A different approach to validating screening assays for developmental toxicity

BACKGROUND: There continue to be many efforts around the world to develop assays that are shorter than the traditional embryofetal developmental toxicity assay, or use fewer or no mammals, or use less compound, or have all three attributes. Each assay developer needs to test the putative assay against a set of performance standards, which traditionally has involved testing the assays against a list of compounds that are generally recognized as “positive” or “negative” in vivo. However, developmental toxicity is highly conditional, being particularly dependent on magnitude (i.e. dose) and timing of exposure, which makes it difficult to develop lists of compounds neatly assigned as developmental toxicants or not. APPROACH: Here we offer an alternative approach for the evaluation of developmental toxicity assays based on exposures. Exposures are classified as “positive” or “negative” in a system, depending on the compound and the internal concentration. Although this linkage to “internal dose” departs from the recent approaches to validation, it fits well with widely accepted principles of developmental toxicology. CONCLUSIONS: This paper introduces this concept, discusses some of the benefits and drawbacks of such an approach, and lays out the steps we propose to implement it for the evaluation of developmental toxicity assays. Birth Defects Res (Part B) 89:526–530, 2010. © 2010 Wiley‐Liss, Inc.     

An in vitro approach to the study of target organ toxicity of drugs and chemicals

Summary A major goal of our laboratory has been the development of primary culture systems that retain differentiated fucntions and responses characteristic of intact tissues in vivo. Specifically, we have developed cellular models of primary cultures of rat heart, liver, and kidney cells to explore the mechanisms by which drugs or chemicals may be toxic to key organs of the body and to develop new techniques by which xenobiotics may be evaluated or identified as potential toxicants to living systems. The purpose of this paper is to describe our rationale and approach to the study of target organ toxicology with in vitro cellular systems.     

Submitochondrial particles asin vitro biosensors of heavy metal toxicity

The effects on mitochondrial respiratory parameters of heavy metals, such as Cu, Ni, Pb, Cd, Zn, Ag, Hg, were recorded by using thein vitro response of submitochondrial particles (SMP) from beef heart mitochondria.The toxicity of these elements was estimated by determining their effects on the energy-coupled reverse electron transfer (RET), which is induced by ATP and succinate at first site level of the respiratory chain in SMP.The RET rate was easily monitored by recording spectrophotometrically at 340 nm the production of NADH, arising from the reduction of exogenous NAD⁺ by RET.The toxicity values were expressed as the toxicant molar concentration which decreases the rate of reduction of NAD⁺ to an extent of 50 percent (EC50). The toxicity increased in the following order: Ni²⁺2+2+< Cd²⁺2+2++.The SMP data were compared with the toxicity values obtained from a variety of biological systems currently used for toxicity testing. The results obtained demonstrate that the SMP test generally provides a good estimate of metal toxicity for several fish and invertebrate species. This is demonstrated by the statistical parameters obtained in the regression analysis. The broadened 95% confidence intervals and, in particular, the poor correlations obtained for some aquatic organisms can be ascribed to the more complex metabolic interactions and competing toxic pathways in aquatic organisms, when compared to SMP.     

Exposure‐Based Validation List for Developmental Toxicity Screening Assays

Validation of alternative assays requires comparison of the responses to toxicants in the alternative assay with in vivo responses. Chemicals have been classified as “positive” or “negative” in vivo, despite the fact that developmental toxicity is conditional on magnitude of exposure. We developed a list of positive and negative developmental exposures, with exposure defined by toxicokinetic data, specifically maternal plasma C_max. We selected a series of 20 chemicals that caused developmental toxicity and for which there were appropriate toxicokinetic data. Where possible, we used the same chemical for both positive and negative exposures, the positive being the C_max at a dose level that produced significant teratogenicity or embryolethality, the negative being the C_max at a dose level not causing developmental toxicity. It was not possible to find toxicokinetic data at the no‐effect level for all positive compounds, and the negative exposure list contains C_max values for some compounds that do not have developmental toxicity up to the highest dose level tested. This exposure‐based reference list represents a fundamentally different approach to the evaluation of alternative tests and is proposed as a step toward application of alternative tests in quantitative risk assessment     

