Review of dose setting for the extended one-generation reproductive toxicity studies (OECD TG 443): Considerations on ECHA's dose level selection recommendations.

During 2020, The European Chemicals Agency (ECHA) began evaluating the OECD Test Guideline 443: Extended One Generation Reproductive Toxicity Study (EOGRTS) to analyze specific aspects related to study design, conduct and toxicological findings. A significant outcome of this ECHA evaluation focused on adequate dose level selection. Subsequently, ECHA published recommendations for DART studies, however, these recommendations seemingly do not align with the principles of the 3Rs, animal welfare or human safety goals, specifically, regarding three aspects. First, the requirement to segregate testing for sexual function and fertility from the ability to produce normally developing offspring increases the risk of inadequate identification of postnatal hazards for development and sexual function and fertility, therefore failing human health protection goals. Second, the current ECHA high-dose level setting recommendations for EOGRTS exceed the MTD (Maximum Tolerated Dose), and therefore compromise the interpretation of the biological response relative to the intrinsic effect of the chemical under evaluation. Third, the combination of these aspects will result in an increase in the number of animals tested, increasing animal welfare concerns. This paper reflects the consensus of subject matter experts, professional, and scientific societies who have authored and signed on to this statement. The signatories encourage ECHA to adopt a revised science-driven approach to the dose selection criteria that strikes a balance between regulatory vigilance and scientific pragmatism.

Sensory irritation and use of the best available science in setting exposure limits: Issues raised by a scientific panel review of formaldehyde human research studies.

As a high production volume chemical with recognized sensory irritation and widespread exposure, the human health risk potential of formaldehyde has been reviewed by many international regulatory agencies and scientific advisory bodies. A scientific panel, the Human Studies Review Board, under the auspices of the EPA's Toxic Substances Control Act (TSCA) program recently reviewed the sensory irritation studies included in the 2022 Draft Integrated Risk Information System (IRIS) Formaldehyde Hazard Assessment in the context of their use in a weight of evidence evaluation of acute inhalation health effects. This panel issued a series of recommendations on the use of these studies for the purposes of calculating exposure limits (e.g., study design preferences; uncertainty adjustment). Considering that these recommendations might reflect topic areas with varying degrees of scientific consensus, this commentary reflects on commonalities and distinctions amongst international formaldehyde exposure limits based on sensory irritation. Notably, each review panel charged with an assessment of the science recommended that no adjustment was needed to account for either exposure duration or human variability. These areas of scientific consensus should be considered as the best available science for the purposes of setting exposure limits in the anticipated TSCA Risk Evaluation on formaldehyde.

Challenges integrating skin sensitization data for assessment of difficult to test substances.

Difficult to test substances, including poorly soluble, mildly irritating, or UVCBs (unknown or variable composition complex reaction products or biological materials), producing weak or borderline in vivo results, face additional challenges in in vitro assays that often necessitate data integration in a weight of evidence (WOE) approach to inform skin sensitization potential. Here we present several case studies on difficult to test substances and highlight the utility of the toxicological priority index (ToxPi) as a data visualization tool to compare skin sensitization biological activity. The case study test substances represent two poorly soluble substances, tetrakis (2-ethylbutyl) orthosilicate and decyl palmitate, and two UVCB substances, alkylated anisole and hydrazinecarboximidamide, 2-[(2-hydroxyphenyl)methylene]-, reaction products with 2 undecanone. Data from key events within the skin sensitization adverse outcome pathway were gathered from publicly available sources or specifically generated. Incorporating the data for these case study test substances as well as data on chemicals of a known sensitization class (sensitizer, irritating non-sensitizer, and non-sensitizer) into ToxPi produced biological activity profiles which were grouped using unsupervised hierarchical clustering. Three of the case study test substances concluded to lack skin sensitization potential by traditional WOE produced biological activity profiles most consistent with non-sensi­tizing substances, whereas the prediction was less definitive for a substance considered positive by traditional WOE. Visualizing the data using bioactivity profiles can provide further support for WOE conclusions in certain circumstances but is unlikely to replace WOE as a stand-alone prediction due to limitations of the method including the impact of missing data points.

