Benchmark Dose Research Articles

PPARα/ACOX1 as a novel target for hepatic lipid metabolism disorders induced by per- and polyfluoroalkyl substances: An integrated approach

BackgroundPer- and polyfluoroalkyl substances (PFAS) are persistent and ubiquitous environmental contaminants with well-documented hepatotoxicity. However, the mechanistic linkage between PFAS exposure and non-alcoholic fatty liver disease (NAFLD) remains largely elusive. ObjectivesThis study aimed to explore PFAS-to-NAFLD link and the relevant molecular mechanisms. MethodsThe cross-sectional analyses using National Health and Nutrition Examination Survey (NHANES) data were conducted to investigate the association between PFAS exposure and NAFLD. A combination of in silico toxicological analyses, bioinformatics approaches, animal experiments, and in vitro assays was used to explore the molecular initiating events (MIEs) and key events (KEs) in PFAS-induced hepatic lipid metabolism disorders. ResultsThe cross-sectional analyses with NHANES data revealed the significant association between PFAS exposure and hepatic steatosis/NAFLD. The in silico toxicological analyses showed that PPARα activation induced by perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS), prototypical representatives of PFAS, is the critical MIE associated with NAFLD-predominant liver diseases. Transcriptome-based bioinformatic annotation and analyses identified that transcriptional upregulation of hepatic acyl-CoA oxidase 1 (ACOX1) in PPARα-regulated peroxisomal β-oxidation pathway was the KE involved with PFOA/PFOS-perturbed hepatic lipid metabolic pathways in humans, mice and rats. The in vivo and in vitro assays further verified that ACOX1-mediated oxidative stress contributed to mitochondrial compromise and lipid accumulation in PFOA/PFOS-exposed mouse hepatocytes, which could be mitigated by co-treatment with ACOX1 inhibitor and mitochondria ROS scavenger. Additionally, we observed that besides PFOA and PFOS, hepatic ACOX1 exhibited good-fit response to short-term exposures of long-chain (C7-C10) perfluoroalkyl carboxylic acids (PFHpA, PFNA, PFDA) and perfluoroalkyl sulfonic acids (PFHpS, PFDS) in human hepatocyte spheroids through benchmark dose (BMD) modeling. ConclusionOur study unveils a novel molecular target for PFAS-induced hepatic lipid metabolic disorders, shedding new light on prediction, assessment, and mitigation of PFAS hepatotoxicity.

Early microRNA responses in rodent liver mediated by furan exposure establish dose thresholds for later adverse outcomes

To reduce reliance on long-term in vivo studies, short-term data linking early molecular-based measurements to later adverse health effects is needed. Although transcriptional-based benchmark dose (BMDT) modeling has been used to estimate potencies and stratify chemicals based on potential to induce later-life effects, dose-responsive epigenetic alterations have not been routinely considered. Here, we evaluated the utility of microRNA (miRNA) profiling in mouse liver and blood, as well as in mouse primary hepatocytes in vitro, to indicate mechanisms of liver perturbation due to short-term exposure of the known rodent liver hepatotoxicant and carcinogen, furan. Benchmark dose modeling of miRNA measurements (BMDmiR) were compared to the referent transcriptional (BMDT) and apical (BMDA) estimates. These analyses indicate a robust dose response for 34 miRNAs to furan and involvement of p53-linked pathways in furan-mediated hepatotoxicity, supporting mRNA and apical measurements. Liver-sourced miRNAs were also altered in the blood and primary hepatocytes. Overall, these results indicate mechanistic involvement of miRNA in furan carcinogenicity and provide evidence of their potential utility as accessible biomarkers of exposure and disease.

Benchmark dose modeling for epidemiological dose-response assessment using prospective cohort studies.

