Quantifying Analogue Suitability for SAR-Based Read-Across Toxicological Assessment.

Structure activity relationship (SAR)-based read-across often is an integral part of toxicological safety assessment, and justification of the prediction presents the most challenging aspect of the approach. It has been established that structural consideration alone is inadequate for selecting analogues and justifying their use, and biological relevance must be incorporated. Here we introduce an approach for considering biological and toxicological related features quantitatively to compute a similarity score that is concordant with suitability for a read-across prediction for systemic toxicity. Fingerprint keys for comparing metabolism, reactivity, and physical chemical properties are presented and used to compare these attributes for 14 case study chemicals each with a list of potential analogues. Within each case study, the sum of these nonstructural similarity scores is consistent with suitability for read-across established using an approach based on expert judgment. Machine learning is applied to determine the contributions from each of the similarity attributes revealing their importance for each structure class. This approach is used to quantify and communicate the differences between a target and a potential analogue as well as rank analogue quality when more than one is relevant. A numerical score with easily interpreted fingerprints increases transparency and consistency among experts, facilitates implementation by others, and ultimately increases chances for regulatory acceptance.

Impact of chemical structure on the in vitro hydrolysis of fatty esters of 2-ethylhexanoic acid or 2-ethylhexanol and extrapolation to the in vivo situation.

Fatty esters of 2-ethylhexanoic acid (EHA) and 2-ethylhexanol (EH) are commonly used in cosmetics. Human liver and skin S9 and human plasma were used to determine the in vitro rates of clearance (CLint) of a series of compounds, with a range of 2-11 carbons on the acid or alcohol moiety and branching at the C2 position. The impact of carbon chain length on in vitro CLint was most prominent for the liver metabolism of esters of EH, while for in vitro skin metabolism it was greater for esters of EHA. The position of the branching also impacted the liver hydrolysis rates, especially for the C3, C4, and C5 esters with lower CLint in vitro rates for esters of EHA relative to those of EH. When the in vitro intrinsic clearance rates were scaled to in vivo rates of hepatic clearance, all compounds approximated the rate for hepatic blood flow, mitigating this dependence of metabolism on structure. This work shows how structural changes to the molecule can affect in vitro metabolism and, furthermore, allows for an estimation of the in vivo metabolism.

A New Approach Methodology (NAM) Based Assessment of Butylated hydroxytoluene (BHT) for Endocrine Disruption Potential.

Butylated hydroxytoluene (BHT) is a synthetic antioxidant widely used in many industrial sectors. BHT is a well-studied compound for which there are many favorable regulatory decisions. However, a recent opinion by the French Agency for Food, Environmental and Occupational Health and Safety (ANSES) hypothesizes a role for BHT in endocrine disruption (ANSES (2021). This opinion is based on observations in mostly rat studies where changes to thyroid physiology are observed. Enzymatic induction of Cytochrome P450-mediated thyroid hormone catabolism has been proposed as a mechanism for these observations, however, a causal relationship has not been proven. Other evidence proposed in the document includes a read across argument to butylated hydroxyanisole (BHA), another Community Rolling Action Plan (CoRAP)-listed substance with endocrine disruption concerns. We tested the hypothesis that BHT is an endocrine disruptor by using a Next Generation Risk Assessment (NGRA) method. Four different cell lines: A549, HCC1428, HepG2, and MCF7 were treated with BHT and a series of BHT analogs at 5 different concentrations, RNA was isolated from cell extracts and run on the L1000 gene array platform. A toxicogenomics-based assessment was performed by comparing BHT's unique genomic signature to a large external database containing signatures of other compounds (including many known endocrine disruptors) to identify if any endocrine disruption-related modes of action (MoAs) are prevalent among BHT and other compounds with similar genomic signatures. In addition, we performed a toxicogenomics-based structure activity relationship (SAR) assessment of BHT and a series of structurally similar analogs to understand if endocrine disruption is a relevant MoA for chemicals that are considered suitable analogs to BHT using the P&G read across framework (Wu et al., 2010). Neither BHT nor any of its analogs connected to compounds that had endocrine activity for estrogens, androgens, thyroid, or steroidogenesis.

