Learning from Safe-by-Design for Safe-and-Sustainable-by-Design: Mapping the current landscape of Safe-by-Design reviews, case studies, and frameworks.

With the introduction of the European Commission's "Safe and Sustainable-by-Design" (SSbD) framework, the interest in understanding the implications of safety and sustainability assessments of chemicals, materials, and processes at early-innovation stages has skyrocketed. Our study focuses on the "Safe-by-Design" (SbD) approach from the nanomaterials sector, which predates the SSbD framework. In this assessment, SbD studies have been compiled and categorized into reviews, case studies, and frameworks. Reviews of SbD tools have been further classified as quantitative, qualitative, or toolboxes and repositories. We assessed the SbD case studies and classified them into three categories: safe(r)-by-modeling, safe(r)-by-selection, or safe(r)-by-redesign. This classification enabled us to understand past SbD work and subsequently use it to define future SSbD work so as to avoid confusion and possibilities of "SSbD-washing" (similar to greenwashing). Finally, the preexisting SbD frameworks have been studied and contextualized against the SSbD framework. Several key recommendations for SSbD based on our analysis can be made. Knowledge gained from existing approaches such as SbD, green and sustainable chemistry, and benign-by-design approaches needs to be preserved and effectively transferred to SSbD. Better incorporation of chemical and material functionality into the SSbD framework is required. The concept of lifecycle thinking and the stage-gate innovation model need to be reconciled for SSbD. The development of high-throughput screening models is critical for the operationalization of SSbD. We conclude that the rapid pace of both SbD and SSbD development necessitates a regular mapping of the newly published literature that is relevant to this field.

A roadmap to strengthen standardisation efforts in risk governance of nanotechnology.

A roadmap was developed to strengthen standardisation activities for risk governance of nanotechnology. Its baseline is the available standardised and harmonised methods for nanotechnology developed by the International Organization for Standardization (ISO), the European Committee for Standardization (CEN), and the Organisation for Economic Co-operation and Development (OECD). In order to identify improvements and needs for new themes in standardisation work, an analysis of the state-of-the-art concepts and interpretations of risk governance of nanotechnology was performed. Eleven overall areas of action were identified, each including a subset of specific topics. Themes addressed include physical chemical characterisation, assessment of hazard, exposure, risk and socio-economic factors, as well as education & training and social dialogue. This has been visualised in a standardisation roadmap spanning a timeframe of ten years and including key outcomes and highlights of the analysis. Furthermore, the roadmap indicates potential areas of action for harmonisation and standardisation (H&S) for nanomaterials and nanotechnology. It also includes an evaluation of the current level (limited, moderate, intense) of ongoing H&S activities and indicates the time horizon for the different areas of action. As the identified areas differ in their state of development, the number and type of actions varied widely amongst the different actions towards achieving standardisation. Thus, priority areas were also identified. The overall objective of these actions is to strengthen risk governance towards a safe use of nanomaterials and nano-related products. Though not explicitly addressed, risk-based legislation and policies are supported via the proposed H&S actions.

Life cycle thinking and safe-and-sustainable-by-design approaches for the battery innovation landscape.

Developments in battery technology are essential for the energy transition and need to follow the framework for safe-and-sustainable-by-design (SSbD) materials, chemicals, products, and processes as set by the EU. SSbD is a broad approach that ensures that chemicals/advanced materials/products/services are produced and used in a way to avoid harm to humans and the environment. Technical and policy-related literature was surveyed for battery technologies and recommendations were provided for a broad SSbD approach that remains firmly grounded in Life Cycle Thinking principles. The approach integrates functional performance and sustainability (safety, social, environmental, and economic) aspects throughout the life cycle of materials, products, and processes, and evaluates how their interactions reflect on SSbD parameters. 22 different types of batteries were analyzed in a life cycle thinking approach for criticality, toxicity/safety, environmental and social impact, circularity, functionality, and cost to ensure battery innovation has a green and sustainable purpose to avoid unintended consequences.

