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.

A weight of evidence evaluation of the mode of action of isoeugenol.

Isoeugenol is one of several phenylpropenoid compounds that is used as a fragrance, food flavoring agent and in aquaculture as a fish anesthetic. Carcinogenicity testing in rats and mice by NTP resulted in clear evidence of carcinogenicity (hepatic adenomas/carcinomas) in male mice only. A nongenotoxic threshold mode of action (MOA) is postulated for isoeugenol and is discussed considering the IPCS MOA and Human Relevance Framework. The weight of evidence indicates that isoeugenol is not genotoxic and that the carcinogenic outcome in male mice relates directly to the metabolism of individual compounds. Benchmark Dose (BMD) modeling was conducted to determine a Point of Departure (POD) and potential threshold of carcinogenicity. The results of the BMD evaluation for isoeugenol resulted in an estimated POD for carcinogenicity in the male mouse of 8 mg/kg with a lower limit of 4 mg/kg, representing a POD for the determination of an acceptable daily intake. With application of uncertainty factors, an ADI of 40 µg/kg is calculated. This daily dose in humans would be protective of human health, including carcinogenicity. A corresponding maximum residual level (MRL) of 3200 µg/kg fish is also estimated based on this POD that considers the threshold MOA.

A Review of Stable Isotope Labeling and Mass Spectrometry Methods to Distinguish Exogenous from Endogenous DNA Adducts and Improve Dose-Response Assessments.

Cancer remains the second most frequent cause of death in human populations worldwide, which has been reflected in the emphasis placed on management of risk from environmental chemicals considered to be potential human carcinogens. The formation of DNA adducts has been considered as one of the key events of cancer, and persistence and/or failure of repair of these adducts may lead to mutation, thus initiating cancer. Some chemical carcinogens can produce DNA adducts, and DNA adducts have been used as biomarkers of exposure. However, DNA adducts of various types are also produced endogenously in the course of normal metabolism. Since both endogenous physiological processes and exogenous exposure to xenobiotics can cause DNA adducts, the differentiation of the sources of DNA adducts can be highly informative for cancer risk assessment. This review summarizes a highly applicable methodology, termed stable isotope labeling and mass spectrometry (SILMS), that is superior to previous methods, as it not only provides absolute quantitation of DNA adducts but also differentiates the exogenous and endogenous origins of DNA adducts. SILMS uses stable isotope-labeled substances for exposure, followed by DNA adduct measurement with highly sensitive mass spectrometry. Herein, the utilities and advantage of SILMS have been demonstrated by the rich data sets generated over the last two decades in improving the risk assessment of chemicals with DNA adducts being induced by both endogenous and exogenous sources, such as formaldehyde, vinyl acetate, vinyl chloride, and ethylene oxide.

An updated mode of action and human relevance framework evaluation for Formaldehyde-Related nasal tumors.

Formaldehyde is a reactive aldehyde naturally present in all plant and animal tissues and a critical component of the one-carbon metabolism pathway. It is also a high production volume chemical used in the manufacture of numerous products. Formaldehyde is also one of the most well-studied chemicals with respect to environmental fate, biology, and toxicology-including carcinogenic potential, and mode of action (MOA). In 2006, a published MOA for formaldehyde-induced nasal tumors in rats concluded that nasal tumors were most likely driven by cytotoxicity and regenerative cell proliferation, with possible contributions from direct genotoxicity. In the past 15 years, new research has better informed the MOA with the publication of in vivo genotoxicity assays, toxicogenomic analyses, and development of ultra-sensitive methods to measure endogenous and exogenous formaldehyde-induced DNA adducts. Herein, we review and update the MOA for nasal tumors, with particular emphasis on the numerous studies published since 2006. These new studies further underscore the involvement of cytotoxicity and regenerative cell proliferation, and further inform the genotoxic potential of inhaled formaldehyde. The data lend additional support for the use of mechanistic data for the derivation of toxicity criteria and/or scientifically supported approaches for low-dose extrapolation for the risk assessment of formaldehyde.

Using mechanistic information to support evidence integration and synthesis: a case study with inhaled formaldehyde and leukemia.