Benchmark dose (BMD) methodology has been employed as a default dose-response modeling approach to determine the toxicity value of chemicals to support regulatory chemical risk assessment. Especially, a relatively standardized BMD analysis framework has been established for modeling toxicological data regarding the formats of input data, dose-response models, definitions of benchmark response, and model uncertainty consideration. However, the BMD approach has not been well developed for epidemiological data mainly because of the diverse designs of epidemiological studies and various formats of data reported in the literature. Although most of the epidemiological BMD analyses were developed to solve a particular question, the methods proposed in two recent studies are able to handle cohort and case-control studies using summary data with consideration of adjustments for confounders. Therefore, the purpose of the present study is to investigate and compare the "effective count"-based BMD modeling approach and adjusted relative risk (RR)-based BMD analysis approach to identify an appropriate BMD modeling framework that can be generalized for analyzing published data of prospective cohort studies for BMD analysis. The two methods were applied to the same set of studies that investigated the association between bladder and lung cancer and inorganic arsenic exposure for BMD estimation. The results suggest that estimated BMDs and BMDLs are relatively consistent; however, with the consideration of established common practice in BMD analysis, modeling adjusted RR values as continuous data for BMD estimation is a more generalizable approach harmonized with the BMD approach using toxicological data.

The Effect of Historical Data-Based Informative Prior on Benchmark Dose Estimation of Toxicogenomics.

High-throughput toxicogenomics as an advanced toolbox of Tox21 plays an increasingly important role in facilitating the toxicity assessment of environmental chemicals. However, toxicogenomic dose-response analyses are typically challenged by limited data, which may result in significant uncertainties in parameter and benchmark dose (BMD) estimation. Integrating historical data via prior distribution using a Bayesian method is a useful but not-well-studied strategy. The objective of this study is to evaluate the effectiveness of informative priors in genomic dose-response modeling and BMD estimation. Specifically, we aim to identify plausible informative priors and evaluate their effects on BMD estimates at both gene and pathway levels. A general informative prior and eight time-specific (from 3 h to 29 d) informative priors for seven commonly used continuous dose-response models were derived. Results suggest that the derived informative priors are sensitive to the specific data sets used for elicitation. Real data-based simulations indicate that BMD estimation with the time-specific informative priors can achieve increased or equivalent accuracy, significantly decreased uncertainty, and a slightly enhanced correlation with the points of departure estimated from apical end points than the counterparts with noninformative priors. Overall, our study systematically examined the effects of historical data-based informative priors on BMD estimates, highlighting the benefits of plausible information priors in advancing the practice of toxicogenomics.

6:2 Chlorinated Polyfluoroalkyl Ether Sulfonates Exert Stronger Thyroid Homeostasis Disruptive Effects in Newborns than Perfluorooctanesulfonate: Evidence Based on Bayesian Benchmark Dose Values from a Population Study.

Growing toxicologic evidence suggests that emerging perfluoroalkyl substances (PFASs), like chlorinated polyfluoroalkyl ether sulfonate (Cl-PFESA), may be as toxic or more toxic than perfluorooctanesulfonate (PFOS) and perfluorooctanoic acid (PFOA). However, further investigations are needed in terms of the human health risk assessment. This study examined the effects of emerging and legacy PFAS exposure on newborn thyroid homeostasis and compared the thyroid disruption caused by 6:2 Cl-PFESA and PFOS using a benchmark dose approach. The health effects of mixture and individual exposure were estimated using the partial least-squares (PLS) model and linear regression, respectively. A Bayesian benchmark dose (BMD) analysis determined the BMD value for adverse effect comparison between 6:2 Cl-PFESA and PFOS. The median (interquartile range) concentrations of 6:2 Cl-PFESA (0.573 [0.351-0.872] ng/mL), PFOS (0.674 [0.462-1.007] ng/mL), and PFOA (1.457 [1.034, 2.405] ng/mL) were found to be similar. The PLS model ranked the PFAS variables' importance in projection (VIP) scores as follows: 6:2 Cl-PFESA > PFOS > PFOA. Linear regression showed that 6:2 Cl-PFESA had a positive association with free triiodothyronine (FT3, P = 0.006) and triiodothyronine (T3, P = 0.014), while PFOS had a marginally significant positive association with FT3 alone (P = 0.042). The BMD analysis indicated that the estimated BMD10 for 6:2 Cl-PFESA (1.01 ng/mL) was lower than that for PFOS (1.66 ng/mL) in relation to a 10% increase in FT3. These findings suggest that 6:2 Cl-PFESA, an alternative to PFOS, has a more pronounced impact on newborns' thyroid homeostasis compared to PFOS and other legacy PFASs.

