Exploring structure/property relationships to health and environmental hazards of polymeric polyisocyanate prepolymer substances-2. Dermal sensitization potential in the mouse local lymph node assay.

The sensitization potencies of twenty custom-designed monomer-depleted polymeric polyisocyanate prepolymer substances and their associated toluene diisocyanate (TDI), methylene diphenyl diisocyanate (MDI), hexamethylene diisocyanate (HDI), and isophorone diisocyanate (IPDI) monomer precursors were investigated by means of the mouse Local Lymph Node Assay (LLNA). These polymeric prepolymers were designed to represent the structural features and physical-chemical properties exhibited by a broad range of commercial polymeric polyisocyanate prepolymers that are produced from the reaction of aromatic and aliphatic diisocyanate monomers with aliphatic polyether and polyester polyols. The normalization of LLNA responses to the applied (15-45-135 mM) concentrations showed that the skin sensitization potency of polymeric polyisocyanate prepolymers is at least 300 times less than that of the diisocyanate monomers from which they are derived. The sensitization potency of the prepolymers was shown to be mainly governed by their hydrophobicity (as expressed by the calculated octanol-water partition coefficient, log Kow) and surfactant properties. Neither hydrophilic (log Kow <0) nor very hydrophobic (log Kow >25) prepolymers stimulated lymphocyte proliferation beyond that of the dosing vehicle control. The findings of this investigation challenge the generally held assumption that all isocyanate (-N=C=O) bearing substances are potential skin (and respiratory) sensitizers. Further, these findings can guide the future development of isocyanate chemistries and associated polyurethane applications toward reduced exposure and health hazard potentials.

In silico predictions of absorption of MDI substances after dermal or inhalation exposures to support a category based read-across assessment.

Methylenediphenyl diisocyanate (MDI) substances used polyurethane production can range from their simplest monomeric forms (e.g., 4,4'-MDI) to mixtures of the monomers with various homologues, homopolymer, and prepolymer derivatives. The relative dermal or inhalation absorption of 39 constituents of these substances in human were predicted using the GastroPlus® program. Predicted dermal uptake and absorption of the three MDI monomers from an acetone vehicle was 84-86% and 1.4-1.5%, respectively, with lower uptake and absorption predicted for the higher MW analogs. Lower absorption was predicted from exposures in a more lipophilic vehicle (1-octanol). Modeled inhalation exposures afforded the highest pulmonary absorption for the MDI monomers (38-54%), with 3-27% for the MW range of 381-751, and <0.1% for the remaining, higher MW derivatives. Predicted oral absorption, representing mucociliary transport, ranged from 5 to 10% for the MDI monomers, 10-25% for constituents of MW 381-751, and ≤3% for constituents with MW > 900. These in silico evaluations should be useful in category-based, worst-case, Read-Across assessments for MDI monomers and modified MDI substances for potential systemic effects. Predictions of appreciable mucociliary transport may also be useful to address data gaps in oral toxicity testing for this category of compounds.

FutureTox IV Workshop Summary: Predictive Toxicology for Healthy Children.

