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An area that requires further research is how best to measure test method performance in validation studies and how to set criteria that should be used to judge the adequacy of this performance. The studies reported here were designed to begin an investigation of these questions. Computer simulations were used to generate data sets similar to those that might be obtained from a large validation study. These data were then analysed using three procedures including determination of the 95% prediction interval (PI), calculation of Pearson's correlation coefficient and calculation of the contingent probability statistics (CPS), sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). The results of this work suggest that of the three approaches examined, quantitative measurements with calculation of the 95% PI provide the most information to allow discrimination between the performance of several different NTMs. The results also suggest that dividing data sets into positive and negative toxicity classifications followed by the calculation of CPS leads to considerable information loss. This loss of information may be so significant that it is not possible in certain circumstances to distinguish between NTMs that are adequate and those that are not. [ABSTRACT FROM AUTHOR]

An approach commonly used to measure new toxicity test method (NTM) performance in validation studies is to divide toxicity results into positive and negative classifications, and then identify true positive (TP), true negative (TN), false positive (FP) and false negative (FN) results. After this step is completed, the contingent probability statistics (CPS), sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) are calculated. Although these statistics are widely used and often the only statistics used to assess the performance of toxicity test methods, there is little specific guidance in the validation literature on what values for these statistics indicate adequate performance. The purpose of this study was to begin developing data-based answers to this question by characterizing the CPS obtained from an NTM whose data have a completely random association with a reference test method (RTM).Determining the CPS of this worst-case scenario is useful because it provides a lower baseline from which the performance of an NTM can be judged in future validation studies. It also provides an indication of relationships in the CPS that help identify random or near-random relationships in the data. The results from this study of randomly associated tests show that the values obtained for the statistics vary significantly depending on the cut-offs chosen, that high values can be obtained for individual statistics, and that the different measures cannot be considered independently when evaluating the performance of an NTM. When the association between results of an NTM and RTM is random the sum of the complementary pairs of statistics (sensitivity+ specificity, NPV+PPV) is approximately 1, and the prevalence (i.e., the proportion of toxic chemicals in the population of chemicals) and PPV are equal. Given that combinations of high sensitivity-low specificity or low specificity-high sensitivity (i.e., the sum of the sensitivity and specificity equal to approximately 1) indicate lack of predictive capacity, an NTM having these performance characteristics should be considered no better for predicting toxicity than by chance alone. [ABSTRACT FROM AUTHOR]

Often, the only measures of toxicity test performance provided in validation studies are the contingent probability statistics (CPS) sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). Sensitivity and specificity are generally used in preference to NPV and PPV since NPV and PPV are assumed to vary with changes in prevalence while sensitivity and specificity are assumed to be independent of changes in prevalence. The purpose of the studies reported here was to test whether or not sensitivity and specificity are actually independent of changes in prevalence. Results derived from these studies indicate that sensitivity and specificity vary significantly depending on the prevalence of toxic substances in the set of chemicals being tested. This means sensitivity and specificity should not always be considered constant indicators of toxicity test performance. [ABSTRACT FROM AUTHOR]

Objective: Increasing scalp hair fullness is a global unmet consumer need. An approach to decrease hair shedding by reducing scalp stratum corneum oxidation via a combination of antioxidant and barrier-enhancing technologies has been previously demonstrated. The purpose of this study was to test the effectiveness of the individual antioxidant piroctone olamine in two different product forms (shampoo or leave-on product) for activity to improve hair retention., Methods: Female subjects with self-perceived hair thinning participated in an 8-week, double-blind, placebo-controlled, randomized clinical study to evaluate either a piroctone olamine (PO) containing shampoo or a PO containing leave on treatment, each relative to their corresponding placebo formulation Too many periods. Results for phototrichograms, TEWL, and biomarker analysis of scalp condition for the shampoo treatments are discussed. Phototrichogram results are shared for the assessment of the leave on treatment., Results: Statistically significant increases in hair amount were observed by phototrichogram after use of both PO-containing products versus placebo formulations. The PO shampoo treatment also significantly decreased oxidative stress on the hair and scalp, and improved scalp condition as assessed by TEWL and scalp biomarker values., Conclusion: These results illustrate the effectiveness of a cosmetic antioxidant to improve scalp condition thereby improving hair retention. The observed improvements in scalp condition are consistent with previous reports with other antioxidant technologies and suggest that the hair retention effect was achieved by preventing oxidative damage to the scalp., (© 2021 Society of Cosmetic Scientists and Societe Francaise de Cosmetologie.)