Non-animal test methods to detect chemicals that cause skin allergies are accepted alternatives to animal testing for this purpose. However, some chemicals are difficult to test using these methods, e.g., substances that cause skin irritation, are not water soluble or are mixtures of different compo­nents. We compiled existing and new data on how four such chemicals activate key elements of the biological pathway leading to allergic skin reactions and compared the resulting patterns with respective patterns of many chemicals confirmed to cause skin allergy, skin irritation or neither. The patterns were visualized and analyzed with a computer software tool. The tool confirmed that three substances were non-sensitizers but did not confirm that the fourth substance was a skin sensitizer as predicted by the standard assessment. This approach, which incorporates all available data types into the assessment of difficult to test chemicals, may further reduce unnecessary animal testing.

The last resort requirement under REACH: From principle to practice.

REACH (Registration, Evaluation, Authorisation and Restriction of Chemicals) is a European Union regulation that aims to protect human health and the environment from the risks posed by chemicals. Article 25 clearly states that: "[i]n order to avoid animal testing, testing on vertebrate animals for the purposes of this Regulation shall be undertaken only as a last resort." In practice, however, the standard information requirements under REACH are still primarily filled using animal studies. This paper presents examples illustrating that animal testing is not always undertaken only as a last resort. Six over-arching issues have been identified which contribute to this: (1) non-acceptance of existing animal or non-animal data, (2) non-acceptance of read-across, (3) inflexible administrative processes, (4) redundancy of testing, (5) testing despite animal welfare concerns and (6) testing for cosmetic-only ingredients. We, members of the Animal-Free Safety Assessment (AFSA) Collaboration, who work together to accelerate the global adoption of non-animal approaches for chemical safety assessment, herein propose several recommendations intended to aid the European Commission, the European Chemicals Agency and registrants to protect human health and the environment while avoiding unnecessary animal tests - truly upholding the last resort requirement in REACH.

Integrating Concentration-Dependent Toxicity Data and Toxicokinetics To Inform Hepatotoxicity Response Pathways.

Failure of animal models to predict hepatotoxicity in humans has created a push to develop biological pathway-based alternatives, such as those that use in vitro assays. Public screening programs (e.g., ToxCast/Tox21 programs) have tested thousands of chemicals using in vitro high-throughput screening (HTS) assays. Developing pathway-based models for simple biological pathways, such as endocrine disruption, has proven successful, but development remains a challenge for complex toxicities like hepatotoxicity, due to the many biological events involved. To this goal, we aimed to develop a computational strategy for developing pathway-based models for complex toxicities. Using a database of 2171 chemicals with human hepatotoxicity classifications, we identified 157 out of 1600+ ToxCast/Tox21 HTS assays to be associated with human hepatotoxicity. Then, a computational framework was used to group these assays by biological target or mechanisms into 52 key event (KE) models of hepatotoxicity. KE model output is a KE score summarizing chemical potency against a hepatotoxicity-relevant biological target or mechanism. Grouping hepatotoxic chemicals based on the chemical structure revealed chemical classes with high KE scores plausibly informing their hepatotoxicity mechanisms. Using KE scores and supervised learning to predict in vivo hepatotoxicity, including toxicokinetic information, improved the predictive performance. This new approach can be a universal computational toxicology strategy for various chemical toxicity evaluations.

An interdisciplinary framework for derivation of occupational exposure limits.

Protecting the health and safety of workers in industrial operations is a top priority. One of the resources used in industry to ensure worker safety is the occupational exposure limit (OEL). OELs are derived from the assessment and interpretation of empirical data from animal and/or human studies. There are various guidelines for the derivation and implementation of OELs globally, with a range of stakeholders (including regulatory bodies, governmental agencies, expert groups and others). The purpose of this manuscript is to supplement existing guidance with learnings from a multidisciplinary team approach within an industry setting. The framework we present is similar in construct to other risk assessment frameworks and includes: (1) problem formulation, (2) literature review, (3) weight of evidence considerations, (4) point of departure selection/derivation, (5) application of assessment factors, and the final step, (6) derivation of the OEL. Within each step are descriptions and examples to consider when incorporating data from various disciplines such as toxicology, epidemiology, and exposure science. This manuscript describes a technical framework by which available data relevant for occupational exposures is compiled, analyzed, and utilized to inform safety threshold derivation applicable to OELs.

