Regulatory Testing Research Articles

Variation among hemp (Cannabis sativus L.) analytical testing laboratories evinces regulatory and quality control issues for the industry

Validated analytical testing of cannabinoid content for regulatory purposes is critical to farming high-cannabidiol (CBD) hemp (Cannabis sativus L.), as these methods are key to determine whether a crop is federally compliant by containing < 0.3% THC or must be destroyed at the time of harvest. This report identifies the sources and extent of variation in reported cannabinoid content after flower selection and conducted a survey of ten accredited hemp regulatory testing laboratories, including one state-owned laboratory. The results indicate that total tetrahydrocannabinol (THC) content is reported inconsistently due to an insufficient standardization of sample preparation and testing methods, thus likely leading to erroneous data. This work represents the early phase of ongoing research to enhance the consistency of cannabinoid analyses of hemp flower samples as an essential tool in Cannabis crop development.

A Transformative Vision for an Omics-Based Regulatory Chemical Testing Paradigm.

Use of molecular data in human and ecological health risk assessments of industrial chemicals and agrochemicals has been anticipated by the scientific community for many years; however, these data are rarely used for risk assessment. Here, a logic framework is proposed to explore the feasibility and future development of transcriptomic methods to refine and replace the current apical endpoint-based regulatory toxicity testing paradigm. Four foundational principles are outlined and discussed that would need to be accepted by stakeholders prior to this transformative vision being realized. Well-supported by current knowledge, the first principle is that transcriptomics is a reliable tool for detecting alterations in gene expression that result from endogenous or exogenous influences on the test organism. The second principle states that alterations in gene expression are indicators of adverse or adaptive biological responses to stressors in an organism. Principle 3 is that transcriptomics can be employed to establish a benchmark dose-based point of departure (POD) from short-term, in vivo studies at a dose level below which a concerted molecular change (CMC) is not expected. Finally, Principle 4 states that the use of a transcriptomic POD (set at the CMC dose level) will support a human health-protective risk assessment. If all four principles are substantiated, this vision is expected to transform aspects of the industrial chemical and agrochemical risk assessment process that are focused on establishing safe exposure levels for mammals across numerous toxicological contexts resulting in a significant reduction in animal use while providing equal or greater protection of human health. Importantly, these principles and approaches are also generally applicable for ecological safety assessment.

Tumorigenicity Assessment of Human Cancer Cell Lines Xenografted on Immunodeficient Mice as Positive Controls of Tumorigenicity Testing.

Recent advances in human pluripotent stem cell (hPSC)-derived cell therapies and genome editing technologies such as CRISPR/Cas9 make regenerative medicines promising for curing diseases previously thought to be incurable. However, the possibility of off-target effects during genome editing and the nature of hPSCs, which can differentiate into any cell type and infinitely proliferate, inevitably raises concerns about tumorigenicity. Tumorigenicity acts as a major obstacle to the application of hPSC-derived and gene therapy products in clinical practice. Thus, regulatory authorities demand mandatory tumorigenicity testing as a key pre-clinical safety step for the products. In the tumorigenicity testing, regulatory guidelines request to include human cancer cell line injected positive control group (PC) animals, which must form tumors. As the validity of the whole test is determined by the tumor-forming rates (typically above 90%) of PC animals, establishing the stable tumorigenic condition of PC animals is critical for successful testing. We conducted several studies to establish the proper positive control conditions, including dose, administration routes, and the selection of cell lines, in compliance with Good Laboratory Practice (GLP) regulations and/or guidelines, which are essential for pre-clinical safety tests of therapeutic materials. We expect that our findings provide insights and practical information to create a successful tumorigenicity test and its guidelines.

A History of Regulatory Animal Testing: What Can We Learn?

The contemporary pharmaceutical industry is voicing growing concerns about the translatability and reproducibility of animal models. In addition, the usefulness of certain of the required regulatory safety tests in animals is being increasingly questioned. It remains difficult, however, to make the move toward alternative testing methods, not least because of legislative demands. A historical analysis was performed, in order to study how the mandatory animal studies in legislative requirements came about. This article reflects on the role that specific public health disasters played in the creation of (more) regulatory requirements for animal testing. It will show how the regulatory changes prompted by the sulfanilamide elixir disaster in the 1930s and the thalidomide disaster in the early 1960s were based on the belief that extensive animal testing would prevent similar future human health tragedies. As scientists increasingly highlight issues with translatability between non-human animals and humans, the belief that current regulatory requirements ensure safety becomes more difficult to maintain. In addition, it means that some of the regulations now in place require animal tests that do not contribute to the safety of a drug, as shown in a third case study of the court case by Vanda industries against the FDA. We finally argue that regulations should be critically examined and altered where necessary, so that they are no longer a barrier in the transition toward animal-free testing and more human-relevant science.