OECD 414 supplementary prenatal developmental toxicity study of sodium molybdate dihydrate in the rat and benchmark dose evaluation

In a continuing investigation of the potential for reproductive and developmental toxicity of molybdenum (Mo), consequent to the previous published OECD studies [1,2] and as directed by the European Chemicals Agency [3], a supplemental rat GLP-compliant Prenatal Developmental Toxicity (PNDT) study was conducted to investigate higher dose levels of sodium molybdate dihydrate (SMD) in an identical study design (OECD 414)[4] to Murray et al. 2014a [1], at dietary concentrations calculated to provide target Mo levels of 80 and 120 mg/kg bw/day (the maximum-tolerated dose). There was no effect on post-implantation loss, litter size, sex ratio or the incidence of external, visceral or skeletal fetal malformations or variations. Fetal weight was reduced proportionate to maternal dose. Minimal differences observed in the ossification status of some extremities of fetuses from females receiving 120 mg Mo/kg bw/day were confirmed as transient by skeletal examination of PND 21 pups from a further group of females receiving the same dose regime. There was no evidence of copper depletion in serum, placenta or liver. A benchmark dose evaluation using continuous and dichotomous approaches by combining the fetal body weight data from this study and the previous study determined that the BMD05 ranged from 47 to 57 mg Mo/kg bw/day, depending on the modelling approach and the BMDL05 estimates ranged from 37 to 47 mg Mo/kg bw/day. These levels are considered a more statistically robust point of departure for risk assessment for reproductive effects than the established NOAEL of 40 mg Mo/kg bw/day.

Benchmark Dose Assessment for Coke Oven Emissions-Induced Mitochondrial DNA Copy Number Damage Effects*

ObjectiveThe study aimed to estimate the benchmark dose (BMD) of coke oven emissions (COEs) exposure based on mitochondrial damage with the mitochondrial DNA copy number (mtDNAcn) as a biomarker. MethodsA total of 782 subjects were recruited, including 238 controls and 544 exposed workers. The mtDNAcn of peripheral leukocytes was detected through the real-time fluorescence-based quantitative polymerase chain reaction. Three BMD approaches were used to calculate the BMD of COEs exposure based on the mitochondrial damage and its 95% confidence lower limit (BMDL). ResultsThe mtDNAcn of the exposure group was lower than that of the control group (0.60 ± 0.29 vs. 1.03 ± 0.31; P < 0.001). A dose–response relationship was shown between the mtDNAcn damage and COEs. Using the Benchmark Dose Software, the occupational exposure limits (OELs) for COEs exposure in males was 0.00190 mg/m3. The OELs for COEs exposure using the BBMD were 0.00170 mg/m3 for the total population, 0.00158 mg/m3 for males, and 0.00174 mg/m3 for females. In possible risk obtained from animal studies (PROAST), the OELs of the total population, males, and females were 0.00184, 0.00178, and 0.00192 mg/m3 respectively. ConclusionBased on our conservative estimate, the BMDL of mitochondrial damage caused by COEs is 0.002 mg/m3. This value will provide a benchmark for determining possible OELs.

