Reduction In Number Of Animals Research Articles

Interval estimators of relative potency in toxicology and radiation countermeasure studies: comparing methods and experimental designs

ABSTRACT The relative potency of one agent to another is commonly represented by the ratio of two quantal response parameters; for example, the LD50 of animals receiving a treatment to the LD50 of control animals, where LD50 is the dose of toxin that is lethal to 50% of animals. Though others have considered interval estimators of LD50, here, we extend Bayesian, bootstrap, likelihood ratio, Fieller’s and Wald’s methods to estimate intervals for relative potency in a parallel-line assay context. In addition to comparing their coverage probabilities, we also consider their power in two types of dose designs: one assigning treatment and control the same doses vs. one choosing doses for treatment and control to achieve same lethality targets. We explore these methods in realistic contexts of relative potency of radiation countermeasures. For larger experiments (e.g., ≥100 animals), the methods return similar results regardless of the interval estimation method or experiment design. For smaller experiments (e.g., < 60 animals), Wald’s method stands out among the others, producing intervals that hold closely to nominal levels and providing more power than the other methods in statistically efficient designs. Using this simple statistical method within a statistically efficient design, researchers can reduce animal numbers.

Continual Trials Spontaneous Recognition Tasks in Mice: Reducing Animal Numbers and Improving Our Understanding of the Mechanisms Underlying Memory.

Spontaneous recognition tasks are widely used as a laboratory measure of memory in animals but give rise to high levels of behavioral noise leading to a lack of reliability. Previous work has shown that a modification of the procedure to allow continual trials testing (in which many trials are run concurrently in a single session) decreases behavioral noise and thus significantly reduces the numbers of rats required to retain statistical power. Here, we demonstrate for the first time that this improved method of testing extends to mice, increasing the overall power of the approach. Moreover, our results show that the new continual trials approach provides the additional benefits of heightened sensitivity and thus provides greater insight into the mechanisms at play. Standard (c57) and transgenic Alzheimer model (TASTPM) mice were tested both at 7 and 10 months of age in both object recognition (OR) and object-location (OL) spontaneous recognition tasks using the continual trials methodology. Both c57 and TASTPM mice showed age-dependent changes in performance in OR. While c57 mice also showed age-related changes in performance of OL, TASTPM mice were unable to perform OL at either age. Significantly, we demonstrate that differences in OL performance in c57s and TASTPM animals is a result of proactive interference rather than an absolute inability to recognize OL combinations. We argue that these continual trials approaches provide overall improved reliability and better interpretation of the memory ability of mice, as well as providing a significant reduction in overall animal use.

Continuous Blood Glucose Monitoring Reveals Enormous Circadian Variations in Pregnant Diabetic Rats.

Diabetes in pregnancy is a major burden with acute and long-term consequences. Its treatment requires adequate diagnosis and monitoring of therapy. Many experimental research on diabetes during pregnancy has been performed in rats. Recently, continuous blood glucose monitoring of non-pregnant diabetic rats revealed an increased circadian variability of blood glucose that made a single blood glucose measurement per day inappropriate to reflect glycemic status. Continuous blood glucose measurement has never been performed in pregnant rats. We wanted to perform continuous blood glucose monitoring in pregnant rats to decipher the influence of pregnancy on blood glucose in diabetic and normoglycemic status. We used the transgenic Tet29 diabetes rat model with an inducible knock down of the insulin receptor via RNA interference upon application of doxycycline (DOX) leading to insulin resistant type II diabetes. All Tet29 rats received a HD-XG telemetry implant (Data Sciences International, USA) that measured blood glucose and activity continuously. Rats were divided into four groups and blood glucose was monitored until end of pregnancy or the corresponding period: Tet29 + DOX (diabetic) non-pregnant, Tet29 + DOX (diabetic) pregnant, Tet29 (normoglycemic) non-pregnant, Tet29 (normoglycemic) pregnant. All analyzed rats displayed a circadian variation in blood glucose concentration. Circadian variability was much more pronounced in pregnant diabetic rats than in normoglycemic pregnant rats. Pregnancy ameliorated variation in blood glucose in diabetic situation. Pregnancy continuously decreased blood glucose during normoglycemic pregnancy. Diabetic rats were less active than normoglycemic rats. We performed a calculation showing that application of continuous blood glucose measurement reduces animal numbers needed to detect a given effect in experimental setting by decreasing variability and SD. Continuous blood glucose monitoring via a telemetry device in pregnant rats provides a more informative picture of the glycemic situation in comparison to single measurements. This could improve diagnosis and therapy of diabetes, decrease animal numbers within experimental settings, and add another physiological parameter (activity) to the analysis that could be helpful in testing therapeutic concepts targeting blood glucose levels and peripheral muscle function. We propose continuous glucose monitoring as a new tool for the evaluation of pregnant diabetic rats.