A New Phased-Array Magnetic Resonance Imaging Receive-Only Coil for HBO2 Studies.

The paper describes a new magnetic resonance imaging (MRI) phased-array receive-only (Rx) coil for studying decompression sickness and disorders of hyperbaricity, including nitrogen narcosis. Functional magnetic resonance imaging (fMRI) is noninvasive, is considered safe, and may allow studying the brain under hyperbaric conditions. All of the risks associated with simultaneous MRI and HBO2 therapy are described in detail, along with all of the mitigation strategies and regulatory testing. One of the most significant risks for this type of study is a fire in the hyperbaric chamber caused by the sparking of the MRI coils as a result of high-voltage RF arcs. RF pulses at 128 MHz elicit signals from human tissues, and RF sparking occurs commonly and is considered safe in normobaric conditions. We describe how we built a coil for HBO2-MRI studies by modifying an eight-channel phased-array MRI coil with all of the mitigation strategies discussed. The coil was fabricated and tested with a unique testing platform that simulated the worst-case RF field of a three-Tesla MRI in a Hyperlite hyperbaric chamber at 3 atm pressure. The coil was also tested in normobaric conditions for image quality in a 3 T scanner in volunteers and SNR measurement in phantoms. Further studies are necessary to characterize the coil safety in HBO2/MRI.

Pesticide risk assessment: honeybee workers are not all equal regarding the risk posed by exposure to pesticides.

Toxicological studies in honeybees have long shown that a single pesticide dose or concentration does not necessarily induce a single response. Inter-individual differences in pesticide sensitivity and/or the level of exposure (e.g., ingestion of pesticide-contaminated matrices) may explain this variability in risk posed by a pesticide. Therefore, to better inform pesticide risk assessment for honeybees, we studied the risk posed by pesticides to two behavioral castes, nurse, and forager bees, which are largely represented within colonies and which exhibit large differences in their physiological backgrounds. For that purpose, we determined the sensitivity of nurses and foragers to azoxystrobin (fungicide) and sulfoxaflor (insecticide) upon acute or chronic exposure. Azoxystrobin was found to be weakly toxic to both types of bees. However, foragers were more sensitive to sulfoxaflor than nurses upon acute and chronic exposure. This phenomenon was not explained by better sulfoxaflor metabolization in nurses, but rather by differences in body weight (nurses being 1.6 times heavier than foragers). Foragers consistently consumed more sugar syrup than nurses, and this increased consumption was even more pronounced with pesticide-contaminated syrup (at specific concentrations). Altogether, the stronger susceptibility and exposure of foragers to sulfoxaflor contributed to increases of 2 and tenfold for the acute and chronic risk quotients, respectively, compared to nurses. In conclusion, to increase the safety margin and avoid an under-estimation of the risk posed by insecticides to honeybees, we recommend systematically including forager bees in regulatory tests.

Arbuscular Mycorrhizal Fungi and the Need for a Meaningful Regulatory Plant Protection Product Testing Strategy.

Arbuscular mycorrhizal fungi (AMF) perform key soil ecosystem services and, because of their symbiotic relationship with plant roots, may be exposed to the plant protection products (PPPs) applied to soils and crops. In 2017, the European Food Safety Authority (EFSA) released a scientific opinion addressing the state of the science on risk assessment of PPPs for in‐soil organisms, recommending the inclusion of AMF ecotoxicological testing in the PPP regulatory process. However, it is not clear how this can be implemented in a tiered, robust, and ecologically relevant manner. Through a critical review of current literature, we examine the recommendations made within the EFSA report and the methodologies available to integrate AMF into the PPP risk assessment and provide perspective and commentary on their agronomic and ecological relevance. We conclude that considerable research questions remain to be addressed prior to the inclusion of AMF into the in‐soil organism risk assessment, many of which stem from the unique challenges associated with including an obligate symbiont within the PPP risk assessment. Finally, we highlight critical knowledge gaps and the further research required to enable development of relevant, reliable, and robust scientific tests alongside pragmatic and scientifically sound guidance to ensure that any future risk‐assessment paradigm is adequately protective of the ecosystem services it aims to preserve. Environ Toxicol Chem 2022;41:1808–1823. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Development of a one-step RT-qPCR assay for the detection of Grapevine leafroll-associated virus 7.