The State of the Art and Challenges of In Vitro Methods for Human Hazard Assessment of Nanomaterials in the Context of Safe-by-Design.

The Safe-by-Design (SbD) concept aims to facilitate the development of safer materials/products, safer production, and safer use and end-of-life by performing timely SbD interventions to reduce hazard, exposure, or both. Early hazard screening is a crucial first step in this process. In this review, for the first time, commonly used in vitro assays are evaluated for their suitability for SbD hazard testing of nanomaterials (NMs). The goal of SbD hazard testing is identifying hazard warnings in the early stages of innovation. For this purpose, assays should be simple, cost-effective, predictive, robust, and compatible. For several toxicological endpoints, there are indications that commonly used in vitro assays are able to predict hazard warnings. In addition to the evaluation of assays, this review provides insights into the effects of the choice of cell type, exposure and dispersion protocol, and the (in)accurate determination of dose delivered to cells on predictivity. Furthermore, compatibility of assays with challenging advanced materials and NMs released from nano-enabled products (NEPs) during the lifecycle is assessed, as these aspects are crucial for SbD hazard testing. To conclude, hazard screening of NMs is complex and joint efforts between innovators, scientists, and regulators are needed to further improve SbD hazard testing.

Challenges in Predicting the Change in the Cumulative Exposure of New Tobacco and Related Products Based on Emissions and Toxicity Dose-Response Data.

Many novel tobacco products have been developed in recent years. Although many may emit lower levels of several toxicants, their risk in the long term remains unclear. We previously published a method for the exposure assessment of mixtures that can be used to compare the changes in cumulative exposure to carcinogens among tobacco products. While further developing this method by including more carcinogens or to explore its application to non-cancer endpoints, we encountered a lack of data that are required for better-substantiated conclusions regarding differences in exposure between products. In this special communication, we argue the case for more data on adverse health effects, as well as more data on the composition of the emissions from tobacco products. Such information can be used to identify significant changes in relevance to health using the cumulative exposure method with different products and to substantiate regulatory decisions.

Safe-and-Sustainable-by-Design Framework Based on a Prospective Life Cycle Assessment: Lessons Learned from a Nano-Titanium Dioxide Case Study.

Safe-and-sustainable-by-design (SSbD) is a concept that takes a systems approach by integrating safety, sustainability, and functionality throughout a product's the life cycle. This paper proposes a framework based on a prospective life cycle assessment for early safety and sustainability assessment. The framework's purpose is to identify environmental sustainability and toxicity hotspots early in the innovation process for future SSbD applicability. If this is impossible, key performance indicators are assessed. Environmental sustainability aspects, such as global warming potential (GWP) and cumulative energy demand (CED), and toxicity aspects, such as human toxicity potential and freshwater ecotoxicity potential, were assessed upon applying the framework on a case study. The case study regarded using nano-titanium dioxide (P25-TiO2) or a modified nano-coated version (Cu2O-coated/P25-TiO2) as photocatalysts to produce hydrogen from water using sunlight. Although there was a decrease in environmental impact (GWP and CED), the modified nano-coated version had a relatively higher level of human toxicity and freshwater eco-toxicity. For the presented case study, SSbD alternatives need to be considered that improve the photocatalytic activity but are not toxic to the environment. This case study illustrates the importance of performing an early safety and environmental sustainability assessment to avoid the development of toxic alternatives.

Risk assessment of components in tobacco smoke and e-cigarette aerosols: a pragmatic choice of dose metrics.