Refinement of a mouse cardiovascular model: Development, application and dissemination.

European and UK legislation requires all animal procedures to be conducted with consideration to reduction, refinement and replacement. In this review, 3Rs developments are discussed in the field of platelet biology and thromboembolism. Platelet research requires the use of animal models, and mice are widely used in the field. When working in vitro, conventional light transmission techniques have been scaled down allowing reduction in animal numbers. In vivo, vascular injury models are widely used and work is ongoing to develop ex vivo approaches that use fewer animals. Thromboembolic mortality models, which inflict considerable pain and suffering, have also been used widely. A published and characterised refinement of this mortality model allows real-time monitoring of radiolabelled platelets under general anaesthesia and reduces both the severity level and the numbers of mice used in a typical experiment. This technique is more sensitive than the mortality approach and has opened up new avenues of research, which would not have been feasible by using death as an end-point. To drive uptake of real-time monitoring, a more simplistic approach has been developed involving micro-sampling and cell counting. Thromboembolic mortality models should therefore be considered obsolete due to the emergence of 3Rs models with improved scientific outcomes and that can be implemented relatively easily.

A novel method for expansion and differentiation of mouse tracheal epithelial cells in culture

Air-liquid interface (ALI) cultures of mouse tracheal epithelial cells (MTEC) are a well-established model to study airway epithelial cells, but current methods require large numbers of animals which is unwanted in view of the 3R principle and introduces variation. Moreover, stringent breeding schemes are frequently needed to generate sufficient numbers of genetically modified animals. Current protocols do not incorporate expansion of MTEC, and therefore we developed a protocol to expand MTEC while maintaining their differentiation capacity. MTEC were isolated and expanded using the ROCK inhibitor Y-27632 in presence or absence of the γ-secretase inhibitor DAPT, a Notch pathway inhibitor. Whereas MTEC proliferated without DAPT, growth rate and cell morphology improved in presence of DAPT. ALI-induced differentiation of expanded MTEC resulted in an altered capacity of basal cells to differentiate into ciliated cells, whereas IL-13-induced goblet cell differentiation remained unaffected. Ciliated cell differentiation improved by prolonging the ALI differentiation or by adding DAPT, suggesting that basal cells retain their ability to differentiate. This technique using expansion of MTEC and subsequent ALI differentiation drastically reduces animal numbers and costs for in vitro experiments, and will reduce biological variation. Additionally, we provide novel insights in the dynamics of basal cell populations in vitro.

Cattle producer willingness to afforest pastureland and sequester carbon

The beef industry can mitigate climate change by reducing greenhouse gas emissions associated with cattle production and increasing carbon sequestration on grazing lands. One alternative for increasing carbon sequestration is to convert pasture to forest while either reducing animal numbers or increasing cattle stocking density on more productive pastureland. This study uses data from a survey of beef cattle producers in the eastern United States to determine: (i) interest in afforesting pastureland; (ii) contingent upon interest, willingness to participate in a hypothetical afforestation program; and (iii) contingent upon willingness to participate, participation intensity in the form of enrolled acres. Less than one-third of producers were interested in afforesting pasture. Producer interest was associated with beliefs about the on- and off-farm effects of afforestation, risk aversion, age, educational attainment, opportunity costs, forest ownership, and previous experience with afforestation. Willingness to participate in the program was influenced by the incentive offered, household income, and opportunity costs. Respondents willing to participate in the program were willing to afforest an average of 55 acres, given the incentive offered. Producers with larger farms were willing to enroll more acres. Extrapolating these results to the population of beef cattle producers in the eastern United States provides a measure of the potential for pasture afforestation and carbon sequestration.