Grapevine leafroll disease (GLD) is one of the most economically important viral diseases of grapevines. GLD is caused by a complex of several ssRNA (+) viruses referred to as Grapevine leafroll-associated viruses (GLRaVs). To date, five different GLRaV species have been identified. One of those species, GLRaV-7, was first reported from a symptomless white-fruited wine grape cultivar from Albania. Since its discovery, GLRaV-7 has been reported from 14 countries. Although serological assays have been developed to detect GLRaV-7, commercially available antibodies produce high background signals making them unsuitable for regulatory testing. Furthermore, while molecular detection assays have been shown to be more sensitive when compared to the serological assays, published molecular assays, except the one Reverse Transcription-quantitaive Polymerase Chain Reaction (RT-qPCR) assay based on heat shock protein 70 homologue (HSP70h) gene, have been reported to be inadequate in detecting all reported isolates of GLRaV-7. Availability of multiple assays provides flexibility to diagnostic laboratories in cases where the chosen assay fails to detect a strain or an isolate of a pathogen due to variation in its targeted region or where additional confirmation of the results is required. In this study, we developed a sensitive and specific RT-qPCR assay, based on a region of p61 gene of GLRaV-7, which detected all available isolates.

Multi-mycotoxin analysis method using liquid chromatography with tandem mass spectrometry and fluorescence detection in Indian medicinal herbs: Development and validation

While medicinal plants are in high demand worldwide for their therapeutic properties, they can constitute a health concern to consumers when contaminated with mycotoxins. The unavailability of standardised methods for multiclass mycotoxin analysis to assess health risks has thus been realised. This study reports a simple, robust and precise method to estimate nine regulated mycotoxins in a range of Indian medicinal plant matrices including giloy (Tinospora cordifolia), ashwagandha (Withania somnifera), safed musli (Chlorophytum borivilianum), satavari (Asparagus racemosus) and tulsi (Ocimum sanctum). The sample preparation method involved extraction of homogenised matrices (12.5 g) using methanol:water (8:2, 100 mL) followed by cleanup through a multi-mycotoxin immunoaffinity column (IAC), which significantly reduced matrix interferences. The method was initially developed and validated using liquid chromatography tandem mass spectrometry (LC-MS/MS) for the simultaneous analysis of aflatoxins (B1, B2, G1, G2), ochratoxin A, zearalenone, deoxynivalenol, T-2 and HT-2 toxin. Later, it was validated using LC-fluorescence (LC-FLD) for aflatoxins, ochratoxin A and zearalenone. The optimised sample preparation protocol and analytical method provided acceptable results. Compared to LC-FLD, it was possible to attain a lower limit of quantification (LOQ) with LC-MS/MS for all the tested analytes except aflatoxins. However, LOQs of both instruments were lower than the maximum limits (MLs), with recoveries ranging between 71 and 110% and precision (RSD) of ≤10% across matrices. Despite matrix-induced signal suppressions in LC-MS/MS analysis, the matrix-matched calibrations corrected all recoveries. Considering its accuracy, reliability, robustness and time-effectiveness, this method is recommended for regulatory testing purposes.

Extending Animal Cruelty Protections to Scientific Research

Extending Animal Cruelty Protections to Scientific Research

Why Due Regard Is More Appropriate than Proportionality Testing in International Investment Law

Abstract Global regulatory standards of due diligence, regulatory coherence, and due regard are emerging in public international law. Investment law has been concerned to settle upon the most appropriate regulatory coherence tests for application in the arbitration of regulatory disputes. Candidates have included proportionality, rationality, and reasonableness tests. This article argues instead for reliance on the due regard standard in conjunction with reasonableness or rationality testing. This will more appropriately reflect the nature of investment treaties as inter-State bargains. Further, responding to arguments for the adoption of proportionality on the basis of comparative public law, the article demonstrates that proportionality is not established as a general head of review in common law jurisdictions including England, Australia, Canada, New Zealand and South Africa. At the same time, the application of the due regard standard can have much in common with procedural proportionality testing as seen among these domestic legal systems and elsewhere.

