Consideration of the variability in control tumor incidence data at the Ramazzini Institute in evaluating treatment-related effects following chemical exposure.

The Ramazzini Institute (RI) has been conducting animal carcinogenicity studies for decades, many of which have been considered by authoritative bodies to determine potential carcinogenicity in humans. Unlike other laboratories, such as the U.S. National Toxicology Program (NTP), the RI does not provide a report or record of historical control data. Transparently documenting historical control data is critical in the interpretation of individual study results within the same laboratory. Historical control data allow an assessment of significant trends, either increasing or decreasing, resulting from changes in laboratory methods or genetic drift. In this investigation: (1) we compiled a dataset of the tumors reported in control groups of Sprague-Dawley rats and Swiss mice based on data included in published RI studies on specific substances, and (2) conducted case studies to compare data from this RI control dataset to the findings from multiple RI studies on sweeteners and corresponding breakdown products. We found considerable variability in the tumor incidence across multiple tumor types when comparing across control groups from RI studies. When compared to the tumor incidence in treated groups from multiple studies, the incidence of some tumors considered to be treatment-related fell within the variability of background incidence from the RI control dataset.

Dermal absorption of cyclic and linear siloxanes: a review.

Cyclic and linear siloxanes are compounds synthesized from silicon consisting of alternating atoms of silicone and oxygen [Si-O] units with organic side chains. The most common cyclic siloxanes are octamethylcyclotetrasiloxane (D4), decamethylcyclopentasiloxane (D5), and dodecamethylcyclohexasiloxane (D6), while the most common linear siloxanes are high molecular weight polydimethylsiloxanes (PDMS) and low molecular weight volatile linear siloxanes known as hexamethyldisiloxane (L2), octamethyltrisiloxane (L3), decamethyltetrasiloxane (L4), dodecamethylpentasiloxane (L5). These compounds (1) exhibit low dermal toxicity, (2) are generally inert and non-reactive, and (3) are compatible with a wide range of chemicals offering beneficial chemical properties which include the following: wash-off or transfer resistance from the skin, sun protection factor (SPF) enhancement, emolliency in cleaning products). Because of these properties, these compounds are incorporated into multiple consumer products for use on the skin, such as cosmetics and health-care products, with over 300,000 tons annually sold into the personal care and consumer products sector. Because of their widespread use in consumer products and potential for human dermal exposure, a comprehensive understanding of the dermal absorption and overall fate of siloxanes following dermal exposure is important. This review summarizes available data associated with the dermal absorption/penetration as well as fate of the most commonly used siloxane substances.

Interspecies scaling of toxicity reference values in human health versus ecological risk assessments: A critical review.

Risk assessments that focus on anthropogenic chemicals in environmental media-whether considering human health or ecological effects-often rely on toxicity data from experimentally studied species to estimate safe exposures for species that lack similar data. Current default extrapolation approaches used in both human health risk assessments and ecological risk assessments (ERAs) account for differences in body weight between the test organisms and the species of interest, but the two default approaches differ in important ways. Human health risk assessments currently employ a default based on body weight raised to the three-quarters power. Ecological risk assessments for wildlife (i.e., mammals and birds) are typically based directly on body weight, as measured in the test organism and receptor species. This review describes differences in the experimental data underlying these default practices and discusses the many factors that affect interspecies variability in chemical exposures. The interplay of these different factors can lead to substantial departures from default expectations. Alternative methodologies for conducting more accurate interspecies extrapolations in ERAs for wildlife are discussed, including tissue-based toxicity reference values, physiologically based toxicokinetic and/or toxicodynamic modeling, chemical read-across, and a system of categorical defaults based on route of exposure and toxic mode of action. Integr Environ Assess Manag 2024;20:749-764. © 2023 SETAC.

PBPK modeling to evaluate maximum tolerated doses: A case study with 3-chloroallyl alcohol.