Assessing the genotoxicity and carcinogenicity of 2-chloroethanol through structure activity relationships and in vitro testing approaches.

The detection of 2-chloroethanol in foods generally follows an assumption that the pesticide ethylene oxide has been used at some stage in the supply chain. In this situation the Pesticide Residues in Food Regulation (EC) 396/2005 requires 2-chloroethanol to be assessed as if equivalent to ethylene oxide, which has been classified as a genotoxic carcinogen. This review investigated whether this is an appropriate risk assessment approach for 2-chloroethanol. This involved an assessment of existing genotoxicity and carcinogenicity data, application of Structure Activity Based Read Across for carcinogenicity assessment, biological reactivity in the ToxTracker assay and micronuclei formation in HepaRG cells. Although we identified there is an absence of a standard oral bioassay for 2-chloroethanol, carcinogenicity weight-of-evidence assessment along with data on relevant structural analogues do not show evidence for carcinogenicity for 2-chloroethanol. The absence of genotoxicity was demonstrated for 2-chloroethanol and suitable analogues. In contrast, ethylene oxide showed reactivity towards markers indicative of direct DNA damage which is consistent with what is known about its mode-of-action. These data facilitate the understanding of 2-chloroethanol and given that it is not a genotoxic carcinogen suggest it must be assessed relative to non-cancer endpoints and a health protective Reference Dose should be established on that basis.

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.

A matched molecular pair (MMP) approach for selecting analogs suitable for structure activity relationship (SAR)-based read across.

One of the most challenging aspects of SAR-based read across is the identification of structurally similar compounds suitable for use as data sources to cover the safety of a target chemical. Matched molecular pair analysis (MMPA) provides a systematic method for mining experimental data for chemical substitutions that may be interpreted in terms of changes in properties. Here we use the relationships between structural substitutions linking a target chemical with an analog determined to be suitable using the expert-judgment based P&G framework of Wu et al. (2010). The relationships are established by applying MMPA to a database of compounds with safety assessed using SAR-based read across to suitable analogs possessing toxicological data. The analysis revealed that only five categories of substitutions per chemical class (aromatic or aliphatic) were necessary to link all molecular pairs. These data are summarized in a workflow outlining a strategy for searching toxicological databases for potential analogs. This approach provides structural comparisons that are interpretable and sensitive to small differences in the local structure of two compounds that may be linked to suitability for read across in contrast to the use of quantitative similarity measures which show little correlation with analog suitability.

New framework for a non-animal approach adequately assures the safety of cosmetic ingredients - A case study on caffeine.

This case study on the model substance caffeine demonstrates the viability of a 10-step read-across (RAX) framework in practice. New approach methodologies (NAM), including RAX and physiologically-based kinetic (PBK) modelling were used to assess the consumer safety of caffeine. Appropriate animal systemic toxicity data were used from the most relevant RAX analogue while assuming that no suitable animal toxicity data were available for caffeine. Based on structural similarities, three primary metabolites of the target chemical caffeine (theophylline, theobromine and paraxanthine) were selected as its most relevant analogues, to estimate a point of departure in order to support a next generation risk assessment (NGRA). On the basis of the pivotal mode of action (MOA) of caffeine and other methylxanthines, theophylline appeared to be the most potent and suitable analogue. A worst-case aggregate exposure assessment determined consumer exposure to caffeine from different sources, such as cosmetics and food/drinks. Using a PBK model to estimate human blood concentrations following exposure to caffeine, an acceptable Margin of Internal Exposure (MOIE) of 27-fold was derived on the basis of a RAX using theophylline animal data, which suggests that the NGRA approach for caffeine is sufficiently conservative to protect human health.

Effect of chain length and branching on the in vitro metabolism of a series of parabens in human liver S9, human skin S9, and human plasma.

