Protecting Human and Animal Health: The Road from Animal Models to New Approach Methods.

Animals and animal models have been invaluable for our current understanding of human and animal biology, including physiology, pharmacology, biochemistry, and disease pathology. However, there are increasing concerns with continued use of animals in basic biomedical, pharmacological, and regulatory research to provide safety assessments for drugs and chemicals. There are concerns that animals do not provide sufficient information on toxicity and/or efficacy to protect the target population, so scientists are utilizing the principles of replacement, reduction, and refinement (the 3Rs) and increasing the development and application of new approach methods (NAMs). NAMs are any technology, methodology, approach, or assay used to understand the effects and mechanisms of drugs or chemicals, with specific focus on applying the 3Rs. Although progress has been made in several areas with NAMs, complete replacement of animal models with NAMs is not yet attainable. The road to NAMs requires additional development, increased use, and, for regulatory decision making, usually formal validation. Moreover, it is likely that replacement of animal models with NAMs will require multiple assays to ensure sufficient biologic coverage. The purpose of this manuscript is to provide a balanced view of the current state of the use of animal models and NAMs as approaches to development, safety, efficacy, and toxicity testing of drugs and chemicals. Animals do not provide all needed information nor do NAMs, but each can elucidate key pieces of the puzzle of human and animal biology and contribute to the goal of protecting human and animal health. SIGNIFICANCE STATEMENT: Data from traditional animal studies have predominantly been used to inform human health safety and efficacy. Although it is unlikely that all animal studies will be able to be replaced, with the continued advancement in new approach methods (NAMs), it is possible that sometime in the future, NAMs will likely be an important component by which the discovery, efficacy, and toxicity testing of drugs and chemicals is conducted and regulatory decisions are made.

HESI workshop summary: Interpretation of developmental and reproductive toxicity endpoints and the impact on data interpretation of adverse events.

The Health and Environmental Sciences Institute Developmental and Reproductive Toxicology (HESI-DART) group held a hybrid in-person and virtual workshop in Washington, DC, in 2022. The workshop was entitled, "Interpretation of DART in Regulatory Contexts and Frameworks." There were 154 participants (37 in person and 117 virtual) across 9 countries. The purpose of the workshop was to capture key consensus approaches used to assess DART risks associated with chemical product exposure when a nonclinical finding is identified. The decision-making process for determining whether a DART endpoint is considered adverse is critical because the outcome may have downstream implications (e.g., increased animal usage, modifications to reproductive classification and pregnancy labeling, impact on enrollment in clinical trials and value chains). The workshop included a series of webinar modules to train and engage in discussions with federal and international regulators, clinicians, academic investigators, nongovernmental organizations, contract research organization scientists, and private sector scientists on the best practices and principles of interpreting DART and new approach methodologies in the context of regulatory requirements and processes. Despite the differences in regulatory frameworks between the chemical and pharmaceutical sectors, the same foundational principles for data interpretation should be applied. The discussions led to the categorization of principles, which offer guidance for the systematic interpretation of data. Step 1 entails identifying any hazard by closely analyzing the data at the study endpoint level, while Step 2 involves assessing risk using weight of evidence. These guiding principles were derived from the collective outcomes of the workshop deliberations.

Evaluation of gadolinium-based contrast agents in juvenile CD-1 mice including behavioral evaluations.

INTRODUCTION: Seven gadolinium-based contrast agents (GBCAs), four linear and three macrocyclic, were evaluated for potential effects on development, including behavior of juvenile CD-1 mice. METHODS: The GBCAs were administered via intravenous injection once daily on postnatal day (PND) 9, 12, 15, 18, and 21 (PND 1 was the day of delivery) at doses up to twice the human equivalent clinical dose (i.e., 0.63 mmol Gd/kg for gadoxetate disodium and 2.5 mmol Gd/kg for the other GBCAs). Mice were bled for evaluation of exposure (plasma) to gadolinium (Gd) on PND 9, 12, and 70. At scheduled euthanasia, the liver, spleen, brain, skin (dorsal surface), bone (left femur), and kidneys were excised from up to six mice/sex/group on PND 10, 22, or 70 for the determination of Gd levels and histopathological analysis. All mice were monitored for toxicity, growth and survival, sexual maturation, and behavior. CONCLUSION: Gd was quantifiable in the brain tissues with levels declining over time. There was no long-term effect on the growth and development for mice exposed to any of the GBCAs. There was no impact on neurodevelopment as assessed by brain histology and validated neurobehavioral tests, including a functional observational battery, motor activity, and learning and memory as evaluated in the Morris water maze. For all GBCAs, the highest dose tested represented the no-observable-adverse-effect level in juvenile mice.