Introduction: A physiologically based pharmacokinetic (PBPK) model for 3-chloroallyl alcohol (3-CAA) was developed and used to evaluate the design of assays for the in vivo genotoxicity of 3-CAA. Methods: Model development was supported by read across from a published PBPK model for ethanol. Read across was motivated by the expectation that 3-CAA, which like ethanol is a primary alcohol, is metabolized largely by hepatic alcohol dehydrogenases. The PBPK model was used to evaluate how two metrics of tissue dosimetry, maximum blood concentration (Cmax; mg/L) and area under the curve (AUC; mg-hr/L) vary with dose of 3-CAA and with dose route (oral gavage, drinking water). Results: The model predicted that oral gavage results in a 6-fold higher Cmax than the same dose administered in drinking water, but in similar AUCs. Predicted Cmax provided the best correlation with severe toxicity (e.g., lethality) from 3-CAA, consistent with the production of a reactive metabolite. Therefore, drinking water administration can achieve higher sustained concentration without severe toxicity in vivo. Discussion: This evaluation is significant because cytotoxicity is a potential confounder of mutagenicity testing. The PBPK model can be used to ensure that studies meet OECD and USEPA test guidelines and that the highest dose used is not associated with severe toxicity. In addition, PBPK modeling provides assurance of target tissue (e.g., bone marrow) exposure even in the absence of laboratory data, by defining the relationship between applied dose and target tissue dose based on accepted principles of pharmacokinetics, relevant physiology and biochemistry of the dosed animals, and chemical-specific information.

Updating the biologically based dose-response model for the nasal carcinogenicity of inhaled formaldehyde in the F344 rat.

Chronic inhalation of formaldehyde by F344 rats causes nasal squamous cell carcinoma (SCC). This outcome is well-characterized: including dose-response and time course data for SCC, mechanistic endpoints, and nasal dosimetry. Conolly et al. (Toxicol. Sci. 75, 432-447, 2003) used these resources to develop a biologically based dose-response (BBDR) model for SCC in F344 rats. This model, scaled up to humans, has informed dose-response conclusions reached by several international regulatory agencies. However, USEPA concluded that uncertainties precluded its use for cancer risk assessment. Here, we describe an updated BBDR model that addresses uncertainties through refined dosimetry modeling, revised analysis of labeling index data, and an extended dataset where both inhaled (exogenous) and endogenous formaldehyde (exogF, endoF) form DNA adducts. Further, since Conolly et al. (ibid) was published, it has become clear that, when controls from all F344 inhalation bioassays are considered, accounting for over 4000 rats, at most one nasal SCC occurred. This low spontaneous incidence constrains possible contribution of endoF to the formation of nasal SCC via DNA reactivity. Further, since both exogF and endoF form DNA adducts, this constraint also applies to exogF. The revised BBDR model therefore drives SCC formation through the cytotoxicity of high concentration exogF. An option for direct mutagenicity associated with DNA adducts is retained to allow estimation of an upper bound on adduct mutagenicity consistent with the lack of a spontaneous SCC incidence. These updates represent an iterative refinement of the 2003 model, incorporating new data and insights to reduce identified model uncertainties.

Incorporation of a recirculating mobile lipid pool description into the cyclic volatile siloxane (cVMS) PBPK model captures terminal clearance of D4 after repeated inhalation exposure in F344 and SD Rats.

The most recent version of the octamethylcyclotetrasiloxane (D4) physiologically based pharmacokinetic (model) was developed using the available kinetic studies in male and female F344 rats. Additional data, which had not been included in the D4 model development, allowed for a more detailed assessment of the loss of D4 following long-term exposure in both SD and F344 rats. This new data demonstrated a deficiency in the published PBPK model predictions of terminal concentrations of D4 in plasma and fat 14 days after the end of exposures for 28-days, 6 h/day, where the model predictions were an order of magnitude lower than the data. To capture this time-point without altering the end-of-exposure peak concentrations in blood and fat required conversion of the one-way (liver to fat) mobile lipoprotein pool (MLP) into a bi-directional pool between liver and fat. Simulation of the D4 pharmacokinetics in the SD rat, as opposed to the F344 rat, also required a reduction of both fold induction of liver metabolism (KMAX: 5- to 2-fold) and maximal rate of metabolism (VMAXC: 5.0-1.54 mg/kg0.75). The revised MLP description was extended to the human D4 model using a parallelogram approach between rat and human MLP parameters to establish the parameters for the current model in the absence of similar long-term clearance data in the human. The revised human D4 model provided good fits to the human inhalation and dermal exposure studies while not appreciably altering cross-species dose metrics based on the free concentration of D4 in blood.