Parabens are antimicrobial compounds used as preservatives in cosmetics, foods, and pharmaceuticals. Paraben exposure occurs through a variety of routes including dermal absorption, ingestion, and inhalation. Ester bond hydrolysis has been shown to be the predominant biotransformation for this chemical class. Here we evaluated a series of parabens of increasing alkyl chain length and branching in addition to the aryl side chain of phenyl paraben (PhP). We evaluated the parabens under full Michaelis-Menten (MM) parameters to obtain intrinsic clearance values and found different trends between human liver and skin, which correlate with the predominant esterase enzymes in those matrices, respectively. In liver, where carboxylesterase 1 (CES1) is the predominant esterase enzyme, the shorter chain parabens were more readily metabolized, while in skin, where carboxylesterase 2 (CES2) is the predominant esterase enzyme, the longer chain parabens were more readily metabolized. Alkyl chain branching reduced the hydrolysis rates relative to those for the straight chain compounds, while the addition of a phenyl group, as in PhP, showed an increase in hydrolysis, producing the highest observed hydrolysis rate for skin. These data summarize the structure-metabolism relationship for a series of parabens and contribute to the safety assessment of this class of compounds.

Metabolism and plasma protein binding of 16 straight- and branched-chain parabens in in vitro liver and skin models.

Parabens are alkyl esters of 4-hydroxybenzoic acid (4-HBA), with short-chain parabens used as antimicrobials in cosmetics. We investigated the impact of chain structure on skin and liver metabolism. Incubations with primary human hepatocytes and human liver S9 indicated that methyl-, ethyl-, propyl- and butylparaben were rapidly metabolized to similar metabolites, including 4-HBA plus the corresponding alcohols. Liver and EpiSkin™ S9 were used to investigate the metabolism of 16 short and long straight- and branched-chain parabens. The rate of hydrolysis generally decreased with increasing chain length in liver S9, whereas the reverse was true for EpiSkin™ S9. Chain length also correlated with the number of metabolites, with more oxidized metabolites detected from longer chain parabens. The identity of the alcohol group impacted metabolism the most, in terms of the rate of metabolism and the contribution of cofactors. The majority of parabens (13/16) exhibited high plasma protein binding (PPB) (>90%); whereas, 4-HBA PPB was 38%. PPB was related to the LogP of the parabens. In conclusion, the major and common paraben metabolite in PHH, liver S9 and EpiSkin™ S9 was 4-HBA. The rate of metabolism, type of metabolite and contribution of hydrolysis was tissue-specific (liver, skin) and was influenced by the chain length (and hence LogP), structural isomeric form (straight vs branched), and/or the identity of the alkyl group. SHORT ABSTRACT: We investigated how the chain structure of parabens affects their metabolism by liver and EpiSkin™ S9. The major and common metabolite in primary human hepatocytes, liver S9 and EpiSkin™ S9 was 4-HBA plus the corresponding alcohols. The rate of metabolism, type of metabolite and contribution of hydrolysis was tissue-specific and influenced by the chain length, structural isomeric form (straight vs branched), and/or the identity of the alkyl group. Most parabens exhibited high PPB (>90%), whereas the PPB of 4-HBA was 38%.

Measurement of the penetration of 56 cosmetic relevant chemicals into and through human skin using a standardized protocol.

OECD test guideline 428 compliant protocol using human skin was used to test the penetration of 56 cosmetic-relevant chemicals. The penetration of finite doses (10 µL/cm2 ) of chemicals was measured over 24 hours. The dermal delivery (DD) (amount in the epidermis, dermis and receptor fluid [RF]) ranged between 0.03 ± 0.02 and 72.61 ± 8.89 µg/cm2 . The DD of seven chemicals was comparable with in vivo values. The DD was mainly accounted for by the amount in the RF, although there were some exceptions, particularly of low DD chemicals. While there was some variability due to cell outliers and donor variation, the overall reproducibility was very good. As six chemicals had to be applied in 100% ethanol due to low aqueous solubility, we compared the penetration of four chemicals with similar physicochemical properties applied in ethanol and phosphate-buffered saline. Of these, the DD of hydrocortisone was the same in both solvents, while the DD of propylparaben, geraniol and benzophenone was lower in ethanol. Some chemicals displayed an infinite dose kinetic profile; whereas, the cumulative absorption of others into the RF reflected the finite dosing profile, possibly due to chemical volatility, total absorption, chemical precipitation through vehicle evaporation or protein binding (or a combination of these). These investigations provide a substantial and consistent set of skin penetration data that can help improve the understanding of skin penetration, as well as improve the prediction capacity of in silico skin penetration models.