Objective: Determining the amount of hair on the scalp has always been an important metric of patient satisfaction for hair growth and hair retention technologies. While simple in concept, this measurement is a difficult, resource intensive task for the dermatologist and the research scientist. Specifically, counting and measuring hair in phototrichogram images is very time consuming and labour intensive. Due to cost, often only a fraction of available images is manually analysed. There is a need for an automated method that can significantly increase speed and throughput while reducing the cost of counting and measuring hair in phototrichogram images., Methods: Recent advances in machine learning and deep convolutional neural networks (deep learning) have led to a revolution in the analysis of image, video, speech, text and other sensor data. Image diagnostics have seen remarkable improvements with completely automated methods outperforming both human experts and human-engineered analysis methods. Deep learning methods can also provide speed and cost benefits. To enable use of a deep learning, we created a data set of 288 manually annotated phototrichogram images with marked location and length of each hair (the training dataset). We designed a custom neural network architecture and custom image processing algorithms to best utilize the available training data and to maximize performance for hair counting and length measurement. The performance of the algorithm was qualified by comparing hair count and length measurements to an independent ground truth method, the semi-manual Canfield's Hair Metrix method., Results: Leveraging deep neural networks, we have developed capability to apply machine learning to reduce the time needed to acquire data from phototrichograms of patients' scalp from months to seconds. Our algorithm enables fast and fully automated hair counting and length measurement. The algorithm shows high agreement with human manually assisted analysis (ground truth)., Conclusions: We have trained and deployed an algorithm utilizing this technology and have demonstrated the reproducibility, accuracy and speed of this algorithm that, once deployed, requires little to no recurring cost or manual intervention for its operation. The method allows fast analysis of large number of images, reducing study cost and significantly reducing study analysis time., (© 2021 Society of Cosmetic Scientists and Societe Francaise de Cosmetologie.)

Objective: Increasing hair fullness is a global unmet need for many men and women. An approach to the problem is to decrease hair fall or shedding by reducing scalp stratum corneum oxidation and barrier damage to increase hair retention. This study evaluated a combination of functional antioxidants and barrier-enhancing cosmetic ingredients to improve scalp condition thereby enabling stronger hair anchorage and longer retention., Methods: Male and female subjects with normal scalp condition and self-perceived hair thinning participated in a 24-week, double-blind, placebo-controlled, randomized clinical study assessing either a regimen of treatment shampoo and leave-on treatment containing functional antioxidant and barrier-enhancing agents or an identical placebo chassis shampoo control. The functional ingredients were piroctone olamine, zinc pyrithione, zinc carbonate, niacinamide, panthenol and caffeine. At baseline and after 8, 16 and 24 weeks of product use, several measurements were taken: hair shedding, total hair count (by phototrichogram), hair samples, TEWL and evaluation of biomarkers of scalp and hair conditions. Subjects also completed self-assessment questionnaires., Results: Statistically significant effects for functional ingredient-containing treatment regimen versus a placebo control shampoo formulation were observed for reduced hair shedding, increased total hair count, reduced TEWL and improvement in scalp biomarker values. Subjects also noticed these improvements assessed via self-assessment questionnaires., Conclusions: These results establish that the use of functional antioxidant and barrier-enhancing agents to further improve scalp condition can enable a reduction in hair shedding and thus an increase in perceived hair fullness. The underlying improvements in scalp condition suggest the hair benefits were achieved as a result of improved scalp skin barrier and scalp condition leading to a viable preventative approach for hair thinning., (© 2021 Society of Cosmetic Scientists and Societe Francaise de Cosmetologie.)