Relative contributions of endogenous and exogenous formaldehyde to formation of deoxyguanosine monoadducts and DNA-protein crosslink adducts of DNA in rat nasal mucosa.

Understanding the dose-response for formaldehyde-induced nasal cancer in rats is complicated by (1) the uneven distribution of inhaled formaldehyde across the interior surface of the nasal cavity and, (2) the presence of endogenous formaldehyde (endoF) in the nasal mucosa. In this work, we used computational fluid dynamics (CFD) modeling to predict flux of inhaled (exogenous) formaldehyde (exogF) from air into tissue at the specific locations where DNA adducts were measured. Experimental work has identified DNA-protein crosslink (DPX) adducts due to exogF and deoxyguanosine (DG) adducts due to both exogF and endoF. These adducts can be considered biomarkers of exposure for effects of endoF and exogF on DNA that may be part of the mechanism of tumor formation. We describe a computational model linking CFD-predicted flux of formaldehyde from air into tissue, and the intracellular production of endoF, with the formation of DPX and DG adducts. We assumed that, like exogF, endoF can produce DPX. The model accurately reproduces exogDPX, exogDG, and endoDG data after inhalation from 0.7 to 15 ppm. The dose-dependent concentrations of exogDPX and exogDG are predicted to exceed the concentrations of their endogenous counterparts at about 2 and 6 ppm exogF, respectively. At all concentrations examined, the concentrations of endoDPX and exogDPX were predicted to be at least 10-fold higher than that of their DG counterparts. The modeled dose-dependent concentrations of these adducts are suitable to be used together with data on the dose-dependence of cell proliferation to conduct quantitative modeling of formaldehyde-induced rat nasal carcinogenicity.

Incorporation of rapid association/dissociation processes in tissues into the monkey and human physiologically based pharmacokinetic models for manganese.

In earlier physiologically based pharmacokinetic (PBPK) models for manganese (Mn), the kinetics of transport of Mn into and out of tissues were primarily driven by slow rates of association and dissociation of Mn with tissue binding sites. However, Mn is known to show rapidly reversible binding in tissues. An updated Mn model for primates, following similar work with rats, was developed that included rapid association/dissociation processes with tissue Mn-binding sites, accumulation of free Mn in tissues after saturation of these Mn-binding sites and rapid rates of entry into tissues. This alternative structure successfully described Mn kinetics in tissues in monkeys exposed to Mn via various routes including oral, inhalation, and intraperitoneal, subcutaneous, or intravenous injection and whole-body kinetics and tissue levels in humans. An important contribution of this effort is showing that the extension of the rate constants for binding and cellular uptake established in the monkey were also able to describe kinetic data from humans. With a consistent model structure for monkeys and humans, there is less need to rely on cadaver data and whole-body tracer studies alone to calibrate a human model. The increased biological relevance of the Mn model structure and parameters provides greater confidence in applying the Mn PBPK models to risk assessment. This model is also well-suited to explicitly incorporate emerging information on the role of transporters in tissue disposition, intestinal uptake, and hepatobiliary excretion of Mn.

Range of the perfluorooctanoate (PFOA) safe dose for human health: An international collaboration.