Acute toxicity tests on three species of the genus Lecane (Rotifera: Monogononta)

Three rotifer species, Lecane hamata L. luna, and L. quadridentata, were submitted to acute toxicity tests to compare their susceptibility to 11 toxicants. In acute tests with 48-h exposure of neonates of less than 24 h old, copper was most toxic with LC₅₀ values in the range of 0.06–0.33 mg l^–1, while acetone was the least toxic with LC₅₀ values in the range of 5000–7000 mg l^–1. Differences in LC₅₀ value of up to 22-fold were found in the susceptibility to lead between the three species. These data indicate large differences in toxicity among members of the same genus, and point out that it is necessary to submit several species to toxicity tests in order to assess the potential effects of toxicants to rotifers. The commonly used Brachionus calyciflorus cannot be considered representative of all freshwater rotifers in this respect.     

Rhodamine 123 as a probe of in vitro toxicity in MDCK cells

The effect of mercuric chloride on Madin-Darby Canine Kidney (MDCK) cells grown in culture was assayed by the mitochondrial-specific fluorescent probe, rhodamine 123. Treatment of cells with mercuric chloride resulted in a dissipation of rhodamine fluorescence from the mitochondria into the cytoplasm, followed by a release into the medium bathing the cells. Toxicity was assayed either by determining the proportion of cells with delocalized rhodamine fluorescence, or by measuring the rhodamine released from or retained in the cells. Quantifying the release or retention of rhodamine 123 is semi-automated and represents a highly sensitive method of using a vital fluorescent dye for in vitro toxicity analysis.     

Developing a statistical support system for environmental hazard evaluation

Estimating the hazard or risk to both human health and the environment has been based almost exclusively on single species toxicity tests low in environmental realism and without validation of their accuracy in more complex systems. While this may be quite appropriate for humans in a large variety of circumstances, there is no substantive body of direct experimental evidence indicating that precise predictions of harm from hazardous materials can be extrapolated from single species laboratory tests (or even multispecies laboratory tests) to the more complex highly variable natural systems. Now added to the hazardous chemical assessment problem is the accidental or deliberate release of genetically engineered microorganisms into the environment that have the additional capability of multiplying and expanding their numbers and also transferring genetic information to other organisms. This paper focuses entirely on hazard evaluation for organisms other than humans, namely predicting the potential risk or probability of harm to natural systems based on laboratory toxicity testing using single species. Not only will the basic risk assessment strategy itself be examined but also the question of determining the statistical reliability of various extrapolations from one level of biological organization to another. ‘For every complex problem, there is a simple, direct solution ... and it is invariably wrong!’ H. L. Mencken     

Characterization of a gastrointestinal tract microscale cell culture analog used to predict drug toxicity

The lining of the gastrointestinal (GI) tract is the largest surface exposed to the external environment in the human body. One of the main functions of the small intestine is absorption, and intestinal absorption is a route used by essential nutrients, chemicals, and pharmaceuticals to enter the systemic circulation. Understanding the effects of digestion on a drug or chemical, how compounds interact with and are absorbed through the small intestinal epithelium, and how these compounds affect the rest of the body is critical for toxicological evaluation. Our goal is to create physiologically realistic in vitro models of the human GI tract that provide rapid, inexpensive, and accurate predictions of the body's response to orally delivered drugs and chemicals. Our group has developed an in vitro microscale cell culture analog (µCCA) of the GI tract that includes digestion, a mucus layer, and physiologically realistic cell populations. The GI tract µCCA, coupled with a multi‐chamber silicon µCCA representing the systemic circulation, is described and challenged with acetaminophen. Proof of concept experiments showed that acetaminophen passes through and is metabolized by the in vitro intestinal epithelium and is further metabolized by liver cells, resulting in liver cell toxicity in a dose‐dependent manner. The µCCA response is also consistent with in vivo measurements in mice. The system should be broadly useful for studies on orally delivered drugs or ingestion of chemicals with potential toxicity. Biotechnol. Bioeng. 2009; 104: 193–205 © 2009 Wiley Periodicals, Inc.     

