During the past few decades, the science of toxicology has been undergoing a transformation from observational to predictive science. New approach methodologies (NAMs), including in vitro assays, in silico models, read-across, and in vitro to in vivo extrapolation (IVIVE), are being developed to reduce, refine, or replace whole animal testing, encouraging the judicious use of time and resources. Some of these methods have advanced past the exploratory research stage and are beginning to gain acceptance for the risk assessment of chemicals. A review of the recent literature reveals a burst of IVIVE publications over the past decade. In this review, we propose operational definitions for IVIVE, present literature examples for several common toxicity endpoints, and highlight their implications in decision-making processes across various federal agencies, as well as international organizations, including those in the European Union (EU). The current challenges and future needs are also summarized for IVIVE. In addition to refining and reducing the number of animals in traditional toxicity testing protocols and being used for prioritizing chemical testing, the goal to use IVIVE to facilitate the replacement of animal models can be achieved through their continued evolution and development, including a strategic plan to qualify IVIVE methods for regulatory acceptance.

Background: The developmental toxicity potential (dTP) concentration from the devTOX quickPredict (devTOX^qP) assay, a metabolomics‐based human induced pluripotent stem cell assay, predicts a chemical's developmental toxicity potency. Here, in vitro to in vivo extrapolation (IVIVE) approaches were applied to address whether the devTOX^qP assay could quantitatively predict in vivo developmental toxicity lowest effect levels (LELs) for the prototypical teratogen valproic acid (VPA) and a group of structural analogues.Methods: VPA and a series of structural analogues were tested with the devTOX^qP assay to determine dTP concentration and we estimated the equivalent administered doses (EADs) that would lead to plasma concentrations equivalent to the in vitro dTP concentrations. The EADs were compared to the LELs in rat developmental toxicity studies, human clinical doses, and EADs reported using other in vitro assays. To evaluate the impact of different pharmacokinetic (PK) models on IVIVE outcomes, we compared EADs predicted using various open‐source and commercially available PK and physiologically based PK (PBPK) models. To evaluate the effect of in vitro kinetics, an equilibrium distribution model was applied to translate dTP concentrations to free medium concentrations before subsequent IVIVE analyses.Results: The EAD estimates for the VPA analogues based on different PK/PBPK models were quantitatively similar to in vivo data from both rats and humans, where available, and the derived rank order of the chemicals was consistent with observed in vivo developmental toxicity. Different models were identified that provided accurate predictions for rat prenatal LELs and conservative estimates of human safe exposure. The impact of in vitro kinetics on EAD estimates is chemical‐dependent. EADs from this study were within range of predicted doses from other in vitro and model organism data.Conclusions: This study highlights the importance of pharmacokinetic considerations when using in vitro assays and demonstrates the utility of the devTOX^qP human stem cell‐based platform to quantitatively assess a chemical's developmental toxicity potency.

Organophosphorus flame retardants (OPFRs) are abundant and persistent in the environment but have limited toxicity information. Their similarity in structure to organophosphate pesticides presents great concern for developmental neurotoxicity (DNT). However, current in vivo testing is not suitable to provide DNT information on the amount of OPFRs that lack data. Over the past decade, an in vitro battery was developed to enhance DNT assessment, consisting of assays that evaluate cellular processes in neurodevelopment and function. In this study, behavioral data of small model organisms were also included. To assess if these assays provide sufficient mechanistic coverage to prioritize chemicals for further testing and/or identify hazards, an integrated approach to testing and assessment (IATA) was developed with additional information from the Integrated Chemical Environment (ICE) and the literature. Human biomonitoring and exposure data were identified and physiologically-based toxicokinetic models were applied to relate in vitro toxicity data to human exposure based on maximum plasma concentration. Eight OPFRs were evaluated, including aromatic OPFRs (triphenyl phosphate (TPHP), isopropylated phenyl phosphate (IPP), 2-ethylhexyl diphenyl phosphate (EHDP), tricresyl phosphate (TMPP), isodecyl diphenyl phosphate (IDDP), tert-butylphenyl diphenyl phosphate (BPDP)) and halogenated FRs ((Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP), tris(2-chloroethyl) phosphate (TCEP)). Two representative brominated flame retardants (BFRs) (2,2′4,4′-tetrabromodiphenyl ether (BDE-47) and 3,3′,5,5′-tetrabromobisphenol A (TBBPA)) with known DNT potential were selected for toxicity benchmarking. Data from the DNT battery indicate that the aromatic OPFRs have activity at similar concentrations as the BFRs and should therefore be evaluated further. However, these assays provide limited information on the mechanism of the compounds. By integrating information from ICE and the literature, endocrine disruption was identified as a potential mechanism. This IATA case study indicates that human exposure to some OPFRs could lead to a plasma concentration similar to those exerting in vitro activities, indicating potential concern for human health.