Many government agencies and expert groups have estimated a dose-rate of perfluorooctanoate (PFOA) that would protect human health. Most of these evaluations are based on the same studies (whether of humans, laboratory animals, or both), and all note various uncertainties in our existing knowledge. Nonetheless, the values of these various, estimated, safe-doses vary widely, with some being more than 100,000 fold different. This sort of discrepancy invites scrutiny and explanation. Otherwise what is the lay public to make of this disparity? The Steering Committee of the Alliance for Risk Assessment (2022) called for scientists interested in attempting to understand and narrow these disparities. An advisory committee of nine scientists from four countries was selected from nominations received, and a subsequent invitation to scientists internationally led to the formation of three technical teams (for a total of 24 scientists from 8 countries). The teams reviewed relevant information and independently developed ranges for estimated PFOA safe doses. All three teams determined that the available epidemiologic information could not form a reliable basis for a PFOA safe dose-assessment in the absence of mechanistic data that are relevant for humans at serum concentrations seen in the general population. Based instead on dose-response data from five studies of PFOA-exposed laboratory animals, we estimated that PFOA dose-rates 10-70 ng/kg-day are protective of human health.

Considerations for Improving Metabolism Predictions for In Vitro to In Vivo Extrapolation.

High-throughput (HT) in vitro to in vivo extrapolation (IVIVE) is an integral component in new approach method (NAM)-based risk assessment paradigms, for rapidly translating in vitro toxicity assay results into the context of in vivo exposure. When coupled with rapid exposure predictions, HT-IVIVE supports the use of HT in vitro assays for risk-based chemical prioritization. However, the reliability of prioritization based on HT bioactivity data and HT-IVIVE can be limited as the domain of applicability of current HT-IVIVE is generally restricted to intrinsic clearance measured primarily in pharmaceutical compounds. Further, current approaches only consider parent chemical toxicity. These limitations occur because current state-of-the-art HT prediction tools for clearance and metabolite kinetics do not provide reliable data to support HT-IVIVE. This paper discusses current challenges in implementation of IVIVE for prioritization and risk assessment and recommends a path forward for addressing the most pressing needs and expanding the utility of IVIVE.

The Conundrum of the PFOA human half-life, an international collaboration.

The Steering Committee of the Alliance for Risk Assessment (ARA) opened a call for scientists interested in resolving what appeared to be a conundrum in estimating of the half-life of perfluorooctanoate (PFOA) in humans. An Advisory Committee was formed from nominations received and a subsequent invitation led to the development of three small independent working groups to review appropriate information and attempt a resolution. Initial findings were shared among these groups and a conclusion developed from the ensuing discussions. Many human observational studies have estimated the PFOA half-life. Most of these studies note the likely occurrence of unmonitored PFOA exposures, which could inflate values of the estimated PFOA half-life. Also, few of these studies estimated the half-life of PFOA isomers, the branched chains of which likely have shorter half-lives. This could deflate values of the estimated linear PFOA half-life. Fortunately, several studies informed both of these potential problems. The majority opinion of this international collaboration is that the studies striking the best balance in addressing some of these uncertainties indicate the likely central tendency of the human PFOA half-life is less than 2 years. The single best value appears to be the geometric mean (GM) of 1.3 years (Zhang et al., 2013, Table 3), based on a GM = 1.7 years in young females (n = 20) and GM = 1.2 years in males of all ages and older females (n = 66). However, a combined median value from Zhang et al. (2013) of 1.8 years also adds value to this range of central tendency. While the Collaboration found this study to be the least encumbered with unmonitored PFOA exposures and branched isomers, more studies of similar design would be valuable. Also valuable would be clarification around background exposures in other existing studies in case adjustments to half-life estimates are attempted.

Read-across and new approach methodologies applied in a 10-step framework for cosmetics safety assessment - A case study with parabens.

Parabens are esters of para-hydroxybenzoic acid that have been used as preservatives in many types of products for decades including agrochemicals, pharmaceuticals, food and cosmetics. This illustrative case study with propylparaben (PP) demonstrates a 10-step read-across (RAX) framework in practice. It aims at establishing a proof-of-concept for the value added by new approach methodologies (NAMs) in read-across (RAX) for use in a next-generation risk assessment (NGRA) in order to assess consumer safety after exposure to PP-containing cosmetics. In addition to structural and physico-chemical properties, in silico information, toxicogenomics, in vitro toxicodynamic, toxicokinetic data from PBK models, and bioactivity data are used to provide evidence of the chemical and biological similarity of PP and analogues and to establish potency trends for observed effects in vitro. The chemical category under consideration is short (C1-C4) linear chain n-alkyl parabens: methylparaben, ethylparaben, propylparaben and butylparaben. The goal of this case study is to illustrate how a practical framework for RAX can be used to fill a hypothetical data gap for reproductive toxicity of the target chemical PP.