Assessment of Regulatory Compliance Testing for Vitamin D in Infant Formula-Impact of Delegated Regulation (EU) 2019/828.

Since the publication of Standard Method Performance Requirements (SMPR®) for vitamin D in infant formula (SMPR 2011.004) by AOAC INTERNATIONAL, revised vitamin D limits have been recommended by the European Food Safety Authority (EFSA) for infant formula and adopted in Commission Delegated Regulation (EU) 2019/828. The vitamin D range introduced, 2-2.5 μg/100 kcal, is significantly narrower than previous limits specified by Codex Standard 72-1981 and requires lower method reproducibility metrics to adequately assess regulatory compliance. The narrower limits for vitamin D present a significant challenge for current-generation reference analytical methods that comply with SMPR 2011.004. We evaluate the impact of Delegated Regulation (EU) 2019/828 on the demonstrated performance of AOAC Method 2016.05/ISO 20636:2018 to assess the likelihood that vitamin D results produced by the method would be found outside the EU limits when testing infant formula that is compliant as manufactured. AOAC Method 2016.05/ISO 20636:2018, specifically data generated during multi-laboratory study, was used as a basis for statistical evaluation of the impact of the narrower EU vitamin D limits. The review of AOAC Method 2016.05/ISO 20636:2018 method performance against the vitamin D regulatory range introduced in (EU) 2019/828 indicates methods capable of performing in alignment with SMPR 2011.004 are likely to produce results that fail to meet EU requirements. Our assessment illustrates the high probability that a well-manufactured product with vitamin D levels within the EU regulatory range would fail to meet the regulatory requirements due to analytical method variability when tested using fit-for-purpose methods. Further, required method performance cannot be expected with the future development of new methods. To avoid this, consideration should be given to aligning proposed regulatory limits with method performance metrics of current-generation compendial methods. Current, state-of-the-art methods cannot consistently verify infant formula product compliance for vitamin D in accordance with (EU) 2019/828.

Comparison of PAC and MOAH for understanding the carcinogenic and developmental toxicity potential of mineral oils

The carcinogenicity and developmental toxicity of unrefined mineral oil is related to its 3–7 ring polycyclic aromatic compounds (PAC) content. Therefore, refining operations focus on the targeted removal PAC from mineral oil that may contain aromatics of low toxicological concern. There are thus, two types of aromatic substances in mineral oil: hazardous and non-hazardous. The first type consists of 3–7 ring PAC which may be naked (unsubstituted) or lowly alkylated. The second type or non-hazardous consists of 1–7 ring aromatics with high degree of alkylation or lack of bay or fjord regions. Although these are toxicologically different, they may both elute in the same fraction when using chromatography. To understand how these two aromatic types are related we have assessed the entire mineral oil refinement process by measuring total mineral oil aromatic hydrocarbons (MOAH) content by chromatography next to regulatory hazard tests which focus on 3–7 ring PAC. MOAH content is positively correlated to its molecular weight resulting in aromatic content bias for high viscosity substances. Hazard to 3–7 ring PAC is best controlled by the validated IP346 or modified Ames test. We explain the concept of high vs low alkylation by shortly reviewing new data on alkylated PAC.

Versatility of a Salmonella Loop-Mediated Isothermal Amplification Assay Using Multiple Platforms and Master Mixes in Animal Food Matrices.

Improvement in Salmonella detection methods greatly enhances the efficiency of various food testing programs. A Salmonella loop-mediated isothermal amplification (LAMP) assay has been validated in animal food through multi-laboratory validation. The study aimed to demonstrate the versatility of this molecular assay while expanding it to multiple platforms and various reagent choices for use in animal food testing. Following the U.S. Food and Drug Administration (FDA)'s Guidelines for the Validation of Analytical Methods for the Detection of Microbial Pathogens in Foods and Feeds, we examined the inclusivity, exclusivity, and LOD of the assay using two platforms (7500 Fast and Genie II) and three LAMP master mixes (GspSSD, GspSSD2.0, and WarmStart) in seven animal food matrixes (dry cat food, dry dog food, cattle feed, dairy feed, horse feed, poultry feed, and swine feed). The FDA's Bacteriological Analytical Manual (BAM) Salmonella culture method was the reference method. Inclusivity and exclusivity data were consistent among all six platform and master mix combinations with a few exceptions. Comparable LODs were observed down to the single-cell level (WarmStart was 10-fold less sensitive). Performance was similar to the BAM method for detecting fractional positive results in seven animal food matrixes. Nonetheless, LAMP time to positive results and annealing/melting temperature differed among master mixes and platforms. The Salmonella LAMP assay was successfully validated in two platforms and three master mixes, making it a flexible tool for use by the FDA's field laboratories in regulatory testing of animal food and for adoption by other food testing programs. We demonstrated the LAMP assay's versatility on two platforms and three master mixes for the rapid and reliable screening of Salmonella in seven animal food matrixes. GspSSD2.0 was the fastest master mix (time to positive results as early as 3.5 min) while Genie II had several attractive features from a user perspective.

