Challenges and opportunities for overcoming dog use in agrochemical evaluation and registration.

Progress in developing new tools, assays, and approaches to assess human hazard and health risk provides an opportunity to re-evaluate the necessity of dog studies for the safety evaluation of agrochemicals. A workshop was held where partic­ipants discussed the strengths and limitations of past use of dogs for pesticide evaluations and registrations. Opportunities were identified to support alternative approaches to answer human safety questions without performing the required 90-day dog study. Development of a decision tree for determining when the dog study might not be necessary to inform pesticide safety and risk assessment was proposed. Such a process will require global regulatory authority participation to lead to its acceptance. The identification of unique effects in dogs that are not identified in rodents will need further evaluation and determination of their relevance to humans. The establishment of in vitro and in silico approaches that can provide critical data on relative species sensitivity and human relevance will be an important tool to advance the decision process. Promising novel tools including in vitro comparative metabolism studies, in silico models, and high-throughput assays able to identify metabolites and mechanisms of action leading to development of adverse outcome pathways will need further development. To replace or eliminate the 90-day dog study, a collaborative, multidisciplinary, international effort that transcends organi­zations and regulatory agencies will be needed in order to develop guidance on when the study would not be necessary for human safety and risk assessment.

A new conceptional model for deriving average dermal absorption estimates from studies with multiple tested concentrations for non-dietary risk assessment of pesticides.

Dermal absorption values are used to translate external dermal exposure into potential systemic exposure for non-dietary risk assessment of pesticides. While the Environmental Protection Agency of the United States of America (US EPA) derives a common dermal absorption factor for active substances covering all related products, the European Food Safety Authority (EFSA) requests specific product-based estimates for individual concentrations covering the intended use rates. The latter poses challenges, because it disconnects exposure dose from applied dose in absorption studies, which may not be suitable in scenarios where concentration is not relevant. We analyzed the EFSA dermal absorption database, collected 33 human in vitro studies from CropLife Europe (CLE) companies, where ≥3 in-use dilution concentrations were tested, and 15 dermal absorption triple pack datasets. This shows that absolute dermal absorption correlates with absolute applied dose on a decadic logarithm-scale, which is concordant with the toxicological axiom that risk is driven by exposure dose. This method is radically different from the current European approach focused on concentrations and offers new insights into the relationship of internal and external exposure doses when utilizing data from in vitro studies. A single average dermal absorption value can be simply derived from studies with multiple tested concentrations, by calculating the y-intercept of a linear model on a decadic logarithm scale while assuming a slope of 1. This simplifies risk assessment and frees resources to explore exposure refinements. It also serves as a basis to harmonize dermal absorption estimation globally for use in exposure-driven risk assessments.

An OECD TG 428 study ring trial with 14C-Caffeine demonstrating repeatability and robustness of the dermal absorption in vitro method.

The dermal absorption potential of 14C-Caffeine applied as a 4 mg/mL concentration (10 µL/cm2 finite dose) was investigated in six laboratories under Good Laboratory Practice conditions using an OECD TG 428-compliant in vitro assay with flow-through cells and split-thickness human skin. Potential sources of variation were reduced by a standardized protocol, test item and skin source. Particularly, skin samples from same donors were distributed over two repeats and between labs in a non-random, stratified design. Very similar recovery was achieved in the various assay compartments between laboratories, repeats and donors, demonstrating that the assay can be robustly and reliably performed. The absorption in one laboratory was 5-fold higher than in the others. This did not clearly correlate with skin integrity parameters but might be associated with an accidental COVID-19 pandemic-related interruption in sample shipment. It is possible that other factors may affect dermal absorption variation not routinely assessed or considered in the current method. The mean receptor fluid recovery, potential absorption (recovery in receptor fluid and skin except tape strips 1 and 2) and mass balance of caffeine was 6.99%, 7.14% and 99.13%, respectively, across all and 3.87%, 3.96% and 99.00% in the subset of five laboratories.

Recommendations on dose level selection for repeat dose toxicity studies.