An in vitro approach to determine the human relevance of anti-spermatogenic effects of 4-methylmorpholine 4-oxide, monohydrate (NMMO) in rat reproductive toxicity studies.

Reduced sperm counts have been observed in male rats in an extended one generation reproductive toxicity study (EOGRTS, OECD 443) following repeated administration of 300 mg/kg/day N-Methylmorpholine N-oxide (NMMO). However, no adverse effects on reproductive organs have been reported in studies conducted with NMMO, and the mode of action (MOA) for the effects of NMMO on spermatogenesis is unknown, which complicates the interpretation of these data for human risk assessment. Here, a New Approach Method (NAM) strategy was used to evaluate NMMO MOA and compare interspecies susceptibility for anti-spermatogenic effects using organotypic in vitro assays combined with in vitro metabolism and in vitro to in vivo extrapolation (IVIVE) biokinetic modeling to compare predicted oral equivalent doses (OEDs) in human and rat. Dose-response data were collected in isolated germ cells and in an ex vivo seminiferous tubule model that recapitulates the interaction between the somatic environment and differentiating germ cells to account for potential direct and indirect effects on germ cells. With regard to direct spermatogenic effects, the human isolated germ cell model showed no toxicity at doses ≤300 µM (OED ≤ 86 mg/kg/day). With regard to indirect effects, the rat ex vivo model demonstrated dose-dependent decreases in secondary spermatocyte populations at OEDs ≥89 mg/kg/day, and reduced expression of RNAs specific to several stages of spermatogenesis (spermatogonia, pachytene spermatocytes, round spermatids) at OED = 267 mg/kg/day, consistent with in vivo observations. In contrast, the monkey ex vivo model did not show dose-dependent decreases in these same RNAs, and often demonstrated increased trends instead. These studies demonstrate clear quantitative and qualitative differences in the rat and primate response to NMMO. Furthermore, effects observed in the rat in vitro culture were not observed in the monkey at concentrations equivalent to in vivo doses of up to 1376 mg/kg/day, which is higher than the in vivo dose limit in the EOGRT study, indicating that the isolated findings on spermatogenesis in the rat studies are not likely to be relevant to humans.

Opportunities and challenges related to saturation of toxicokinetic processes: Implications for risk assessment.

Top dose selection for repeated dose animal studies has generally focused on identification of apical endpoints, use of the limit dose, or determination of a maximum tolerated dose (MTD). The intent is to optimize the ability of toxicity tests performed in a small number of animals to detect effects for hazard identification. An alternative approach, the kinetically derived maximum dose (KMD), has been proposed as a mechanism to integrate toxicokinetic (TK) data into the dose selection process. The approach refers to the dose above which the systemic exposures depart from being proportional to external doses. This non-linear external-internal dose relationship arises from saturation or limitation of TK process(es), such as absorption or metabolism. The importance of TK information is widely acknowledged when assessing human health risks arising from exposures to environmental chemicals, as TK determines the amount of chemical at potential sites of toxicological responses. However, there have been differing opinions and interpretations within the scientific and regulatory communities related to the validity and application of the KMD concept. A multi-stakeholder working group, led by the Health and Environmental Sciences Institute (HESI), was formed to provide an opportunity for impacted stakeholders to address commonly raised scientific and technical issues related to this topic and, more specifically, a weight of evidence approach is recommended to inform design and dose selection for repeated dose animal studies. Commonly raised challenges related to the use of TK data for dose selection are discussed, recommendations are provided, and illustrative case examples are provided to address these challenges or refute misconceptions.