Inverse relationship between transfer efficiencies into reproductive system and exposure concentration for organic pollutants: Implications for hazard assessment

Organic pollutants impact fertility and reproductive outcomes. Assessing their risk required understanding the great variation of their parental transfer efficiency. To determine whether the proportion of organic pollutants transferred to the reproductive system varies with the level of parental contamination, we analyzed large-scale datasets on organic environmental contaminants found in parental blood, cord blood, and semen. Our results demonstrated that although chemical concentrations in seminal plasma and cord blood significantly increased with those in parental blood, the proportion of parental contaminants transferred to the reproductive system decreased as the chemical concentration in parental blood increased. Using a hierarchical model, a negative association between transfer efficiencies and parental contamination levels was revealed. It is estimated that 41.78% and 46.23% of the variance in transfer efficiencies can be explained by paternal and maternal blood concentrations, respectively. In comparison, chemical class accounted for 25.22% and 18.95% of the variance, while the remaining inconsistencies between studies contributed only 3.38% and 2.56% of the variance for paternal and maternal transfers, respectively. We observed no significant interaction between parental blood concentration and chemical properties. Thus, our findings implied that the transfer efficiency of organic pollutants is not just an intrinsic chemical property and the inverse association has significant implications for health risk assessments and benchmark dose estimations related to environmental pollutants.

Genotoxicity assessment: opportunities, challenges and perspectives for quantitative evaluations of dose-response data.

Genotoxicity data are mainly interpreted in a qualitative way, which typically results in a binary classification of chemical entities. For more than a decade, there has been a discussion about the need for a paradigm shift in this regard. Here, we review current opportunities, challenges and perspectives for a more quantitative approach to genotoxicity assessment. Currently discussed opportunities mainly include the determination of a reference point (e.g., a benchmark dose) from genetic toxicity dose-response data, followed by calculation of a margin of exposure (MOE) or derivation of a health-based guidance value (HBGV). In addition to new opportunities, major challenges emerge with the quantitative interpretation of genotoxicity data. These are mainly rooted in the limited capability of standard in vivo genotoxicity testing methods to detect different types of genetic damage in multiple target tissues and the unknown quantitative relationships between measurable genotoxic effects and the probability of experiencing an adverse health outcome. In addition, with respect to DNA-reactive mutagens, the question arises whether the widely accepted assumption of a non-threshold dose-response relationship is at all compatible with the derivation of a HBGV. Therefore, at present, any quantitative genotoxicity assessment approach remains to be evaluated case-by-case. The quantitative interpretation of in vivo genotoxicity data for prioritization purposes, e.g., in connection with the MOE approach, could be seen as a promising opportunity for routine application. However, additional research is needed to assess whether it is possible to define a genotoxicity-derived MOE that can be considered indicative of a low level of concern. To further advance quantitative genotoxicity assessment, priority should be given to the development of new experimental methods to provide a deeper mechanistic understanding and a more comprehensive basis for the analysis of dose-response relationships.

Benchmark Dose Approach to DNA and Liver Damage by Chlorpyrifos and Imidacloprid in Male Rats: The Protective Effect of a Clove-Oil-Based Nanoemulsion Loaded with Pomegranate Peel Extract.

Pesticides are widely used around the world to increase crop production. They also have negative impacts on animals, humans, and the ecosystem. This is the first report evaluating a novel pomegranate-extract-loaded clove-oil-based nanoemulsion (PELCN) and its potential for reducing oxidative stress and DNA damage, as well as its hepatoprotective effects against imidacloprid (IM) and chlorpyrifos (CPF) toxicity in male rats. The benchmark dose (BMD) approach was also used to study the dose-response toxicity of IM and CPF. IM and CPF were administered daily for 28 days at doses of 14, 28, and 54 mg/kg body weight (bw) of IM and 1, 2, and 4 mg/kg bw of CPF via drinking water. The PELCN was administered orally at a dose of 50 mg/kg bw/day of pomegranate extract, 500 mg/kg bw of the clove oil nanoemulsion, and IM or CPF at high doses in the drinking water. In male rats, IM and CPF caused a reduction in body weight gain and hepatotoxic effects as evidenced by increases in the liver enzymes AST, ALT, and ALP. They caused oxidative damage in the liver of male rats as indicated by the decreased liver activity of the GST, GPX, SOD, and CAT enzymes and decreased serum TAC. IM and CPF produced a significant dose-dependent increase in DNA damage in hepatocyte cells, resulting in moderate to severe liver damage with cells that are more inflammatory and have enlarged sinusoids and compacted nuclei. IM had a higher BMD than CPF for both body and liver weight, suggesting that CPF was more dose-dependently toxic than IM. Albumin was a highly sensitive liver biomarker for IM, while total protein was a biomarker for the CPF-treated rats. GPx was an extremely sensitive biomarker of oxidative stress in the IM treatment, while CAT and GPx were highly sensitive parameters in the CPF-treated rats. Therefore, at comparable doses, CPF has a higher potential to cause liver damage and oxidative stress than IM. The hepatotoxicity of IM and CPF can be mitigated by administering a nanoemulsion containing clove oil and pomegranate extract. The nanoemulsion acts as a protector against the oxidative stress caused by these insecticides, especially at high doses. The nanoemulsion based on clove oil increases the bioavailability and stability of the pomegranate extract, which has antioxidant properties.