Derivation of Predicted No Effect Concentrations (PNECs) for aquatic systems is the primary deterministic form of hazard extrapolation used in environmental risk assessment. Depending on the data availability, different regulatory jurisdictions apply application factors (AFs) to the most sensitive measured endpoint to derive the PNEC for a chemical. To assess differences in estimated PNEC values, two PNEC determination methodologies were applied to a curated public database using the EnviroTox Platform (www.EnviroToxdatabase.org). PNECs were derived for 3647 compounds using derivation procedures based on example US EPA and a modified European Union chemical registration procedure to allow for comparisons. Ranked probability distributions of PNEC values were developed and 5th percentile values were calculated for the entire dataset and scenarios where full acute or full chronic data sets were available. The lowest PNEC values indicated categorization based on chemical attributes and modes of action would lead to improved extrapolations. Full acute or chronic datasets gave measurably higher 5th percentile PNEC values. Algae were under-represented in available ecotoxicity data but drove PNECs disproportionately. Including algal inhibition studies will be important in understanding chemical hazards. The PNEC derivation logic flows are embedded in the EnviroTox Platform providing transparent and consistent PNEC derivations and PNEC distribution calculations., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Algal toxicity studies are required by regulatory agencies for a variety of purposes including classification and labeling and environmental risk assessment of chemicals. Algae are also frequently the most sensitive taxonomic group tested. Acute to chronic ratios (ACRs) have been challenging to derive for algal species because of the complexities of the underlying experimental data including: a lack of universally agreed upon algal inhibition endpoints; evolution of experimental designs over time and by different standardization authorities; and differing statistical approaches (e.g., regression versus hypothesis-based effect concentrations). Experimental data for developing globally accepted algal ACRs have been limited because of data availability, and in most regulatory frameworks an ACR of 10 is used regardless of species, chemical type or mode of action. Acute and chronic toxicity (inhibition) data on 17 algal species and 442 chemicals were compiled from the EnviroTox database (https://envirotoxdatabase.org/) and a proprietary database of algal toxicity records. Information was probed for growth rate, yield, and final cell density endpoints focusing primarily on studies of 72 and 96 h duration. Comparisons of acute and chronic data based on either single (e.g., growth rate) and multiple (e.g., growth rate, final cell density) endpoints were used to assess acute and chronic relationships. Linear regressions of various model permutations were used to compute ACRs for multiple combinations of taxa, chemicals, and endpoints, and showed that ACRs for algae were consistently around 4 (ranging from 2.43 to 5.62). An ACR of 4 for algal toxicity is proposed as an alternative to a default value of 10, and recommendations for consideration and additional research and development are provided., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

A standard protocol was used to determine partition (K) and diffusion (D) coefficients in dermatomed human skin and isolated human skin layers for 50 compounds relevant to cosmetics ingredients. K values were measured in dermatomed skin, isolated dermis, whole epidermis, intact stratum corneum (SC), delipidized SC and SC lipids by direct measurements of the radioactivity in the tissue layers/lipid component vs. buffer samples. D determinations were made in dermatomed skin, isolated dermis, whole epidermis and intact SC using a non-linear regression of the cumulative receptor fluid content of radiolabeled compound, fit to the solution of Fick's 2nd Law. Correlation analysis was completed between K, D, and physicochemical properties. The amount of interindividual (donor) and intraindividual (replicate) variability in the K and D data was characterized for each skin layer and chemical. These data can be further used to help inform the factors that influence skin bioavailability and to help improve in silico models of dermal penetration., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Identifying chemicals with narcotic potency is an important aspect of assessing the safety of consumer products that may be accidentally ingested. A rapid and efficient assay of narcotic potency is desired for assessing chemicals with such suspected activity., Objectives: This purpose of this research was to develop a non-mammalian vertebrate, high throughput, neurobehavioral method to assess the narcotic potency of chemicals using larval zebrafish., Methods: Larval zebrafish were acutely exposed to chemicals beginning at 5 days post fertilization (5 dpf). Locomotor activity, elicited by regular, periodic photostimulation, was quantified using a video tracking apparatus. Narcotic potency was determined as the molar concentration at which photostimulated locomotor activity was reduced by 50% (IC 50 ). Toxicity was assessed based on observations of morbidity or mortality. Recovery was assessed following removal of test material by serial dilution and reassessment of photostimulated behavior 24 hr later (6 dpf)., Results: A total of 21 chemicals were assessed. Etomidate, a human narcotic analgesic agent, was used as a reference material. Investigating a series of eleven linear, primary alcohols (C6 to C16), a relationship between narcotic potency and carbon number was observed; narcotic potency increased with carbon number up to C12, consistent with historical studies. For a set of technical grade surfactants, nonionic surfactants (i.e., alcohol ethoxylates) were observed to be narcotic agents while anionic surfactants produced evidence of reduced locomotor activity only in combination with toxicity. Of the solvents evaluated, only ethanol exhibited narcotic activity with an IC 50 of 261 mM and was the least potent of the chemicals investigated. Etomidate was the most potent material evaluated with an IC 50 of 0.39 μM., Conclusions: The larval zebrafish neurobehavioral assay provides a method capable of estimating the narcotic potency of chemicals and can identify if toxicity contributes to observed neurobehavioral effects in the test organism., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Application factors are routinely applied in the extrapolation of laboratory aquatic toxicity data to ensure protection from exposure to chemicals in the natural environment. The magnitude of the application factor is both a scientific and a policy decision, but in any case, it should be rooted in scientific knowledge so as to not be arbitrary. Information-rich chemicals are often subjected to species sensitivity distribution (SSD) analysis to transparently describe certain aspects of assessment uncertainty and are normally subjected to much smaller application factors than screening information data sets. We describe a new set of tools useful to assess the quality of SSDs. Twenty-two data sets and 19 chemicals representing agrochemicals, biocides, surfactants, metals, and common wastewater contaminants were compiled to demonstrate how the tools can be used. "Add-one-in" and "leave-one-out" simulations were used to investigate SSD robustness and develop quantitative evidence for the use of application factors. Theoretical new toxicity data were identified for add-one-in simulations based on the expected probabilities necessary to lower the hazardous concentration to 5% of a species (HC5) by a factor of 2, 3, 5, or 10. Simulations demonstrate the basis for application factors in the range of 1 to 5 for well-studied chemicals with high-quality SSDs. Leave-one-out simulations identify the fact that the most influential values in the SSD come from the extremes of the sensitive and tolerant toxicity values. Mesocosm and field data consistently demonstrate that HC5s are conservative, further justifying the use of small application factors for high-quality SSDs. Environ Toxicol Chem 2019;38:1526-1541. © 2019 SETAC., (© 2019 SETAC.)