Methods for testing solubility of hydraulic calcium silicate cements for root-end filling

Regulatory testing of hydraulic cements used in dentistry and standard test methods for root-end filling materials do not exist. The aim of this study was to identify a simple, reproducible method for testing the solubility of materials that set with water (hydraulic) used as root-end filling materials in dentistry. Commercial and prototype hydraulic cements were characterized by scanning electron microscopy and X-ray diffraction analyses and their solubilities were determined using ISO 6876; 2012 standard, a modified ISO 6876 method with media alternative to water and a new method measuring the percentage mass loss and volume change of materials (micro-CT method) from a single surface exposed to three solutions. The solubility testing was performed by three operators to enable an intra-laboratory comparison. The solubility data obtained from the two commercial and two prototype materials varied depending on the method used, with the ISO 6876 method identifying differences in solubility of the materials (p < 0.05) but when modified with alternative solutions, no differences were found (p > 0.05). The changes in solution thus effected the solubility of the tested materials. Inter-operator differences were observed with the weight changes determined from the new method indicating this method was not robust. The weight and volume assessments using the new method were not solution-dependent. The advantage of the proposed method compared with the ISO standard is its simplicity, enabling a number of tests to be performed on the same set of samples that also more closely mimics the clinical environment.

3,6'- and 1,6'-Dithiopomalidomide Mitigate Ischemic Stroke in Rats and Blunt Inflammation.

(1) Background: An important concomitant of stroke is neuroinflammation. Pomalidomide, a clinically available immunomodulatory imide drug (IMiD) used in cancer therapy, lowers TNF-α generation and thus has potent anti-inflammatory actions. Well-tolerated analogs may provide a stroke treatment and allow evaluation of the role of neuroinflammation in the ischemic brain. (2) Methods: Two novel pomalidomide derivatives, 3,6′-dithiopomalidomide (3,6′-DP) and 1,6′-dithiopomalidomide (1,6′-DP), were evaluated alongside pomalidomide in a rat middle cerebral artery occlusion (MCAo) stroke model, and their anti-inflammatory actions were characterized. (3) Results: Post-MCAo administration of all drugs lowered pro-inflammatory TNF-α and IL1-β levels, and reduced stroke-induced postural asymmetry and infarct size. Whereas 3,6′- and 1,6′-DP, like pomalidomide, potently bound to cereblon in cellular studies, 3,6′-DP did not lower Ikaros, Aiolos or SALL4 levels—critical intermediates mediating the anticancer/teratogenic actions of pomalidomide and IMiDs. 3,6′-DP and 1,6′-DP lacked activity in mammalian chromosome aberration, AMES and hERG channel assays –critical FDA regulatory tests. Finally, 3,6′- and 1,6′-DP mitigated inflammation across rat primary dopaminergic neuron and microglia mixed cultures challenged with α-synuclein and mouse LPS-challenged RAW 264.7 cells. (4) Conclusion: Neuroinflammation mediated via TNF-α plays a key role in stroke outcome, and 3,6′-DP and 1,6′-DP may prove valuable as stroke therapies and thus warrant further preclinical development.

Application of an Accessible Interface for Pharmacokinetic Modeling and In Vitro to In Vivo Extrapolation