BACKGROUND: Risk assessment of individual tobacco smoke components is important for the purpose of prioritization or selecting chemicals for monitoring products. Smoking is characterized by a highly varying, intermittent exposure and the challenge is to choose the most appropriate dose metric. METHODS: Generally, average daily exposure estimates are used as dose metric, without considering temporal determinants. The applicability hereof is discussed in the context of choosing dose metrics for local respiratory tract effects and for systemic effects in a smoking scenario or for the use of e-cigarettes. RESULTS: Using average daily exposure estimates for the smoking scenario can lead to erroneous risk evaluations for several reasons. Inhaled peak air concentrations during a puff can be two to three orders of magnitude higher than the calculated average daily inhaled concentration, which may impact the assessment of both systemic and local health effects. A pragmatic risk assessment is proposed, based on the Margin of Exposure (MoE) approach. The choice of an appropriate dose metric, such as inhaled concentration, inhaled dose or absorbed dose, depends on the type of effect. Temporal characteristics should be considered in the final step of the MoE approach, as is illustrated by two cases, glycerol and benzene. CONCLUSION: The choice of an appropriate dose metric and inclusion of temporal determinants are important aspects in the risk assessment of individual smoke components. The proposed MoE approach provides the opportunity to weigh smoking-related exposure characteristics during the final step of the risk evaluation.

Modernizing innovation governance to meet policy ambitions through trusted environments.

A vision for modernization of nanotechnology innovation governance is a Safe Innovation Approach (SIA). SIA combines two concepts: Safe-by-Design (SbD) and Regulatory Preparedness (RP). SbD aims to motivate industry to integrate safety considerations early in the innovation process and onwards. RP aspires to improve the anticipation capabilities of regulators and develop legislation that can keep pace with innovations. The pace, scope and complexity of nanotechnology present novel challenges for governance, especially law and regulation. A possible option forward for nanotechnology is to move towards a more goal-based governance system including anticipatory regulation. Anticipatory regulation and experimentation can be considered as an agile approach with emphasis on flexibility, collaboration and innovation. SIA can be seen as part of experimentation in support of agile regulatory practices. A trusted environment is needed in which innovators, regulators and other stakeholders are motivated to understand each other's concerns and together develop solutions to anticipate and address safety whilst also facilitating the development of safe, sustainable and socially beneficial innovations. Trust drivers to facilitate trusted environments include focusing on the public interest, competence, respect, integrity, inclusion, fairness and openness. Here, we explore the concept of building trusted environments in the context of the SIA for nanotechnologies.

Safe-by-Design part II: A strategy for balancing safety and functionality in the different stages of the innovation process.

Manufactured nanomaterials have the potential to impact an exceedingly wide number of industries and markets ranging from energy storage, electronic and optical devices, light-weight construction to innovative medical approaches for diagnostics and therapy. In order to foster the development of safer nanomaterial-containing products, two main aspects are of major interest: their functional performance as well as their safety towards human health and the environment. In this paper a first proposal for a strategy is presented to link the functionality of nanomaterials with safety aspects. This strategy first combines information on the functionality and safety early during the innovation process and onwards, and then identifies Safe-by-Design (SbD) actions that allow for optimisation of both aspects throughout the innovation process. The strategy encompasses suggestions for the type of information needed to balance functionality and safety to support decision making in the innovation process. The applicability of the strategy is illustrated using a literature-based case study on carbon nanotube-based transparent conductive films. This is a first attempt to identify information that can be used for balancing functionality and safety in a structured way during innovation processes.

A Method for Comparing the Impact on Carcinogenicity of Tobacco Products: A Case Study on Heated Tobacco Versus Cigarettes.

Comparing the harmful health effects related to two different tobacco products by applying common risk assessment methods to each individual compound is problematic. We developed a method that circumvents some of these problems by focusing on the change in cumulative exposure (CCE) of the compounds emitted by the two products considered. The method consists of six steps. The first three steps encompass dose-response analysis of cancer data, resulting in relative potency factors with confidence intervals. The fourth step evaluates emission data, resulting in confidence intervals for the expected emission of each compound. The fifth step calculates the change in CCE, probabilistically, resulting in an uncertainty range for the CCE. The sixth step estimates the associated health impact by combining the CCE with relevant dose-response information. As an illustrative case study, we applied the method to eight carcinogens occurring both in the emissions of heated tobacco products (HTPs), a novel class of tobacco products, and tobacco smoke. The CCE was estimated to be 10- to 25-fold lower when using HTPs instead of cigarettes. Such a change indicates a substantially smaller reduction in expected life span, based on available dose-response information in smokers. However, this is a preliminary conclusion, as only eight carcinogens were considered so far. Furthermore, an unfavorable health impact related to HTPs remains as compared to complete abstinence. Our method results in useful information that may help policy makers in better understanding the potential health impact of new tobacco and related products. A similar approach can be used to compare the carcinogenicity of other mixtures.