We propose a framework on sample size for species sensitivity distribution (SSD) analyses, with perspectives on Bayesian, frequentist, and even nonparametric approaches to estimation. The intent of a statistical sample size analysis is to ensure that the implementation of a statistical model will satisfy a minimum performance standard when relevant conditions are met. It requires that a statistical model be fully specified and that the means of measuring its performance as a function of sample size be detailed. Defining the model conditions under which sample size is calculated is often the most difficult, and important, aspect of sample size analysis because if the model is not representative, then the sample size analysis will provide incorrect guidance. Definitive guidance on sample size requires general agreement on representative models and their performance from stakeholders in important domains such as chemical safety assessments involving government regulators and industry; the present study provides an initial framework that could be used to this end in the future. In addition, our analysis provides immediate value for understanding how well current SSD analyses perform under a few basic models, sample sizes, and quantitative performance criteria. The results confirm that many analyses are adequately sized to estimate hazardous concentration percentile values (typically the 5th percentile for chemical hazard assessments). However, on the low end of sizes seen in common practice, hazardous concentration estimates can be more than 1 order of magnitude greater than the model-defined value. Environ Toxicol Chem 2019;38:1514-1525. © 2019 SETAC., (© 2019 SETAC.)

A database was compiled for algal Organisation for Economic Co-operation and Development (OECD) test guideline 201, for Daphnia magna OECD test guideline 202, for the acute fish toxicity (AFT) OECD test guideline 203, and for the fish embryo toxicity (FET) OECD test guideline 236 to assess the suitability and applicability of the FET test in a threshold approach context. In the threshold approach, algal and Daphnia toxicity are assessed first, after which a limit test is conducted at the lower of the 2 toxicity values using fish. If potential fish toxicity is indicated, a full median lethal concentration assay is performed. This tiered testing strategy can significantly reduce the number of fish used in toxicity testing because algae or Daphnia are typically more sensitive than fish. A total of 165 compounds had AFT and FET data available, and of these, 82 had algal and Daphnia acute toxicity data available. Algae and Daphnia were more sensitive 75 to 80% of the time. Fish or FET tests were most sensitive 20 and 16% of the time, respectively, when considered as the sole fish toxicity indicator and 27% of the time when both were considered simultaneously. When fish were the most sensitive trophic level, different compounds were identified as the most toxic in FET and to AFT tests; however, the differences were not so large that they resulted in substantially different outcomes when potencies were binned using the United Nations categories of aquatic toxicity under the Globally Harmonized System for classification and labeling. It is recommended that the FET test could be used to directly replace the AFT test in the threshold approach or could be used as the definitive test if an AFT limit test indicated toxicity potential for a chemical. Environ Toxicol Chem 2019;38:671-681. © 2019 SETAC., (© 2019 SETAC.)

