Sean C. Gehen

Acute toxicity testing of chemicals—Opportunities to avoid redundant testing and use alternative approaches

Assessment of the acute systemic oral, dermal, and inhalation toxicities, skin and eye irritancy, and skin sensitisation potential of chemicals is required under regulatory schemes worldwide. In vivo studies conducted to assess these endpoints can sometimes be associated with substantial adverse effects in the test animals, and their use should always be scientifically justified. It has been argued that while information obtained from such acute tests provides data needed to meet classification and labelling regulations, it is of limited value for hazard and risk assessments. Inconsistent application of in vitro replacements, protocol requirements across regions, and bridging principles also contribute to unnecessary and redundant animal testing. Assessment of data from acute oral and dermal toxicity testing demonstrates that acute dermal testing rarely provides value for hazard assessment purposes when an acute oral study has been conducted. Options to waive requirements for acute oral and inhalation toxicity testing should be employed to avoid unnecessary in vivo studies. In vitro irritation models should receive wider adoption and be used to meet regulatory needs. Global requirements for sensitisation testing need continued harmonisation for both substance and mixture assessments. This paper highlights where alternative approaches or elimination of tests can reduce and refine animal use for acute toxicity requirements.

Pharmaceutical toxicology: Designing studies to reduce animal use, while maximizing human translation

Evaluation of the safety of new chemicals and pharmaceuticals requires the combination of information from various sources (e.g. in vitro, in silico and in vivo) to provide an assessment of risk to human health and the environment. The authors have identified opportunities to maximize the predictivity of this information to humans while reducing animal use in four key areas; (i) accelerating the uptake of in vitro methods; (ii) incorporating the latest science into safety pharmacology assessments; (iii) optimizing rodent study design in biological development and (iv) consolidating approaches in developmental and reproductive toxicology. Through providing a forum for open discussion of novel proposals, reviewing current research and obtaining expert opinion in each of the four areas, the authors have developed recommendations on good practice and future strategy.

Chlorpyrifos: weight of evidence evaluation of potential interaction with the estrogen, androgen, or thyroid pathways.

Chlorpyrifos was selected for EPAs Endocrine Disruptor Screening Program (EDSP) based on widespread use and potential for human and environmental exposures. The purpose of the program is to screen chemicals for their potential to interact with the estrogen, androgen, or thyroid pathways. A battery of 11 assays was completed for chlorpyrifos in accordance with test guidelines developed for EDSP Tier 1 screening. To determine potential endocrine activity, a weight-of-evidence (WoE) evaluation was completed for chlorpyrifos, which included the integration of EDSP assay results with data from regulatory guideline studies and the published literature. This WoE approach was based on the OECD conceptual framework for testing and assessment of potential endocrine-disrupting chemicals and consisted of a systematic evaluation of data, progressing from simple to complex across multiple levels of biological organization. The conclusion of the WoE evaluation is that chlorpyrifos demonstrates no potential to interact with the estrogen, androgen, or thyroid pathways at doses below the dose levels that inhibit cholinesterase. Therefore, regulatory exposure limits for chlorpyrifos, which are based on cholinesterase inhibition, are sufficient to protect against potential endocrine alterations. Based on the results of this WoE evaluation, there is no scientific justification for pursuing additional endocrine testing for chlorpyrifos.

Alternative approaches for identifying acute systemic toxicity: Moving from research to regulatory testing

Acute systemic toxicity testing provides the basis for hazard labeling and risk management of chemicals. A number of international efforts have been directed at identifying non-animal alternatives for in vivo acute systemic toxicity tests. A September 2015 workshop, Alternative Approaches for Identifying Acute Systemic Toxicity: Moving from Research to Regulatory Testing, reviewed the state-of-the-science of non-animal alternatives for this testing and explored ways to facilitate implementation of alternatives. Workshop attendees included representatives from international regulatory agencies, academia, nongovernmental organizations, and industry. Resources identified as necessary for meaningful progress in implementing alternatives included compiling and making available high-quality reference data, training on use and interpretation of in vitro and in silico approaches, and global harmonization of testing requirements. Attendees particularly noted the need to characterize variability in reference data to evaluate new approaches. They also noted the importance of understanding the mechanisms of acute toxicity, which could be facilitated by the development of adverse outcome pathways. Workshop breakout groups explored different approaches to reducing or replacing animal use for acute toxicity testing, with each group crafting a roadmap and strategy to accomplish near-term progress. The workshop steering committee has organized efforts to implement the recommendations of the workshop participants.

Retrospective evaluation of the impact of functional immunotoxicity testing on pesticide hazard identification and risk assessment

Abstract Conduct of a T-cell-dependent antibody response (TDAR) assay in rodents according to Environmental Protection Agency (EPA) Test Guideline OPPTS 870.7800 is now required for chemical pesticide active ingredients registered in the United States. To assess potential regulatory impact, a retrospective analysis was developed using TDAR tests conducted on 78 pesticide chemicals from 46 separate chemical classes. The objective of the retrospective analysis was to examine the frequency of positive responses and determine the potential for the TDAR to yield lower endpoints than those utilized to calculate reference doses (RfDs). A reduction in the TDAR response was observed at only the high-dose level in five studies, while it was unaltered in the remaining studies. Importantly, for all 78 pesticide chemicals, the TDAR no-observed-adverse-effect levels (TDAR NOAELs) were greater than the NOAELS currently in use as risk assessment endpoints. The TDAR NOAELs were higher than the current EPA-selected endpoints for the chronic RfD, short-term, intermediate and long-term exposure scenarios by 3–27,000, 3–1,688, 3–1,688 and 4.9–1,688 times, respectively. Based on this analysis, conduct of the TDAR assay had minimal impact on hazard identification and did not impact human health risk assessments for the pesticides included in this evaluation. These data strongly support employment of alternative approaches including initial weight-of-evidence analysis for immunotoxic potential prior to conducting functional immunotoxicity testing for pesticide active ingredients.