Glyphosate, the most heavily used herbicide world‐wide, is applied to plants in complex formulations that promote absorption. The National Toxicology Program reported in 1992 that glyphosate, administered to rats and mice at doses up to 50,000 ppm in feed for 13 weeks, showed little evidence of toxicity, and no induction of micronuclei was observed in the mice in this study. Subsequently, mechanistic studies of glyphosate and glyphosate‐based formulations (GBFs) that have focused on DNA damage and oxidative stress suggest that glyphosate may have genotoxic potential. However, few of these studies directly compared glyphosate to GBFs, or effects among GBFs. To address these data gaps, we tested glyphosate, glyphosate isopropylamine (IPA), and (aminomethyl)phosphonic acid (AMPA, a microbial metabolite of glyphosate), 9 high‐use agricultural GBFs, 4 residential‐use GBFs, and additional herbicides (metolachlor, mesotrione, and diquat dibromide) present in some of the GBFs in bacterial mutagenicity tests, and in human TK6 cells using a micronucleus assay and a multiplexed DNA damage assay. Our results showed no genotoxicity or notable cytotoxicity for glyphosate or AMPA at concentrations up to 10 mM, while all GBFs and herbicides other than glyphosate were cytotoxic, and some showed genotoxic activity. An in vitro to in vivo extrapolation of results for glyphosate suggests that it is of low toxicological concern for humans. In conclusion, these results demonstrate a lack of genotoxicity for glyphosate, consistent with observations in the NTP in vivo study, and suggest that toxicity associated with GBFs may be related to other components of these formulations.

An extensive battery of 17 in vitro assays has been developed for assessing developmental neurotoxicity (DNT), with the aim of replacing or supplementing traditional in vivo guideline studies for risk assessment, as these mechanistic assays provide advantages over costly, lengthy in vivo studies. However, 1 major challenge in employing in vitro assays is the translation of in vitro bioactive concentrations into in vivo doses that can be compared with human exposures. This study describes an in vitro to in vivo extrapolation (IVIVE) approach to derive human-relevant administered equivalent doses based on chemical partitioning into DNT target organs during the critical period of brain development. We used data from chemicals previously found to elicit bioactivity in a subset (7 of 17) of the in vitro DNT battery assays conducted at the US Environmental Protection Agency. Three physiologically based pharmacokinetic modeling platforms were evaluated for their suitability for this DNT-IVIVE approach. Chemical predictions for administered equivalent doses were compared against in vivo effect levels, where available, and found to be within 3-fold for 78% of chemicals. To provide metrics for risk assessment considerations, administered equivalent doses were compared with predicted human exposures. Overall, this DNT-IVIVE approach was found to be relatively transferable among modeling platforms, albeit with varying limitations and considerations that should be taken into account for specific contexts of use.

Traditional chemical risk assessment is often based on published mammalian in vivo toxicity data, which are used to identify a point-of-departure (PoD) to derive the minimal risk level (MRL) and similar health guidance values. However, time and resource requirements prohibit efficient multi-target-organ toxicity assessments for a large number of environmental chemical pollutants. In vitro high-throughput screening assays and other new approach methodologies could address this problem by using reverse dosimetry to contextualize activity concentrations obtained from the in vitro assays to in vivo settings. In this study, we selected sample priority of diverse chemicals for which both curated high-throughput screening (cHTS) assay data and acute oral MRLs were available for neurotoxicity, hepatotoxicity, and developmental toxicity. We obtained in vitro activity concentrations for these chemicals and conducted in vitro to in vivo extrapolation (IVIVE) to estimate the daily equivalent administered dose (EAD) that would result in rat or human plasma concentrations equivalent to the in vitro activity concentrations. The range of EAD values across various cHTS assays was then compared with in vivo PoDs, MRLs, and predicted human exposure levels. Although variations existed depending on toxicity endpoints evaluated, our results showed that PoDs for a majority of chemicals can be predicted using such data. Our results also demonstrated that a majority of MRL and EAD values fall well below predicted human exposure levels. In summary, our findings demonstrate the usefulness and limitations of using cHTS data and IVIVE approaches for the derivation of health guidance values.