Formaldehyde is one of the most comprehensively studied chemicals, with over 30 years of research focused on understanding the development of cancer following inhalation. The causal conclusions regarding the potential for leukemia are largely based on the epidemiological literature, with little consideration of cancer bioassays, dosimetry studies, and mechanistic research, which challenge the biological plausibility of the disease. Recent reanalyzes of the epidemiological literature have also raised significant questions related to the purported associations between formaldehyde and leukemia. Because of this, considerable scientific debate and uncertainty remain on whether there is a causal association between formaldehyde inhalation exposure and leukemia. Further complexity in evaluating this association is related to the endogenous production of formaldehyde. Multiple modes of action (MOA) have been postulated for the development of leukemia following formaldehyde inhalation that includes unsupported hypotheses of direct or indirect toxicity to the target cell population. Herein, the available evidence relevant to evaluating the postulated MOAs for leukemia following formaldehyde inhalation exposure is organized in the IPCS MOA Framework. The integration of all the available evidence clearly highlights the limited amount of data that support any of the postulated MOAs and demonstrates a significant amount of research supporting the null hypothesis that there is no causal association between formaldehyde inhalation exposure and leukemia. These analyses result in a lack of confidence in any of the postulated MOAs, increasing confidence in the conclusion that there is a lack of biological plausibility for a causal association between formaldehyde inhalation exposure and leukemia.

A critical review of the literature to conduct a toxicity assessment for oral exposure to methyl salicylate.

Methyl salicylate is the predominant constituent of oil of wintergreen and is used as a pesticide, a denaturant, an external analgesic, a fragrance ingredient, and a flavoring agent in products such as chewing gum, baked goods, syrups, candy, beverages, ice cream, and tobacco products; and it occurs naturally in some vegetables and berries. Methyl salicylate is of interest to the tobacco industry as oil of wintergreen is used as a flavorant in tobacco products. The purpose of this investigation was to conduct a critical review of the available literature for oral exposure to methyl salicylate, incorporating an analysis of the quality of the studies available and the current understanding of the mode of action. Following a review of all of the available literature, the most appropriate data sets for dose-response modeling were reported by Gulati et al. in which significant changes in reproductive/development endpoints were reported to occur after exposure to 500 mg/kg/d of methyl salicylate in male and female mice. Benchmark dose modeling was performed and the most sensitive endpoint, the number of litters per mating pair, was associated with a BMDL of 220 mg/kg/d. This BMDL was chosen as the point of departure and adjusted by a body weight scaling factor to derive a human equivalent dose. Based on the uncertainty factor analysis, the POD for methyl salicylate was adjusted by a UF of 3 for interspecies uncertainty to derive an allowable daily intake of 11 mg/kg/d.

Predicting lung dosimetry of inhaled particleborne benzo[a]pyrene using physiologically based pharmacokinetic modeling.

Benzo[a]pyrene (BaP) is a by-product of incomplete combustion of fossil fuels and plant/wood products, including tobacco. A physiologically based pharmacokinetic (PBPK) model for BaP for the rat was extended to simulate inhalation exposures to BaP in rats and humans including particle deposition and dissolution of absorbed BaP and renal elimination of 3-hydroxy benzo[a]pyrene (3-OH BaP) in humans. The clearance of particle-associated BaP from lung based on existing data in rats and dogs suggest that the process is bi-phasic. An initial rapid clearance was represented by BaP released from particles followed by a slower first-order clearance that follows particle kinetics. Parameter values for BaP-particle dissociation were estimated using inhalation data from isolated/ventilated/perfused rat lungs and optimized in the extended inhalation model using available rat data. Simulations of acute inhalation exposures in rats identified specific data needs including systemic elimination of BaP metabolites, diffusion-limited transfer rates of BaP from lung tissue to blood and the quantitative role of macrophage-mediated and ciliated clearance mechanisms. The updated BaP model provides very good prediction of the urinary 3-OH BaP concentrations and the relative difference between measured 3-OH BaP in nonsmokers versus smokers. This PBPK model for inhaled BaP is a preliminary tool for quantifying lung BaP dosimetry in rat and humans and was used to prioritize data needs that would provide significant model refinement and robust internal dosimetry capabilities.

A global human health risk assessment for Decamethylcyclopentasiloxane (D5).

Decamethylcyclopentasiloxane (D5) is a low-molecular-weight cyclic siloxane used primarily as an intermediate in the production of several widely-used industrial and consumer products and intentionally added to consumer products, personal products and some dry cleaning solvents. The global use requires consideration of consumer use information and risk assessment requirements from various sources and authoritative bodies. A global "harmonized" risk assessment was conducted to meet requirements for substance-specific risk assessments conducted by regulatory agencies such as USEPA's Integrated Risk Information System (IRIS), Health Canada and various independent scientific committees of the European Commission, as well as provide guidance for chemical safety assessments under REACH in Europe, and other relevant authoritative bodies. This risk assessment incorporates global exposure information combined with a Monte Carlo analysis to determine the most significant routes of exposure, utilization of a multi-species, multi-route physiologically based pharmacokinetic (PBPK) model to estimate internal dose metrics, benchmark modeling to determine a point of departure (POD), and a margin of safety (MOS) evaluation to compare the estimates of intake with the POD. Because of the specific pharmacokinetic behaviors of D5 including high lipophilicity, high volatility with low blood-to-air partition coefficients and extensive metabolic clearance that regulate tissue dose after exposure, the use of a PBPK model was essential to provide a comparison of a dose metric that reflects these processes. The characterization of the potential for adverse effects after exposure to D5 using a MOS approach based on an internal dose metric removes the subjective application of uncertainty factors that may be applied across various regulatory agencies and allows examination of the differences between internal dose metrics associated with exposure and those associated with adverse effects.