Prior to registering and marketing any new pharmaceutical, (agro)chemical or food ingredient product manufacturers must, by law, generate data to ensure human safety. Safety testing requirements vary depending on sector, but generally repeat-dose testing in animals form the basis for human health risk assessments. Dose level selection is an important consideration when designing such studies, to ensure that exposure levels that lead to relevant hazards are identified. Advice on dose level selection is provided in test guidelines and allied guidance documents, but it is not well harmonised, particularly for selection of the highest dose tested. This paper further builds on concepts developed in a technical report by the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) which recommends pragmatic approaches to dose selection considering regulatory requirements, animal welfare and state of the art scientific approaches. Industry sectors have differing degrees of freedom to operate regarding dose level selection, depending on the purpose of the studies and the regulatory requirements/legislation, and this is reflected in the overall recommended approaches. An understanding of systemic exposure should be utilised where possible (e.g., through toxicokinetic approaches) and used together with apical endpoints from existing toxicity studies to guide more appropriate dose level selection. The highest dose should be limited to a reasonable level, causing minimal but evident toxicity to the test animals without significantly compromising their well-being. As the science of predictive human exposure further develops and matures, this will provide exciting and novel opportunities for more human-relevant approaches to dose level selection.

Predicting nonlinear relationships between external and internal concentrations with physiologically based pharmacokinetic modeling.

Although external concentrations are more readily quantified and often used as the metric for regulating and mitigating exposures to environmental chemicals, the toxicological response to an environmental chemical is more directly related to its internal concentrations than the external concentration. The processes of absorption, distribution, metabolism, and excretion (ADME) determine the quantitative relationship between the external and internal concentrations, and these processes are often susceptible to saturation at high concentrations, which can lead to nonlinear changes in internal concentrations that deviate from proportionality. Using generic physiologically-based pharmacokinetic (PBPK) models, we explored how saturable absorption or clearance influence the shape of the internal to external concentration (IEC) relationship. We used the models for hypothetical chemicals to show how differences in kinetic parameters can impact the shape of an IEC relationship; and models for styrene and caffeine to explore how exposure route, frequency, and duration impact the IEC relationships in rat and human exposures. We also analyzed available plasma concentration data for 2,4-dichlorophenoxyacetic acid to demonstrate how a PBPK modeling approach can be an alternative to common statistical methods for analyzing dose proportionality. A PBPK modeling approach can be a valuable tool used in the early stages of a chemical safety assessment program to optimize the design of longer-term animal toxicity studies or to interpret study results.

Opportunities and challenges related to saturation of toxicokinetic processes: Implications for risk assessment.

Top dose selection for repeated dose animal studies has generally focused on identification of apical endpoints, use of the limit dose, or determination of a maximum tolerated dose (MTD). The intent is to optimize the ability of toxicity tests performed in a small number of animals to detect effects for hazard identification. An alternative approach, the kinetically derived maximum dose (KMD), has been proposed as a mechanism to integrate toxicokinetic (TK) data into the dose selection process. The approach refers to the dose above which the systemic exposures depart from being proportional to external doses. This non-linear external-internal dose relationship arises from saturation or limitation of TK process(es), such as absorption or metabolism. The importance of TK information is widely acknowledged when assessing human health risks arising from exposures to environmental chemicals, as TK determines the amount of chemical at potential sites of toxicological responses. However, there have been differing opinions and interpretations within the scientific and regulatory communities related to the validity and application of the KMD concept. A multi-stakeholder working group, led by the Health and Environmental Sciences Institute (HESI), was formed to provide an opportunity for impacted stakeholders to address commonly raised scientific and technical issues related to this topic and, more specifically, a weight of evidence approach is recommended to inform design and dose selection for repeated dose animal studies. Commonly raised challenges related to the use of TK data for dose selection are discussed, recommendations are provided, and illustrative case examples are provided to address these challenges or refute misconceptions.

PBPK model reporting template for chemical risk assessment applications.