With increasing antibiotic resistance observed amongst clinical isolates of Neisseria gonorrhoeae, the second most prevalent sexually transmitted bacterial disease in the United States, there is still a need for antimicrobial susceptibility testing (AST). The current method recommended by the Clinical and Laboratory Standards Institute is agar dilution. In this study, we show that a commercially available version of Fastidious Broth is capable of supporting N. gonorrhoeae in the evaluation of minimum inhibitory concentrations of 4 antibiotics (ceftriaxone, azithromycin, ciprofloxacin, and tetracycline), when comparing the agar dilution (AD) versus microbroth dilution (MBD) method and the susceptibilities obtained for 32 N. gonorrhoeae isolates. Herein, 3 out of the 4 antibiotics tested showed 94% or greater essential agreement (EA) and 91% or greater categorical agreement (CA) respectively, when comparing the MBD versus AD methods., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Physiologically based pharmacokinetic (PBPK) models are developed from compound-independent information to describe important anatomical and physiological characteristics of an individual or population of interest. Modeling pediatric populations is challenging because of the rapid changes that occur during growth, particularly in the first few weeks and months after birth. Neonates who are born premature pose several unique challenges in PBPK model development. To provide appropriate descriptions for body weight (BW) and height (Ht) for age and appropriate incremental gains in PBPK models of the developing preterm and full term neonate, anthropometric measurements collected longitudinally from 1,063 preterm and 158 full term neonates were combined with 2,872 cross-sectional measurements obtained from the NHANES 2007-2010 survey. Age-specific polynomial growth equations for BW and Ht were created for male and female neonates with corresponding gestational birth ages of 25, 28, 31, 34, and 40 weeks. Model-predicted weights at birth were within 20% of published fetal/neonatal reference standards. In comparison to full term neonates, postnatal gains in BW and Ht were slower in preterm subgroups, particularly in those born at earlier gestational ages. Catch up growth for BW in neonates born at 25, 28, 31, and 34 weeks gestational age was complete by 13, 8, 6, and 2 months of life (males) and by 10, 6, 5, and 2 months of life (females), respectively. The polynomial growth equations reported in this paper represent extrauterine growth in full term and preterm neonates and differ from the intrauterine growth standards that were developed for the healthy unborn fetus., (© 2018 The Authors. Birth Defects Research Published by Wiley Periodicals, Inc.)

The fish acute toxicity test method is foundational to aquatic toxicity testing strategies, yet the literature lacks a concise sample size assessment. Although various sources address sample size, historical precedent seems to play a larger role than objective measures. We present a novel and comprehensive quantification of the effect of sample size on estimation of the median lethal concentration (LC50), covering a wide range of scenarios. The results put into perspective the practical differences across a range of sample sizes, from n = 5/concentration up to n = 23/concentration. We also provide a framework for setting sample size guidance illustrating ways to quantify the performance of LC50 estimation, which can be used to set sample size guidance given reasonably difficult (or worst-case) scenarios. There is a clear benefit to larger sample size studies: they reduce error in the determination of LC50s, and lead to more robust safe environmental concentration determinations, particularly in cases likely to be called worst-case (shallow slope and true LC50 near the edges of the concentration range). Given that the use of well-justified sample sizes is crucial to reducing uncertainty in toxicity estimates, these results lead us to recommend a reconsideration of the current de minimis 7/concentration sample size for critical studies (e.g., studies needed for a chemical registration, which are being tested for the first time, or involving difficult test substances). Environ Toxicol Chem 2018;37:1565-1578. © 2018 SETAC., (© 2018 SETAC.)

We previously disclosed the discovery of rationally designed N-((1-(4-(propylsulfonyl)piperazin-1-yl)cycloalkyl)methyl)benzamide inhibitors of glycine transporter-1 (GlyT-1), represented by analogues 10 and 11. We describe herein further structure-activity relationship exploration of this series via an optimization strategy that primarily focused on the sulfonamide and benzamide appendages of the scaffold. These efforts led to the identification of advanced leads possessing a desirable balance of excellent in vitro GlyT-1 potency and selectivity, favorable ADME and in vitro pharmacological profiles, and suitable pharmacokinetic and safety characteristics. Representative analogue (+)-67 exhibited robust in vivo activity in the cerebral spinal fluid glycine biomarker model in both rodents and nonhuman primates. Furthermore, rodent microdialysis experiments also demonstrated that oral administration of (+)-67 significantly elevated extracellular glycine levels within the medial prefrontal cortex (mPFC).