FutureTox IV, a Society of Toxicology Contemporary Concepts in Toxicology workshop, was held in November 2018. Building upon FutureTox I, II, and III, this conference focused on the latest science and technology for in vitro profiling and in silico modeling as it relates to predictive developmental and reproductive toxicity (DART). Publicly available high-throughput screening data sets are now available for broad in vitro profiling of bioactivities across large inventories of chemicals. Coupling this vast amount of mechanistic data with a deeper understanding of molecular embryology and post-natal development lays the groundwork for using new approach methodologies (NAMs) to evaluate chemical toxicity, drug efficacy, and safety assessment for embryo-fetal development. NAM is a term recently adopted in reference to any technology, methodology, approach, or combination thereof that can be used to provide information on chemical hazard and risk assessment to avoid the use of intact animals (U.S. Environmental Protection Agency [EPA], Strategic plan to promote the development and implementation of alternative test methods within the tsca program, 2018, https://www.epa.gov/sites/production/files/2018-06/documents/epa_alt_strat_plan_6-20-18_clean_final.pdf). There are challenges to implementing NAMs to evaluate chemicals for developmental toxicity compared with adult toxicity. This forum article reviews the 2018 workshop activities, highlighting challenges and opportunities for applying NAMs for adverse pregnancy outcomes (eg, preterm labor, malformations, low birth weight) as well as disorders manifesting postnatally (eg, neurodevelopmental impairment, breast cancer, cardiovascular disease, fertility). DART is an important concern for different regulatory statutes and test guidelines. Leveraging advancements in such approaches and the accompanying efficiencies to detecting potential hazards to human development are the unifying concepts toward implementing NAMs in DART testing. Although use of NAMs for higher level regulatory decision making is still on the horizon, the conference highlighted novel testing platforms and computational models that cover multiple levels of biological organization, with the unique temporal dynamics of embryonic development, and novel approaches for estimating toxicokinetic parameters essential in supporting in vitro to in vivo extrapolation.

Practical learnings from an epidemiology study on TDI-related occupational asthma: Part II-Exposure without respiratory protection to TWA-8 values indicative of peak events is a good indicator of risk.

The anonymized data of an epidemiology study on the incidence of toluene diisocyanate (TDI)-related occupational asthma in three US-based TDI production facilities have been reanalyzed to identify where to best focus exposure reduction efforts in industrial practice in order to reduce the risk of sensitization to TDI. In Part I, it was demonstrated that cumulative exposure is not a good indicator of the risk of developing TDI-related occupational asthma. In this Part II, an alternative model was developed based on net exposure parameters (i.e. samples taken when no respiratory protection was used). A statistically significant relationship was determined between asthma incidence and the frequency of exposure to TDI levels indicative of peak events that are expressed as time-weighted average-8 (TWA-8) values greater than 3 ppb during which no respiratory protection was used. This relationship suggests a threshold to induction of TDI-related asthma. The findings also highlight the importance of a comprehensive program for controlling workplace atmosphere in the plant by technical measures (e.g. selection of equipment, cleaning procedures) and controlling exposure by organizational measures and situational awareness (e.g. training, use of in-the-field direct reading indicators) during high potential exposure scenarios (e.g. line breaking, spills) to encourage or enforce the appropriate use of respiratory protection.

Practical learnings from an epidemiology study on TDI-related occupational asthma: Part I-Cumulative exposure is not a good indicator of risk.

The anonymized data of an epidemiology study on incidence of toluene diisocyanate (TDI)-related occupational asthma in three US-based TDI production facilities have been reanalyzed to identify where to best focus exposure reduction efforts in industrial practice to reduce the risk of sensitization to TDI. Since the induction of sensitization has sometimes been attributed to cumulative exposure, this relationship was examined first. Gross cumulative exposure values (i.e. not taking into account whether respiratory protection was used or not) and net cumulative exposure values (i.e. accounting for the use of respiratory protection) per participant were calculated based on the duration of their study participation and the average time-weighted average value of the exposure group to which they belonged. These two sets of cumulative exposure data were compared with asthma incidence using logistic regression. Incidence was zero among workers who rarely come into contact with open plant systems (e.g. during maintenance or spills). Notwithstanding, no statistically significant relationship between asthma incidence and either gross or net cumulative exposure could be determined. This is shown to be consistent with the results of several other epidemiology studies on TDI-related occupational asthma. In conclusion, cumulative exposure values are not a good indicator of the risk of developing TDI-related occupational asthma.

Solution structure analysis of the HPV16 E6 oncoprotein reveals a self-association mechanism required for E6-mediated degradation of p53.