Evaluation of gadolinium-based contrast agents in pregnant CD-1 mice and subsequent in utero exposure of the developing offspring, including behavioral evaluations.

INTRODUCTION: The offspring of CD-1 mice exposed during pregnancy to one of seven gadolinium-based contrast agents (GBCAs) were evaluated for potential effects on postnatal development and behavior. The GBCAs, comprising four linear (gadopentetate dimeglumine, gadodiamide, gadobenate dimeglumine, and gadoxetate disodium) and three macrocyclic (gadoterate meglumine, gadoteridol, and gadobutrol), were administered via intravenous injection once daily from Gestation Day 6 through 17 following confirmed mating (Day 0) at doses of at least twice the human equivalent recommended clinical dose (i.e., 0.63 mmol Gd/kg for gadoxetate disodium and 2.5 mmol Gd/kg for the other GBCAs). All dams were allowed to deliver naturally. F0 generation females were monitored for maternal toxicity and gadolinium (Gd) levels in blood and brain. Offspring were evaluated for Gd levels in blood and brain at birth and on Day 70 postpartum. F1 generation mice were evaluated for survival and growth preweaning. Selected pups/litter were evaluated postweaning for sexual maturation, growth, and behavior. Gd was quantifiable in the brain of the F1 offspring on PND 1, with levels declining over time. There was no long-term effect of any GBCA on the growth and development of any offspring. There was no impact on neurodevelopment, as assessed by brain histology and validated neurobehavioral tests, including a battery of functional observational tests, motor activity, and learning and memory as evaluated in the Morris water maze. CONCLUSION: At the end of the postweaning period, the highest dose tested was considered the no-observable-adverse-effect level (NOAEL) in the F0 and F1 offspring for all tested GBCAs.

Alternatives to Monkey Reproductive Toxicology Testing for Biotherapeutics.

Embryofetal toxicity studies are conducted to support inclusion of women of childbearing potential in clinical trials and to support labeling for the marketed pharmaceutical product. For biopharmaceuticals, which frequently lack activity in the rodent or rabbit, the nonhuman primate is the standard model to evaluate embryofetal toxicity. These studies have become increasingly challenging to conduct due to the small number of facilities capable of performing them and a shortage of sexually mature monkeys. The low number of animals per group and the high rate of spontaneous abortion in cynomolgus monkeys further complicate interpretation of the data. Recent FDA guidance has proposed a weight of evidence (WoE) approach to support product labeling for reproductive toxicity of products intended to be used for the treatment of cancer (Oncology Pharmaceuticals: Reproductive Toxicity Testing and Labeling Recommendations), an approach that has also supported the approval of biotherapeutics for non-cancer indications. Considerations to determine the appropriateness and content of a WoE approach to support product labeling for embryofetal risk include known class effects in humans; findings from genetically modified animals with or without drug administration; information from surrogate compounds; literature-based assessments about the developmental role of the pharmaceutical target; and the anticipated exposure during embryofetal development. This paper summarizes the content of a session presented at the 42nd annual meeting at the American College of Toxicology, which explored the conditions under which alternative approaches may be appropriate to support product labeling for reproductive risk, and how sponsors can best justify the use of this approach.

Sodium molybdate dihydrate does not exhibit developmental or reproductive toxicity in Sprague-Dawley rats maintained on a marginal copper diet.

