and 0.3–2.65% FA, respectively. The average BM LA level across the world, including mainland China, is below the maximum 28% FA, and no toxicological or long-term safety data are available on LA levels > 28% FA. Although recommended IF LA/ALA ratios range from 5:1 to 15:1, ratios closer to 5:1 seem to promote a higher endogenous synthesis of DHA. However, even those infants fed IF with more optimal LA/ALA ratios do not reach the DHA levels observed in breastfed infants, and the levels of DHA present are not sufficient to have positive effects on vision. Current evidence suggests that there is no benefit to going beyond the maximum LA level of 28% FA in IF. To achieve the DHA levels found in BM, the addition of DHA to IF is necessary, which is in line with regulations in China and the EU. Virtually all intervention studies investigating LA levels and safety were conducted in Western countries in the absence of added DHA. Therefore, well-designed intervention trials in infants across the globe are required to obtain clarity about optimal and safe levels of LA and LA/ALA ratios in IF.

Invited Perspective: Challenges in Evaluating the Effect of Per- and Polyfluoroalkyl Substance Mixtures on Polycystic Ovarian Syndrome

Invited Perspective: Challenges in Evaluating the Effect of Per- and Polyfluoroalkyl Substance Mixtures on Polycystic Ovarian Syndrome

Long-term oral administration of Asarum heterotropoides f. mandshuricum (Maxim.) Kitag. decoction and its aristolochic acid analogs do not cause renal toxicity in mice.

Asarum heterotropoides f. mandshuricum (Maxim.) Kitag. (AH) is widely used to treat influenza, COVID-19, allergic rhinitis, headache, toothache, rheumatoid arthritis, and peptic ulcer. However, its clinical use is controversial due to the concern of aristolochic acid nephropathy (AAN) caused by its component aristolochic acid analogs (AAs). The chronic toxicity of AH decoction and its main components AA IVa (AA-IVa) and aristolactam I (AL-I) was evaluated in mice. AAs contents in AH were quantitated by liquid chromatography-mass spectrometry. A parallel design was employed to examine the potential chronic toxicity of AH decoction at doses equivalent to 0.5, 1.6, and 5.0g/kg AH (approximately 10-100 times the clinical doses for humans) and its major AA components at doses equivalent to that in 5.0g/kg AH to mice after consecutive daily oral administration for 12 and 24 weeks, and at 32 weeks after withdrawal for 8 weeks. AH crude herb contained 2.18μg/g of AA-I, 48.49μg/g of AA-IVa, and 14.0μg/g of AL-I. AH decoction contained 5.45μg/g of AA-IVa and 2.71μg/g of AL-I. None of AA-II and AA-IIIa were detected in AH. After long-term administration of AH decoction and its major components AA-IVa and AL-I, mice showed no signs of illness or body weight changes. In addition, biochemical and pathohistological examinations showed that long-term administration of AH decoction and its major components AA-IVa and AL-I did not alter 1) serum levels of glutamic-pyruvic transaminase, glutamic oxalacetic transaminase, alkaline phosphatase, creatinine, and urea nitrogen, 2) renal tissue mRNA expression of kidney injury molecule 1 and neutrophil gelatinase-associated lipocalin, and 3) pathological morphology in the mouse liver, kidney, stomach, and bladder. AH has no obvious toxicity to mice and is relatively safe when it is used in the form of decoction. AA-IVa and AL-I, the two major AAs in AH, are not toxic to mice at the dose equivalent to that in the high dose of AH decoction. Considering the limited toxicological data on AH, we recommend that AH decoction medication should not overdose and the duration should not be too long.

FEMA GRAS assessment of derivatives of basil, nutmeg, parsley, tarragon and related allylalkoxybenzene-containing natural flavor complexes.

