Predicting the future: opportunities and challenges for the chemical industry to apply 21st-century toxicity testing.

Interest in applying 21st-century toxicity testing tools for safety assessment of industrial chemicals is growing. Whereas conventional toxicology uses mainly animal-based, descriptive methods, a paradigm shift is emerging in which computational approaches, systems biology, high-throughput in vitro toxicity assays, and high-throughput exposure assessments are beginning to be applied to mechanism-based risk assessments in a time- and resource-efficient fashion. Here we describe recent advances in predictive safety assessment, with a focus on their strategic application to meet the changing demands of the chemical industry and its stakeholders. The opportunities to apply these new approaches is extensive and include screening of new chemicals, informing the design of safer and more sustainable chemical alternatives, filling information gaps on data-poor chemicals already in commerce, strengthening read-across methodology for categories of chemicals sharing similar modes of action, and optimizing the design of reduced-risk product formulations. Finally, we discuss how these predictive approaches dovetail with in vivo integrated testing strategies within repeated-dose regulatory toxicity studies, which are in line with 3Rs principles to refine, reduce, and replace animal testing. Strategic application of these tools is the foundation for informed and efficient safety assessment testing strategies that can be applied at all stages of the product-development process.

A critical assessment of the methodologies to investigate the role of inhibition of apoptosis in rodent hepatocarcinogenesis.

Non-genotoxic carcinogens act by promoting the clonal expansion of preneoplastic cells by directly or indirectly stimulating cell division or inhibiting cell loss in the target organ. The specific mode-of-action (MoA) by which some non-genotoxic carcinogens ultimately cause cancer is not completely understood. To date, there are several proposed MoAs for non-genotoxic carcinogens, and some of these propose inhibition of apoptosis as one of the key events. In general, inhibition of apoptosis is considered a necessary step for cell survival and in theory can occur in combination or in association with other key promotional events, such as cell proliferation, oxidative stress and inhibition of intercellular communication to promote carcinogenesis. However, the evidence supporting the role of inhibition of apoptosis as a necessary step in promoting specific chemically induced tumors is often debated. To address this evidence, we reviewed studies that utilized prototypical nuclear receptor-mediated hepatocarcinogens. Based on this review, it is proposed that the ability to determine the importance of inhibition of apoptosis as a key event in the MoA for tumor promotion is hampered by the limitations of the methods utilized for its detection. This review provides an assessment of the strengths and limitations of the current methodology used for detection of apoptosis and provides suggestions for improving its detection, thereby strengthening the weight of evidence supporting inhibition of apoptosis as a key event in a MoA for tumor promotion.

Incorporating new technologies into toxicity testing and risk assessment: moving from 21st century vision to a data-driven framework.

Based on existing data and previous work, a series of studies is proposed as a basis toward a pragmatic early step in transforming toxicity testing. These studies were assembled into a data-driven framework that invokes successive tiers of testing with margin of exposure (MOE) as the primary metric. The first tier of the framework integrates data from high-throughput in vitro assays, in vitro-to-in vivo extrapolation (IVIVE) pharmacokinetic modeling, and exposure modeling. The in vitro assays are used to separate chemicals based on their relative selectivity in interacting with biological targets and identify the concentration at which these interactions occur. The IVIVE modeling converts in vitro concentrations into external dose for calculation of the point of departure (POD) and comparisons to human exposure estimates to yield a MOE. The second tier involves short-term in vivo studies, expanded pharmacokinetic evaluations, and refined human exposure estimates. The results from the second tier studies provide more accurate estimates of the POD and the MOE. The third tier contains the traditional animal studies currently used to assess chemical safety. In each tier, the POD for selective chemicals is based primarily on endpoints associated with a proposed mode of action, whereas the POD for nonselective chemicals is based on potential biological perturbation. Based on the MOE, a significant percentage of chemicals evaluated in the first 2 tiers could be eliminated from further testing. The framework provides a risk-based and animal-sparing approach to evaluate chemical safety, drawing broadly from previous experience but incorporating technological advances to increase efficiency.

Assessment of the developmental and reproductive toxicity of diiodomethyl-p-tolylsulfone in rats and rabbits.