Groups of 24 weanling female Sprague-Dawley rats were administered molybdenum (Mo) as sodium molybdate dihydrate (SMD) in drinking water at target dose levels of 0, 20, or 40 mg Mo/kg bw/day and fed a semi-purified marginal copper (6.2 ppm Cu) AIN-93 G diet for 8 weeks prior to mating, through cohabitation and pregnancy until Gestation Day 21. The objective was to confirm the reproductive and developmental effects of SMD reported by Fungwe et al. (1990) at estimated doses as low as 1.5 mg Mo/kg bw/day in a similarly designed study with marginal Cu diet (6.3 ppm). There were no test material-related effects at 20 or 40 mg Mo/kg bw/day on mortality, clinical observations, body weight, body weight gain, food consumption, estrous cycling, reproductive performance, maternal macroscopic pathology, ovarian or uterine parameters, litter size, resorptions, fetal sex ratio, fetal weight, or external fetal malformations or variations. Water consumption was increased compared to controls at both dose levels during the pre-mating and gestation periods, with no apparent adverse impact. There was no evidence of copper depletion in serum at any dose level. In conclusion, the no-observed-adverse-effect levels (NOAELs) for systemic, maternal reproductive, and developmental toxicity in this marginal Cu diet study are 40 mg Mo/kg bw/day, consistent with the results of guideline developmental and reproductive toxicity studies of SMD. The results of Fungwe et al. were not replicated, even at higher dose levels of Mo, and their inconsistencies with guideline toxicity studies of Mo are not explained by the marginal dietary Cu level.

Developmental and reproduction toxicity of piperonyl butoxide part 2 developmental safety of piperonyl butoxide in the NZW rabbit.

Piperonyl butoxide (PBO) was developed in the 1940s. PBO increases the effectiveness of pyrethrins, thus it is called a synergist. Herein, the findings from a guideline developmental toxicity study in rabbits conducted in 1986 are reported. Inseminated New Zealand White rabbits were randomly assigned to a control and three treatment groups of 16 does each. Dose levels of 50, 100 and 200 mg/kg/day were selected based on a dosage-range study to avoid excessive maternal toxicity and administered orally (gavage) as a single daily dose on days 7-19 of gestation at a volume of 0.5 mL/kg. The control group received the vehicle only, Mazola® corn oil. Cesarean sections were performed on all surviving females on gestation day 29 and fetuses were evaluated. Survival for all study groups was 100%. Treatment-related maternotoxicity was manifested at the 100 and 200 mg/kg/day levels as decreased defecation and dose-related body weight losses during the treatment period (gestation days 7-13 and 7-19). The Cesarean section parameter values and fetal morphological observations of the treated groups did not differ significantly from the concurrent control group and were within the historical control range for this rabbit strain. No maternal or fetal adverse effects were seen at the 50 mg/kg/day dose level. Although maternal toxicity resulting from treatment was apparent at the 100 and 200 mg/kg/day dose levels, neither fetotoxicity nor teratogenicity were elicited in rabbits by piperonyl butoxide at dose levels as high as 200 mg/kg/day.

Developmental and reproduction toxicity of piperonyl butoxide part 3 two generation (two litter) reproduction study of piperonyl butoxide administered in the diet to CD ® (Sprague Dawley) rats.

Piperonyl butoxide (PBO) an insecticide synergist was evaluated in a guideline multigenerational toxicity study in rats. F0 and F1 adult generations consisted of groups of 26 male and 26 female CD (Sprague Dawley) rats that were exposed to PBO in the diet at concentrations of 0, 300, 1000 or 5000 ppm for 85 (F0) or 83 (F1) days prior to cohabitation and throughout two mating periods (F1a, F2a and F1b, F2b). Exposure to test diets continued through the mating, gestation, and lactation periods for the females. F2 generation pups were euthanized following weaning. There were no effects on survival, clinical observations, gross or histological findings, fertility, pup viability, lactation indices or sex ratio in adults or pups in any generation. All effects of PBO occurred in the 5000-ppm exposure group. These effects included reduced body weight gains for F0 and F1 males and females during pre-cohabitation resulting in reduced body weights during both gestation periods. Food consumption of the F1b group males was slightly or significantly less than control values from week 3 onward. F1a generation pup weights were reduced on days 4, 7, 14 and 21 postpartum. Pup weights in the F1 and F2 generations were significantly reduced on days 14 and 21 postpartum when diets were being consumed by pups. The no-observable-adverse-effect level (NOAEL) for general toxicity was 1000 ppm based on reductions in body weights (parental and offspring) at 5000 ppm; and the NOAEL for reproductive toxicity was 5000 ppm with no direct effects on reproduction.

Developmental and reproduction toxicity of piperonyl butoxide part 1 developmental safety of piperonyl butoxide in the CD® (Sprague Dawley) rat.