In 2015, the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA) initiated a program for the re-evaluation of the safety of over 250 natural flavor complexes (NFCs) used as flavoring ingredients in food. In this publication, tenth in the series, NFCs containing a high percentage of at least one naturally occurring allylalkoxybenzene constituent with a suspected concern for genotoxicity and/or carcinogenicity are evaluated. In a related paper, ninth in the series, NFCs containing anethole and/or eugenol and relatively low percentages of these allylalkoxybenzenes are evaluated. The Panel applies the threshold of toxicological concern (TTC) concept and evaluates relevant toxicology data on the NFCs and their respective constituent congeneric groups. For NFCs containing allylalkoxybenzene constituent(s), the estimated intake of the constituent is compared to the TTC for compounds with structural alerts for genotoxicity and when exceeded, a margin of exposure (MOE) is calculated. BMDL10 values are derived from benchmark dose analyses using Bayesian model averaging for safrole, estragole and methyl eugenol using EPA's BMDS software version 3.2. BMDL10 values for myristicin, elemicin and parsley apiole were estimated by read-across using relative potency factors. Margins of safety for each constituent congeneric group and MOEs for each allylalkoxybenzene constituent for each NFC were determined that indicate no safety concern. The scope of the safety evaluation contained herein does not include added use in dietary supplements or any products other than food. Ten NFCs, derived from basil, estragon (tarragon), mace, nutmeg, parsley and Canadian snakeroot were determined or affirmed as generally recognized as safe (GRAS) under their conditions of intended use as flavor ingredients based on an evaluation of each NFC and the constituents and congeneric groups therein.

A 13-week subchronic toxicity study of heme iron in SD rats.

Heme iron (HI) has been widely used as a food additive and supplement to support iron fortification. However, no sufficient toxicological data to evaluate the safety of HI have been reported. In the current study, we performed a 13-week subchronic toxicity study of HI in male and female Crl:CD(SD) rats. Rats were orally administered HI in the diet at concentrations of 0%, 0.8%, 2%, and 5%. Observations of general condition, body weight (bw) and food consumption, urinalysis, hematology, serum biochemistry, and macroscopic and histopathological examination were performed. The results showed that HI had no adverse effects on any of the examined parameters. Therefore, we concluded that the no-observed-adverse-effect level (NOAEL) for HI was estimated to be 5% for both sexes (2,890mg/kgbw/day for males and 3,840mg/kgbw/day for females). Since the iron content of HI used in this study was in a range of 2.0-2.6%, iron content at NOAEL for HI was calculated to be 57.8-75.1mg/kgbw/day for males and 76.8-99.8mg/kgbw/day for females.

Farm Fields as Toxic Sinkhole

Farm Fields as Toxic Sinkhole

Child and adolescent mortality associated with pesticide toxicity in Cape Town, South Africa, 2010–2019: a retrospective case review

BackgroundPoisoning of children after exposure to pesticides is a major public health concern, particularly in countries with poorer urban populations, such as South Africa. This may stem from the illegal distribution and domestic use of street pesticides, which are highly hazardous agricultural pesticides. The aim of this study was to profile paediatric deaths due to acute pesticide poisoning in the west-metropole of Cape Town, South Africa; to identify whether the active ingredients were highly hazardous pesticides according to the FAO and WHO; and to inform policy and public health interventions to prevent future exposures and mortality.MethodsA retrospective and descriptive analysis of forensic post-mortem records (2010 to 2019) was conducted to identify cases of paediatric deaths (< 18 years old) in the west metropole of Cape Town, involving pesticide poisoning admitted to the Salt River mortuary (one out of 16 mortuaries in the Western Cape province). Demographic, circumstantial, autopsy, and toxicological information was captured. Descriptive statistics, together with chi-square tests, Fisher’s probability tests, and Mann–Whitney U tests were used to analyse the data.ResultsIn total, 54 paediatric pesticide deaths were identified, including 22 (40.7%) males and 32 (59.3%) females, out of 5,181 paediatric unnatural deaths admitted over the 10-year period. The median age of the decedents was 8.3 years (range: 1 day to 17.9 years), with the majority under five years (42.6%) or between 15 and 18 years old (40.7%). All incidents occurred in peri-urban areas of Cape Town, with most individuals being admitted to hospital (88.9%) for a median survival time of 4.8 h. Toxicological analysis was requested in 50 cases (92.6%) with the organophosphate pesticides terbufos (n = 29), methamidophos (n = 2) and diazinon (n = 2) detected most frequently. Adolescent (15–18 years) suicides (29.6%) and accidental child deaths (< 4 years) (18.5%) were common.ConclusionsTerbufos and methamidophos are highly hazardous pesticide (HHP) active ingredients registered in South Africa for agricultural uses, yet commonly sold as street pesticides for domestic use in lower socioeconomic areas. Reducing access and availability of toxic pesticides, especially through the illegal selling of street pesticides, and providing low toxic alternatives to poorer communities, may support mortality reduction initiatives. Mortality and toxicology data provide important, often overlooked, surveillance tools for informing policy and public health interventions to reduce toxic pesticide harm in local communities.