Intrinsic relative potency of a series of pyrrolizidine alkaloids characterized by rate and extent of metabolism.

1,2-Unsaturated pyrrolizidine alkaloids (PAs) are sometimes present in foods or herbal supplements/medicines as impurities and pose potential concerns for liver genotoxicity/carcinogenicity. PAs display a strong structure toxicity relationship, however, current regulatory approaches to risk assessment take the precautionary approach of assuming all PAs display the same potency as the most toxic congeners lasiocarpine (LAS) and riddelliine (RID). Here we explore the relative potencies of a series of structurally diverse PAs by measuring DNA adduct formation in vitro in a rat sandwich culture hepatocyte (SCH) cell system. The adducts generated are consistent with those identified in vivo as biomarkers of PA exposure and potential liver-tumor formation. DNA reactive PAs require metabolic activation to form intermediates that bind DNA, therefore, adduct formation is a direct reflection of reactive metabolite formation. Since the area under the concentration versus time curve (AUC) for the depletion of parent PA from the extracellular media is a measure of PA exposure, the ratio of adducts/AUC provides a measure of hepatocyte exposure to DNA-binding metabolites corresponding to an intrinsic potency for DNA adduct formation. Intrinsic potencies relative to potencies for LAS compare well with existing relative potency data further affirming that PA toxicity varies considerably with chemical structure.

Use of connectivity mapping to support read across: A deeper dive using data from 186 chemicals, 19 cell lines and 2 case studies.

We previously demonstrated that the Connectivity Map (CMap) (Lamb et al., 2006) concept can be successfully applied to a predictive toxicology paradigm to generate meaningful MoA-based connections between chemicals (De Abrew et al., 2016). Here we expand both the chemical and biological (cell lines) domain for the method and demonstrate two applications, both in the area of read across. In the first application we demonstrate CMap's utility as a tool for testing biological relevance of source chemicals (analogs) during a chemistry led read across exercise. In the second application we demonstrate how CMap can be used to identify functionally relevant source chemicals (analogs) for a structure of interest (SOI)/target chemical with minimal knowledge of chemical structure. Finally, we highlight four factors: promiscuity of chemical, dose, cell line and timepoint as having significant impact on the output. We discuss the biological relevance of these four factors and incorporate them into a work flow.

Relative potency of fifteen pyrrolizidine alkaloids to induce DNA damage as measured by micronucleus induction in HepaRG human liver cells.

Plant-based 1,2-unsaturated Pyrrolizidine Alkaloids (PAs) can be found as contaminants in foods like teas, herbs and honey. PAs are responsible for liver genotoxicity/carcinogenicity following metabolic activation, making them a relevant concern for safety assessment. Current regulatory risk assessments take a precautionary approach and assume all PAs are as potent as the known most potent representatives: lasiocarpine and riddelliine. Our study investigated whether genotoxicity potency differed as a consequence of structural differences, assessing micronuclei in vitro in HepaRG cells which express metabolising enzymes at levels similar to primary human hepatocytes. Benchmark Dose (BMD) analysis was used to calculate the critical effect dose for 15 PAs representing 6 structural classes. When BMD confidence intervals were used to rank PAs, lasiocarpine was the most potent PA and plotted distinctly from all other PAs examined. PA-N-oxides were least potent, notably less potent than their corresponding parent PA's. The observed genotoxic potency compared favorably with existing in vitro data when metabolic competency was considered. Although further consideration of biokinetics will be needed to develop a robust understanding of relative potencies for a realistic risk assessment of PA mixtures, these data facilitate understanding of their genotoxic potencies and affirm that not all PAs are created equal.