Development of a new in vitro system for continuous in vitro exposure of lung tissue to complex atmospheres: Application to diesel exhaust toxicology

The purpose of this study was the development of a new incubation system that can allow continuous exposure of lung tissue to complex atmospheres as a tool for the assessment of aerial environmental lung toxicology. To assess the pertinence of this new exposure system, we studied the impact of diesel engine exhausts as a complex atmosphere containing both gaseous and particulate fractions and have been able to discriminate between the toxicological impacts of the gaseous phase and particulate matter from diesel exhausts. Continuous flow-through rotating chambers with controlled pO₂, pCO₂, and hygrometry have been designed in which lung slices are positioned in rolling inserts that allow free access of atmosphere to the exposed lung tissue. Under control conditions, cell viability was preserved for at least 48 h as assessed by intracellular ATP, GSH, and K⁺ levels and slice O₂ consumption levels. Short-term exposure (1 h) to diesel whole exhausts did not affect intracellular potassium or slice O₂ consumption, while intracellular ATP and GSH levels were markedly decreased. Exposure to filtered exhausts showed less marked effects on both ATP and GSH levels. Superoxide dismutase activity was decreased in a similar way by both total and filtered exhausts while Se⁺-dependent glutathione peroxidase activity was induced by filtered exhausts to a larger extent than after total exhaust exposure, showing different response patterns of lung tissue after exposure to whole or filtered exhausts. In conclusion, this newly designed model opens a promising area in in vitro environmental lung toxicology testing.     

Establishing and maintaining laboratory-based microcosms of riffle insect communities: their potential for multispecies toxicity tests

The purpose of this study was to determine which of the following artificial stream designs would be most logistically simple yet effective in maintaining riffle insects during a 30 d bioassay: 1) static and no current (S-NC); 2) flow-through and no current (FT-NC); 3) static with current (S-C); or 4) flow-through with current (FT-C). Flow-through and current, when provided, were 12 ml min^–1 and 30 cm sec^–1, respectively. Streams were covered by emergence traps, and daylight equivalent lights provided a natural photoperiod. The four stream designs were evaluated in triplicate based on changes in insect species-abundances after 30 d. Test organisms were transferred to the artificial streams in rock-filled containers previously colonized for 30 d in a third-order mountain stream riffle. Additional colonized substrates were sampled immediately to provide an estimate of initial densities placed in the artificial streams. Hess samples were taken directly from the source riffle to evaluate how well the artificial substrates reflected species-abundances on the natural substrate. Adults were collected from the artificial streams every 48–72 h to determine the percentage of initial densities that emerged. After 30 d (7 Aug–6 Sept, 1986), all organisms remaining in the streams were censused. Designs were evaluated using combined densities of adults and young.Relative to benthic samples taken directly from the source riffle, the artificial substrates selected for collector-filterers and against collector-gatherers. The FT-C and S-C stream designs maintained most taxa at or above initial densities, and even in the FT-NC and S-NC streams densities of some taxa were not significantly different (P 0.05) from initial densities. Emergent adults comprised a large proportion of mayfly and chironomid densities and must be monitored during bioassays with aquatic insects. These results indicate that microcosms of riffle insect communities can be maintained for at least 30 d with moderate current and minimal flow-through.     

