A new approach methodology using kinetically-derived maximum dose levels in risk assessment - A case study with afidopyropen.

We present a case study for afidopyropen (AF; insecticide) to characterize chronic dietary human health risk using a Risk 21-based approach. Our objective is to use a well-tested pesticidal active ingredient (AF) to show how a new approach methodology (NAM), using the kinetically-derived maximum dose (KMD) and with far less animal testing, can reliably identify a health-protective point of departure (PoD) for chronic dietary human health risk assessments (HHRA). Chronic dietary HHRA involves evaluation of both hazard and exposure information to characterize risk. Although both are important, emphasis has been placed on a checklist of required toxicological studies for hazard characterization, with human exposure information only considered after evaluation of hazard data. Most required studies are not used to define the human endpoint for HHRA. The information presented demonstrates a NAM that uses the KMD determined by saturation of a metabolic pathway, which can be used as an alternative POD. In these cases, the full toxicological database may not need to be generated. Demonstration that the compound is not genotoxic and that the KMD is protective of adverse effects in 90-day oral rat and reproductive/developmental studies is sufficient to support the use of the KMD as an alternative POD.

Afidopyropen: Challenges and impact of a toxicokinetic study designed to identify a point of inflection from dose-proportionality.

Afidopyropen is an insecticide that acts as a transient receptor potential vanilloid subtype (TRPV) channel modulator in chordotonal organs of target insects and has been assessed for a wide range of toxicity endpoints including chronic toxicity and carcinogenicity in rats and mice. The current study evaluates the toxicokinetic properties of afidopyropen and its plasma metabolites in rats at dose levels where the pharmacokinetics (PK) are linear and nonlinear in an attempt to identify a point of inflection. Based on the results of this study and depending on the analysis method used, the kinetically derived maximum dose (KMD) is estimated to be between 2.5 and 12.5 mg/kg bw/d with linearity observed at doses below 2.5 mg/kg bw/d. A defined point of inflection could not be determined. These data demonstrate that consideration of PK is critical for improving the dose-selection in toxicity studies as well as to enhance human relevance of the interpretation of animal toxicity studies. The study also demonstrates the technical difficulty in obtaining a defined point of inflection from in vivo PK data.

Assessing chemical carcinogenicity: hazard identification, classification, and risk assessment. Insight from a Toxicology Forum state-of-the-science workshop.

The Toxicology Forum convened an international state-of-the-science workshop Assessing Chemical Carcinogenicity: Hazard Identification, Classification, and Risk Assessment in December 2020. Challenges related to assessing chemical carcinogenicity were organized under the topics of (1) problem formulation; (2) modes-of-action; (3) dose-response assessment; and (4) the use of new approach methodologies (NAMs). Key topics included the mechanisms of genotoxic and non-genotoxic carcinogenicity and how these in conjunction with consideration of exposure conditions might inform dose-response assessments and an overall risk assessment; approaches to evaluate the human relevance of modes-of-action observed in rodent studies; and the characterization of uncertainties. While the scientific limitations of the traditional rodent chronic bioassay were widely acknowledged, knowledge gaps that need to be overcome to facilitate the further development and uptake of NAMs were also identified. Since one single NAM is unlikely to replace the bioassay, activities to combine NAMs into integrated approaches for testing and assessment, or preferably into defined approaches for testing and assessment that include data interpretation procedures, were identified as urgent research needs. In addition, adverse outcome pathway networks can provide a framework for organizing the available evidence/data for assessing chemical carcinogenicity. Since a formally accepted decision tree to guide use of the best and most current science to advance carcinogenicity risk assessment is currently unavailable, a Decision Matrix for carcinogenicity assessment could be useful. The workshop organizers developed and presented a decision matrix to be considered within a carcinogenicity hazard and risk assessment that is offered in tabular form.

The Relationship Between Internal and External Dose: Some General Results Based on a Generic Compartmental Model.

Statements on how the internal-to-external-dose (IED) relationship looks like are often based on qualitative toxicokinetic arguments. For example, the recently proposed kinetically derived maximum dose (KMD) states that the IED relationship must have an inflection point, due to saturation of underlying processes like metabolism or absorption. However, such statements lack a solid quantitative foundation. Therefore, we derived expressions for the IED relationship for a number of scenarios based on a generic compartmental model involving saturation. The scenarios included repeated or single dose, and saturable metabolism or saturable absorption. For some of these scenarios, an explicit expression for the IED relationship can be derived, for others only implicit expressions can be established, which need to be evaluated numerically. The results show that saturable processes will lead to an IED relationship that is nonlinear over the whole dose range, ie, it can be approximated by a linear relationship at the lower end, whereas the approximation will become gradually poorer with increasing doses. The finding that saturation does not lead to an inflection point in the IED relationship, as assumed in the KMD, implies that the KMD is not a valid approach for selecting the top dose in toxicological studies. An additional use of our results is that the derived explicit expressions of the IED relationship can be fitted to IED data, and, possibly, for extrapolation outside the observed dose range.