Application of the KeratinoSens™ assay for assessing the skin sensitization potential of agrochemical active ingredients and formulations.

Assessment of skin sensitization potential is an important component of the safety evaluation process for agrochemical products. Recently, non-animal approaches including the KeratinoSens™ assay have been developed for predicting skin sensitization potential. Assessing the utility of the KeratinoSens™ assay for use with multi-component mixtures such as agrochemical formulations has not been previously evaluated and is a significant need. This study was undertaken to evaluate the KeratinoSens™ assay prediction potential for agrochemical formulations. The assay was conducted for 8 agrochemical active ingredients (AIs) including 3 sensitizers (acetochlor, meptyldinocap, triclopyr), 5 non-sensitizers (aminopyralid, clopyralid, florasulam, methoxyfenozide, oxyfluorfen) and 10 formulations for which in vivo sensitization data were available. The KeratinoSens™ correctly predicted the sensitization potential of all the AIs. For agrochemical formulations it was necessary to modify the standard assay procedure whereby the formulation was assumed to have a common molecular weight. The resultant approach correctly predicted the sensitization potential for 3 of 4 sensitizing formulations and all 6 non-sensitizing formulations when compared to in vivo data. Only the meptyldinocap-containing formulation was misclassified, as a result of high cytotoxicity. These results demonstrate the promising utility of the KeratinoSens™ assay for evaluating the skin sensitization potential of agrochemical AIs and formulations.

Chapter 87 – Toxicology of Triazolopyrimidine Herbicides

Publisher Summary The triazolopyrimidines are herbicides used for the preemergent and postemergent control of broadleaf weeds in a variety of crops. The empirical formula for cloransulam-methyl is C15H13ClFN5O5S, with a molecular weight of 429.8. It is a solid at room temperature, with a low vapor pressure. They have very low mammalian toxicity as determined by acute, short-term, long-term (chronic), genotoxicity, reproduction, developmental, and neurotoxicity tests. The kidneys and/or liver are the primary organs affected by repeated exposure, and, even then, in most cases the effects represent adaptive responses to very high levels of exposure. No studies are available on intentional human exposure. However, the risk to humans from exposure to cloransulam-methyl following normal use patterns is low. No detectable residues were found either in soybeans or, in most cases, in soybean forage or hay at a limit of detection of 0.005 ppm, and accumulation is unlikely based on plant and animal data. In conclusion, the triazolopyrimides are a structurally related class of herbicides that act to inhibit the enzyme acetolactate synthase (ALS) in plants. In repeat-dose toxicity studies, the liver and kidneys have been identified as target organs with effects that were often adaptive in nature generally observed only at excessively high-dose levels. In addition, the triazolopyrimidines were shown to be rapidly absorbed and excreted, have a low potential for bioaccumulation, and in general are not extensively metabolized.

Pathway-based predictive approaches for non-animal assessment of acute inhalation toxicity

New approaches are needed to assess the effects of inhaled substances on human health. These approaches will be based on mechanisms of toxicity, an understanding of dosimetry, and the use of in silico modeling and in vitro test methods. In order to accelerate wider implementation of such approaches, development of adverse outcome pathways (AOPs) can help identify and address gaps in our understanding of relevant parameters for model input and mechanisms, and optimize non-animal approaches that can be used to investigate key events of toxicity. This paper describes the AOPs and the toolbox of in vitro and in silico models that can be used to assess the key events leading to toxicity following inhalation exposure. Because the optimal testing strategy will vary depending on the substance of interest, here we present a decision tree approach to identify an appropriate non-animal integrated testing strategy that incorporates consideration of a substances physicochemical properties, relevant mechanisms of toxicity, and available in silico models and in vitro test methods. This decision tree can facilitate standardization of the testing approaches. Case study examples are presented to provide a basis for proof-of-concept testing to illustrate the utility of non-animal approaches to inform hazard identification and risk assessment of humans exposed to inhaled substances.

A retrospective analysis of in vivo eye irritation, skin irritation and skin sensitisation studies with agrochemical formulations: Setting the scene for development of alternative strategies

&NA; Analysis of the prevalence of health effects in large scale databases is key in defining testing strategies within the context of Integrated Approaches on Testing and Assessment (IATA), and is relevant to drive policy changes in existing regulatory toxicology frameworks towards non‐animal approaches. A retrospective analysis of existing results from in vivo skin irritation, eye irritation, and skin sensitisation studies on a database of 223 agrochemical formulations is herein published. For skin or eye effects, high prevalence of mild to non‐irritant formulations (i.e. per GHS, CLP or EPA classification) would generally suggest a bottom‐up approach. Severity of erythema or corneal opacity, for skinor eye effects respectively, were the key drivers for classification, consistent with existing literature. The reciprocal predictivity of skin versus eye irritation and the good negative predictivity of the GHS additivity calculation approach (>85%) provided valuable non‐testing evidence for irritation endpoints. For dermal sensitisation, concordance on data from three different methods confirmed the high false negative rate for the Buehler method in this product class. These results have been reviewed together with existing literature on the use of in vitro alternatives for agrochemical formulations, to propose improvements to current regulatory strategies and to identify further research needs. HighlightsRetrospective analysis of local toxicity data from 223 agrochemical formulations.Low prevalence of skin and eye irritants, suggesting bottom‐up approaches.GHS additivity showed good negative predictivity for skin and eye irritation.The reciprocal negative predictivity of skin versus eye irritation confirmed.