Physiologically-based pharmacokinetic (PBPK) modeling analysis does not stand on its own for regulatory purposes but is a robust tool to support drug/chemical safety assessment. While the development of PBPK models have grown steadily since their emergence, only a handful of models have been accepted to support regulatory purposes due to obstacles such as the lack of a standardized template for reporting PBPK analysis. Here, we expand the existing guidances designed for pharmaceutical applications by recommending additional elements that are relevant to environmental chemicals. This harmonized reporting template can be adopted and customized by public health agencies receiving PBPK model submission, and it can also serve as general guidance for submitting PBPK-related studies for publication in journals or other modeling sharing purposes. The current effort represents one of several ongoing collaborations among the PBPK modeling and risk assessment communities to promote, when appropriate, incorporating PBPK modeling to characterize the influence of pharmacokinetics on safety decisions made by regulatory agencies.

Dermal absorption study OECD TG 428 mass balance recommendations based on the EFSA database.

The European Food Safety Authority (EFSA) guidance (EFSA, 2017) for dermal absorption (DA) studies recommends stringent mass balance (MB) limits of 95-105%. EFSA suggested that test material can be lost after penetration and requires that for chemicals with <5% absorption the non-recovered material must be added to the absorbed dose if MB is <95%. This has huge consequences for low absorption pesticides. Indeed, one third of the MBs in the EFSA DA database are outside the refined criteria. This is also true for DA data generated by Cosmetics Europe (Gregoire et al., 2019), indicating that this criterion is often not achieved even when using highly standardized protocols. While EFSA hypothesizes that modern analytical and pipetting techniques would enable to achieve this criterion, no scientific basis was provided. We describe how protocol procedures impact MB and evaluate the EFSA DA database to demonstrate that MB is subject to random variation. Generic application of "the addition rule" skews the measured data and increases the DA estimate, which results in unnecessary risk assessment failure. In conclusion, "missing material" is just a random negative deviation to the nominal dose. We propose a data-driven MB criterion of 90-110%, fully in line with OECD recommendations.

Metabolism and disposition of [14C]-methylcyclosiloxanes in rats.

Octamethylcyclotetrasiloxane (D4) and decamethylcyclopentasiloxane (D5) are low molecular weight cyclic volatile methyl siloxanes (cVMSs) primarily used as intermediates or monomers in the production of high molecular weight silicone polymers. The use of D4 as a direct ingredient in personal care products has declined significantly over the past 20 years, although it may be present as a residual impurity in a variety of consumer products. D5 is still used as an intentional ingredient in cosmetics, consumer products and in dry cleaning. Persons who may be exposed include occupational exposure for workers, and potential inhalation or dermal exposure for consumers and the general public. Because of the diverse use, especially of D5, and the potential for human exposure, a comprehensive program was undertaken to understand the kinetics, metabolism, enzyme induction and toxicity of D4 and D5 in rats following relevant routes of exposure. Physiologically based pharmacokinetic (PBPK) models utilizing these studies have been reported for D4 and D5 in the rat and human following dermal and inhalation exposures, with the oral uptake component of the model being limited in its description. Data from high dose oral studies in corn oil and simethicone vehicles and neat were used in the D4/D5 harmonized PBPK model development. It was uncertain if the inability to adequately describe the oral uptake was due to unrealistic high doses or unique aspects of the chemistry of D4/D5. Low dose studies were used to provide data to refine the description of oral uptake in the model by exploring the dose dependency and the impact of a more realistic food-like vehicle. Absorption, distribution, metabolism and elimination (ADME) of D4 and D5 was determined following a single low oral gavage dose of 14C-D4 and 14C-D5 at 30 and 100mg/kg body weight (bw), respectively, in a rodent liquid diet. Comparison of the low vs. high dose oral gavage administration of D4 and D5 demonstrated dose-dependent kinetic behavior. Data and modeling results suggest differences in metabolism between low and high dose administration indicating high dose administration results in or approaches non-linear saturated metabolism. These low dose data sets were used to refine the D4/D5 multi-route harmonized PBPK model to allow for a better description of the disposition and toxicokinetics of D4/D5 following oral exposure. With a refined oral uptake description, the model could be used in risk assessment to better define the internal dose of D4 and D5 following exposure to D4 and D5 via multiple routes.

