Current ecotoxicity testing needs among selected U.S. federal agencies

U.S. regulatory and research agencies use ecotoxicity test data to assess the hazards associated with substances that may be released into the environment, including but not limited to industrial chemicals, pharmaceuticals, pesticides, food additives, and color additives. These data are used to conduct hazard assessments and evaluate potential risks to aquatic life (e.g., invertebrates, fish), birds, wildlife species, or the environment. To identify opportunities for regulatory uses of non-animal replacements for ecotoxicity tests, the needs and uses for data from tests utilizing animals must first be clarified. Accordingly, the objective of this review was to identify the ecotoxicity test data relied upon by U.S. federal agencies. The standards, test guidelines, guidance documents, and/or endpoints that are used to address each of the agencies’ regulatory and research needs regarding ecotoxicity testing are described in the context of their application to decision-making. Testing and information use, needs, and/or requirements relevant to the regulatory or programmatic mandates of the agencies taking part in the Interagency Coordinating Committee on the Validation of Alternative Methods Ecotoxicology Workgroup are captured. This information will be useful for coordinating efforts to develop and implement alternative test methods to reduce, refine, or replace animal use in chemical safety evaluations

Mismatch repair deficiency predicts response of solid tumors to PD-1 blockade.

The genomes of cancers deficient in mismatch repair contain exceptionally high numbers of somatic mutations. In a proof-of-concept study, we previously showed that colorectal cancers with mismatch repair deficiency were sensitive to immune checkpoint blockade with antibodies to programmed death receptor-1 (PD-1). We have now expanded this study to evaluate the efficacy of PD-1 blockade in patients with advanced mismatch repair-deficient cancers across 12 different tumor types. Objective radiographic responses were observed in 53% of patients, and complete responses were achieved in 21% of patients. Responses were durable, with median progression-free survival and overall survival still not reached. Functional analysis in a responding patient demonstrated rapid in vivo expansion of neoantigen-specific T cell clones that were reactive to mutant neopeptides found in the tumor. These data support the hypothesis that the large proportion of mutant neoantigens in mismatch repair-deficient cancers make them sensitive to immune checkpoint blockade, regardless of the cancers\u27 tissue of origin

Data from: Differential sensitivity to in vitro inhibition of cytochrome P450 aromatase (CYP19) activity among 18 freshwater fishes

There is significant concern regarding potential impairment of fish reproduction associated with endocrine disrupting chemicals. Aromatase (CYP19) is a steroidogenic enzyme involved in the conversion of androgens to estrogens. Inhibition of aromatase by chemicals can result in reduced concentrations of estrogens leading to adverse reproductive effects. These effects have been extensively investigated in a small number of laboratory model fishes, such as fathead minnow (Pimephales promelas), Japanese medaka (Oryzias latipes), and zebrafish (Danio rerio). But, differences in sensitivity among species is largely unknown. Therefore, this study took a first step towards understanding potential differences in sensitivity to aromatase inhibitors among fishes. Specifically, a standard in vitro aromatase inhibition assay using subcellular fractions of whole tissue homogenates was used to evaluate the potential sensitivity of eighteen phylogenetically diverse species of freshwater fish to the nonsteroidal aromatase inhibitor fadrozole. Sensitivity to fadrozole ranged by more than 52-fold among these species. Five species were further investigated for sensitivity to up to four additional nonsteroidal aromatase inhibitors, letrozole, imazalil, prochloraz, and propiconazole. Potencies of each of these chemicals relative to fadrozole ranged by up to two orders of magnitude among the five species. Fathead minnow, Japanese medaka, and zebrafish were among the least sensitive to all the investigated chemicals; therefore, ecological risks of aromatase inhibitors derived from these species might not be adequately protective of more sensitive native fishes. This information could guide more objective ecological risk assessments of native fishes to chemicals that inhibit aromatase

Doering et al Supplementary Data

The data file includes supplementary data, including detailed information on the species used, basal aromatase activity values, maximal percent inhibition, assay replication, Calculated IC50 values, correlations, and comparison to previously published IC50s

Determination of Metabolic Stability Using Cryopreserved Hepatocytes from Rainbow Trout (Oncorhynchus mykiss).

Trout provide a relatively easy source of hepatocytes that can be cryopreserved and used for a range of applications including toxicity testing and determination of intrinsic clearance. Standard protocols for isolating, cryopreserving, and thawing rainbow trout hepatocytes are described, along with procedures for using fresh or cryopreserved hepatocytes to assess metabolic stability of xenobiotics in fish by means of a substrate depletion approach. Variations on these methods, troubleshooting tips, and directions for use of extrapolation factors to express results in terms of in vivo intrinsic clearance are included. These protocols have been developed for rainbow trout, but can be adapted to other fish species with appropriate considerations

Which Molecular Features Affect the Intrinsic Hepatic Clearance Rate of Ionizable Organic Chemicals in Fish?