Regulatory respiratory data refinement with reduced animal usage

IntroductionAssessment of effects of potential drug candidates on the respiratory system is part of the regulatory preclinical safety assessment conducted prior to first in human trials (FTIH). Commonly, this is carried out utilizing head out plethysmography (HOP) or whole body plethysmography (WBP) which record only ventilatory parameters. When dosing via the inhaled route a more thorough respiratory assessment, including a direct measure of airway mechanics, is desirable. The aim of the present work was to improve the strategy for respiratory safety testing by a) evaluating a telemetered pleural pressure – HOP (PP-HOP) model and b) evaluating a crossover study design protocol in the WBP model to reduce variability and animal usage. MethodsFor the PP- HOP model, rats were surgically implanted with a telemetry device for measurement of pleural pressure. Animals were placed in HOP tubes and respiratory function assessed when exposed to methacholine at doses of 0 (saline only), 0.42, 1.6 and 3.8 mg/kg. WBP assessment was performed in rats in a crossover study design when treated with theophylline at doses of 0 (saline only), 3, 10 and 30 mg/kg. ResultsData from the PP-HOP study confirmed the expected changes in ventilatory parameters and airway mechanics in response to inhaled methacholine, including an increase in pulmonary resistance and decrease in tidal volume.Data from the WBP crossover study demonstrated similar sensitivity and statistical power to detect changes in respiratory rate and tidal volume to a standard parallel group design. ConclusionMeasurement of PP-HOP in a stand-alone safety pharmacology study in conjunction with HOP assessment conducted as part of a toxicology study, represents an improved respiratory testing strategy for inhaled drugs. For compounds administered by other routes, we conclude that use of WBP using a crossover dosing design is a suitable alternative to parallel dosing groups, with a significant reduction in animal numbers and no loss of statistical power.

Vaccine safety testing using magnetic resonance imaging in suckling pigs

Safety testing is one major part of the licensing procedure for veterinary vaccines and demands a large number of animals. Magnetic resonance imaging (MRI) was tested as an alternative, which may lead to a reduction in numbers of animals required for safety testing, and, correspondingly to a detailed description of the three-dimensional extent of the local tissue reaction repetitively in live pigs. In previous pig studies the following questions arose:(1)Can MRI be used in suckling pigs in terms of safety testing?(2)Does the injection of 2 ml saline solution lead to a volume effect resulting in tissue alterations comparable to a vaccine response?(3)Is the local reaction size affected by the tattoo marking of the injection point for the final pathomorphologic examination?To answer these questions the following study was performed by comparing two vaccine groups of suckling piglets (8 animals per group; A and B) with two control groups (4 animals per group; C and D). One control group was injected with a saline solution (C) and the other was only tattoo marked (D). The animals were examined using MRI at days 1, 8, 15, 22, 29, 36, and 43 post vaccination, ending with a final pathomorphologic examination. Pathomorphologic examination confirmed MRI findings. Saline solution does not result in a local tissue reaction as detected after injecting vaccines. Tattoo marking causes no local tissue reaction, neither in MRI nor in pathomorphologic examination. Therefore, MRI can be used as an alternative method for safety testing of vaccines in pigs of different age categories offering repetitive measurements of local tissue reactions. Involved cells might be examined only in a final pathomorphologic examination at the end of the trial on a reduced number of animals.

AN ETHNOZOOLOGICAL ASSESSMENT OF TRADITIONALLY USED ANIMAL-BASED THERAPIES IN ATTAPPADY OF PALAKKAD DISTRICT, KERALA, INDIA.

