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.

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.

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.

Comparing rat and rabbit embryo-fetal developmental toxicity data for 379 pharmaceuticals: on systemic dose and developmental effects.

A database of embryo-fetal developmental toxicity (EFDT) studies of 379 pharmaceutical compounds in rat and rabbit was analyzed for species differences based on toxicokinetic parameters of area under the curve (AUC) and maximum concentration (Cmax) at the developmental lowest adverse effect level (dLOAEL). For the vast majority of cases (83% based on AUC of n = 283), dLOAELs in rats and rabbits were within the same order of magnitude (less than 10-fold different) when compared based on available data on AUC and Cmax exposures. For 13.5% of the compounds the rabbit was more sensitive and for 3.5% of compounds the rat was more sensitive when compared based on AUC exposures. For 12% of the compounds the rabbit was more sensitive and for 1.3% of compounds the rat was more sensitive based on Cmax exposures. When evaluated based on human equivalent dose (HED) conversion using standard factors, the rat and rabbit were equally sensitive. The relative extent of embryo-fetal toxicity in the presence of maternal toxicity was not different between species. Overall effect severity incidences were distributed similarly in rat and rabbit studies. Individual rat and rabbit strains did not show a different general distribution of systemic exposure LOAELs as compared to all strains combined for each species. There were no apparent species differences in the occurrence of embryo-fetal variations. Based on power of detection and given differences in the nature of developmental effects between rat and rabbit study outcomes for individual compounds, EFDT studies in two species have added value over single studies.

Oral 1-Generation Rat Reproduction Study of Isobornyl Acetate: An Evaluation Through Sexual Maturity in the F1 Generation.

Reproductive toxicity of isobornyl acetate (IA), a widely used fragrance ingredient, was investigated in a 1-generation reproduction study in which 25 Crl: CD (Sprague-Dawley) rats/sex/group were gavaged with dosages of 0 (corn oil vehicle), 30, 100, or 300 mg/kg/d during premating, mating, gestation, and lactation. After weaning, 25 F1 generation pups/sex/dosage group were randomly selected for evaluation until sexual maturity. The following parameters were evaluated in P generation males and females: viability, clinical signs, body weights, feed consumption, mating and fertility, organ weights, gross and microscopic observations, sperm assessments (motility and concentration), natural delivery and litter observations, and ovarian follicle counts. In F1 generation pups, viability, body weights, sexual maturation, anogenital distance (days 1 and 22 postpartum), nipple eruption (day 12 postpartum), and gross necropsy observations were recorded. Isobornyl acetate did not adversely affect any of the investigated parameters. Based on the results of this investigation, the no observable adverse effect level (NOAEL) for toxicity of IA is considered to be 300 mg/kg/d. Increased incidences of excess salivation occurred in P generation male and female rats at 100 and/or 300 mg/kg/d throughout the dosage period, and low incidences of urine-stained abdominal fur were seen in females at 300 mg/kg/d during the gestation period. These clinical signs were not considered as adverse effects of IA administration. Thus, the NOAEL for reproductive toxicity in the P generation rats and the NOAEL for viability and growth of the F1 generation offspring is considered to be ≥300 mg/kg/d.

Comparison of rat and rabbit embryo-fetal developmental toxicity data for 379 pharmaceuticals: on the nature and severity of developmental effects.