Unique aspects of childhood exposure to products need childs specific exposure data. This study developed a probabilistic exposure model for lotion transfer to diapered skin through normal use of baby wipes in children up to 48 months of age. Monte Carlo simulations used baby wipe diary data from the US, Germany and the UK, body weight data from the US, and lotion transfer data from single and multiple wipes adjusting for separate diaper changes. The models predicted a declining number of wipes used/day with a reduction in lotion transfer as age and body weight increased. Experimental testing on multiple sequential wipes used on an overlapping area showed a reduction in lotion deposition by 23.9% after the first wipe. Overall, the weighted population average over the approximate diapering period of 0-36 months across the three geographies at 50th, 90th, & 95th percentiles, were between 130, 230, 260 mg/kg/day, respectively, and 150, 270, 310 mg/kg/day depending on whether a reduction due to overlap is implemented. The statistical model represents an effective strategy to determine exposure to baby wipes lotion for exposure based risk assessment., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

To further define the utility of the Ishikawa cells as a reliable in vitro model to determine the potential estrogenic activity of chemicals of interest, transcriptional changes induced by genistein (GES) in Ishikawa cells at various doses (10 pM, 1 nM, 100 nM, and 10 μM) and time points (8, 24, and 48 h) were identified using a comprehensive microarray approach. Trend analysis indicated that the expression of 5342 unique genes was modified by GES in a dose- and time-dependent manner (P ≤ 0.0001). However, the majority of gene expression changes induced in Ishikawa cells were elicited by the highest dose of GES evaluated (10 μM). The GES' estrogenic activity was identified by comparing the Ishikawa cells' response to GES versus 17 α-ethynyl estradiol (EE, at equipotent doses, ie, 10 μM vs 1 μM, respectively) and was defined by changes in the expression of 284 unique genes elicited by GES and EE in the same direction, although the magnitude of the change for some genes was different. Further, comparing the response of the Ishikawa cells exposed to high doses of GES and EE versus the response of the juvenile rat uterus exposed to EE, we identified 66 unique genes which were up- or down regulated in a similar manner in vivo as well as in vitro Genistein elicits changes in multiple molecular pathways affecting various biological processes particularly associated with cell organization and biogenesis, regulation of translation, cell proliferation, and intracellular transport; processes also affected by estrogen exposure in the uterus of the rat. These results indicate that Ishikawa cells are capable of generating a biologically relevant estrogenic response and offer an in vitro model to assess this mode of action., (© The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Exposure to topically applied substances occurs routinely in premature and hospitalized infant care. Safety determinations are most accurate when exposures are based on appropriately designed studies to capture variations in practice patterns and population heterogeneity. Current safety assessments may not reflect actual practice resulting in overly conservative or understated default assumptions for toxicological determinations. We quantified the amount of baby wipes lotion transferred to premature and term neonatal skin as grams/kg body weight/day. We observed the soil type and number of wipes used for skin cleansing and measured lotion transfer from one wipe applied to freshly clean, dry skin. A Bayesian imputation approach was applied to compute lotion exposure and produce summary statistics. Model covariates were age and weight at evaluation, gender, soil type, soil amount, and number of diaper changes per day. Lotion transfer was measured for 66 premature and 55 term neonates with 449 and 254 evaluations, respectively. The wipes per day was 12.52 overall (all infants and soils), 12.78 for premature and 12.21 for term neonates. Lotion transfer was 0.20 g/kg/day (95th percentile) overall, 0.21 for premature and 0.19 for term neonates. The statistical and experimental methodology represents an effective strategy for determining exposure and assessing risk., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