The present study is an attempt to collect and document the ethnozoological knowledge possessed by the traditional tribal people of Attappady hills of Western Ghats in Palakkad district. The study area consisted of three categories of tribes namely, Irula, Kurumba and Muduga. Irulas contribute the majority followed by Mudugas and Kurumbas respectively. The study involved collection of information regarding the topic from all the three categories of tribes. Informations were collected by direct personal interviews with traditional healers belonging to tribal community. The mentioned animals were identified by their local names and previous studies in ethnozoology and available biodiversity records of regions in and around Attappady which involves Silent Valley National Park. The lack of biodiversity records posed difficulty in identifying the animal mentioned by the healers. The collected data was analyzed mathematically by calculating Informant Consensus Factor (ICF) to know the category of ailment for which more treatment is available, Fidelity level of animal species to identify the most preferred species for zootherapeutics, andInformant Agreement Index (IAR) to determine the agreement between informants for the use of a particular animal species for the treatment of a particular ailment category. ICF value obtained is highest for orthopedic ailments, FL is highest for Varanus bengalensis and Rusa unicolor and IAR is highest for seven species. The reduction in number of animals, lack of efficiency in implementing forest laws and their cultural taboos regarding the interaction with the outside world have resulted in the deterioration of traditional knowledge among the tribal population itself and also, due to legal issues, they have switched over almost completely to floral medicine. Therefore, they have a very little knowledge on the practice of zootherapeutics and ethnozoology.

Lacking quality in research: Is behavioral neuroscience affected more than other areas of biomedical science?

There are many reasons why novel therapeutics fail in clinical trials but these failures are often attributed to lacking quality of preclinical data. These problems are not limited to any specific therapeutic area, academic or industrial research and are due in large part to several generic factors influencing research quality (e.g., related to definition of pre-specified endpoints, principles of study design and analysis, biased reporting, and lack of proper training). Yet, neuroscience drug discovery is often said to be affected more than the other fields. Within neuroscience, behavioral studies are the most blamed for being poorly designed, underpowered and mis-reported and there are indeed several factors that may be rather unique for behavioral research, such as a multitude of environmental conditions that are difficult to control and that are often not reported, ethical concerns about in vivo research and the pressure to reduce animal numbers, contributing to under-powered studies, and the complexity of study design and analysis, creating too much room for post hoc data massaging and selective reporting. Also, the blood-brain barrier as a frequently neglected complicating factor has to be considered in CNS research. The importance of these factors is increasingly recognized and urgent efforts are needed to demonstrate that behavioral methods of preclinical neuroscience research deliver results that can be as robust as with the non-behavioral methods Until this goal is achieved, behavioral neuroscience and neuroscience in general may be losing young talent, CNS drug discovery may lack the needed investment and this field may indeed be amongst the most affected by the current preclinical data quality crisis.

Discriminating modes of toxic action in mice using toxicity in BALB/c mouse fibroblast (3T3) cells

The objective of this study was to determine whether toxicity in mouse fibroblast cells (3T3 cells) could predict toxicity in mice. Synthesized data on toxicity was subjected to regression analysis and it was observed that relationship of toxicities between mice and 3T3 cells was not strong (R2 = 0.41). Inclusion of molecular descriptors (e.g. ionization, pKa) improved the regression to R2 = 0.56, indicating that this relationship is influenced by kinetic processes of chemicals or specific toxic mechanisms associated to the compounds. However, to determine if we were able to discriminate modes of action (MOAs) in mice using the toxicities generated from 3T3 cells, compounds were first classified into “baseline” and “reactive” guided by the toxic ratio (TR) for each compound in mice. Sequence, binomial and recursive partitioning analyses provided strong predictions of MOAs in mice based upon toxicities in 3T3 cells. The correct classification of MOAs based on these methods was 86%. Nearly all the baseline compounds predicted from toxicities in 3T3 cells were identified as baseline compounds from the TR in mice. The incorrect assignment of MOAs for some compounds is hypothesized to be due to experimental uncertainty that exists in toxicity assays for both mice and 3T3 cells. Conversely, lack of assignment can also arise because some reactive compounds have MOAs that are different in mice compared to 3T3 cells. The methods developed here are novel and contribute to efforts to reduce animal numbers in toxicity tests that are used to evaluate risks associated with organic pollutants in the environment.