Effects of the real-life metal(oid)s mixture on female reproductive function: Less is different

The current study aimed to examine the effect of toxic metal(oid) mixtures (lead (Pb), cadmium (Cd), arsenic (As), mercury (Hg), cadmium (Cd), chromium (Cr), and nickel (Ni)) on female reproductive function in Wistar rats after the 28- and 90-day exposure to dose levels calculated on the basis of the previously conducted human study. Experimental groups included: 2 controls (28- and 90-day), treated groups - doses based on: median– F2 (28) and F2(90) and 95th percentile concentrations in the general human population – F3(28) and F3(90); calculated lower Benchmark dose confidence limit (BMDL) for effects on hormone levels – F1(28) and F1(90) and a group given the doses calculated on the basis of the reference values from the literature (F4(28)). Blood and ovarian samples were collected for sex hormones and ovary redox status analysis. After 28-day exposure, changes were present both in prooxidants and antioxidants. However, after the 90-day exposure redox status imbalance was majorly caused by the disturbance of antioxidants. Changes in some parameters were observed even after exposure to the lowest doses. After 28-day exposure, the strongest dose-response relationship was found between hormones: LH and FSH and toxic metal(oid)s and, after 90-day exposure, between investigated redox status parameters: sulfhydryl groups, ischemia-modified albumin and nuclear factor erythroid 2–related factor 2 (Nrf2) and toxic metal(oid)s. Low obtained BMDLs and narrow Benchmark intervals for toxic metal(oid)s and some of the parameters might confirm the “no-threshold” paradigm. This study indicates possible detrimental effects of prolonged exposure to real-life mixtures of toxic metal(oid) on female reproductive function.

Reproductive and immune effects emerge at similar thresholds of PFHxS in deer mice

Although perfluorohexane sulfonate (PFHxS) is structurally similar to perfluorooctane sulfonate (PFOS) and also widely detected in humans and the environment, comparatively fewer toxicity data exists on this 6-chain perfluoroalkyl sulfonic acid. In this study, repeated oral doses of PFHxS were administered to deer mice (Peromyscus maniculatus) to evaluate subchronic toxicity and potential effects on reproduction and development. Maternal oral exposure to PFHxS caused increased stillbirths, which is relevant for ecological risk assessment, and resulted in a benchmark dose lower limit (BMDL) of 5.72 mg/kg-d PFHxS. Decreased plaque formation, which is relevant for human health risk assessment, occurred in both sexes of adult animals (BMDL = 8.79 mg/kg-d PFHxS). These data are the first to suggest a direct link between PFHxS and decreased functional immunity in an animal model. Additionally, female animals exhibited increased liver:body weight and animals of both sexes exhibited decreased serum thyroxine (T4) levels. Notably, since reproductive effects were used to support 2016 draft health advisories and immune effects were used in 2022 drinking water health advisories released by the United States Environmental Protection Agency for PFOS and perfluorooctanoic acid (PFOA), these novel data can potentially support advisories for PFHxS because relevant points of departure emerge at similar thresholds in a wild mammal and corroborate the general understanding of per- and polyfluoroalkyl substances (PFAS).