Developed as an insecticide synergist in the early 1940s, PBO increases the effectiveness of pyrethrins. Herein, the findings from a guideline developmental toxicity study in rat conducted in 1991 are reported. Timed-pregnant CD® (Sprague Dawley) rats were randomly assigned to a control and three treatment groups of 25 females each. A single daily dose of 200, 500 and 1000 mg/kg/day was administered orally (gavage) on days 6-15 of gestation. A vehicle group received deionized water. Cesarean sections were performed on all surviving females on gestation day 21 and fetuses were evaluated. All rats survived to GD 21 of gestation. Pregnancy rates in each group ranged from 88 % to 96 %. One dam in the 500 mg/kg/day dose had a single conceptus litter (one early resorption). Adverse clinical observations (urogenital wetness and staining) occurred in the 1000 mg/kg/day dose group. Maternal body weight decrease and food reductions occurred over the dosing period in the 500 and 1000 mg/kg/day groups. There were no treatment-related maternal necropsy findings. Terminal body weights and gravid uterine weights were comparable among the groups. Corrected body weight gain was decreased (>10 %) at 500 and 1000 mg/kg/day. Increased liver weights and relative liver weights were observed in the 1000 mg/kg/day dose group. There were no treatment-related effects on early resorptions, late resorptions, live fetuses per litter or sex ratio, or fetal weight per litter among the dose groups and no fetal malformations or variations attributed to PBO at any dose level.

Regulatory Perspectives on Juvenile Animal Toxicologic Pathology.

The Society of Toxicologic Pathology's Annual Virtual Symposium (2021) included a session on "Regulatory Perspectives on Juvenile Animal Toxicologic Pathology." The following narrative summarizes the key concepts from the four talks included in this symposium session chaired by Drs Deepa Rao and Alan Hoberman. These encompass an overview of various global regulations impacting the conduct of juvenile animal studies in pharmaceutical drug development and chemical toxicity assessments in a talk by Dr Alan Hoberman. Given the numerous regulatory guidances and legal statutes that have covered the conduct of juvenile animal studies and the recent harmonization of these guidances for pharmaceuticals, Dr Paul Brown provided an update on the harmonization of these guidances for pharmaceuticals, in the recently finalized version of the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use S11 guidance document, "Nonclinical Safety Testing in Support of Development of Pediatric Medicines." The first two talks on regulations were followed by two talks focused on an evaluation of the postnatal development of two major organ systems relevant in juvenile animals. Dr Aurore Varela covered study design and endpoints impacting the skeletal system (bone), while Dr Brad Bolon presented a talk on the study design and conduct of neuropathology evaluations for the developing nervous system.

Correction to: Letter to the editor regarding "safety of safety evaluation of pesticides: developmental neurotoxicity of chlorpyrifos and chlorpyrifos-methyl" by Mie et al. (environmental health. 2018. 17:77).

Following publication of the original article.

Addendum to Iwai and Hoberman (2014)-Reassessment of Developmental Toxicity of PFHxA in Mice.

This article presents a supplemental data analysis and evaluation of the findings from an oral (gavage) combined developmental and perinatal/postnatal reproduction toxicity study of the ammonium salt of perfluorohexanoic acid (CASRN: 21615-47-4) in Crl: CD-1(ICR) mice. The original study has been cited as supporting a lowest-observed-adverse-effects level of 175 mg/kg/d and no-observed-adverse-effects level of 35 mg/kg/d for developmental effects from perfluorohexanoic acid (PFHxA, CASRN: 307-24-4) in mice. The statistical analysis reported in 2014 was accurate in terms of quantifying statistical significance within phase 2 of the study. However, given the low incidence of findings, the purpose of this article is to extend the analysis and interpretation of findings by pooling the control group information from both phases of the same study, comparing the study findings to the incidence rates for stillbirths and postpartum viability for this species and strain of mouse observed for similar studies conducted by the same laboratory, and evaluating data on the incidence and range of spontaneous eye abnormalities reported in the literature. Based on this supplemental evaluation, the original study supports a NOAEL of 175 mg/kg/d for PFHxA in mice, which is a factor of 5-fold higher than previously reported. Furthermore, to the extent that this study may be considered in the selection of a point of departure for PFHxA in mice, it is noted that 175 mg/kg/d for maternal exposure is an unbounded NOAEL for developmental effects, meaning that the study did not establish a dose at which developmental effects may occur.