A Methodological Safe-by-Design Approach for the Development of Nanomedicines.

Safe-by-Design (SbD) concepts foresee the risk identification and reduction as well as uncertainties regarding human health and environmental safety in early stages of product development. The EU's NANoREG project and further on the H2020 ProSafe initiative, NanoReg2, and CALIBRATE projects have developed a general SbD approach for nanotechnologies (e.g., paints, textiles, etc.). Based on it, the GoNanoBioMat project elaborated a methodological SbD approach (GoNanoBioMat SbD approach) for nanomedicines with a focus on polymeric nanobiomaterials (NBMs) used for drug delivery. NBMs have various advantages such as the potential to increase drug efficacy and bioavailability. However, the nanoscale brings new challenges to product design, manufacturing, and handling. Nanomedicines are costly and require the combination of knowledge from several fields. In this paper, we present the GoNanoBioMat SbD approach, which allows identifying and addressing the relevant safety aspects to address when developing polymeric NBMs during design, characterization, assessment of human health and environmental risk, manufacturing and handling, and combines the nanoscale and medicine field under one approach. Furthermore, regulatory requirements are integrated into the innovation process.

A test strategy for the assessment of additive attributed toxicity of tobacco products.

The new EU Tobacco Product Directive (TPD) prohibits tobacco products containing additives that are toxic in unburnt form or that increase overall toxicity of the product. This paper proposes a strategy to assess additive attributed toxicity in the context of the TPD. Literature was searched on toxicity testing strategies for regulatory purposes from tobacco industry and governmental institutes. Although mainly traditional in vivo testing strategies have been applied to assess toxicity of unburnt additives and increases in overall toxicity of tobacco products due to additives, in vitro tests combined with toxicogenomics and validated using biomarkers of exposure and disease are most promising in this respect. As such, tests are needed that are sensitive enough to assess additive attributed toxicity above the overall toxicity of tobacco products, which can associate assay outcomes to human risk and exposure. In conclusion, new, sensitive in vitro assays are needed to conclude whether comparable testing allows for assessment of small changes in overall toxicity attributed to additives. A more pragmatic approach for implementation on a short-term is mandated lowering of toxic emission components. Combined with risk assessment, this approach allows assessment of effectiveness of harm reduction strategies, including banning or reducing of additives.

Empirical analysis of BMD metrics in genetic toxicology part I: in vitro analyses to provide robust potency rankings and support MOA determinations.

Genetic toxicity testing has traditionally been used for hazard identification, with dichotomous classification of test results serving to identify genotoxic agents. However, the utility of genotoxicity data can be augmented by employing dose-response analysis and point of departure determination. Via interpolation from a fitted dose-response model, the benchmark dose (BMD) approach estimates the dose that elicits a specified (small) effect size. BMD metrics and their confidence intervals can be used for compound potency ranking within an endpoint, as well as potency comparisons across other factors such as cell line or exposure duration. A recently developed computational method, the BMD covariate approach, permits combined analysis of multiple dose-response data sets that are differentiated by covariates such as compound, cell type or exposure regime. The approach provides increased BMD precision for effective potency rankings across compounds and other covariates that pertain to a hypothesised mode of action (MOA). To illustrate these applications, the covariate approach was applied to the analysis of published in vitro micronucleus frequency dose-response data for ionising radiations, a set of aneugens, two mutagenic azo compounds and a topoisomerase II inhibitor. The ionising radiation results show that the precision of BMD estimates can be improved by employing the covariate method. The aneugen analysis provided potency groupings based on the BMD confidence intervals, and analyses of azo compound data from cells lines with differing metabolic capacity confirmed the influence of endogenous metabolism on genotoxic potency. This work, which is the first of a two-part series, shows that BMD-derived potency rankings can be employed to support MOA evaluations as well as facilitate read across to expedite chemical evaluations and regulatory decision-making. The follow-up (Part II) employs the combined covariate approach to analyse in vivo genetic toxicity dose-response data focussing on how improvements in BMD precision can impact the reduction and refinement of animal use in toxicological research.