The Natural History and Rehabilitative Outcomes of Hearing Loss in Congenital Cytomegalovirus: A Systematic Review.

OBJECTIVE: The purpose of this study was to examine the literature regarding the natural history and rehabilitative outcomes of sensorineural hearing loss from congenital cytomegalovirus infections. DATA SOURCES AND STUDY ELIGIBILITY CRITERIA: A systematic search was performed in PubMed, PsychINFO, CINAHL, and Web of Science to identify peer-reviewed research. Eligible studies were those containing original peer-reviewed research in English addressing either the natural history or rehabilitative outcomes of sensorineural hearing loss (SNHL) in congenital cytomegalovirus (cCMV). STUDY APPRAISAL AND SYNTHESIS METHODS: Two investigators independently reviewed all articles and extracted data. Bias was assessed using the Cochrane Collaboration's tool and the Newcastle-Ottawa Assessment Scale. RESULTS: Thirty-six articles were reviewed. Universal screening identifies 0.2 to 1% of newborns with cCMV infection. SNHL ranged from 8 to 32% of infants and was more prevalent in symptomatic versus asymptomatic cases. Nine to 68% of hearing loss occurs in a late or delayed fashion. In 7 to 71% of cases hearing loss is progressive. Cochlear implantation (CI) is a viable option for patients with cCMV associated hearing loss and leads to improvements in hearing and language. There is limited literature comparing rehabilitation outcomes in cCMV and non-cCMV CI recipients. CONCLUSION: Late onset and progressive hearing loss is seen in children who develop hearing loss from cCMV. Frequent audiologic follow-up is necessary considering the natural history of cCMV hearing loss. Universal screening should be pursued due to the number of asymptomatic children, at birth, who develop late onset/delayed hearing loss. CI is an effective means of improving speech and language in this population.

Structure activity relationship (SAR) toxicological assessments: The role of expert judgment.

Structure activity relationships (SAR) and read-across are widely used animal alternative approaches for filling toxicological data gaps. A framework describing the use of expert judgment in evaluating analogs for SAR has been published and widely cited, however, reliance on expert judgment can introduce inconsistent results across experts and hinder transparency. Here we explore the use of a quantitative similarity score between an analog and a Structure of Interest (SOI) to see if these scores correlate with the expert judgement-based suitability rankings. We find these global similarity scores representing a "whole-molecule" view of similarity to be insensitive to differences in local structure which may be important for toxicity, and, therefore, cannot be substituted for expert judgement-based similarity rankings. In this paper, we suggest that the next step in the progression of SAR approaches retains the insights from expert judgment, but facilitates consistency and transparency through the development of rating "rules". This report outlines and defines analog rating rules for several compound categories. While not comprehensive, the exercises demonstrate the development of rules for categories with a large spread in molecular weight and alkyl chain length and explains the advantages that we see in this approach compared to relying solely on a computational approach or an unstructured expert judgement approach. These rules may be incorporated into analog searching work flows to define boundaries for analogs "suitable" for read-across.

Promotion of early pediatric hearing detection through patient navigation: A randomized controlled clinical trial.

OBJECTIVES/HYPOTHESIS: To assess the efficacy of a patient navigator intervention to decrease nonadherence to obtain audiological testing following failed screening, compared to those receiving the standard of care. METHODS: Using a randomized controlled design, guardian-infant dyads, in which the infants had abnormal newborn hearing screening, were recruited within the first week after birth. All participants were referred for definitive audiological diagnostic testing. Dyads were randomized into a patient navigator study arm or standard of care arm. The primary outcome was the percentage of patients with follow-up nonadherence to obtain diagnostic testing. Secondary outcomes were parental knowledge of infant hearing testing recommendations and barriers in obtaining follow-up testing. RESULTS: Sixty-one dyads were enrolled in the study (patient navigator arm = 27, standard of care arm = 34). The percentage of participants nonadherent to diagnostic follow-up during the first 6 months after birth was significantly lower in the patient navigator arm compared with the standard of care arm (7.4% vs. 38.2%) (P = .005). The timing of initial follow-up was significantly lower in the navigator arm compared with the standard of care arm (67.9 days after birth vs. 105.9 days, P = .010). Patient navigation increased baseline knowledge regarding infant hearing loss diagnosis recommendations compared with the standard of care (P = .004). CONCLUSIONS: Patient navigation decreases nonadherence rates following abnormal infant hearing screening and improves knowledge of follow-up recommendations. This intervention has the potential to improve the timeliness of delivery of infant hearing healthcare; future research is needed to assess the cost and feasibility of larger scale implementation. LEVEL OF EVIDENCE: 1b. Laryngoscope, 127:S1-S13, 2017.