Cultivation of the heart urchin Echinocardium cordatum and validation of its use in marine toxicity testing for environmental risk assessment

To study environmental risk assessment, echinoderms provide a useful model for ecotoxicological testing. However, limited knowledge of the life history of field collected heart urchins is a problem and the use of cultured urchins has been investigated here. The present study describes a culture method for the heart urchin Echinocardium cordatum under controlled laboratory conditions, providing organisms with a low biological variation. Based on our optimized growth protocol both larvae and juveniles have a growth rate comparable to E. cordatum in the wild. The toxicological response of cultured and field-collected E. cordatum was compared in standard saltwater toxicity bioassays. Using ammonium chloride as a water-soluble reference toxicant, mean 96 h LC₅₀ values for cultured heart urchins versus field collected animals were 37.4 ± 7.6 mg NH₄+/l (n = 5) versus 22.5 ± 4.9 mg NH₄+/l (n = 19), respectively. Additional toxicity experiments with tributyl tin (TBT) spiked sediments revealed 14d LC₅₀ values of 1,242 (95% confidence interval 986–1,564) and 964 (95% confidence interval 843–1,102) µg Sn/kg dw respectively in cultured and field collected E. cordatum. From this it was concluded that cultured heart urchins are less sensitive to TBT than field collected E. cordatum. Furthermore in whole sediment toxicity tests, survival of cultured sea urchins was higher or at least similar to that of field collected E. cordatum. The increased sensitivity of field urchins compared to cultured urchins in various toxicity tests may be due to multiple environmental stressors reducing their overall performance. Overall it was demonstrated that the use of cultured E. cordatum provides a significant advance for urchin-based bioassays for marine environmental toxicity testing, resulting in a more homogeneous, vital population with experimental data displaying reduced variability.     

Implications of a statistical occurrence model for mixture toxicity estimation

This study provides a method for characterizing the effects of concentration variability and correlation among co-acting compounds on mixture toxicity, considering the implications of missing chemical data. The method is explored by developing a set of multiple occurrence scenarios for mixtures of related chemicals. The calculations are performed for hypothetical mixtures of a group of ten synthetic antibiotics that have been tested on marine bacterium to fit dose-response relationships for long-term bioluminescence inhibition of Vibrio fischeri. Mixture toxicities are computed and compared for the assumptions of independent joint action theory and concentration/dose addition theory. The study results show that higher variability in concentrations is associated with higher effective (average) mixture toxicity, in this application by as much as a factor of ten for mixtures with highly variable component concentrations. Moreover, omitting the most toxic compounds caused mixture toxicities to be underestimated by a factor of approximately two. We recommend a pre-assessment of the effect of different chemical occurrence patterns and variability on mixture toxicity to help prioritize needs for further co-occurrence data and toxicity studies.     

The integration of investigative toxicology in the drug discovery process

Summary— Integrating toxicology early in the drug discovery process adds value by providing the earliest possible identification of a compound's potential for toxicological and pathological effects relevant to intended clinical use. With this approach true ‘lead’ candidates, with a high probability of clinical success, are identified and advanced while reducing effort and resources expended on compounds without the requisite therapeutic index. Resources are focussed on the speed of getting a discovery ‘lead’ into early clinical development, defining the mechanisms of observed preclinical toxicity and their relevance to human use, and developing early safety data with in vitro test systems ahead of in vivo systems where possible, thus reducing animal use.     

Lost in translation: The search for an in vitro screen for spermatogenic toxicity

The last two decades have seen an increasing search for in vitro models that can replace the use of animals for safety testing. We adapted the methods from a recent nonquantitative report of spermatogenesis occurring in ex vivo mouse testis explants and tried to develop them into a screening assay. The model consisted of small pieces of neonatal mouse testis (testis “chunks”), explanted and placed on pillars of agarose or chamber inserts, and cultured at the air–liquid interface. A peripheral torus‐shaped zone in these explants would often contain tubules showing spermatogenesis, while the middle of each chunk was often necrotic, depending on the thickness of the tissue. The endpoint was histology: what proportion of tubules in the “permissive torus” actually contained healthy pachytene spermatocytes or spermatids? Extensive statistical modeling revealed that a useful predictive model required more than 60% of these tubules to show spermatogenesis. Separately, the logistics of running this as a predictive assay require that the controls consistently produce ≥ 60% tubules with pachytenes and round spermatids, and achieving this level of spermatogenesis reliably and consistently every week proved ultimately not possible. Extensive trials with various media additions and amendments proved incapable of maintaining the frequency of spermatogenic tubules at consistently ≥ 60%. Congruent with Schooler's “decline effect”; generally, the more often we ran these cultures, the worse the performance became. We hope that future efforts in this area may use our experience as a starting point on the way to a fully productive in vitro model of spermatogenesis.     