Regulatory non-clinical safety testing of human pharmaceuticals typically requires embryo-fetal developmental toxicity (EFDT) testing in two species (one rodent and one non-rodent). The question has been raised whether under some conditions EFDT testing could be limited to one species, or whether the testing in a second species could be decided on a case-by-case basis. As part of a consortium initiative, we built and queried a database of 379 compounds with EFDT studies (in both rat and rabbit animal models) conducted for marketed and non-marketed pharmaceuticals for their potential for adverse developmental and maternal outcomes, including EFDT incidence and the nature and severity of adverse findings. Manifestation of EFDT in either one or both species was demonstrated for 282 compounds (74%). EFDT was detected in only one species (rat or rabbit) in almost a third (31%, 118 compounds), with 58% (68 compounds) of rat studies and 42% (50 compounds) of rabbit studies identifying an EFDT signal. For 24 compounds (6%), fetal malformations were observed in one species (rat or rabbit) in the absence of any EFDT in the second species. In general, growth retardation, fetal variations, and malformations were more prominent in the rat, whereas embryo-fetal death was observed more often in the rabbit. Discordance across species may be attributed to factors such as maternal toxicity, study design differences, pharmacokinetic differences, and pharmacologic relevance of species. The current analysis suggests that in general both species are equally sensitive on the basis of an overall EFDT LOAEL comparison, but selective EFDT toxicity in one species is not uncommon. Also, there appear to be species differences in the prevalence of various EFDT manifestations (i.e. embryo-fetal death, growth retardation, and dysmorphogenesis) between rat and rabbit, suggesting that the use of both species has a higher probability of detecting developmental toxicants than either one alone.

Micro-CT imaging: Developing criteria for examining fetal skeletons in regulatory developmental toxicology studies - A workshop report.

During the past two decades the use and refinements of imaging modalities have markedly increased making it possible to image embryos and fetuses used in pivotal nonclinical studies submitted to regulatory agencies. Implementing these technologies into the Good Laboratory Practice environment requires rigorous testing, validation, and documentation to ensure the reproducibility of data. A workshop on current practices and regulatory requirements was held with the goal of defining minimal criteria for the proper implementation of these technologies and subsequent submission to regulatory agencies. Micro-computed tomography (micro-CT) is especially well suited for high-throughput evaluations, and is gaining popularity to evaluate fetal skeletons to assess the potential developmental toxicity of test agents. This workshop was convened to help scientists in the developmental toxicology field understand and apply micro-CT technology to nonclinical toxicology studies and facilitate the regulatory acceptance of imaging data. Presentations and workshop discussions covered: (1) principles of micro-CT fetal imaging; (2) concordance of findings with conventional skeletal evaluations; and (3) regulatory requirements for validating the system. Establishing these requirements for micro-CT examination can provide a path forward for laboratories considering implementing this technology and provide regulatory agencies with a basis to consider the acceptability of data generated via this technology.

Tavaborole, a Novel Boron-Containing Small Molecule Pharmaceutical Agent for Topical Treatment of Onychomycosis: I. Reproductive and Developmental Toxicity Studies.

Tavaborole is a topical antifungal agent approved by the US Food and Drug Administration for the treatment of toenail onychomycosis. As part of the nonclinical development program, reproductive and developmental toxicity studies were conducted (rat oral fertility and early embryonic development, rat (oral) and rabbit (dermal) embryo-fetal development). There were no effects on fertility or reproductive performance at doses up to 300 mg/kg/d (107 times the maximum recommended human dose [MRHD] based on mean area under the plasma concentration-time curve comparisons). In the rat embryo-fetal development toxicity studies, teratogenicity was not observed at doses up to 100 mg/kg/d (29 times the MRHD). However, several treatment-related skeletal malformations and variations were observed at 300 mg/kg/d (570 times the MRHD). In rabbit embryo-fetal development toxicity studies dosed via oral or dermal administration, the no observable adverse effect level for maternal toxicity and embryo-fetal toxicity was 50 mg/kg/d (16 times the MRHD) and 5% (26 times the MRHD), respectively.

Tavaborole, a Novel Boron-Containing Small Molecule Pharmaceutical Agent for Topical Treatment of Onychomycosis: II. Prenatal and Postnatal Developmental Toxicity and Maternal Function Study.