Greater knowledge of biotransformation rates for ionizable organic compounds (IOCs) in fish is required to properly assess the bioaccumulation potential of many environmentally relevant contaminants. In this study, we measured in vitro hepatic clearance rates for 50 IOCs using a pooled batch of liver S9 fractions isolated from rainbow trout (Oncorhynchus mykiss). The IOCs included four types of strongly ionized acids (carboxylates, phenolates, sulfonates, and sulfates), three types of strongly ionized bases (primary, secondary, tertiary amines), and a pair of quaternary ammonium compounds (QACs). Included in this test set were several surfactants and a series of beta-blockers. For linear alkyl chain IOC analogues, biotransformation enzymes appeared to act directly on the charged terminal group, with the highest clearance rates for tertiary amines and sulfates and no clearance of QACs. Clearance rates for C12-IOCs were higher than those for C8-IOC analogues. Several analogue series with multiple alkyl chains, branched alkyl chains, aromatic rings, and nonaromatic rings were evaluated. The likelihood of multiple reaction pathways made it difficult to relate all differences in clearance to specific molecular features the tested IOCs. Future analysis of primary metabolites in the S9 assay is recommended to further elucidate biotransformation pathways for IOCs in fish

Reliability of In Vitro Methods Used to Measure Intrinsic Clearance of Hydrophobic Organic Chemicals by Rainbow Trout: Results of an International Ring Trial.

In vitro assays are widely employed to obtain intrinsic clearance estimates used in toxicokinetic modeling efforts. However, the reliability of these methods is seldom reported. Here we describe the results of an international ring trial designed to evaluate two in vitro assays used to measure intrinsic clearance in rainbow trout. An important application of these assays is to predict the effect of biotransformation on chemical bioaccumulation. Six laboratories performed substrate depletion experiments with cyclohexyl salicylate, fenthion, 4-n-nonylphenol, deltamethrin, methoxychlor, and pyrene using cryopreserved hepatocytes and liver S9 fractions from trout. Variability within and among laboratories was characterized as the percent coefficient of variation (CV) in measured in vitro intrinsic clearance rates (CLIN VITRO, INT; ml/h/mg protein or 106 cells) for each chemical and test system. Mean intralaboratory CVs for each test chemical averaged 18.9% for hepatocytes and 14.1% for S9 fractions, whereas interlaboratory CVs (all chemicals and all tests) averaged 30.1% for hepatocytes and 22.4% for S9 fractions. When CLIN VITRO, INT values were extrapolated to in vivo intrinsic clearance estimates (CLIN VIVO, INT; l/d/kg fish), both assays yielded similar levels of activity (<4-fold difference for all chemicals). Hepatic clearance rates (CLH; l/d/kg fish) calculated using data from both assays exhibited even better agreement. These findings show that both assays are highly reliable and suggest that either may be used to inform chemical bioaccumulation assessments for fish. This study highlights several issues related to the demonstration of assay reliability and may provide a template for evaluating other in vitro biotransformation assays

Intra- and Interlaboratory Reliability of a Cryopreserved Trout Hepatocyte Assay for the Prediction of Chemical Bioaccumulation Potential

Measured rates of intrinsic clearance determined using cryopreserved trout hepatocytes can be extrapolated to the whole animal as a means of improving modeled bioaccumulation predictions for fish. To date, however, the intra- and interlaboratory reliability of this procedure has not been determined. In the present study, three laboratories determined in vitro intrinsic clearance of six reference compounds (benzo­[<i>a</i>]­pyrene, 4-nonylphenol, di-<i>tert</i>-butyl phenol, fenthion, methoxychlor and <i>o</i>-terphenyl) by conducting substrate depletion experiments with cryopreserved trout hepatocytes from a single source. <i>O</i>-terphenyl was excluded from the final analysis due to nonfirst-order depletion kinetics and significant loss from denatured controls. For the other five compounds, intralaboratory variability (% CV) in measured in vitro intrinsic clearance values ranged from 4.1 to 30%, while interlaboratory variability ranged from 27 to 61%. Predicted bioconcentration factors based on in vitro clearance values exhibited a reduced level of interlaboratory variability (5.3–38% CV). The results of this study demonstrate that cryopreserved trout hepatocytes can be used to reliably obtain in vitro intrinsic clearance of xenobiotics, which provides support for the application of this in vitro method in a weight-of-evidence approach to chemical bioaccumulation assessment