Estimating arsenic biotransfer factors from feed to chicken: a viable approach to animal feed risk assessment

Reliable biotransfer factor (BTF) data are critical for animal-feed studies in human risk assessment. A wide range of BTF values have been reported (0.0015–0.83 d/kg), expressed as the ratio of total arsenic (tAs) concentration (µg/kg) in chicken to daily intake rate (µg/d) of tAs in feed (tAs/tAs). Data on inorganic As (iAs) for chicken meat and tAs concentrations for feed were obtained from our related study. Using the linear regression technique developed in this study, we estimated the BTF as 0.016 d/kg, as iAs/tAs (R 2 > 0.7702) and 0.55 d/kg as tAs/tAs (R 2 = 0.9743) for whole chicken meat. From a mass-balance perspective, we suggest that tAs be used as the denominator for the BTF unit. To illustrate our feed-risk assessment method, we analyzed commercial feeds for tAs concentration (n = 79). Consumption data for the general population (n = 2479) were obtained from a Taiwanese total diet study. Bivariate Monte Carlo simulations (n = 10,000) showed that the 95th percentile (P95) of estimated daily intake (EDI) was 0.002 µg/kg body weight (bw)/d as iAs (< the benchmark dose lower limit of 3.0 µg/kg bw/d). Our results thus show that the commercial chicken feeds assessed in this study are of low health concern for the general Taiwanese population. We discuss the factors that may have affected the assessment, such as the type of animals investigated, type of feeds, feed tested, type of chemical species used for BTF estimation and statistical approach.

Health risk assessment of perfluorooctane sulfonate and perfluorooctanoic acid exposure in China based on epidemiological data

BackgroundHealth risk assessment based on epidemiological data for perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) are evidenced. Whereas information about health-based guidance values (HBGVs) and health risk for Chinese population related to PFOS and PFOA is scarce. ObjectivesTo provide candidate HBGVs for PFOS and PFOA based on Chinese epidemiological data and assess the health risk related to excessive exposure. MethodsWe reviewed scientific literature and collected available original data from epidemiological studies conducted in China. Critical endpoints and data were selected and used in benchmark dose (BMD) analysis to obtain lower confidence limits of BMD (BMDLs) of PFOS and PFOA. Physiologically based pharmacokinetic (PBPK) models were applied to calculate the point of departure (PODs) for PFOS and PFOA, which led to the determination of the HBGVs for the said chemicals. Margin of exposure (MOE) method was used to evaluate the health risk of population based on exposure data among Chinese and HBGVs. ResultsAlanine aminotransferase (ALT), total cholesterol (TC) and birth weight (BW) were selected as critical endpoints. Epidemiological data from 4,224 adults, 2,153 paired mother-newborn, 2,518 newborns, 439 pregnant women and 733 children in China were included in this study. BMDLs and PODs for PFOS are varied while BMDLs for PFOA were similar. PODs for PFOS (1.56 ng/kg/day) and PFOA (1.52 ng/kg/day) based on BW using National Birth Cohort Study were selected as HBGVs. Most MOEs for PFOS and PFOA among Chinese population were larger than 1 and smaller than 100, indicating moderate concern. Around 16% MOEs for PFOS were smaller than 1, implying high concern. ConclusionThe HBGVs for PFOS and PFOA were 1.56 ng/kg/day and 1.52 ng/kg/day, respectively. Health risk of Chinese population related to PFOS and PFOA exposure should be concerned and more studies should be conducted to evaluate the risk of the chemicals.

Effects of Cyanobacterial Harmful Algal Bloom Toxin Microcystin-LR on Gonadotropin-Dependent Ovarian Follicle Maturation and Ovulation in Mice.