The viral oncoprotein E6 is an essential factor for cervical cancers induced by "high-risk" mucosal HPV. Among other oncogenic activities, E6 recruits the ubiquitin ligase E6AP to promote the ubiquitination and subsequent proteasomal degradation of p53. E6 is prone to self-association, which long precluded its structural analysis. Here we found that E6 specifically dimerizes through its N-terminal domain and that disruption of the dimer interface strongly increases E6 solubility. This allowed us to raise structural data covering the entire HPV16 E6 protein, including the high-resolution NMR structures of the two zinc-binding domains of E6 and a robust data-driven model structure of the N-terminal domain homodimer. Interestingly, homodimer interface mutations that disrupt E6 self-association also inactivate E6-mediated p53 degradation. These data suggest that E6 needs to self-associate via its N-terminal domain to promote the polyubiquitination of p53 by E6AP.

Development of a method for the determination of bisphenol A at trace concentrations in human blood and urine and elucidation of factors influencing method accuracy and sensitivity.

Bisphenol A (BPA) is an industrial chemical used to make polymers including some used in food contact applications. Virtually complete presystemic clearance of orally administered BPA occurs in humans by metabolism to BPA-glucuronide (BPA-G), but some biomonitoring studies report low concentrations of free (parent) BPA in human blood and urine. Trace contamination of BPA from exogenous sources or hydrolysis of BPA-G to free BPA, either during or after biomonitoring specimen collection, may have contributed to the reported concentrations of free BPA. An analytical method for the determination of free BPA in human blood and urine was developed and validated in two independent laboratories, using the latest generation of high-performance liquid chromatography-tandem mass spectrometry instrumentation to ensure the desired high sensitivity and selectivity. The method was designed to account for and/or eliminate background contamination from all sources and demonstrated that contamination could occur from devices used for specimen collection or storage, as well as other sources. The method employed an internal standard (BPA-d(8)) and demonstrated accuracy and reproducibility in both matrices fortified with BPA or a surrogate analyte ((13)C-BPA) at a low quantitation limit (0.1-0.2 ng/mL). For validation, five replicate samples were analyzed to evaluate reproducibility. Importantly, it was demonstrated that the conditions of the method did not result in the hydrolysis of BPA-G to free BPA, another possible source of error in BPA analysis. Application of the principles defined by this method will be critical to assure valid analytical results in any future biomonitoring studies.

Developmental neurotoxicity study of dietary bisphenol A in Sprague-Dawley rats.

This study was conducted to determine the potential of bisphenol A (BPA) to induce functional and/or morphological effects to the nervous system of F(1) offspring from dietary exposure during gestation and lactation according to the Organization for Economic Cooperation and Development and U.S. Environmental Protection Agency guidelines for the study of developmental neurotoxicity. BPA was offered to female Sprague-Dawley Crl:CD (SD) rats (24 per dose group) and their litters at dietary concentrations of 0 (control), 0.15, 1.5, 75, 750, and 2250 ppm daily from gestation day 0 through lactation day 21. F(1) offspring were evaluated using the following tests: detailed clinical observations (postnatal days [PNDs] 4, 11, 21, 35, 45, and 60), auditory startle (PNDs 20 and 60), motor activity (PNDs 13, 17, 21, and 61), learning and memory using the Biel water maze (PNDs 22 and 62), and brain and nervous system neuropathology and brain morphometry (PNDs 21 and 72). For F(1) offspring, there were no treatment-related neurobehavioral effects, nor was there evidence of neuropathology or effects on brain morphometry. Based on maternal and offspring body weight reductions, the no-observed-adverse-effect level (NOAEL) for systemic toxicity was 75 ppm (5.85 and 13.1 mg/kg/day during gestation and lactation, respectively), with no treatment-related effects at lower doses or nonmonotonic dose responses observed for any parameter. There was no evidence that BPA is a developmental neurotoxicant in rats, and the NOAEL for developmental neurotoxicity was 2250 ppm, the highest dose tested (164 and 410 mg/kg/day during gestation and lactation, respectively).