Buprenorphine and opioid analgesics: Dispensation and discontinuity among accidental overdose fatalities in the Indianapolis metropolitan area, 2016–2021

BackgroundThis study describes overall trends and sociodemographic disparities in buprenorphine and opioid analgesic uptake and prescribing patterns prior to fatal overdose events. MethodsWe examined toxicology data from all accidental overdose deaths from 2016 to 2021 (N = 2682) in a large metropolitan area. These data were linked at the individual-level with a prescription drug monitoring program (PDMP). ResultsFewer than half of all deaths had any kind of PDMP record (39.9 %, n = 1070). Among those with a buprenorphine prescription, 10.6 % (n = 35) of decedents had a buprenorphine dispensation within 7 days of their death, while the majority (64.7 %, n = 214) were dispensed buprenorphine more than 30 days prior to death. Evidence existed of racial disparities among those with any buprenorphine uptake, whereby Black individuals (7.3 %, n = 24) had significantly fewer any dispensations compared to White individuals (92.7 %, n = 307). Among those with an opioid analgesic prescription, about 12.2 % (n = 90) were dispensed within 7 days of death, with the majority (68.5 %, n = 506) occurring more than 30 days prior to death. Like buprenorphine dispensations, Black individuals were prescribed a significantly smaller proportion of opioid analgesics (21.9 %, n = 162) versus White individuals (77.7 %, n = 574). Buprenorphine was detected in 78.5 % of deaths where fentanyl was present in the toxicology record, significantly greater when compared to opioid analgesics (57.5 %). ConclusionConsistent with prior research, our findings suggest prescription opioid analgesics may protect against fatal overdoses. Access to buprenorphine treatment did not keep pace with the rising lethality of the overdose crisis, and in recent years, a smaller percentage of the people at risk of fatal overdose availed themselves of MOUD preceding their death.

The perturbation of parabens on the neuroendocrine system in zebrafish larvae

Parabens, as the synthetic preservatives, have caused universal environmental contamination and human exposure. Whether parabens could disturb neuroendocrine system was still ambiguous. In this study, the effects of four commonly-used parabens, i.e. methyl paraben (MeP), ethyl paraben (EtP), propyl paraben (PrP) and butyl paraben (BuP), were tested on the neuroendocrine system of zebrafish larvae by investigating the swimming behavior, the related hormones and biomarkers in the hypothalamic-pituitary-interrenal (HPI) axis. The results showed that all test chemicals significantly reduced the swimming distance and mean velocity of zebrafish larvae. The adrenocorticotropic hormone (ACTH) levels in zebrafish larvae were significantly increased, while the cortisol levels were obviously decreased by paraben exposure. The transcriptional analysis showed that the expressions of the target genes including gr, mr and crhr2 in the HPI axis were mostly down-regulated. The exploration of the initial molecular event showed that parabens could bind with the glucocorticoid receptor (GR) and trigger its transactivation, according to MDA-kb2 luciferase assay and molecular docking analysis. The interaction of parabens with the GR included the hydrogen bond and hydrophobic interaction. The findings herein revealed the potential deleterious effects of parabens on the neuroendocrine system of zebrafish larvae, thus accumulating the in vivo toxicological data on this kind of food preservatives.

Water-related atmospheric agents and solubility: two parameters of validation in toxicological screening on clothing worn by skeletal remains

In forensic toxicology, when conventional matrices are no longer available, alternative matrices can be used to assess toxicological investigations. Clothes worn by skeletal remains may be a good unconventional matrix for toxicological analyses considering that they have absorbed decomposition fluids and blood from a body. We hypothesized a scenario in which a skeleton, wearing clothes, was discovered in an open environment. From this starting point, an experimental study was developed on different textiles (cotton, wool, and polyester) to evaluate whether water-related atmospheric agents and molecule solubility can largely influence the detection of molecules of toxicological interest on this specific matrix, together with the characteristics of different garments chosen. The experimental study was performed on blood spots, previously spiked with 6-monoacetylmorphine and morphine, accurately placed on different textiles and washed with different quantities of deionized water adjusted at pH 5.6 with formic acid to simulate different rainfall conditions. Toxicological analyses were performed via Solid-Phase Extraction and High-Performance Liquid Chromatography—Tandem Mass Spectrometry analyses (Thermo Scientific™ TSQ Fortis™ II Triple-Quadrupole Mass Spectrometer). From the experimental study morphine could not be detected on 100% cotton and 100% wool fabric after the passing of 500 mL of deionized water and in 100% synthetic polyester textile after washing with 250 mL of deionized water. In conclusion, when toxicological analyses are carried out on unconventional matrices as textiles worn by corpses exposed to different environmental conditions, it is of great importance, in using such substrates as evidence for the presence of molecules of toxicological interest, to evaluate chemical-physical characteristics of each analyte under investigation in order to correctly interpret the toxicological data obtained.