Adverse outcome pathway (AOP): α2u-globulin nephropathy and kidney tumors in male rats.

The National Research Council's vision of using adverse outcome pathways (AOPs) as a framework to assist with toxicity assessment for regulatory requirements of chemical assessment has continued to gain traction since its release in 2007. The need to expand the AOP knowledge base has gained urgency, with the U.S. Environmental Protection Agency's directive to eliminate reliance on animal toxicity testing by 2035. To meet these needs, our goal was to elucidate the AOP for male-rat-specific kidney cancer. Male-rat-specific kidney tumors occur through the ability of structurally diverse substances to induce α2u-globulin nephropathy (α2u-N), a well-studied mode of action (MoA) not relevant in humans that results in kidney tumor formation in male rats. An accepted AOP may help facilitate the differentiation from other kidney tumors MoAs. Following identification and review of relevant in vitro and in vivo literature, both the MIE and subsequent KEs were identified. Based on the weight of evidence from the various resources, the confidence in this AOP is high. Uses of this AOP include hazard identification, development of in vitro assays to determine if the MoA is through α2u-N and not relevant to humans resulting in decreased use of animals, and regulatory applications.

Applying evidence-based methods to the development and use of adverse outcome pathways

The workshop "Application of evidence-based methods to construct mechanistic frameworks for the development and use of non-animal toxicity tests" was organized by the Evidence-based Toxicology Collaboration and hosted by the Grading of Recommendations Assessment, Development and Evaluation Working Group on June 12, 2019. The purpose of the workshop was to bring together international regulatory bodies, risk assessors, academic scientists, and industry to explore how systematic review methods and the adverse outcome pathway framework could be combined to develop and use mechanistic test methods for predicting the toxicity of chemical substances in an evidence-based manner. The meeting covered the history of biological frameworks, the way adverse outcome pathways are currently developed, the basic principles of systematic methodology, including systematic reviews and evidence maps, and assessment of cer­tainty in models, and adverse outcome pathways in particular. Specific topics were discussed via case studies in small break-out groups. The group concluded that adverse outcome pathways provide an important framework to support mechanism-based assessment in environmental health. The process of their development has a few challenges that could be addressed with systematic methods and automation tools. Addressing these challenges will increase the transparency of the evidence behind adverse outcome pathways and the consistency with which they are defined; this in turn will increase their value for supporting public health decisions. It was suggested to explore the details of applying systematic methods to adverse outcome pathway development in a series of case studies and workshops.

Application of adverse outcome pathway networks to integrate mechanistic data informing the choice of a point of departure for hydrogen sulfide exposure limits.

Acute exposure to hydrogen sulfide initiates a series of hallmark biological effects that occur progressively at increasing exposure levels: odor perception, conjunctivitis, olfactory paralysis, "knockdown," pulmonary edema, and apnea. Although effects of exposure to high concentrations of hydrogen sulfide are clear, effects associated with chronic, low-level exposure in humans is under debate, leading to uncertainty in the critical effect used in regulatory risk assessments addressing low dose exposures. This study integrates experimental animal, observational epidemiology, and occupational exposure evidence by applying a pathway-based approach. A hypothesized adverse outcome pathway (AOP) network was developed from 34 studies, composed of 4 AOPs sharing 1 molecular initiating events (MIE) and culminating in 4 adverse outcomes. A comparative assessment of effect levels and weight of evidence identified an AOP leading to a biologically-plausible, low-dose outcome relative to the other outcomes (nasal lesions, 30 ppm versus olfactory paralysis, >100 ppm; neurological effects, >80 ppm; pulmonary edema, >80 ppm). This AOP (i.e. AOP1) consists of the following key events: cytochrome oxidase inhibition (>10 ppm), neuronal cell loss (>30 ppm), and olfactory nasal lesions (defined as both neuronal cell loss and basal cell hyperplasia; >30 ppm) in rodents. The key event relationships in this pathway were supported by moderate empirical evidence and have high biological plausibility due to known mechanistic understanding and consistency in observations for diverse chemicals.