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.

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.

A comprehensive physiologically based pharmacokinetic (PBPK) model for nicotine in humans from using nicotine-containing products with different routes of exposure.

Physiologically based pharmacokinetic (PBPK) modeling can be a useful tool for characterizing nicotine pharmacokinetics (PK) from use of tobacco products. We expand a previously published PBPK model to simulate a nicotine PK profile, following single or multiple use of various tobacco products [cigarettes, smokeless tobacco, and electronic nicotine delivery systems, or a nicotine inhaler (NICOTROL)] The uptake route in the model was designed to allow for three uptake compartments: buccal cavity (BC), upper respiratory tract (URT) (conducting and transitional airways) and lower respiratory tract (alveolar region). Within each region, the model includes product-specific descriptions of the flux of nicotine into plasma, as well as the flux of nicotine from the BC and URT to the gastrointestinal tract. These descriptions are based on regional deposition and diffusion models of nicotine into plasma, which depends on the product type. Regional deposition flux combined with regional differences in physiological parameters (e.g., blood perfusion ratio and tissue thickness) play a key role in the product-specific PK profile of nicotine. The current model describes the slower flux of nicotine into plasma across the BC and URT, as well as the rapid flux known to occur in the alveolar region. Overall, the addition of the BC and respiratory tract compartments to the nicotine model provided simulation results that are comparable to the nicotine time-course plasma concentrations reported from clinical studies for the four product categories simulated.

Integration of evidence to evaluate the potential for neurobehavioral effects following exposure to USFDA-approved food colors.

California's Office of Environmental Health Hazard Assessment was tasked with conducting risk assessments for United States Food and Drug Administration-approved food dyes relative to neurobehavioral concerns. The purpose of this assessment was to evaluate the evidence for neurodevelopment effects based on three streams of evidence: 1) studies identified by OEHHA for consideration in a quantitative risk assessment; 2) studies relevant to understanding mechanisms of neurobehavioral effects; 3) an in silico assessment of the bioavailability of USFDA-approved food dyes. The results indicate a lack of adequate or consistent evidence of neurological effects, supported by a lack of bioavailability and brain penetration predicted by the in silico assessment. Further, the mechanistic evidence supports a lack of activity from in vitro neurotransmitter assays, and a lack of evidence to support molecular initiating events or key events in adverse outcome pathways associated with neurodevelopmental effects, supporting a lack of biological plausibility for neurobehavioral effects following food exposures to colors. These conclusions are consistent with other authoritative bodies, such as JECFA and EFSA, that have determined (i) other effects are more appropriate for estimating acceptable daily intakes and (ii) evidence from the neurobehavioral studies lack the strength to be relied upon for quantitative risk assessment.

A pharmacokinetic model of the intracellular dosimetry of inhaled nickel.

The potential associations between exposure to nickel compounds and cancer have been evaluated in both animal and epidemiological studies of occupationally exposed workers. The results of the epidemiological studies suggest that not all nickel compounds are equally carcinogenic, an observation supported by the animal bioassay results. Given the complexity and the differences in the modes of uptake of different forms of nickel by cells and the subsequent delivery of nickel to the nucleus, it would be expected that some forms of nickel would be more potent than others. A physiologically based pharmacokinetic (PBPK) model would be useful in estimating the cellular exposure to nickel resulting from inhalation of the different forms of nickel. To this end, a preliminary model of a tracheobronchial epithelial cell was developed to describe the differences in the extracellular and intracellular kinetics of the different classes of nickel compounds. Data available in the published literature were used to define the initial model parameters. The resulting cellular dosimetry model was able to describe kinetic data on three forms of nickel (soluble chloride and insoluble sulfide and subsulfide). This preliminary model development effort has identified critical data gaps that could be filled by additional research. The ultimate goal will be to integrate a refined cellular dosimetry model with published lung deposition/clearance and systemic distribution/clearance models for nickel. The use of such an integrated PBPK model would allow for more biologically based risk estimates for the inhalation of the different nickel compounds, as well as mixtures of these compounds.

Toxicology of octamethylcyclotetrasiloxane (D4).