Exposure to polypropylene microplastics via diet and water induces oxidative stress in Cyprinus carpio

The occurrence of accumulation of microplastics in humans and wildlife has become a serious concern on a global scale, especially in the last decade. Although there are many studies on microplastics, their biological effects and toxicity on freshwater fish have not been fully revealed. In order to evaluate the potential toxic effects of PP (polypropylene) microplastics in freshwater fish, we performed 1-day, 2-day, 3-day, 4-day, 5-day, 6-day, and 7-day microplastic exposure to different concentrations of the microplastics through water and diet on Cyprinus carpio. Fish samples were divided into 3 groups; Group-A with different PP microplastic concentrations in their water (ALow:1.0 g/L and AHigh:2.5 g/L), Group-B with different PP microplastic concentrations in their diet (BLow:100 mg/g and BHigh:250 mg/g), and Group-C (Control group) free of PP microplastics in their diet and water. The results showed that although microplastics did not cause death in C. carpio, they caused oxidative stress in comparing the MP exposed groups to the control groups. When indices of oxidative stress of fish individuals in all treatment groups were compared with the control group, it was determined that MDA (malondialdehyde) and GSH (glutathione) levels increased, while TPC (total protein content) and CAT (catalase) levels decreased depending on the concentrations and exposure times. Significant differences were observed between the control and treatment groups in the indices of oxidative stress (P<0.05). This study provided basic toxicological data to elucidate and quantify the effects of PP microplastics on freshwater fish. In addition, this study is the first study to indicate that microplastic exposure of carp via diet and water causes oxidative stress in gill tissues and causes changes in CAT, MDA, GSH, and TPC levels. The findings also provide useful reference data for improving knowledge of the effects of microplastics on organisms in freshwater systems.

Retrospective analysis of forensic toxicology data from 2021 to identify patterns of various forensic cases

PurposeKnowing and analyzing the characteristics and trends of forensic toxicology cases in a certain area is particularly important for a local government to establish an effective prevention and control system. The purpose of this work was to summarize data from forensic toxicology cases received by the Academy of Forensic Sciences (AFS) in 2021. MethodsAs requested by the police or according to the details of cases, samples were systematically screened or analyzed by various methods. Liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry were used to identify medicinal drugs, drugs of abuse, pesticides, poison gases, etc. ResultsAFS received a total of 17,758 cases in 2021, of which 314 cases underwent autopsy. The main cause of death was sudden death, and the manner of death was mainly accident. Among 13,744 drug abuse cases, the number of positive cases was 1721, with a positive rate of 12.5%, and synthetic cannabinoids were the most frequently detected substances. In 3640 traffic cases, 85.3% of drivers were suspected of drunk driving. In 103 poisoning cases, hydrogen sulfide poisoning and pesticide suicide account for a large proportion. In drug-facilitated crimes, zolpidem was the most frequently tested component. In 55 case of dog poisonings, the main poisons were cyanides and succinylcholine, and the main poisoning tools were poisonous baits and poisonous syringe darts. ConclusionsThis study profiles the toxicological characteristics of forensic toxicological cases conducted at the AFS in 2021 and provides a scientific basis for poisoning cases and drug abuse prevention.

Abstract 1880: A novel CD137 agonist exhibits potent antitumor efficacy with a good safety profile