Evaluating the MoA/human relevance framework for F-344 rat liver epithelioid granulomas with mineral oil hydrocarbons.

Toxicology feeding studies of mineral oil hydrocarbons (MOHs), within the carbon number range C22-C28, results in species-specific epithelioid granulomas in the liver of F-344 rats but not in other rat strains, or species. While MOH has been detected, and some pathological effects have been shown to occur in other organs/tissues of F-344 rats and other rat strains/species, it is generally accepted that the effect of toxicological concern is species-specific inflammatory liver granuloma. As oil retention and other MOH-related nontoxic pathological changes in the liver are observed in humans, some have hypothesized that the potential for oil accumulation over a lifetime, through dietary sources, may predispose humans to similar liver effects as observed in F-344 rats. To address this concern, a mode of action/human relevance framework (MoA/HRF) analysis for MOH-induced epithelioid granuloma in the F-344 rat model was developed. The key events for the development of liver epithelioid granulomas were identified as increased MOH intestinal absorption, preferential tissue retention and ultimately formation of necrotic granulomas encased by infiltrating inflammatory lymphocytes. The hypothesized MoA was evaluated using the modified Bradford Hill considerations for causality and was considered to be established in the F-344 rodent model. However, key strain/species differences in the rate of intestinal absorption, tissue retention of MOH and inflammatory response to MOH in the liver were identified. Overall, the F-344 rat MoA was not considered to be relevant to humans, consistent with data showing no evidence for the formation of epithelioid granulomas with humans even in cases of massive ingestion of MOHs.

A quantitative screening-level approach to incorporate chemical exposure and risk into alternative assessment evaluations.

As the general public and retailers ask for disclosure of chemical ingredients in the marketplace, a number of hazard screening tools were developed to evaluate the so-called "greenness" of individual chemical ingredients and/or formulations. The majority of these tools focus only on hazard, often using chemical lists, ignoring the other part of the risk equation: exposure. Using a hazard-only focus can result in regrettable substitutions, changing 1 chemical ingredient for another that turns out to be more hazardous or shifts the toxicity burden to others. To minimize the incidents of regrettable substitutions, BizNGO describes "Common Principles" to frame a process for informed substitution. Two of these 6 principles are: "reduce hazard" and "minimize exposure." A number of frameworks have emerged to evaluate and assess alternatives. One framework developed by leading experts under the auspices of the US National Academy of Sciences recommended that hazard and exposure be specifically addressed in the same step when assessing candidate alternatives. For the alternative assessment community, this article serves as an informational resource for considering exposure in an alternatives assessment using elements of problem formulation; product identity, use, and composition; hazard analysis; exposure analysis; and risk characterization. These conceptual elements build on practices from government, academia, and industry and are exemplified through 2 hypothetical case studies demonstrating the questions asked and decisions faced in new product development. These 2 case studies-inhalation exposure to a generic paint product and environmental exposure to a shampoo rinsed down the drain-demonstrate the criteria, considerations, and methods required to combine exposure models addressing human health and environmental impacts to provide a screening level hazard and exposure (risk) analysis. This article informs practices for these elements within a comparative risk context to improve alternatives assessment evaluation and decision making. Integr Environ Assess Manag 2017;13:1007-1022. © 2017 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals, Inc. on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Development of a screening tool to prioritize testing for the carcinogenic hazard of residual aromatic extracts and related petroleum streams.