Tavaborole is a topical antifungal agent approved by the US Food and Drug Administration for the treatment of toenail onychomycosis. The effects of tavaborole on gestation, parturition (delivery, labor), offspring development, and survival during the perinatal and postnatal periods were assessed in mated female rats. Females (F0 generation) were administered single daily oral (gavage) doses of 15, 60, or 100 mg/kg/d from gestation day 6 through lactation day 20. The females were allowed to deliver naturally and rear their offspring until lactation day 21, at which time the F0 females were euthanized. One male and female from each litter were selected (F1 generation) and retained for assessments, including growth, neurobehavior, fertility, and their ability to produce an F2 generation. Reproductive and offspring parameters were determined for the F1 and F2 generations, as applicable. F1 females and F2 pups were euthanized on postnatal day 7. In the F0 females, decreased activity was observed in the 100 mg/kg/d dose group. Excess salivation was observed in the 60 and 100 mg/kg/d dose groups (slight to moderate), however, this finding was not considered adverse. There were no tavaborole-related effects on the growth, viability, development, neurobehavioral assessments, or reproductive performance of the F1 generation. Survivability and mean body weight of the F2 pups were unaffected. The no observed adverse effect level (NOAEL) for maternal toxicity (F0 generation) was 60 mg/kg/d, based on the decreased activity observed in the 100 mg/kg/d dose group. The NOAEL for the offspring effects was ≥100 mg/kg/d, based on the lack of test article-related changes.

Oral (Gavage) Combined Developmental and Perinatal/Postnatal Reproduction Toxicity Study of Ammonium Salt of Perfluorinated Hexanoic Acid in Mice.

The reproductive toxicity potential of Ammonium Salt of Perfluorinated Hexanoic Acid (PFHxA Ammonium Salt) in pregnant Crl: CD1(ICR) mice was investigated. Twenty females/group were administered the test substance or vehicle once daily from gestation day 6 through 18. Phase 1 doses: 0, 100, 350, and 500 mg/kg/d; phase 2: 0, 7, 35, and 175 mg/kg/d. Parameters evaluated include mortality, viability, body weights, clinical signs, abortions, premature deliveries, pregnancy and fertility, litter observations, maternal behavior, and sexual maturity in the F1 generation. The level of PFHxA Ammonium Salt was measured in the liver of F0 and F1 mice. At doses of 350 and 500 mg/kg/d maternal mortalities, excess salivation and changes in body weight gains occurred. Pup body weights were reduced on postpartum day (PPD) 0 in all the dosage groups, but persisted only in the 350 and 500 mg/kg/d groups. Additional effects at 300 and 500 mg/kg/d included stillbirths, reductions in viability indices, and delays in physical development. Levels of PFHxA Ammonium Salt in the livers of the 100 mg/kg/d dams were all below the lower limit of quantization (0.02 µg/mL); in the 350 mg/kg/d group, 3 of the 8 samples had quantifiable analytical results. In phase 2 no PFHxA Ammonium Salt was found in the liver. Adverse effects occurred only in the 175 mg/kg/d group and consisted of increased stillborn pups, pups dying on PPD 1, and reduced pup weights on PPD 1. Based on these data, the maternal and reproductive no observable adverse effect level of PFHxA Ammonium Salt is 100 mg/kg/d.

Reproductive toxicity evaluation of the dental resin monomer triethylene glycol dimethacrylate (CASRN 109-16-0) in mice.