Estimating the carcinogenic potency of chemicals from the in vivo micronucleus test.

In this study, we investigated the applicability of using in vivo mouse micronucleus (MN) data to derive cancer potency information. We also present a new statistical methodology for correlating estimated potencies between in vivo MN tests and cancer studies, which could similarly be used for other systems (e.g. in vitro vs. in vivo genotoxicity tests). The dose-response modelling program PROAST was used to calculate benchmark doses (BMDs) for estimating the genotoxic and carcinogenic potency for 48 compounds in mice; most of the data were retrieved from the National Toxicology Program (NTP) database, while some additional data were retrieved from the Carcinogenic Potency Database and published studies. BMD05s (doses with 5% increase in MN frequency) were derived from MN data, and BMD10s (doses with 10% extra cancer risk) were derived from carcinogenicity data, along with their respective lower (BMDL) and upper (BMDU) confidence bounds. A clear correlation between the in vivo MN BMD05s and the cancer BMD10s was observed when the lowest BMD05 from the in vivo MN was plotted against the lowest BMD10 from the carcinogenicity data for each individual compound. By making a further selection of BMDs related to more or less equally severe cancer lesions, the correlation was considerably improved. Getting a general scientific consensus on how we can quantitatively compare different tumour lesion types and investigating the impact of MN study duration are needed to refine this correlation analysis. Nevertheless, our results suggest that a BMD derived from genotoxicity data might provide a prediction of the tumour potency (BMD10) with an uncertainty range spanning roughly a factor of 100.

Correlation of In Vivo Versus In Vitro Benchmark Doses (BMDs) Derived From Micronucleus Test Data: A Proof of Concept Study.

In this study, we explored the applicability of using in vitro micronucleus (MN) data from human lymphoblastoid TK6 cells to derive in vivo genotoxicity potency information. Nineteen chemicals covering a broad spectrum of genotoxic modes of action were tested in an in vitro MN test using TK6 cells using the same study protocol. Several of these chemicals were considered to need metabolic activation, and these were administered in the presence of S9. The Benchmark dose (BMD) approach was applied using the dose-response modeling program PROAST to estimate the genotoxic potency from the in vitro data. The resulting in vitro BMDs were compared with previously derived BMDs from in vivo MN and carcinogenicity studies. A proportional correlation was observed between the BMDs from the in vitro MN and the BMDs from the in vivo MN assays. Further, a clear correlation was found between the BMDs from in vitro MN and the associated BMDs for malignant tumors. Although these results are based on only 19 compounds, they show that genotoxicity potencies estimated from in vitro tests may result in useful information regarding in vivo genotoxic potency, as well as expected cancer potency. Extension of the number of compounds and further investigation of metabolic activation (S9) and of other toxicokinetic factors would be needed to validate our initial conclusions. However, this initial work suggests that this approach could be used for in vitro to in vivo extrapolations which would support the reduction of animals used in research (3Rs: replacement, reduction, and refinement).

Potential harmful health effects of inhaling nicotine-free shisha-pen vapor: a chemical risk assessment of the main components propylene glycol and glycerol.