Residual aromatic extracts (RAE) are petroleum substances with variable composition predominantly containing aromatic hydrocarbons with carbon numbers greater than C25. Because of the high boiling nature of RAEs, the aromatics present are high molecular weight, with most above the range of carcinogenic polycyclic aromatic hydrocarbons (PAHs). However, refinery distillations are imperfect; some PAHs and their heteroatom-containing analogs (collectively referred to as polycyclic aromatic content or PAC) may remain in the parent stream and be extracted into the RAE, and overall PAC content is related to the carcinogenic potential of an RAE. We describe here a real-time analytical chemistry-based tool to assess the carcinogenic hazard of RAE via the development of a functional relationship between carcinogenicity and boiling point. Samples representative of steps along the RAE manufacturing process were obtained from five refineries to evaluate relationships between mutagenicity index (MI), PAC ring content and gas chromatographic distillation (GCD) curves. As expected, a positive linear relationship between MI and PAC ring content occurred, most specifically for 3-6 ring PAC (R2=0.68). A negative correlation was found between MI and temperature at 5% vaporization by GCD (R2=0.72), indicating that samples with greater amounts of lower boiling constituents were more likely to be carcinogenic. The inverse relationship between boiling range and carcinogenicity was further demonstrated by fractionation of select RAE samples (MI=0.50+0.07; PAC=1.70+0.51wt%; n=5) into low and high boiling fractions, where lower boiling fractions were both more carcinogenic than the higher boiling fractions (MI=2.36±0.55 and 0.17±0.11, respectively) and enriched in 3-6 ring PACs (5.20+0.70wt% and 0.97+0.35wt%, respectively). The criteria defining carcinogenicity was established as 479°C for the 5% vaporization points by GCD, with an approximate 95% probability of a future sample having an MI below the recommended limit of 0.4 for RAEs. Overall, these results provide a cost-efficient and real-time tool by which the carcinogenic potential of RAEs can be assessed at the refinery level, ultimately providing a means to readily monitor and minimize the carcinogenic potential of RAEs.

Pathway-based toxicity: history, current approaches and liver fibrosis and steatosis as prototypes.

The Human Toxicology Project Consortium (HTPC) was created to accelerate implementation of the science and policies required to achieve a pathway-based foundation for toxicology as articulated in the 2007 National Research Council report, Toxicity Testing in the 21st Century: a Vision and a Strategy. The HTPC held a workshop, "Building Shared Experience to Advance Practical Application of Pathway-Based Toxicology: Liver Toxicity Mode-of-Action," in January, 2013, in Baltimore, MD, to further the science of pathway-based approaches to liver toxicity. This review was initiated as a thought-starter for this workshop and has since been updated to include insights from the workshop and other activities occurring in 2013. The report of the workshop has been published elsewhere in this journal (Willett et al., 2014).

Developing scientific confidence in HTS-derived prediction models: lessons learned from an endocrine case study.

High throughput (HTS) and high content (HCS) screening methods show great promise in changing how hazard and risk assessments are undertaken, but scientific confidence in such methods and associated prediction models needs to be established prior to regulatory use. Using a case study of HTS-derived models for predicting in vivo androgen (A), estrogen (E), thyroid (T) and steroidogenesis (S) endpoints in endocrine screening assays, we compare classification (fitting) models to cross validation (prediction) models. The more robust cross validation models (based on a set of endocrine ToxCast™ assays and guideline in vivo endocrine screening studies) have balanced accuracies from 79% to 85% for A and E, but only 23% to 50% for T and S. Thus, for E and A, HTS results appear promising for initial use in setting priorities for endocrine screening. However, continued research is needed to expand the domain of applicability and to develop more robust HTS/HCS-based prediction models prior to their use in other regulatory applications. Based on the lessons learned, we propose a framework for documenting scientific confidence in HTS assays and the prediction models derived therefrom. The documentation, transparency and the scientific rigor involved in addressing the elements in the proposed Scientific Confidence Framework could aid in discussions and decisions about the prediction accuracy needed for different applications.

Subchronic and developmental toxicity of aromatic extracts.