Metabolism of 14C-octamethylcyclotetrasiloxane ([14C]D4) or 14C-decamethylcyclopentasiloxane ([14C]D5) orally gavaged in rainbow trout (Oncorhynchus mykiss).

Critical factors (uptake, distribution, metabolism and elimination) for understanding the bioaccumulation/biomagnification potential of Octamethylcyclotetrasiloxane (D4) and Decamethylcyclopentasiloxane (D5) siloxanes in fish were investigated to address whether these chemicals meet the "B" criteria of the Persistent, Bioaccumulative, and Toxic (PBT) classification. A metabolism study was conducted in rainbow trout whereby a 15mg [14C]D4/kg bw or [14C]D5/kg bw as a single bolus oral dose was administered via gavage. Of the administered dose, 79% (D4) and 78% (D5) was recovered by the end of the study (96-h). Eighty-two percent and 25% of the recovered dose was absorbed based on the percentage of recovered dose in carcass (69% and 17%), tissues, bile and blood (12% and 8%) and urine (1%) for D4 and D5, respectively. A significant portion of the recovered dose (i.e. 18% for D4 and 75% for D5) was eliminated in feces. Maximum blood concentrations were 1.6 and 1.4µg D4 or D5/g blood at 24h post-dosing, with elimination half-lives of 39h (D4) and 70h (D5). Modeling of parent and metabolite blood concentrations resulted in estimated metabolism rate constants (km(blood)) of 0.15 (D4) and 0.17day-1(D5). Metabolites in tissues, bile, blood, and urine totaled a minimum of 2% (D4) and 14% (D5) of the absorbed dose. The highest concentration of 14C-activity in the fish following D4 administration was in mesenteric fat followed by bile, but the opposite was true for D5. Metabolites were not detected in fat, only parent chemical. In bile, 94% (D4) and 99% (D5) of the 14C-activity was due to metabolites. Metabolites were also detected in the digestive tract, liver and gonads. Approximately 40% of the 14C-activity detected in the liver was due to the presence of metabolites. Urinary elimination represented a minor pathway, but all the 14C-activity in the urine was associated with metabolites. Clearance may occur via enterohepatic circulation of metabolic products in bile with excretion via the digestive tract and urinary clearance of polar metabolites.

Assessment of metabolic stability using the rainbow trout (Oncorhynchus mykiss) liver S9 fraction.

Standard protocols are given for assessing metabolic stability in rainbow trout using the liver S9 fraction. These protocols describe the isolation of S9 fractions from trout livers, evaluation of metabolic stability using a substrate depletion approach, and expression of the result as in vivo intrinsic clearance. Additional guidance is provided on the care and handling of test animals, design and interpretation of preliminary studies, and development of analytical methods. Although initially developed to predict metabolism impacts on chemical accumulation by fish, these procedures can be used to support a broad range of scientific and risk assessment activities including evaluation of emerging chemical contaminants and improved interpretation of toxicity testing results. These protocols have been designed for rainbow trout and can be adapted to other species as long as species-specific considerations are modified accordingly (e.g., fish maintenance and incubation mixture temperature). Rainbow trout is a cold-water species. Protocols for other species (e.g., carp, a warm-water species) can be developed based on these procedures as long as the specific considerations are taken into account.

In vitro study to determine decontamination of 3,5-dichloro-2,4,6-trifluoropyridine (DCTFP) from human skin.