Cyanobacterial harmful algal blooms (CyanoHABs) originate from the excessive growth or bloom of cyanobacteria often referred to as blue-green algae. They have been on the rise globally in both marine and freshwaters in recently years with increasing frequency and severity owing to the rising temperature associated with climate change and increasing anthropogenic eutrophication from agricultural runoff and urbanization. Humans are at a great risk of exposure to toxins released from CyanoHABs through drinking water, food, and recreational activities, making CyanoHAB toxins a new class of contaminants of emerging concern. We investigated the toxic effects and mechanisms of microcystin-LR (MC-LR), the most prevalent CyanoHAB toxin, on the ovary and associated reproductive functions. Mouse models with either chronic daily oral or acute intraperitoneal exposure, an engineered three-dimensional ovarian follicle culture system, and human primary ovarian granulosa cells were tested with MC-LR of various dose levels. Single-follicle RNA sequencing, reverse transcription-quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, western blotting, immunohistochemistry (IHC), and benchmark dose modeling were used to examine the effects of MC-LR on follicle maturation, hormone secretion, ovulation, and luteinization. Mice exposed long term to low-dose MC-LR did not exhibit any differences in the kinetics of folliculogenesis, but they had significantly fewer corpora lutea compared with control mice. Superovulation models further showed that mice exposed to MC-LR during the follicle maturation window had significantly fewer ovulated oocytes. IHC results revealed ovarian distribution of MC-LR, and mice exposed to MC-LR had significantly lower expression of key follicle maturation mediators. Mechanistically, in both murine and human granulosa cells exposed to MC-LR, there was reduced protein phosphatase 1 (PP1) activity, disrupted PP1-mediated PI3K/AKT/FOXO1 signaling, and less expression of follicle maturation-related genes. Using both invivo and in vitro murine and human model systems, we provide data suggesting that environmentally relevant exposure to the CyanoHAB toxin MC-LR interfered with gonadotropin-dependent follicle maturation and ovulation. We conclude that MC-LR may pose a nonnegligible risk to women's reproductive health by heightening the probability of irregular menstrual cycles and infertility related to ovulatory disorders. https://doi.org/10.1289/EHP12034.

Benchmark dose estimation based on oxidative damage in Chinese workers exposed to benzene series compounds

This study evaluated the effects of BTEX exposure on oxidative stress; it analyzed the correlation between oxidative stress and peripheral blood counts and estimated the benchmark dose (BMD) of BTEX compounds. This study recruited 247 exposed workers and 256 controls; physical examination data were collected and serum oxidative stress levels were measured. Relationships between BTEX exposure and biomarkers were analyzed using Mann-Whitney U, generalized linear model, and chi-square trend tests. Environmental Protection Agency Benchmark Dose Software was used to calculate the BMD and lower confidence limit of the BMD (BMDL) for BTEX exposure. The total antioxidant capacity (T-AOC) correlated positively with peripheral blood counts, and negatively with the cumulative exposure dose. On using T-AOC as the outcome variable, the estimated BMD and BMDL for BTEX exposure were 3.57 mg/m3 and 2.20 mg/m3, respectively. Based on T-AOC, the calculated occupational exposure limit of BTEX was 0.055 mg/m3.

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.

46 Solubility and the Toxicity of Metal Oxide Nanoparticles: Looking Through the Lens of Toxicogenomics and DNA Damage

Abstract Metal oxide nanoparticles (MONPs) are amongst the most widely utilized classes of nanomaterials worldwide. The mechanisms underlying their pulmonary toxicity are dependant on the physical-chemical properties of the particles. Solubility plays a critical role, as proportions of nanoparticles and dissolved metals change over time; however their relative contribution to the resulting toxicity has yet to be thoroughly investigated. The objective of this study was to mechanistically evaluate the impact of solubility on the toxic potential of MONPs. In this study, mouse lung epithelial cells were exposed for 2 – 48 Hrs to copper, zinc, nickel, aluminum and titanium oxides (CuO, ZnO, NiO, Al2O3, TiO2) as MONPs and microparticles, and corresponding metal chloride salts. Viability was assessed at each timepoint using Trypan Blue staining. DNA damage was measured after 2 and 4 Hrs exposure via the CometChip assay, while transcriptomic changes were measured using microarrays after 2, 24, and 48 Hrs of exposure. Benchmark dose modelling of viability and transcriptomics data indicates that ZnO and CuO MONPs induced more pronounced cytotoxicity and gene expression, earlier, than NiO, Al2O3, and TiO2 MONPs. With respect to DNA damage potential, NiO, CuO and ZnO MONPs showed the highest potency, with ZnCl2 being the only chloride to induce DNA damage at either timepoint. The metal chlorides induced similar pathway perturbations as their respective MONPs, while corresponding microparticle oxides (except ZnO microparticles) exhibited muted responses. This presentation will further explore these findings, with an emphasis on the effect of solubility on toxicity.