Octamethylcyclotetrasiloxane (D4) is a volatile cyclic siloxane used primarily as a monomer or intermediate in the production of some silicon-based polymers widely used in industrial and consumer applications and may be present as a residual impurity in a variety of consumer products. A robust toxicological data set exists for D4. Treatment-related results from a chronic inhalation study conducted in rats are limited to mild effects on the respiratory tract, increases in liver weight, increases in the incidence of uterine endometrial epithelial hyperplasia, and a dose-related trend in the incidence of endometrial adenomas. The observed increases in liver weight appear to be related to the induction of hepatic metabolizing enzymes, similar to those that are induced in the presence of phenobarbital. D4 is not mutagenic or genotoxic in standard in vitro and in vivo tests; therefore, the benign uterine tumors observed likely occur by a non-genotoxic mechanism. Results from mechanistic studies suggest that D4 has very weak estrogenic and antiestrogenic activity, as well as dopamine agonist-like activity. In rats, D4 exposure delays ovulation and hypothesized to prolong exposure of the uterine endometrium to endogenous estrogen. Though this mode of action may play a role in the development of benign uterine tumors in the rat, it is considered unlikely to occur in the human due to the marked differences in cycle regulatory mechanisms. Reproductive effects were observed following D4 exposure in female rats. These effects appear to be related to a delay of the luteinizing hormone (LH) surge, which fails to induce complete ovulation in the rat. However, based on differences in ovulatory control in rats and humans, it appears these effects may be species-specific with no risk or relevance to human health. Results from pharmacokinetic studies indicate that dermal absorption of D4 is limited, due to its high volatility and, if absorbed via dermal, oral or inhalation exposure, the majority of D4 is rapidly cleared from the body, indicating bioaccumulation is unlikely.

A global human health risk assessment for octamethylcyclotetrasiloxane (D4).

Octamethylcyclotetrasiloxane (D4) is a low-molecular-weight volatile cyclic siloxane, primarily used as an intermediate in the production of some widely-used industrial and consumer silicone based polymers and may be present as a component in a variety of consumer products. A global "harmonized" risk assessment was conducted to meet requirements for substance-specific risk assessments conducted by regulatory agencies such as USEPA's Integrated Risk Information System (IRIS), Health Canada's Chemical Management Program (CMP) and various independent scientific committees of the European Commission (e.g. the Scientific Committee on Consumer Safety (SCCS), the Scientific Committee on Health and Environmental Risks (SCHER)), as well as to provide guidance for chemical safety assessments under REACH in Europe. This risk assessment incorporates global exposure information combined with a Monte Carlo analysis to determine the most significant routes of exposure. Utilization of a multi-species, multi-route physiologically based pharmacokinetic (PBPK) model was included to estimate internal dose metrics, benchmark modeling was used to determine a point of departure (POD), and a margin of safety (MOS) evaluation was used to compare the estimates of intake with the POD. Because of the specific pharmacokinetic behaviors of D4 including high lipophilicity, high volatility with low blood-to-air partition coefficients and an extensive metabolic clearance that regulates tissue dose after exposure, the use of a PBPK model was essential to provide a comparison of a dose metric that reflects these processes. The characterization of the potential for adverse effects after exposure to D4 using a MOS approach based on an internal dose metric removes the subjective application of varying uncertainty factors from various regulatory agencies and allows examination of the differences between internal dose metrics associated with exposure and those associated with adverse effects.

Refinement of the oral exposure description in the cyclic siloxane PBPK model for rats and humans: Implications for exposure assessment.

The multi-compound, and multi-dose (MC-MD) route physiologically based pharmacokinetic (PBPK) model for cyclic siloxanes reported by McMullin et al. (2016) brought together the series of models for octamethylcyclotetrasiloxane (D4) and decamethylcyclopentasiloxane (D5) in rat and human into a unified code structure that would allow simulation of both compounds following the inhalation and dermal routes of exposure. The refined MC-MD PBPK model presented here expands upon this effort to include representation of rat kinetic data in plasma, tissues and exhaled breath for the parent compounds after oral bolus administration. Additional refinements were made with regards to hepatic induction of metabolism in the liver and allometric scaling of rate constants for the deep tissue compartments which will allow the MC-MD model to be used in uncertainty analysis. Overall, the refined MC-MD model was able to reproduce both parent D4 and D5 kinetic data in rat and human after inhalation exposure (rat and human) or dermal exposure (human). The inclusion of sequestered (i.e., lipid associated) oral absorption into plasma after oral bolus dosing successfully described the lack of exhalation as well as the initial distribution of siloxane to the liver which was higher than simple partitioning from plasma would allow. The refined MC-MD PBPK model presented here can be incorporated into uncertainty and variability analysis and cross-species dosimetry for both D4 and D5.

Systematic review in chemical risk assessment - A chemical industry perspective.

This commentary provides a perspective from the chemicals industry on the potential usefulness of systematic review approaches in chemical risk assessment.