Problems associated with selecting the most sensitive species for toxicity testing

When the field of aquatic toxicity testing began its first major expansion about 40 years ago, it was uncommon to use more than one test species (usually a fish). Later, it became customary to use individual microorganisms (usually algae) and macroinvertebrates as well. Most attention was then given to the response of the most sensitive species in that test series when calculating the biologically safe concentration acceptable for use in natural systems. However, in recent years, there has been an attempt to equate the most sensitive species in a laboratory test series to the most sensitive species in natural systems. Since laboratory test species represent only a tiny part of natural systems and since response variability is well established, that can be a dangerous assumption. The purpose of this discussion is to re-examine the scientific support for this practice.     

The role of maternal toxicity in lovastatin-induced developmental toxicity

The role of maternal toxicity in lovastatin-induced developmental toxicity in rats was examined in a series of studies. The first study administered lovastatin at 100, 200, 400, or 800 mg/kg/day (mkd) orally to mated rats from Gestation Day (GD) 6 through 20. Maternal toxicity was observed as transient dose-related body weight losses at the initiation of dosing; there were also deaths and/or morbidity at 400 and 800 mkd. These toxicities occurred in conjunction with forestomach lesions. Mean fetal weights were decreased in all groups (-5 to -16%), and the incidence of skeletal malformations, variations, and incomplete ossifications was increased. The 2 highest doses produced the most severe maternal and developmental effects. Using the same dosages, the second study avoided gestational maternal weight losses and morbidity by starting treatment 14 days before mating with dosing continued to GD 20. There were transient dose-related body weight losses after the start of dosing and deaths in the 400- and 800-mkd groups; however, there was no evidence of maternal toxicity during gestation. Developmental toxicity was evident only as slight, but generally significant (p< or =0.05) decreases in mean fetal weights in groups given > or =200 mkd (-2 to -5%). Significantly, no skeletal abnormalities were observed. A third study administered the pharmacologically active metabolite of lovastatin subcutaneously at dose levels that matched oral maternal drug exposures. In the high-dose group, maternal weight gain and mean fetal weight were slightly decreased but there were no treatment-related skeletal abnormalities. Finally, a series of toxicokinetic studies assessed whether the 2 different developmental toxicity profiles were due to differences in drug exposure between the developmentally toxic and non-toxic dosing regimes. The data showed that groups with no skeletal abnormalities had maternal and embryonic/fetal drug concentrations similar to or even greater than the groups with fetal abnormalities. These results indicate that fetal skeletal abnormalities observed at lovastatin dose levels > or =100 mkd are not due to a direct teratogenic effect, but are the result of excessive maternal toxicity, which most likely involves a nutritional deficiency associated with forestomach lesions and reduced maternal food intake.     

3种除草剂对七星瓢虫的急性毒性与初级风险评估

为了评估除草剂对七星瓢虫Coccinella septempunctata的毒性影响和初级风险,采用药膜法进行了3种除草剂的急性接触毒性测试,并评估其初级风险。结果表明：10%噁唑酰草胺乳油、10%噁嗪草酮悬浮剂和20%异噁唑草酮悬浮剂对七星瓢虫的急性接触毒性分别为LR₅₀>3.61×10² g a.i./ha, LR₅₀>1.485×10² g a.i./ha, LR₅₀>3.60×10² g a.i./ha。10%噁唑酰草胺乳油、10%噁嗪草酮悬浮剂和20%异噁唑草酮悬浮剂的相对安全系数<5,属于中等及以下风险农药,对七星瓢虫的农田内和农田外喷雾场景风险均可接受(危害商分别为HQ_in<0.997,HQ_in<2.000,HQ_in<1.000,HQ_in均<5;HQ_off<0.020,HQ     