A multi-institutional study benchmarking the zebrafish developmental assay for prediction of embryotoxic plasma concentrations from rat embryo-fetal development studies.

Predicting embryotoxicity of pharmaceutical compounds or industrial chemicals is crucial for public safety. Conventional studies which monitor embryo-fetal development in rats and rabbits are costly and time consuming. Alternative assays which are simpler and less costly are being pursued. The purpose of this research was to assess the capacity for the zebrafish development assay to predict mammalian plasma levels that are embryotoxic. Previously published data on rat plasma levels associated with embryotoxicity were used to guide concentration ranges for each of 25 chemicals dissolved in the media bathing developing zebrafish embryos. Embryotoxic media concentrations were compared to embryotoxic rat plasma concentrations. Assays were conducted in parallel at multiple sites as a consortium effort through the Health and Environmental Sciences Institute (HESI). Considering results from all sites, the zebrafish embryo development assay predicted (within 1-log) the rat maternal exposure levels associated with embryotoxicity 75% of the time.

A two-generation reproductive toxicity study of sodium molybdate dihydrate administered in drinking water or diet to Sprague-Dawley rats.

In an OECD Test Guideline 416 multigenerational study, groups of 24 male and 24 female Sprague-Dawley rats were administered sodium molybdate dihydrate at 0, 5, 17, or 40 mg molybdenum (Mo)/kg bw/day in the drinking water or 40 mg Mo/kg bw/day in the diet over two generations to assess reproductive toxicity. No adverse effect on reproductive function was observed at any dose level in either generation as indicated by no significant dose-related effect on estrus cycles, sperm parameters, mating, fertility, gestation, litter size, pup survival, growth or postnatal development. Systemic toxicity, including decreased body weight, food consumption (males only) and water consumption, was observed among both sexes given 40 mg Mo/kg bw/day in the diet. Serum levels of Mo and copper were increased in a dose-related manner. The No Observed Adverse Effect Levels (NOAEL) are 17 mg Mo/kg bw/day for systemic toxicity and 40 mg Mo/kg bw/day for reproductive toxicity.

The urgency for optimization and harmonization of thyroid hormone analyses and their interpretation in developmental and reproductive toxicology studies.

In recent years several OECD test guidelines have been updated and some will be updated shortly with the requirement to measure thyroid hormone levels in the blood of mammalian laboratory species. There is, however, an imperative need for clarification and guidance regarding the collection, assessment, and interpretation of thyroid hormone data for regulatory toxicology and risk assessment. Clarification and guidance is needed for 1) timing and methods of blood collection, 2) standardization and validation of the analytical methods, 3) triggers for additional measurements, 4) the need for T4 measurements in postnatal day (PND) 4 pups, and 5) the interpretation of changes in thyroid hormone levels regarding adversity. Discussions on these topics have already been initiated, and involve expert scientists from a number of international multisector organizations. This paper provides an overview of existing issues, current activities and recommendations for moving forward.

Rethinking developmental toxicity testing: Evolution or revolution?

BACKGROUND: Current developmental toxicity testing adheres largely to protocols suggested in 1966 involving the administration of test compound to pregnant laboratory animals. After more than 50 years of embryo-fetal development testing, are we ready to consider a different approach to human developmental toxicity testing? METHODS: A workshop was held under the auspices of the Developmental and Reproductive Toxicology Technical Committee of the ILSI Health and Environmental Sciences Institute to consider how we might design developmental toxicity testing if we started over with 21st century knowledge and techniques (revolution). We first consider what changes to the current protocols might be recommended to make them more predictive for human risk (evolution). RESULTS: The evolutionary approach includes modifications of existing protocols and can include humanized models, disease models, more accurate assessment and testing of metabolites, and informed approaches to dose selection. The revolution could start with hypothesis-driven testing where we take what we know about a compound or close analog and answer specific questions using targeted experimental techniques rather than a one-protocol-fits-all approach. Central to the idea of hypothesis-driven testing is the concept that testing can be done at the level of mode of action. It might be feasible to identify a small number of key events at a molecular or cellular level that predict an adverse outcome and for which testing could be performed in vitro or in silico or, rarely, using limited in vivo models. Techniques for evaluating these key events exist today or are in development. DISCUSSION: Opportunities exist for refining and then replacing current developmental toxicity testing protocols using techniques that have already been developed or are within reach.