726 Are there only 2Rs in agricultural animal research and production?

Care of agricultural animals is a topic increasingly addressed through a lens and language identified as animal welfare. Welfare inherently is about how animals should be treated. Thus, animal care oversight is unavoidably normative; meaning the topic is undeniably one of ethics. Oversight of USA animal care arose first in publically funded laboratory animal use with the adoption of principles, codes and standards of practices. More recently, oversight has expanded to include agricultural animal research. While the goal of ensuring appropriate animal treatment is common to both lab and agricultural research, there are differences. Consequentially, principles initially implemented for lab animals with specific purpose and positive outcome can be shown to have a counter intuitive outcome when broadly applied. As an example, the 3Rs (Replacement, Reduction, Refinement) were initially specific to lab animal research but are mandated for all animal research today. Lab animals serve only one purpose. They are solely research subjects. Agricultural research animals have utility beyond serving as data points. Specifically, when not contrary to health food standards, they are marketed as food. Thus, an IACUC that attempts to Reduce the herd size may be acting in a manner contrary to both better research numbers and also the maintenance of brood cow numbers necessary for a viable operation. Attempts to expand the application of the 3Rs beyond public institutions and to private animal agriculture raise even more questions. Production of beef or pork and not poultry would considerably Reduce the number of animals used in food production. But a question that remains unanswerable is how to compare the value of an animal life of one species to another. Thus, a measure of total harm using animal numbers alone becomes problematic for both science and ethics. Futurists suggest it may become possible to Replace the food animal altogether. But is a steak without the steer a higher moral position? Is the Replacement of animal discomfort and death through their non-existence more ethically defensible? Yes, Refinement in care animals regardless of purpose should be employed to attain appropriate animal quality of life. Additionally, Reduction of animal numbers would be consistent with better welfare, e.g., sexing offspring in dairy breeding. In summary, the 3Rs proposed by Russell and Burch in 1959 have continued application to lab animal oversight, but a simplistic application of these principles to animal agriculture can be shown to not always be the most defensible position.

The current status of exposure-driven approaches for chemical safety assessment: A cross-sector perspective.

For the purposes of chemical safety assessment, the value of using non-animal (in silico and in vitro) approaches and generating mechanistic information on toxic effects is being increasingly recognised. For sectors where in vivo toxicity tests continue to be a regulatory requirement, there has been a parallel focus on how to refine studies (i.e. reduce suffering and improve animal welfare) and increase the value that in vivo data adds to the safety assessment process, as well as where to reduce animal numbers where possible. A key element necessary to ensure the transition towards successfully utilising both non-animal and refined safety testing is the better understanding of chemical exposure. This includes approaches such as measuring chemical concentrations within cell-based assays and during in vivo studies, understanding how predicted human exposures relate to levels tested, and using existing information on human exposures to aid in toxicity study design. Such approaches promise to increase the human relevance of safety assessment, and shift the focus from hazard-driven to risk-driven strategies similar to those used in the pharmaceutical sectors. Human exposure-based safety assessment offers scientific and 3Rs benefits across all sectors marketing chemical or medicinal products. The UK's National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) convened an expert working group of scientists across the agrochemical, industrial chemical and pharmaceutical industries plus a contract research organisation (CRO) to discuss the current status of the utilisation of exposure-driven approaches, and the challenges and potential next steps for wider uptake and acceptance. This paper summarises these discussions, highlights the challenges - particularly those identified by industry - and proposes initial steps for moving the field forward.