Benchmark dose calculations for PFAS exposure based on two data sets on immunotoxic effects

BackgroundExposure to perfluorinated alkylate substances (PFAS) is associated with harmful effects on human health, including developmental immunotoxicity. This outcome was chosen as the critical effect by the European Food Safety Authority (EFSA), which calculated a new joint reference dose for four PFAS using a Benchmark Dose (BMD) analysis of a study of 1-year old children. However, the U.S. Environmental Protection Agency (EPA) recently proposed much lower exposure limits.MethodsWe explored the BMD methodology for summary and individual data and compared the results with and without grouping for two data sets available. We compared the performance of different dose-response models including a hockey-stick model and a piecewise linear model. We considered different ways of testing the assumption of equal weight-based toxicity of the four PFAS and evaluated more flexible models with exposure indices allowing for differences in toxicity.ResultsResults relying on full and decile-based data were in good accordance. However, BMD results for the larger study were lower than observed by EFSA for the smaller study. EFSA derived a lower confidence limit for the BMD of 17.5 ng/mL for the sum of serum-PFAS concentration, while similar calculations in the larger cohort yielded values of about 1.5 ng/mL. As the assumption of equal weight-based toxicity of the four PFAS seems questionable, we confirmed dose-dependencies that allowed potency differences between PFAS. We also found that models linear in the parameters for the BMD analysis showed superior coverage probabilities. In particular, we found the piecewise linear model to be useful for Benchmark analysis.ConclusionsBoth data sets considered could be analyzed on a decile basis without important bias or loss of power. The larger study showed substantially lower BMD results, both for individual PFAS and for joint exposures. Overall, EFSA’s proposed tolerable exposure limit appears too high, while the EPA proposal is in better accordance with the results.

Benchmark dose estimation for benzene-exposed workers in China: Based on quantitative and multi-endpoint genotoxicity assessments

Based on previous exposure studies, benzene (BZ) has been classified as a human carcinogen and occupational exposure limit (OELs) for BZ has been set to be about 1 ppm around the world. However, health hazards have still been reported with exposure below the OEL. Thus, the OEL needs to be updated to reduce health risk. The overall aim of our study was therefore to generate new OEL for BZ via a benchmark dose (BMD) approach and based on quantitative and multi-endpoint genotoxicity assessments. Genotoxicities were determined using the novel human PIG-A gene mutation assay, the micronucleus (MN) test and the COMET assay in benzene-exposed workers. Among the 104 workers with below current OELs, they exhibited significantly higher PIG-A mutant frequencies (MFs) (15.96 ± 14.41 × 10−6) and MN frequencies (11.55 ± 6.83‰) than those among the controls (PIG-A MFs: 5.46 ± 4.56 × 10−6, MN frequencies: 4.51 ± 1.58 ‰), but no difference in the COMET assay. A significant association was also observed between BZ exposure doses and PIG-A MFs and MN frequencies (P < 0.001). Our results indicate that health hazards were induced among workers with below OEL exposures. Based on results from the PIG-A and MN assays, the lower confidence limit of the BMD (BMDL) were calculated to be 8.71 mg/m3-year and 0.44 mg/m3-year, respectively. Based on these calculations, the OEL for BZ was determined to be lower than 0.07 ppm. This value can be considered by regulatory agencies to set new exposure limits and to better protect workers.