In situ toxicity tests of fishes in acid waters

Toxicity of waters within the North Branch of the Moose River to various life stages of lake trout (Salvelinus namaycush), brook trout (Salvelinus fontinalis), creek chub (Semotilus atromaculatus), and blacknose dace (Rhinichthys atratulus) were examined in situ. Study sites were selected that were expected to range from toxic to favourable water quality. For example, pH varied from 4.25 to 7.17, inorganic monomeric Al ranged from ND (< 0.01 mg/l) to 0.40 mg/l, and Ca, from 0.41 to 4.27 mg/l.Toxicity tests were conducted at times when the life stages would naturally occur in these waters and were continued until a range of mortality was observed at the various sites. These experiments suggested that the extent of the drainage system that is toxic to fish increases during snowmelt and major runoff events. Summer base flow water quality was generally the least toxic.Critical life stages were upon hatching and as early feeding fry. In general, young of the year fish were the most tolerant life stage tested. Yearling and adult fish, however, were very sensitive. Blacknose dace were the most sensitive fish of the four species tested, and brook trout were the most tolerant.Hydrogen ion (H⁺) concentration was the most toxic variable in the majority of tests. Inorganic monomeric Al was the most toxic in several, and the combination of H⁺ and Al seemed to cause increased toxicity in many instances. A three-variable model employing hours of exposure, H⁺ concentration, and inorganic monomeric Al predicted mortality quite well. A simple two-variable model using H⁺ and color was nearly as good (R² from 0.49 to 0.94).Documented differences in toxicity among sites and species agreed with observed patterns of fish distribution. These in situ results indicated that laboratory estimates of safe levels of pH and concentrations of Al can result in mortality of fishes in surface waters subject to acidification.     

Use of skin cell cultures for in vitro assessment of corrosion and cutaneous irritancy

Skin cell culture is one of the most promising tools for in vitro evaluation of both cutaneous irritancy and corrosion. New culture methodologies, including three-dimensional reconstruction of skin, allow the evaluation of a wide range of compounds and complex formulations. A number of tests have already been developed for the evaluation of cytotoxicity and many end-points are now currently used, including cell viability, alteration of cell growth or cell function. In recent years parameters more closely related to in vivo irritancy effects such as synthesis of inflammatory mediators and/or their release by keratinocytes after exposure to potential skin irritants have been evaluated. This paper reviews technological aspects and results of validation using skin cell culture for in vitro assessment of corrosion and skin irritancy. Advantages and limits of skin cell cultures are also presented. Current questions about the validation process of cutaneous irritation and corrosion are also considered.     

Goldilocks’ Determination of What New In Vivo Data are “Just Right” for Different Common Drug Development Scenarios,Part 1

As alternative models and scientific advancements improve the ability to predict developmental toxicity, the challenge is how to best use this information to support safe use of pharmaceuticals in humans. While in vivo experimental data are often expected, there are other important considerations that drive the impact of developmental toxicity data to human risk assessment and product labeling. These considerations include three key elements: (1) the drug's likelihood of producing off‐target toxicities, (2) risk tolerance of adverse effects based on indication and patient population, and (3) how much is known about the effects of modulating the target in pregnancy and developmental biology. For example, there is little impact or value of a study in pregnant monkeys to inform the risk assessment for a highly specific monoclonal antibody indicated for a life‐threatening indication against a target known to be critical for pregnancy maintenance and fetal survival. In contrast, a small molecule to a novel biological target for a chronic lifestyle indication would warrant more safety data than simply in vitro studies and a literature review. Rather than accounting for innumerable theoretical possibilities surrounding each potential submission's profile, we consolidated most of the typical situations into eight possible scenarios across these three elements, and present a discussion of these scenarios here. We hope that this framework will facilitate a rational approach to determining what new information is required to inform developmental toxicity risk of pharmaceuticals in context of the specific needs of each program while reducing animal use where possible.