Biometrical evaluation of the performance of the revised OECD Test Guideline 402 for assessing acute dermal toxicity

A comprehensive biometrical assessment was conducted to compare the performance of multiple test designs for acute dermal systemic toxicity to support the animal welfare update to the original OECD Test Guideline (TG) 402 for acute dermal toxicity. The test designs evaluated included: (1) two, three, or five animals per dose group (2) evident toxicity or lethality endpoints and (3) absence or presence of a one-animal sighting study. The revision of TG 402 respected the 3R principles (replace, reduce, refine) of animal testing. The results demonstrate that the TG 402 test design can be optimised with reduced animal numbers per test group, such that a scenario of two animals per group following a sighting study at a starting dose of 200 mg/kg bw (unless further information is available to better define the starting dose) would provide a classification which in most cases is conservative, without compromising both the statistical ability of the study to assess dermal toxicity, or the relevant classification outcome.

Creation of resin rodent skulls to reduce animal numbers for stereotactic surgery practice.

Creation of resin rodent skulls to reduce animal numbers for stereotactic surgery practice.

Refinement of Mouse Protocols for the Study of Platelet Thromboembolic Responses In Vivo.

Mouse models of thromboembolism are frequently used to investigate platelet function in vivo and, according to European Union (EU) legislation, must be conducted in the context of replacement, refinement and reduction. We have previously developed a refined real-time mouse model of thromboembolism as an alternative to models of thromboembolic mortality which inflict considerable pain and suffering. Real-time monitoring involves infusion of radiolabelled platelets into the circulation of anaesthetized mice, and platelet aggregation is measured as increases in platelet-associated counts in the pulmonary vasculature following injection of platelet agonists. This gives a definitive data set on the tissue localization and extent of platelet activation. We developed an additional, more simplistic alternative to mortality models based on blood microsampling which entails the measurement of circulating platelet counts following agonist stimulation. Blood microsamples were collected from the tail vein of anaesthetized mice at three different time points leading to a reduction in animal numbers. Platelet counts significantly dropped 1 minute after stimulation with collagen or thrombin and were restored over 10 minutes. These results correlate with those obtained via real-time monitoring and were confirmed by immunohistochemistry. Pre-treatment of mice with aspirin significantly inhibited the decrease in platelet counts following collagen. These data suggest that blood microsampling may be implemented as a simplistic refined alternative to mortality models of thromboembolism when specialized monitoring equipment, or use of radioactive isotopes for real-time monitoring, which remains the ‘gold standard’, is not feasible. Microsampling refines and reduces animal procedures in compliance with current EU legislation.

European bison: changes in species acceptance following plans for translocation

The prioritizing of human demands in human-dominated landscapes shapes human–wildlife conflicts. Damage to crops is the main cause of conflict with herbivores. In Lithuania, according to the Plan for the Protection of the European Bison (Bison bonasus L.), the translocation of animals from a free-ranging herd in central Lithuania is planned and has been widely announced in local media. We aimed to examine if the publication of such plans changed species acceptance and if the translocation was supported by local inhabitants. In 2014, after the translocation plan was announced, we interviewed 663 respondents living in the area with the free-ranging bison herd. We found that bison damage was considered more serious than had been expressed in a previous survey in 2008–2009, while overall species acceptance and tolerated distance between the bison and respondents’ living places had not changed, with fear of the animals becoming less expressed. Lethal means and reduction of animal numbers were not favoured, though the numbers of respondents that did favour this was higher in 2014 than in 2008–2009. Among the possible measures of population management that did not harm bison or leave them in captivity, translocation was least popular. Three factors explained 64.2% of bison acceptance, namely (1) respondent concerns about income sources, (2) sustainability of agricultural practices due to bison presence and (3) attractiveness of the locality with respect to child safety and price of real estate.

Automated Microsampling Technologies and Enhancements in the 3Rs.