This in vitro study determined the decontamination potential of soap and water, D-TAM skin cleanser, corn oil and the O'Dell reactive skin decontamination system to remove 3,5-dichloro-2,4,6-trifluoropyridine (DCTFP) from human skin after short exposure periods (10 and 30 min). The main result turned out to be the rapid volatility of DCTFP where half of the dose evaporated within 10 min and most of the dose was evaporated after 30 min. This rapid volatility was confirmed in an additional study where DCTFP rapidly evaporated from inert plastic disks (70% loss in 10 min). Despite rapid evaporation and short exposure periods, some DCTFP entered into the human skin epidermis, dermis and receptor fluid. Less DCTFP reached the receptor fluid with the 10 min decontamination (0.13%) than the 30 min decontamination (0.27%). Statistically, all tested decontamination systems performed the same (P>0.05). For human risk the volatility of DCTFP seems paramount. For skin decontamination any of the tested systems will work. An important point is that they should be used within minutes of skin exposure. This study exhibits the dynamics of evaporation, substantivity (skin binding), percutaneous absorption and decontamination of a volatile chemical.

Inhalation dosimetry modeling with decamethylcyclopentasiloxane in rats and humans.

Decamethylcyclopentasiloxane (D(5)), a volatile cyclic methyl siloxane (VCMS), is used in industrial and consumer products. Inhalation pharmacokinetics of another VCMS, octamethylcyclotetrasiloxane (D(4)), have been extensively investigated and successfully modeled with a multispecies physiologically based pharmacokinetic (PBPK) model. Here, we develop an inhalation PBPK description for D(5), using the D(4) model structure as a starting point, with the objective of understanding factors that regulate free blood and tissue concentrations of this highly lipophilic vapor after inhalation in rats and humans. Compared with D(4), the more lipophilic D(5) required deep compartments in lung, liver, and plasma to account for slow release from tissues after cessation of exposures. Simulations of the kinetics of a stable D(5) metabolite, HO-D(5), required diffusion-limited uptake in fat, a deep tissue store in lung, and its elimination by fecal excretion and metabolism to linear silanols. The combined D(5)/HO-D(5) model described blood and tissue concentrations of parent D(5) and elimination of total radioactivity in single and repeat exposures in male and female rats at 7 and 160 ppm. In humans, D(5) kinetic data are more sparse and the model structure though much simplified, still required free and bound blood D(5) to simulate exhaled air and blood time courses from 1 h inhalation exposures at 10 ppm in five human volunteers. This multispecies PBPK model for D(5) highlights complications in interpreting kinetic studies where chemical in blood and tissues represents various pools with only a portion free. The ability to simulate free concentrations is essential for dosimetry based risk assessments for these VCMS.

The effect of route, vehicle, and divided doses on the pharmacokinetics of chlorpyrifos and its metabolite trichloropyridinol in neonatal Sprague-Dawley rats.

There is a paucity of data on neonatal systemic exposure using different dosing paradigms. Male CD (Sprague-Dawley derived) rats at postnatal day (PND) 5 were dosed with chlorpyrifos (CPF, 1 mg/kg) using different routes of exposure, vehicles, and single versus divided doses. Blood concentrations of CPF and its primary metabolite, trichloropyridinol, were measured at multiple times through 24 h. Groups included were single gavage bolus versus divided gavage doses in corn oil (one vs. three times in 24h), single gavage bolus versus divided gavage doses in rat milk, and sc administration in dimethyl sulfoxide (DMSO). These data were compared with lactational exposure of PND 5 pups from dams exposed to CPF in the diet at 5 mg/kg/day for 4 weeks or published data from dams exposed to daily gavage with CPF at 5 mg/kg/day. Maternal blood CPF levels were an order of magnitude lower from dietary exposure than gavage (1.1 vs. 14.8 ng/g), and blood CPF levels in PND 5 pups that nursed dietary-exposed or gavage-exposed dams were below the limit of detection. Single gavage doses of 1 mg/kg CPF in corn oil vehicle in pups resulted in CPF blood levels of 49 ng/g and in milk vehicle about 9 ng/g. Divided doses led to lower peak CPF levels. A bolus dose of 1 mg/kg CPF in DMSO administered sc appeared to have substantially altered pharmacokinetics from orally administered CPF. To be meaningful for risk assessment, neonatal studies require attention to the exposure scenario, since route, vehicle, dose, and frequency of administration result in different systemic exposure to the test chemical and its metabolites.