Abstract Background: CD137 (TNFRSF9, 4-1BB) is a member of the tumor necrosis factor receptor superfamily that functions as a costimulatory molecule of immunocytes and is mainly expressed on the surface of immune cells including activated T and natural killer (NK) cells. Agonists against CD137 have shown promising therapeutic activity in mouse tumor models but have suffered setback in the clinic due to notably hepatotoxicity, or suboptimal agonistic potency. Here, we developed a fully human agonistic anti-CD137 monoclonal IgG4 antibody, ZG033, with an exclusive epitope, to achieve a better efficacy and safety profile for immunotherapy. Methods: The antigen binding specificity and affinity of ZG033 were determined by ELISA, surface plasmon resonance (SPR). X-ray crystallography was conducted to resolve the binding epitopes of the respective binding arms. The activities were determined using cell-based and reporter gene assays. In vivo antitumor activities were evaluated in human 4-1BB transgenic mice bearing with syngeneic tumors, and possible toxicity was evaluated in humanized mice and cynomolgus monkeys. Cytokine release assay was conducted by incubating ZG033 with PBMCs from healthy donors. The pharmacokinetic (PK) behavior of ZG033 were characterized in cynomolgus monkeys. A phase 1 study was initiated to evaluate the safety and preliminary clinical activities of ZG033, as well as the immunogenicity and pharmacokinetics. Results: ZG033 showed high specificity for human CD137 without cross-species reactivity across mouse, rat and exhibited potent CD137 agonist activity dependent on FcγR crosslinking. The binding affinity of ZG033 determined by surface plasmon resonance (SPR) was moderate (KD=10 nM). X-ray crystallography revealed that the Fab of ZG033 binds CD137 at an epitope overlap with the ligand binding interface. ZG033 increased CD137-driven NFκB reporter gene activation and the production of IFN-γ by human T cells in a FcγR-dependent manner. In human CD137 transgenic mice, ZG033 showed robust antitumor activity and induced durable antigen-specific immunological memory that prevented the tumor formation in the rechallenged mice. There were no signs of toxicity in human cytokine release assay which confirmed that ZG033 did not induce pro-inflammatory cytokines in the absence of TCR stimulation. Toxicology data in mice showed that ZG033 was well-tolerated with normal ALT and AST levels. ZG033 was further confirmed to well tolerated up to 180 mg/kg (MTD≥180mg/kg) dose without systemic toxicity in cynomolgus monkeys. Patients with advanced tumors, who are refractory after exhausting available therapies, were enrolled for ZG033 treatment. We will report the results from early dose escalation part of the study. Conclusion: These data demonstrate that ZG033 is an FcγR crosslinking-dependent CD137 agonist that displays a favorable safety profile and has potential in either mono- or combinational immunotherapies. Citation Format: Yang Huang, Liangwei Li, Wenting Liu, Dayan Zhang, Pengfei Zhou, Qing Fang, Weiming Zhou, Fengrong Wang, Guodong Shen, Liansheng Cheng. A novel CD137 agonist exhibits potent antitumor efficacy with a good safety profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1880.

Abstract 5657: HK010, a novel anti-PDL1 × CD137 bispecific antibody, exhibits potent anti-tumor immunity and low toxicity

Abstract Background: Checkpoint inhibitors targeting the PD-1/PD-L1 have changed the treatment and prognosis for patients with advanced solid tumors. There remains a unmet need for additional therapies due to treatment resistance. Tumor-necrosis superfamily members, such as CD137 and CD40, have been shown to synergize with the checkpoint inhibitors in preclinical studies. However, agonists antibodies have exhibited limited clinical activity for severe liver toxicity. Here we have developed a Fc-silenced bispecific antibody targeting PD-L1 and CD137, HK010, which could overcome these limitations by tumor-directed T-cell activation and checkpoint blockade. Methods: The antigen binding specificity and affinity of HK010 were determined by enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) and flow cytometry. The functional activity of HK010 was determined using in vitro cell-based assays and reporter gene assay. Transgenic mice expressing human PD-L1 and 4-1BB were transplanted with human PD-L1-expressing murine MC38 cells to assess in vivo antitumor activity. Cytokine release assay was conducted by incubating HK010 with peripheral blood mononuclear cells (PBMCs) from healthy donors (n=6). The pharmacokinetic (PK) behavior and safety profiles of HK010 were characterized in cynomolgus monkeys. Results: HK010 is a IgG4 PD-L1 × 4-1BB bsAb, which was designed to maintain a high affinity for human PD-L1 and a low affinity for human 4-1BB (KD PD-L1: 2.27 nM; KD 4-1BB: 493 nM) to achieve strong blocking of PD-1/PD-L1 and appropriate agonism of 4-1BB. HK010 retained full blockade activity on PD-1/PD-L1 signaling and enhances T-cell proliferation and IFN-γ production in vitro primary cell assays and Mixed lymphoid reaction (MLR). In addition, it led to a dose-dependent increase in the CD137-driven NFκB reporter gene activation through the bridging of PD-L1 on target cells and CD137 on effector cells. In humanized PD-L1/4-1BB transgenic mice bearing with MC38/hPDL1 tumor, HK010 showed robust single agent anti-tumor activity and induced durable antigen-specific immunological memory that prevents the growth of the same tumor cells in the mice re-challenged. There were no signs of toxicity in human cytokine release assay for HK010. Toxicology data in cynomolgus monkeys showed that HK010 was well-tolerated up to 150 mg/kg (MTD≥150mg/kg) dose without systemic toxicity. Furthermore, HK010 was confirmed to be highly safe in the 5-week repeated-dose toxicity study with no observed treatment-related mortality, abnormality in hematological indexes, important organs. Conclusion: These data demonstrate that HK010, an anti-PD-L1 × CD137 bispecific antibody, may exert a strong localized anti-tumor therapeutic efficacy with a low risk of liver toxicity through tumor-directed T-cell activation and checkpoint blockade in tumors. Citation Format: Liangwei Li, Wenting Liu, Guodong Shen, Dayan Zhang, Weiming Zhou, Fengrong Wang, Xiaoli Zeng, Yang Huang, Liansheng Cheng. HK010, a novel anti-PDL1 × CD137 bispecific antibody, exhibits potent anti-tumor immunity and low toxicity. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5657.