Data collected in vivo is essential for advising decisions on drug screening and development and basic research, and animal models are used extensively for acquiring experimental measurements. Traditionally, collection of specimens has been invasive, stressful to animal subjects, labor intensive, time-consuming and costly, and required many animals when using small models with low fluid volumes, such as rats or mice. Utilizing automated microsampling (AMS) alone or in an integrative pharmacology approach to evaluate multiple physiological, pharmacokinetic, and pharmacodynamic endpoints simultaneously in the same animal accomplishes multiple experimental goals. Use of AMS robotics and associated instrumentation can assist in achieving significant reduction and refinement of animal use. Automated robotic instrumentation for specimen collection from living animal models can now be used to provide better quality pharmacokinetic and pharmacodynamic data, reduce time, provide more data with less variability, reduce animal use, and refine animal model use to reduce pain and stress. Instrumentation in common use for automated sampling and dosing is briefly discussed. In parallel with advances in automated instrumentation, recent advances in analytical detection methods complement the use of automated technology for data and specimen collection. Methods requiring much smaller volumes can now be utilized. Microsampling (small biological samples in volumes of 5-100 μL) can facilitate reduction in animal numbers while minimizing stresses associated with excessive fluid volume removal. Innovations in automation, microsampling techniques, and analytical methods have facilitated advances in data collection that allow for more robust and accurate data, reduction in animal use, and refinement in techniques that improve animal welfare. These innovations offer opportunities for wider application in nonclinical investigations by collection of multiple data streams simultaneously from individual animals. Many benefits are achievable through the use integrative pharmacology designs utilizing AMS, including decreased time for completion of composite data collection, decreased personnel resources, lower costs, improved safety, higher quality and multiple data-sets, and improvements in aspects of the 3Rs.

Influence of Study Design on Developmental and Reproductive Toxicology Study Outcomes.

Regulatory studies of developmental and reproductive toxicity (DART) studies have remained largely unchanged for decades, with exposures occurring at various phases of the reproductive cycle and toxicity evaluations at different ages/times depending on the study purpose. The National Toxicology Program has conducted studies examining the power to detect adverse effects where there is a prenatal exposure, but evaluations occur postnatally. In these studies, examination is required of only 1 male and female pup from each litter beyond weaning. This provides poor resolving power to detect rare events (e.g., reproductive tract malformations). If an adverse effect is detected, there is little confidence in the shape of the dose-response curve (and the Benchmark Dose or No Observed Adverse Effect Level [NOAEL]). We have developed a new protocol to evaluate DART, the modified one generation study, with exposure commencing with pregnant animals and retention of 4 males and females from each litter beyond weaning to improve statistical power. These animals can be allocated to specific cohorts that examine subchronic toxicity, teratology, littering, and neurobehavioral toxicity in the same study. This approach also results in a reduction in animal numbers used, compared with individual stand-alone studies, and offers increased numbers of end points evaluated compared with recent Organization for Economic Cooperation and Development proposals.

3-Dimensional Patient-Derived Lung Cancer Assays Reveal Resistance to Standards-of-Care Promoted by Stromal Cells but Sensitivity to Histone Deacetylase Inhibitors.

There is a growing recognition that current preclinical models do not reflect the tumor microenvironment in cellular, biological, and biophysical content and this may have a profound effect on drug efficacy testing, especially in the era of molecular-targeted agents. Here, we describe a method to directly embed low-passage patient tumor-derived tissue into basement membrane extract, ensuring a low proportion of cell death to anoikis and growth complementation by coculture with patient-derived cancer-associated fibroblasts (CAF). A range of solid tumors proved amenable to growth and pharmacologic testing in this 3D assay. A study of 30 early-stage non-small cell lung cancer (NSCLC) specimens revealed high levels of de novo resistance to a large range of standard-of-care agents, while histone deacetylase (HDAC) inhibitors and their combination with antineoplastic drugs displayed high levels of efficacy. Increased resistance was seen in the presence of patient-derived CAFs for many agents, highlighting the utility of the assay for tumor microenvironment-educated drug testing. Standard-of-care agents showed similar responses in the 3D ex vivo and patient-matched in vivo models validating the 3D-Tumor Growth Assay (3D-TGA) as a high-throughput screen for close-to-patient tumors using significantly reduced animal numbers. Mol Cancer Ther; 15(4); 753-63. ©2016 AACR.