Use of the kinetically-derived maximum dose concept in selection of top doses for toxicity studies hampers proper hazard assessment and risk management.

The KMD (kinetically-derived maximum dose) is an increasingly advocated concept that uses toxicokinetic data in the top dose selection for toxicity testing. Application of this concept may have serious regulatory implications though, especially in the European Union. The basic assumption is that the relationship between internal and external dose (IED) shows an inflection point where linearity transits into non-linearity due to saturation of underlying processes; top doses in toxicity tests should not be above the inflection point, provided human exposures are well below this point. A critical analysis of the KMD concept and its underlying assumptions shows, however, that the IED relationship is non-linear over the whole dose range, without any point of inflection. The KMD concept thus aims to estimate a non-existing point, rendering it invalid for use in toxicity testing. Moreover, the concept ignores the key question in toxicology: What kind of toxic effects occur at which doses? These and several other reservations against the KMD concept are discussed and illustrated with three existing applications of the KMD approach. Hence, we recommend to abolish the KMD concept for selecting top doses in toxicity testing. This requires the updating of regulations, guidance documents and OECD test guidelines.

Use of toxicokinetic data for afidopyropen to determine the dose levels in developmental toxicity studies.

Afidopyropen is an insecticide that acts as a TRPV channel modulator in chordotonal organs of target insects and has been assessed for a wide range of toxicity endpoints including developmental toxicity in rats and rabbits. The GLP developmental toxicity study in rabbits did not produce evidence of maternal or fetal toxicity at the highest dose tested (32 mg/kg/day) but pharmacokinetics (PK) in pregnant rabbits in this study exhibited onset of PK nonlinearity from 5 mg/kg/day on, as measured by plasma Cmax and AUC. The NOAEL (32 mg/kg/day) is 9000X higher than maximum expected human dietary exposures to afidopyropen; the dose range where nonlinear PK were observed (5-15 mg/kg/day) is 1400-4200X higher. As nonlinearity occurred between 5 and 15 mg/kg/day, 32 mg/kg/day is concluded to be a sufficiently high dose (kinetically derived maximum dose) for a prenatal developmental toxicity study. As recognized by regulatory dose-selection guidance, onset of saturated PK is evidence of excessive biological stress to test animals rendering any effects at such doses of questionable relevance for human risk assessment. These data demonstrate that consideration of PK is critical for improving the dose-selection in developmental toxicity studies to enhance human relevance of animal toxicity studies.

Metabolic Basis for Nonlinearity in 1,3-Dichloropropene Toxicokinetics and Use in Setting a Kinetically-derived Maximum Inhalation Exposure Concentration in Mice.

1,3-Dichloropropene (1,3-D) showed a statistically increased incidence of bronchioloalveolar adenomas in male B6C3F1 mice at 60 ppm air concentration during previous chronic inhalation testing. No tumors were observed in female mice, nor in either sex of F344 rats up to 60 ppm, the highest dose tested. Therefore, to understand if lung tumors observed in high dose male mice are due to saturation of metabolic clearance, the linearity of 1,3-D concentrations in mouse blood was investigated on day 15 of repeated nose-only inhalation exposure to 0, 10, 20, 40, 60, 90, and 120 ppm (6 h/d, 7 d/week). Additional groups were included at 20, 60, and 120 ppm for blood collection at 1.5 and 3 h of exposure and up to 25 or 40 min post-exposure to determine area-under-the-curve. The data provide multiple lines of evidence that systemic exposures to 1,3-D in the mouse become nonlinear at inhalation exposure levels of 30 ppm or above. A reduction in minute volume occurred at the highest exposure concentration. The glutathione (GSH)-dependent metabolism of 1,3-D results in significant depletion of GSH at repeated exposure levels of 30 ppm and above. This loss of GSH results in decreased metabolic clearance of this test material, with a concomitant increase of the 1,3-D isomers in circulating blood at exposure concentrations ≥30 ppm. Shifts in the ratio of cis- and trans-1,3-D also support nonlinear toxicokinetics well below 60 ppm. Based on this data, a kinetically derived maximum dose for 1,3-D in mice for repeated exposures should be at or below 30 ppm. These results support non-relevance of 1,3-D-induced benign pulmonary tumorigenicity in mice for human health risk assessment.

Reproductive and developmental toxicity testing: Examination of the extended one-generation reproductive toxicity study guideline.