Integrating in silico models to enhance predictivity for developmental toxicity.

Application of in silico models to predict developmental toxicity has demonstrated limited success particularly when employed as a single source of information. It is acknowledged that modelling the complex outcomes related to this endpoint is a challenge; however, such models have been developed and reported in the literature. The current study explored the possibility of integrating the selected public domain models (CAESAR, SARpy and P&G model) with the selected commercial modelling suites (Multicase, Leadscope and Derek Nexus) to assess if there is an increase in overall predictive performance. The results varied according to the data sets used to assess performance which improved upon model integration relative to individual models. Moreover, because different models are based on different specific developmental toxicity effects, integration of these models increased the applicable chemical and biological spaces. It is suggested that this approach reduces uncertainty associated with in silico predictions by achieving a consensus among a battery of models. The use of tools to assess the applicability domain also improves the interpretation of the predictions. This has been verified in the case of the software VEGA, which makes freely available QSAR models with a measurement of the applicability domain.

Rural Family Perspectives and Experiences with Early Infant Hearing Detection and Intervention: A Qualitative Study.

Infant hearing loss has the potential to cause significant communication impairment. Timely diagnosis and intervention is essential to preventing permanent deficits. Many infants from rural regions are delayed in diagnosis and treatment of hearing loss. The purpose of this study is to characterize the barriers in timely infant hearing healthcare for rural families following newborn newborn hearing screening (NHS) testing. Using stratified purposeful sampling, the study design involved semi-structured phone interviews with parents/guardians of children who failed NHS testing in the Appalachian region of Kentucky between 2012 and 2014 to describe their experiences with early hearing detection and intervention program. Thematic qualitative analysis was performed on interview transcripts to identify common recurring themes in content. 40 parents/guardians participated in the study and consisted primarily of mothers. Demographic data revealed limited educational levels of the participants and 70 % had state-funded insurance coverage. Participants reported barriers in timely infant hearing healthcare that included poor communication of hearing screening results, difficulty in obtaining outpatient testing, inconsistencies in healthcare information from primary care providers, lack of local resources, insurance-related healthcare delays, and conflict with family and work responsibilities. Most participants expressed a great desire to obtain timely hearing healthcare for their children and expressed a willingness to use resources such as telemedicine to obtain that care. There are multiple barriers to timely rural infant hearing healthcare. Minimizing misinformation and improving access to care are priorities to prevent delayed diagnosis and treatment of hearing loss.

A strategy for safety assessment of chemicals with data gaps for developmental and/or reproductive toxicity.

Alternative methods for full replacement of in vivo tests for systemic endpoints are not yet available. Read across methods provide a means of maximizing utilization of existing data. A limitation for the use of read across methods is that they require analogs with test data. Repeat dose data are more frequently available than are developmental and/or reproductive toxicity (DART) studies. There is historical precedent for using repeat dose data in combination with a database uncertainty factor (UF) to account for missing DART data. We propose that use of the DART decision tree (Wu et al., 2013), in combination with a database UF, provides a path forward for DART data gap filling that better utilizes all of the data. Our hypothesis was that chemical structures identified by the DART tree as being related to structures with known DART toxicity would potentially have lower DART NOAELs compared to their respective repeat dose NOAELs than structures that lacked this association. Our analysis supports this hypothesis and as a result also supports that the DART decision tree can be used as part of weight of evidence in the selection of an appropriate DART database UF factor.