BACKGROUND: A shisha-pen is an electronic cigarette variant that is advertised to mimic the taste of a water pipe, or shisha. The aim of this study was to assess the potential harmful health effects caused by inhaling the vapor of a nicotine-free shisha-pen. METHODS: Gas chromatography analysis was performed to determine the major components in shisha-pen vapor. Risk assessment was performed using puff volumes of e-cigarettes and "normal" cigarettes and a 1-puff scenario (one-time exposure). The concentrations that reached the airways and lungs after using a shisha-pen were calculated and compared to data from published toxicity studies. RESULTS: The main components in shisha-pen vapor are propylene glycol and glycerol (54%/46%). One puff (50 to 70 mL) results in exposure of propylene glycol and glycerol of 430 to 603 mg/m(3) and 348 to 495 mg/m(3), respectively. These exposure concentrations were higher than the points of departure for airway irritation based on a human study (propylene glycol, mean concentration of 309 mg/m(3)) and a rat study (glycerol, no-observed adverse effect level of 165 mg/m(3)). CONCLUSIONS: Already after one puff of the shisha-pen, the concentrations of propylene glycol and glycerol are sufficiently high to potentially cause irritation of the airways. New products such as the shisha-pen should be detected and risks should be assessed to inform regulatory actions aimed at limiting potential harm that may be caused to consumers and protecting young people to take up smoking.

IWGT report on quantitative approaches to genotoxicity risk assessment I. Methods and metrics for defining exposure-response relationships and points of departure (PoDs).

This report summarizes the discussion, conclusions, and points of consensus of the IWGT Working Group on Quantitative Approaches to Genetic Toxicology Risk Assessment (QWG) based on a meeting in Foz do Iguaçu, Brazil October 31-November 2, 2013. Topics addressed included (1) the need for quantitative dose-response analysis, (2) methods to analyze exposure-response relationships & derive point of departure (PoD) metrics, (3) points of departure (PoD) and mechanistic threshold considerations, (4) approaches to define exposure-related risks, (5) empirical relationships between genetic damage (mutation) and cancer, and (6) extrapolations across test systems and species. This report discusses the first three of these topics and a companion report discusses the latter three. The working group critically examined methods for determining point of departure metrics (PoDs) that could be used to estimate low-dose risk of genetic damage and from which extrapolation to acceptable exposure levels could be made using appropriate mode of action information and uncertainty factors. These included benchmark doses (BMDs) derived from fitting families of exponential models, the No Observed Genotoxic Effect Level (NOGEL), and "threshold" or breakpoint dose (BPD) levels derived from bilinear models when mechanistic data supported this approach. The QWG recognizes that scientific evidence suggests that thresholds below which genotoxic effects do not occur likely exist for both DNA-reactive and DNA-nonreactive substances, but notes that small increments of the spontaneous level cannot be unequivocally excluded either by experimental measurement or by mathematical modeling. Therefore, rather than debating the theoretical possibility of such low-dose effects, emphasis should be placed on determination of PoDs from which acceptable exposure levels can be determined by extrapolation using available mechanistic information and appropriate uncertainty factors. This approach places the focus on minimization of the genotoxic risk, which protects against the risk of the development of diseases resulting from the genetic damage. Based on analysis of the strengths and weaknesses of each method, the QWG concluded that the order of preference of PoD metrics is the statistical lower bound on the BMD > the NOGEL > a statistical lower bound on the BPD. A companion report discusses the use of these metrics in genotoxicity risk assessment, including scaling and uncertainty factors to be considered when extrapolating below the PoD and/or across test systems and to the human.

IWGT report on quantitative approaches to genotoxicity risk assessment II. Use of point-of-departure (PoD) metrics in defining acceptable exposure limits and assessing human risk.

This is the second of two reports from the International Workshops on Genotoxicity Testing (IWGT) Working Group on Quantitative Approaches to Genetic Toxicology Risk Assessment (the QWG). The first report summarized the discussions and recommendations of the QWG related to the need for quantitative dose-response analysis of genetic toxicology data, the existence and appropriate evaluation of threshold responses, and methods to analyze exposure-response relationships and derive points of departure (PoDs) from which acceptable exposure levels could be determined. This report summarizes the QWG discussions and recommendations regarding appropriate approaches to evaluate exposure-related risks of genotoxic damage, including extrapolation below identified PoDs and across test systems and species. Recommendations include the selection of appropriate genetic endpoints and target tissues, uncertainty factors and extrapolation methods to be considered, the importance and use of information on mode of action, toxicokinetics, metabolism, and exposure biomarkers when using quantitative exposure-response data to determine acceptable exposure levels in human populations or to assess the risk associated with known or anticipated exposures. The empirical relationship between genetic damage (mutation and chromosomal aberration) and cancer in animal models was also examined. It was concluded that there is a general correlation between cancer induction and mutagenic and/or clastogenic damage for agents thought to act via a genotoxic mechanism, but that the correlation is limited due to an inadequate number of cases in which mutation and cancer can be compared at a sufficient number of doses in the same target tissues of the same species and strain exposed under directly comparable routes and experimental protocols.