Aromatic extracts (AEs; distillate AEs [DAEs] and residual AEs [RAEs]) are complex, highly viscous liquid petroleum streams with variable compositions derived by extraction of aromatic compounds from distillate and residual petroleum fractions from a vacuum distillation tower, respectively. The DAEs generally contain significant amounts of polycyclic aromatic compounds (PACs) and are carcinogenic. The RAEs typically contain lower concentrations of biologically active PACs. The PACs in refinery streams can cause effects in repeated-dose and developmental toxicity studies. In a 13-week dermal study, light paraffinic DAE had several dose-related effects involving multiple organs; no-observed-effect level was <5 mg/kg/d, with no overt toxicity. Predicted dose-responses at 10% (PDR10s), modeled doses causing a 10% effect on sensitive end points based on PAC content, ranged from 25 to 78 mg/kg/d for untested paraffinic DAEs. The no observed adverse effect level (NOAEL) for developmental toxicity for light paraffinic DAE was 5 mg/kg/d. Statistically significant developmental effects at higher doses were associated with maternal effects. The PDR10s for developmental toxicity of paraffinic DAEs ranged from 7 to >2000 mg/kg/d, reflecting differences due to variation in PACs. The NOAELs for RAEs were 500 mg/kg for 90-day studies and 2000 mg/kg for developmental toxicity. Reproductive toxicity is not considered to be a sensitive end point for AEs based on the toxicity tests with DAEs, RAEs, and other PAC-containing petroleum substances. In vivo micronucleus tests on heavy paraffinic DAE, RAEs, and a range of other petroleum substances have been negative. The exception to this general trend was a marginally positive response with light paraffinic DAE. Most DAEs are considered unlikely to produce chromosomal effects in vivo.

Acute, subchronic, and developmental toxicological properties of lubricating oil base stocks.

Lubricating oil base stocks (LOBs) are substances used in the manufacture of finished lubricants and greases. They are produced from residue remaining after atmospheric distillation of crude oil that is subsequently fractionated by vacuum distillation and additional refining steps. Initial LOB streams that have been produced by vacuum distillation but not further refined may contain polycyclic aromatic compounds (PACs) and may present carcinogenic hazards. In modern refineries, LOBs are further refined by multistep processes including solvent extraction and/or hydrogen treatment to reduce the levels of PACs and other undesirable constituents. Thus, mildly (insufficiently) refined LOBs are potentially more hazardous than more severely (sufficiently) refined LOBs. This article discusses the evaluation of LOBs using statistical models based on content of PACs; these models indicate that insufficiently refined LOBs (potentially carcinogenic LOBs) can also produce systemic and developmental effects with repeated dermal exposure. Experimental data were also obtained in ten 13-week dermal studies in rats, eight 4-week dermal studies in rabbits, and seven dermal developmental toxicity studies with sufficiently refined LOBs (noncarcinogenic and commonly marketed) in which no observed adverse effect levels for systemic toxicity and developmental toxicity were 1000 to 2000 mg/kg/d with dermal exposures, typically the highest dose tested. Results in both oral and inhalation developmental toxicity studies were similar. This absence of toxicologically relevant findings was consistent with lower PAC content of sufficiently refined LOBs. Based on data on reproductive organs with repeated dosing and parameters in developmental toxicity studies, sufficiently refined LOBs are likely to have little, if any, effect on reproductive parameters.

Building shared experience to advance practical application of pathway-based toxicology: liver toxicity mode-of-action.

A workshop sponsored by the Human Toxicology Project Consortium (HTPC), "Building Shared Experience to Advance Practical Application of Pathway-Based Toxicology: Liver Toxicity Mode-of-Action" brought together experts from a wide range of perspectives to inform the process of pathway development and to advance two prototype pathways initially developed by the European Commission Joint Research Center (JRC): liver-specific fibrosis and steatosis. The first half of the workshop focused on the theory and practice of pathway development; the second on liver disease and the two prototype pathways. Participants agreed pathway development is extremely useful for organizing information and found that focusing the theoretical discussion on a specific AOP is extremely helpful. In addition, it is important to include several perspectives during pathway development, including information specialists, pathologists, human health and environmental risk assessors, and chemical and product manufacturers, to ensure the biology is well captured and end use is considered.