Time-dependent genomic response in primary human uroepithelial cells exposed to arsenite for up to 60 days.

Evidence from both in vivo and in vitro studies suggests that gene expression changes from long-term exposure to arsenite evolve markedly over time, including reversals in the direction of expression change in key regulatory genes. In this study, human uroepithelial cells from the ureter segments of 4 kidney-donors were continuously treated in culture with arsenite at concentrations of 0.1 or 1 µM for 60 days. Gene expression at 10, 20, 30, 40, and 60 days was determined using Affymetrix human genome microarrays and signal pathway analysis was performed using GeneGo Metacore. Arsenic treated cells continued to proliferate for the full 60-day period, whereas untreated cells ceased proliferating after approximately 30 days. A peak in the number of gene changes in the treated cells compared to untreated controls was observed between 30 and 40 days of exposure, with substantially fewer changes at 10 and 60 days, suggesting remodeling of the cells over time. Consistent with this possibility, the direction of expression change for a number of key genes was reversed between 20 and 30 days, including CFOS and MDM2. While the progression of gene changes was different for each subject, a common pattern was observed in arsenic treated cells over time, with early upregulation of oxidative stress responses (HMOX1, NQ01, TXN, TXNRD1) and down-regulation of immune/inflammatory responses (IKKα). At around 30 days, there was a transition to increased inflammatory and proliferative signaling (AKT, CFOS), evidence of epithelial-to-mesenchymal transition (EMT), and alterations in DNA damage responses (MDM2, ATM). A common element in the changing response of cells to arsenite over time appears to involve up-regulation of MDM2 by inflammatory signaling (through AP-1 and NF-κB), leading to inhibition of P53 function.

Why is elevation of serum cholesterol associated with exposure to perfluoroalkyl substances (PFAS) in humans? A workshop report on potential mechanisms.

Serum concentrations of cholesterol are positively correlated with exposure to perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS) in humans. The associated change in cholesterol is small across a broad range of exposure to PFOA and PFOS. Animal studies generally have not indicated a mechanism that would account for the association in humans. The extent to which the relationship is causal is an open question. Nonetheless, the association is of particular importance because increased serum cholesterol has been considered as an endpoint to derive a point of departure in at least one recent risk assessment. To gain insight into potential mechanisms for the association, both causal and non-causal, an expert workshop was held Oct 31 and Nov 1, 2019 to discuss relevant data and propose new studies. In this report, we summarize the relevant background data, the discussion among the attendees, and their recommendations for further research.

Using quantitative modeling tools to assess pharmacokinetic bias in epidemiological studies showing associations between biomarkers and health outcomes at low exposures.

Biomarkers of exposure can be measured at lower and lower levels due to advances in analytical chemistry. Using these sensitive methods, some epidemiology studies report associations between biomarkers and health outcomes at biomarker levels much below those associated with effects in animal studies. While some of these low exposure associations may arise from increased sensitivity of humans compared with animals or from species-specific responses, toxicology studies with drugs, commodity chemicals and consumer products have not generally indicated significantly greater sensitivity of humans compared with test animals for most health outcomes. In some cases, these associations may be indicative of pharmacokinetic (PK) bias, i.e., a situation where a confounding factor or the health outcome itself alters pharmacokinetic processes affecting biomarker levels. Quantitative assessment of PK bias combines PK modeling and statistical methods describing outcomes across large numbers of individuals in simulated populations. Here, we first provide background on the types of PK models that can be used for assessing biomarker levels in human population and then outline a process for considering PK bias in studies intended to assess associations between biomarkers and health outcomes at low levels of exposure. After providing this background, we work through published examples where these PK methods have been applied with several chemicals/chemical classes - polychlorinated biphenyls (PCBs), perfluoroalkyl substances (PFAS), polybrominated biphenyl ethers (PBDE) and phthalates - to assess the possibility of PK bias. Studies of the health effects of low levels of exposure will be improved by developing some confidence that PK bias did not play significant roles in the observed associations.