Anchoring molecular mechanisms to the adverse outcome pathway for skin sensitization: Analysis of existing data.

Allergic contact dermatitis (ACD) is a hypersensitivity immune response induced by small protein-reactive chemicals. Currently, the murine local lymph node assay (LLNA) provides hazard identification and quantitative estimation of sensitizing potency. Given the complexity of ACD, a single alternative method cannot replace the LLNA, but it is necessary to combine methods through an integrated testing strategy (ITS). In the development of an ITS, information regarding mechanisms and molecular processes involved in skin sensitization is crucial. The recently published adverse outcome pathway (AOP) for skin sensitization captures mechanistic knowledge into key events that lead to ACD. To understand the molecular processes in ACD, a systematic review of murine in vivo studies was performed and an ACD molecular map was constructed. In addition, comparing the molecular map to the limited human in vivo toxicogenomic data available suggests that certain processes are similarly triggered in mice and humans, but additional human data will be needed to confirm these findings and identify differences. To gain insight in the molecular mechanisms represented by various human in vitro systems, the map was compared to in vitro toxicogenomic data. This analysis allows for comparison of emerging in vitro methods on a molecular basis, in addition to mathematical predictive value. Finally, a survey of the current in silico, in chemico, and in vitro methods was used to indicate which AOP key event is modeled by each method. By anchoring emerging classification methods to the AOP and the ACD molecular map, complementing methods can be identified, which provides a cornerstone for the development of a testing strategy that accurately reflects the key events in skin sensitization.

Quantitative dose-response analysis of ethyl methanesulfonate genotoxicity in adult gpt-delta transgenic mice.

The assumption that mutagens have linear dose-responses recently has been challenged. In particular, ethyl methanesulfonate (EMS), a DNA-reactive mutagen and carcinogen, exhibited sublinear or thresholded dose-responses for LacZ mutation in transgenic Muta™Mouse and for micronucleus (MN) frequency in CD1 mice (Gocke E and Müller L [2009]: Mutat Res 678:101-107). In order to explore variables in establishing genotoxicity dose-responses, we characterized the genotoxicity of EMS using gene mutation assays anticipated to have lower spontaneous mutant frequencies (MFs) than Muta™Mouse. Male gpt-delta transgenic mice were treated daily for 28 days with 5 to 100 mg/kg EMS, and measurements were made on: (i) gpt MFs in liver, lung, bone marrow, kidney, small intestine, and spleen; and (ii) Pig-a MFs in peripheral blood reticulocytes (RETs) and total red blood cells. MN induction also was measured in peripheral blood RETs. These data were used to calculate Points of Departure (PoDs) for the dose responses, i.e., no-observed-genotoxic-effect-levels (NOGELs), lower confidence limits of threshold effect levels (Td-LCIs), and lower confidence limits of 10% benchmark response rates (BMDL10 s). Similar PoDs were calculated from the published EMS dose-responses for LacZ mutation and CD1 MN induction. Vehicle control gpt and Pig-a MFs were 13-40-fold lower than published vehicle control LacZ MFs. In general, the EMS genotoxicity dose-responses in gpt-delta mice had lower PoDs than those calculated from the Muta™Mouse and CD1 mouse data. Our results indicate that the magnitude and possibly the shape of mutagenicity dose responses differ between in vivo models, with lower PoDs generally detected by gene mutation assays with lower backgrounds.