Regulatory toxicology testing has traditionally relied on in vivo methods to inform decision-making. However, scientific, practical, and ethical considerations have led to an increased interest in the use of in vitro and in silico methods to fill data gaps. While in vitro experiments have the advantage of rapid application across large chemical sets, interpretation of data coming from these non-animal methods can be challenging due to the mechanistic nature of many assays. In vitro to in vivo extrapolation (IVIVE) has emerged as a computational tool to help facilitate this task. Specifically, IVIVE uses physiologically based pharmacokinetic (PBPK) models to estimate tissue-level chemical concentrations based on various dosing parameters. This approach is used to estimate the administered dose needed to achieve in vitro bioactivity concentrations within the body. IVIVE results can be useful to inform on metrics such as margin of exposure or to prioritize potential chemicals of concern, but the PBPK models used in this approach have extensive data requirements. Thus, access to input parameters, as well as the technical requirements of applying and interpreting models, has limited the use of IVIVE as a routine part of in vitro testing. As interest in using non-animal methods for regulatory and research contexts continues to grow, our perspective is that access to computational support tools for PBPK modeling and IVIVE will be essential for facilitating broader application and acceptance of these techniques, as well as for encouraging the most scientifically sound interpretation of in vitro results. We highlight recent developments in two open-access computational support tools for PBPK modeling and IVIVE accessible via the Integrated Chemical Environment (https://ice.ntp.niehs.nih.gov/), demonstrate the types of insights these tools can provide, and discuss how these analyses may inform in vitro-based decision making.

Rethinking our chemical legacy and reclaiming our planet

Rethinking our chemical legacy and reclaiming our planet

Cobalt-bearing copper slags from Luanshya (Zambian Copperbelt): Mineralogy, geochemistry, and potential recovery of critical metals

Waste materials such as metallurgical slags can be considered as potential resources of valuable elements, including technologically critical metals. Copper slags from Luanshya, the oldest mining and smelting site in the Zambian Copperbelt, produced by smelting local Cu ores in reverberatory furnaces between 1932 and 1999, were deposited on two large dumps in the vicinity of the former ore processing area. The slags exhibit high concentrations of Co (247–5990 ppm, median: 2370 ppm) and Cu (1320–95,300 ppm, median: 8550 ppm). This multi-method mineralogical investigation indicates that the slags are predominantly composed of olivine, clinopyroxene, silicate glass, and spinel-family oxides. Copper-(Fe) sulfides, cobaltpentlandite [(Co,Fe)9S8], Fe sulfides, and metallic Cu prills embedded in the silicate matrix are the major hosts of Cu and Co. The EU regulatory leaching test (EN 12457-2) indicated that the release of contaminants is relatively low and only Cu slightly exceeded the EU limit values for landfilling of inert waste. The secondary phases (Cu hydroxosulfates, Fe (oxyhydr)oxides) observed on the slag surfaces confirm that the slags undergo a weathering process on the dumps. Kinetic abiotic extraction tests were carried out in 0.5 mol/l sulfuric acid at 25 °C and with a pulp density of 1% to determine the release of metals from the milled Luanshya slags under conditions simulating hydrometallurgical recovery via agitation leaching. The Cu and Co extractability correlated with the bulk concentration of these elements, and the highest extraction yields after 24 h of leaching corresponded to 46% of the total Cu and 67% of the total Co. Despite the dramatic increase in Co prices on the global market, the Co recovery from the Luanshya slags appears to be non-economical due to the high costs of the necessary mechanical and chemical processing of the slag materials and the obtained extracts.

Effects of 3D printed surface topography and normal force on implant expulsion

3D printing is a critical method for manufacturing metallic implants as it enables direct fabrication of intricate geometries and porous structures inaccessible to other manufacturing methods. Some common 3D printed porous structures are strut based (e.g. octet truss), triply periodic minimal surfaces (TPMS) (e.g. gyroid) or randomized (e.g. stochastic). When designed to be on the surface of bone interfacing implants, the surface porous region impacts short-term adhesion and friction, ultimately affecting implant stability prior to and during long-term osseointegration. In many orthopedic procedures, expulsion resistance is an essential design requirement, to prevent the risk of the implant migrating from the implantation site. While expulsion tests are universal, they are a poorly understood method to examine the bone-implant interface in determining the performance of an orthopedic implant. In this foundational study, we examine the expulsion behavior of metallic samples in synthetic Sawbone with systematically varied surface topography at increasing applied normal forces. The applied normal force and size of the sample were shown to have the strongest influence on expulsion force followed by surface structure. Compared to a polished sample control, certain 3D printed surface structures are up to 10x more expulsion resistant and should be considered in implants where prevention of implant migration before and during osseointegration is critical. Nonlinear relationships were discovered that reveal “crossover” in expulsion resistance as a function of applied load revealing that the ranking of the relative expulsion resistance of different samples can depend on the normal force selected. This new fundamental understanding has broad implications on both the design and potential standardized regulatory testing of textured orthopedic implants with tailored topologies.