An important aspect of safety assessment of chemicals (industrial and agricultural chemicals and pharmaceuticals) is determining their potential reproductive and developmental toxicity. A number of guidelines have outlined a series of separate reproductive and developmental toxicity studies from fertilization through adulthood and in some cases to second generation. The Extended One-Generation Reproductive Toxicity Study (EOGRTS) is the most recent and comprehensive guideline in this series. EOGRTS design makes toxicity testing progressive, comprehensive, and efficient by assessing key endpoints across multiple life-stages at relevant doses using a minimum number of animals, combining studies/evaluations and proposing tiered-testing approaches based on outcomes. EOGRTS determines toxicity during preconception, development of embryo/fetus and newborn, adolescence, and adults, with specific emphasis on the nervous, immunological, and endocrine systems, EOGRTS also assesses maternal and paternal toxicity. However, EOGRTS guideline is complex, criteria for selecting doses is unclear, and monitoring systemic dose during the course of the study for better interpretation and human relevance is not clear. This paper discusses potential simplification of EOGRTS, suggests procedures for relevant dose selection and monitors systemic dose at multiple life-stages for better interpretation of data and human relevance.

Rethinking guideline toxicity testing.

The guidelines for risk assessment of plant protection products (PPPs) and other non-pharmaceuticals were developed over three decades ago and have generally not been updated to incorporate advancements in toxicology and exposure sciences. These guidelines recommend using maximum-tolerated-dose (MTD) even when human relevance of such high doses is mostly limited due to orders of magnitude margin-of-exposure. Conducting animal studies at such high doses often requires further mode-of-action (MoA) studies elucidating human relevance. In order to improve data, ILSI/HESI-ACSA technical committee proposed a tiered approach with emphasis on determining systemic dose of parent and/or metabolite(s) in test animals as biological effects are reflective of systemic rather than administered dose. Any deviation from linearity in systemic dose (saturation of absorption or elimination) in animal studies may have profound toxic effect(s) not expected to occur in likely human exposure scenarios and should be avoided. Toxicity studies should ideally be conducted at kinetically linear doses or slightly above the point of departure from linearity or kinetically-derived maximum dose (KMD) as the systemic dose nonlinearity is a more sensitive parameter occurring much earlier than the MTD endpoints. Therefore, determining systemic dose, especially KMD, in study animals is an improvement to hazard assessment of PPPs and other non-pharmaceuticals allowing toxicologists to better understand findings in animals at systemically linear as well as nonlinear doses to likely human exposures which can easily be accomplished using core study animals as outlined below. Determining systemic dose in studies will also increase the understanding of initial potential MoA of a PPPs and other non-pharmaceuticals and reduce the use of animals by avoiding unnecessary additional MoA studies.

An F1-extended one-generation reproductive toxicity study in Crl:CD(SD) rats with 2,4-dichlorophenoxyacetic acid.

2,4-Dichlorophenoxyacetic acid (2,4-D) was assessed for systemic toxicity, reproductive toxicity, developmental neurotoxicity (DNT), developmental immunotoxicity (DIT), and endocrine toxicity. CD rats (27/sex/dose) were exposed to 0, 100, 300, 600 (female), or 800 (male) ppm 2,4-D in diet. Nonlinear toxicokinetic behavior was shown at high doses; the renal clearance saturation threshold for 2,4-D was exceeded markedly in females and slightly exceeded in males. Exposure was 4 weeks premating, 7 weeks postmating for P1 males and through lactation for P1 females. F1 offspring were examined for survival and development, and at weaning, pups were divided in cohorts, by sex and dose, and by systemic toxicity (10), DNT (10), DIT (20), and reproductive toxicity (≥ 23). Remaining weanlings were evaluated for systemic toxicity and neuropathology (10-12). Body weight decreased during lactation in high-dose P1 females and in F1 pups. Kidney was the primary target organ, with slight degeneration of proximal convoluted tubules observed in high-dose P1 males and in high-dose F1 males and females. A slight intergenerational difference in kidney toxicity was attributed to increased intake of 2,4-D in F1 offspring. Decreased weanling testes weights and delayed preputial separation in F1 males were attributed to decreased body weights. Endocrine-related effects were limited to slight thyroid hormone changes and adaptive histopathology in high-dose GD 17 dams seen only at a nonlinear toxicokinetic dose. 2,4-D did not cause reproductive toxicity, DNT, or DIT. The "No Observed Adverse Effect Level" for systemic toxicity was 300 ppm in both males (16.6 mg/kg/day) and females (20.6 mg/kg/day), which is approximately 6700- to 93 000-fold higher than that reported for 2,4-D exposures in human biomonitoring studies.