The toxicological effects of heavy fuel oil category substances.

Heavy fuel oil (HFO) category substances are used to manufacture HFO, a product used in industrial boilers and marine diesel engines. Commercial HFOs and blending stream components are substances of complex and variable composition, composed of C20 to >C50 hydrocarbons, although lower molecular weight material may be added to reduce viscosity and improve flow characteristics. An HFO blending stream (catalytically cracked clarified oil [CCCO]) was tested for target organ and developmental toxicity in rats following repeated dermal administration at doses of 5, 25, or 50 mg/kg/d. In the repeated dose study, there was evidence of increased liver weights, reduced thymus weights, and reductions in hematological parameters with an overall no observed adverse effect level (NOAEL) of 5 mg/kg/d. In the developmental toxicity test, there were significant reductions in fetal survival, significant increases in resorption frequency, and significantly reduced fetal weights with an overall NOAEL of 5 mg/kg/d. These target organ and developmental effects are associated with the types and levels of aromatic constituents in these substances. Among HFO blending streams, CCCOs have the highest levels of aromatics and, because they produce the characteristic toxicological effects at the lowest levels, are considered as "reasonable worst-case examples" for this group of substances. Other HFO category members with lower levels of aromatics produce similar effects but have higher NOAELs. The potential for target organ and developmental effects of other HFO category members can be predicted from information on the types and levels of the aromatic constituents present in these substances.

Characterization of the noncancer hazards of gas oils.

Gas oils, used to manufacture diesel fuel and residential heating oil, are complex hydrocarbon substances with carbon numbers of C9-C30 and boiling ranges of approximately 150 °C to 450 °C. Target organ (liver enlargement, reduced thymus weights, and reductions in hematological parameters) and developmental (reduced fetal viability, increased resorption frequency, and reduced fetal weights) effects are associated with aromatic constituents present in some gas oils. Two types of gas oils were tested for repeated-dose and developmental toxicity following repeated dermal administration. A blend of commercial diesel fuels containing 26% aromatics, primarily single-ring compounds, did not cause either target organ or developmental effects at levels up to 600 mg/kg/d. "Cracked" gas oils containing higher levels of aromatic constituents were also tested. Because of limited sample availability, 2 cracked gas oil samples were tested, one for systemic effects and the other for developmental toxicity. The sample tested in the repeated-dose toxicity study (81% aromatics including approximately 10% 3-ring compounds) produced increased liver weights, reduced thymus weights, and reductions in hematological parameters. The overall no observed adverse effect level (NOAEL) was 100 mg/kg/d. The sample tested for developmental toxicity (65% aromatics including approximately 5% 3-ring compounds) resulted in significant reductions in fetal survival, significant increases in resorption frequency, and significant reductions in fetal weights with an overall NOAEL of 100 mg/kg/d. In summary, gas oils may or may not cause target organ and/or developmental effects depending on the levels and types of aromatic constituents that they contain.

Use and validation of HT/HC assays to support 21st century toxicity evaluations.

Advances in high throughput and high content (HT/HC) methods such as those used in the fields of toxicogenomics, bioinformatics, and computational toxicology have the potential to improve both the efficiency and effectiveness of toxicity evaluations and risk assessments. However, prior to use, scientific confidence in these methods should be formally established. Traditional validation approaches that define relevance, reliability, sensitivity and specificity may not be readily applicable. HT/HC methods are not exact replacements for in vivo testing, and although run individually, these assays are likely to be used as a group or battery for decision making and use robotics, which may be unique in each laboratory setting. Building on the frameworks developed in the 2010 Institute of Medicine Report on Biomarkers and the OECD 2007 Report on (Q)SAR Validation, we present constructs that can be adapted to address the validation challenges of HT/HC methods. These are flexible, transparent, and require explicit specification of context and purpose of use such that scientific confidence (validation) can be defined to meet different regulatory applications. Using these constructs, we discuss how anchoring the assays and their prediction models to Adverse Outcome Pathways (AOPs) could facilitate the interpretation of results and support scientifically defensible fit-for-purpose applications.