Diiodomethyl-p-tolylsulfone (DIMPTS) was tested in developmental toxicity (DT) and reproductive toxicity studies. In the rat DT study, DIMPTS was administered at 0, 100, 300 or 1000 mg/kg/day. Maternal toxicity as evidenced by reductions in body weight gain or feed consumption at 1000 and, to a lesser extent, 300 mg/kg/day. The only developmental effect was umbilical hernia at 1000 mg/kg/day; therefore, NOELs for maternal and developmental toxicity were 100 and 300 mg/kg/day, respectively. In the rabbit DT study, NZW rabbits were gavaged with 0, 0.05, 0.5 and 2mg/kg/day DIMPTS. The NOEL for maternal toxicity was 0.5mg/kg/day, based on thyroid weight increase with histopathology. There were no observed developmental effects. In the two-generation study, CD rats were fed 0, 2.5, 10 or 40 mg/kg/day DIMPTS. Increased thyroid weight and histopathology were observed at all doses with associated pituitary findings in males. Reproductive toxicity at 40 mg/kg/day consisted of increased postimplantation loss, decreased gestation survival and two cases of dystocia, while litter size, pup survival/weight were affected at 10 and 40 mg/kg/day. The NOEL for parental toxicity could not be determined, while the NOEL for reproductive toxicity was 2.5mg/kg/day. The maternal thyroid and reproductive effects in this study were consistent with iodine toxicity.

Interpretation of skeletal variations for human risk assessment: delayed ossification and wavy ribs.

Delayed (or incomplete) ossification of developing fetal bones and wavy ribs are some of the most common skeletal variations encountered in regulatory guideline developmental toxicity studies. Although they tend to be regarded as minor effects, they can be quite sensitive and consequently may influence the study lowest-observed-adverse-effect levels (LOAELs), and thus, impact classification, labeling, and risk assessment. In this study, we review the underlying mechanisms of these skeletal variations, evaluate different scenarios in which they have been observed, offer guidance for their interpretation, and comment on their use for risk assessment. Both minor delays in ossification and wavy ribs seem to be readily repairable via postnatal skeletal remodeling, are not mechanistically linked to malformation, and often are seen in the presence of maternal or fetal toxicity. As such, these minor variations would not generally be considered adverse in and of themselves but should be interpreted in the context of other maternal and fetal findings, information available on normal skeletogenesis patterns, mode of action of the test agent, and historical control incidence using a weight of evidence approach.

Assessment of interactions of diverse ternary mixtures in an estrogen receptor-alpha reporter assay.

This study used an MCF-7 cell based ER-alpha reporter gene assay to assess chemical interactions within the following ternary mixtures: (1) three synthetic pesticides, methoxychlor (MXC), o,p-DDT, and dieldrin; (2) three polyaromatic hydrocarbons, benzo[a]pyrene (BAP), 1,2-benzanthracene (BENZ), and chrysene (CHRY); and (3) an endogenous estrogen, [17beta-estradiol, (E(2))]; a phytoestrogen, genistein (GEN); and a synthetic estrogen, o,p-DDT. A full factorial design in which four concentrations of each chemical were assessed in all possible combinations (64 treatment groups) was utilized. In addition, mixtures were tested in both a low range (concentrations near the individual chemical response thresholds) and a high range ( approximately 2-10x higher) experiment. A response surface was estimated using a nonlinear mixed model, and the cumulative response in each mixture was evaluated for departure from additivity. The mixture of E(2), GEN, and DDT exhibited antagonistic interactions (p < 0.001) in both concentration ranges. However, specific interactions between E(2)/GEN and E(2)/DDT differed between the low and high range concentrations. The BAP/BENZ/CHRY mixture did not depart significantly from additivity (p = 0.66) in either concentration range, although response levels were generally low. The MXC/DDT/dieldrin mixture did not depart significantly from additivity in either the high (p = 0.065), or low dose range (p = 0.506), with generally minimal responses dominated by MXC and DDT. This methodology has allowed for a rigorous statistical evaluation of potential departures from additive interactions in endocrine active mixtures. In no case was a significantly greater-than-additive (synergistic) interaction observed.