Abstract 6584: Potential carcinogenic effects of electronic nicotine delivery systems through epigenetic reprogramming in oral, brain, and lung stem cells

Abstract Recent scientific studies exhibited that the electronic nicotine delivery systems (ENDS) aerosols comprise numerous carcinogens namely propylene glycol, diacetyl, formaldehyde, acetaldehyde, crotonaldehyde (2-butenal), acetone, acetyl propionyl (2,3-pentanedione), acrolein, nicotine, arsenic, cadmium and radioisotopes, such as Polonium-210 and lead-210. However, there was no such investigation for clarifying the mechanism of carcinogenic effects and epigenetic reprogramming of human oral, brain, and lung stem cells. In this study, we investigated the carcinogenic effects of ENDS aerosols, and the mechanism of epigenetic reprogramming using the Comparative Toxico-genomics Database (CTD), NIH Toxicology Data Network (TOXNET), the database of the National Institute of Environmental Health Sciences (NIEHS), AACR Genomics Evidence Neoplasia Information Exchange (GENIE) database, and other bioinformatics databases. We found from CTD analysis that the chemical gene interactions showed approximately 3% of genes highly expressed by propylene glycol and diacetyl from a total of 98 genes. The AACR GENIE database analysis showed EGFR, is associated with gliomas (191 clinical data, DFCI glioma cohort), found among 98 genes regulated by propylene glycol and diacetyl. The CTD analysis also exhibited the chemical gene interactions of highly expressed 9 common genes (with miR140 by propylene glycol, and formaldehyde from a total of 3983 genes; 5 common genes by propylene glycol, acetaldehyde, from a total of 275 genes; 1 common gene by propylene glycol, crotonaldehyde (2-butenal), from a total of 227 genes; 7 common genes by propylene glycol, acrolein from total 2185 genes; 18 common genes by propylene glycol, and nicotine from total 1244 genes; 11 common genes by propylene glycol, and arsenic from total 4983 genes; 9 common genes with HDAC4, CD86, miR21 by propylene glycol, and cadmium from total 5242 genes. The ENDs aerosols contain Polonium-210 and lead-210 (concentration; 13 ± 2 Bq/kg), which have been reported as Group 1 human carcinogens and emit alpha particles. We found that alpha particles caused DNA lesions, and gene mutations in human oral, brain, and lung stem cells and resulted in long-term chromosomal instability in primary human T lymphocytes by analyzing recent works of literature. The epigenetic mechanisms of histone modifications and these potential carcinogens have been revealed by the database of NIH Roadmap Epigenomics Mapping Consortium and our analysis discovered that Mitogen-activated protein kinase 1(MAPK1) is epigenetically reprogrammed with close interaction of teen genes. Therefore, it is important to find the actual epigenetic mechanism of transcriptional regulation by these potential carcinogens on oral, lung, and brain stem cells, and our current study revealed this important relationship. Citation Format: Akilah Shepard, Oneilia Yearde, Juan Sanchez, Ryan Amador, Gabriel Xirinachs, Erika Hernandez, Elizabeth Corrales, Swarnava Das, Marco Ruiz, Madhumita Das, Rose M. Stiffin, Marilyn Sherman, Alessandra Manzon, Ayivi Huisso, Jayanta K. Das. Potential carcinogenic effects of electronic nicotine delivery systems through epigenetic reprogramming in oral, brain, and lung stem cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6584.