The reproductive toxicity potential of the resin monomer triethylene glycol dimethacrylate (TEGDMA; Chemical Abstracts Service Registry Number 109-16-0) was investigated in male and female Crl:CD1(ICR) mice, 4 dosage groups, 25 mice/sex/group. Formulations of TEGDMA (0, 0.01, 0.1, or 1.0 mg/kg/d) in reverse osmosis-processed deionized water were intubated once daily beginning 28 days before cohabitation and continuing through mating (males) or through gestation day 17 (females). The following parameters were evaluated: viability, clinical signs, body weights, estrous cyclicity, necropsy observations, organ weights, sperm concentration/motility/morphology, cesarean-sectioning and litter observations, and histopathological evaluation of select tissues. No deaths or clinical signs related to TEGDMA occurred. No significant changes in male and female body weights and body weight gains were recorded for any of the administered dosages of TEGMDA. All mating and fertility parameters and all litter and fetal data were considered to be unaffected by dosages of TEGMDA as high as 1 mg/kg/d. Gross or histopathologic tissue changes attributable to the test article were not observed. Reproductive and developmental no observed adverse effect levels (NOAELs) for TEGMDA were 1.0 mg/kg/d, the highest dose tested. Comparison of conservatively estimated TEGDMA exposures from dental treatments to the NOAEL of 1.0 mg/kg/d identified in this study indicates margins of exposure of at least 120- to 3000-fold depending on the exposure scenario. The results of this study support the continued safe use of TEGDMA in polymeric dental products applied according to the manufacturers' instructions.

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.

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.

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.

Reproductive toxicity evaluation of the dental resin monomer bisphenol a glycidyl methacrylate (CAS 1565-94-2) in mice.

The reproductive toxicity potential of the dental resin monomer bisphenol A glycidyl methacrylate (BisGMA; CASRN 1565-94-2) was investigated in male and female Crl: CD1(ICR) mice, 4 dosage groups, and 25 mice/sex/group. Formulations of BisGMA (0, 0.008, 0.08, or 0.8 mg/kg/d) in 0.8% ethanol in deionized water were intubated once daily beginning 28 days before cohabitation and continuing through mating (males) or through gestation day 17. The following parameters were evaluated: viability, clinical signs, body weights, estrous cyclicity, necropsy observations, organ weights, sperm concentration/motility/morphology, cesarean sectioning and litter observations, and histopathological evaluation of select tissues. No deaths or clinical signs related to BisGMA occurred. No significant changes in male and female body weights and body weight gains were recorded at any of the administered dosages of BisGMA. All mating and fertility parameters, and all litter and fetal data, were considered to be unaffected by dosages of BisGMA as high as 0.8 mg/kg/d. Gross or histopathologic tissue changes attributable to the test article were not observed. Reproductive and developmental no observed effect levels (NOAELs) for BisGMA were 0.8 mg/kg/d, the highest dose tested. Comparison of this NOAEL value to published probabilistic estimates of human BisGMA exposure from dental products suggests a margin of safety of at least 280- to nearly 2000-fold. Under the conditions of this study, BisGMA is not a reproductive toxicant.

Uterotrophic assay of percutaneous lavender oil in immature female rats.

The estrogenic potential of lavender oil was evaluated in a percutaneous uterotrophic bioassay in immature female rats. Four groups of 10 immature female rats each were randomly selected on postpartum day (PPD) 16. During the 3-day treatment period (PPDs 19-21), the immature rats were separated from the dams, caged in groups of 5 in a litter box for 6 hours, and administered the vehicle control article (corn oil) or lavender oil at 20 or 100 mg/kg per day. All dosages were administered as a 5 mL/kg volume in a Hilltop Chamber (25 mm diameter; absorbent material removed) placed on the shaved back of each immature rat, and secured with micropore tape and Vetrap. A positive control group was gavaged twice daily with 2.5 µg/kg per day of 17α-ethinyl estradiol. Daily observations included viability, clinical signs, body weights, and body weight gains. All rats were euthanized 24 hours after the third and final treatment, the uteri and ovaries were removed, and the paired ovaries and wet and blotted uterine weights were recorded. No unscheduled deaths occurred. No skin reactions were observed. Both dosages of lavender oil significantly reduced body weight gains after the third day of treatment, but terminal body weights and mean absolute and relative uterine weights did not differ significantly from vehicle control values. Positive controls showed significant increases in body weight and increased mean absolute and relative uterine weights as expected. Based on these data, lavender oil, at dosages of 20 or 100 mg/kg, was not active in the rat uterotrophic assay and gave no evidence of estrogenic activity.