High-content data have the potential to inform mechanism of action for toxicants. However, most data to support this notion have been generated in vivo. Because many cell lines and primary cells maintain a differentiated cell phenotype, it is possible that cells grown in culture may also be useful in predictive toxicology via high-content approaches such as whole-genome microarray. We evaluated global changes in gene expression in primary rat hepatocytes exposed to two concentrations of ten hepatotoxicants: acetaminophen (APAP), β-naphthoflavone (BNF), chlorpromazine (CPZ), clofibrate (CLO), bis(2-ethylhexyl)phthalate (DEHP), diisononyl phthalate (DINP), methapyrilene (MP), valproic acid (VPA), phenobarbital (PB) and WY14643 at two separate time points. These compounds were selected to cover a range of mechanisms of toxicity, with some overlap in expected mechanism to address the question of how predictive gene expression analysis is, for a given mode of action. Gene expression microarray analysis was performed on cells after 24h and 48h of exposure to each chemical using Affymetrix microarrays. Cluster analysis suggests that the primary hepatocyte model was capable of responding to these hepatotoxicants, with changes in gene expression that appear to be mode of action-specific. Among the different methods used for analysis of the data, a combination method that used pathways (MOAs) to filter total probesets provided the most robust analysis. The analysis resulted in the phthalates clustering closely together, with the two other peroxisome proliferators, CLO and WY14643, eliciting similar responses at the whole-genome and pathway levels. The Cyp inducers PB, MP, CPZ and BNF also clustered together. VPA and APAP had profiles that were unique. A similar analysis was performed on externally available (TG-GATES) in vivo data for 6 of the chemicals (APAP, CLO, CPZ, MP, MP and WY14643) and compared to the in vitro result. These results indicate that transcription profiling using an in vitro assay may offer pertinent biological data to support predictions of in vivo hepatotoxicity potential., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

The OECD validation study of the zebrafish embryo acute toxicity test (ZFET) for acute aquatic toxicity testing evaluated the ZFET reproducibility by testing 20 chemicals at 5 different concentrations in 3 independent runs in at least 3 laboratories. Stock solutions and test concentrations were analytically confirmed for 11 chemicals. Newly fertilised zebrafish eggs (20/concentration and control) were exposed for 96h to chemicals. Four apical endpoints were recorded daily as indicators of acute lethality: coagulation of the embryo, lack of somite formation, non-detachment of the tail bud from the yolk sac and lack of heartbeat. Results (LC50 values for 48/96h exposure) show that the ZFET is a robust method with a good intra- and inter-laboratory reproducibility (CV<30%) for most chemicals and laboratories. The reproducibility was lower (CV>30%) for some very toxic or volatile chemicals, and chemicals tested close to their limit of solubility. The ZFET is now available as OECD Test Guideline 236. Considering the high predictive capacity of the ZFET demonstrated by Belanger et al. (2013) in their retrospective analysis of acute fish toxicity and fish embryo acute toxicity data, the ZFET is ready to be considered for acute fish toxicity for regulatory purposes., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Reconstructed 3D human epidermal skin models are being used increasingly for safety testing of chemicals. Based on EpiDerm™ tissues, an assay was developed in which the tissues were topically exposed to test chemicals for 3h followed by cell isolation and assessment of DNA damage using the comet assay. Inter-laboratory reproducibility of the 3D skin comet assay was initially demonstrated using two model genotoxic carcinogens, methyl methane sulfonate (MMS) and 4-nitroquinoline-n-oxide, and the results showed good concordance among three different laboratories and with in vivo data. In Phase 2 of the project, intra- and inter-laboratory reproducibility was investigated with five coded compounds with different genotoxicity liability tested at three different laboratories. For the genotoxic carcinogens MMS and N-ethyl-N-nitrosourea, all laboratories reported a dose-related and statistically significant increase (P < 0.05) in DNA damage in every experiment. For the genotoxic carcinogen, 2,4-diaminotoluene, the overall result from all laboratories showed a smaller, but significant genotoxic response (P < 0.05). For cyclohexanone (CHN) (non-genotoxic in vitro and in vivo, and non-carcinogenic), an increase compared to the solvent control acetone was observed only in one laboratory. However, the response was not dose related and CHN was judged negative overall, as was p-nitrophenol (p-NP) (genotoxic in vitro but not in vivo and non-carcinogenic), which was the only compound showing clear cytotoxic effects. For p-NP, significant DNA damage generally occurred only at doses that were substantially cytotoxic (>30% cell loss), and the overall response was comparable in all laboratories despite some differences in doses tested. The results of the collaborative study for the coded compounds were generally reproducible among the laboratories involved and intra-laboratory reproducibility was also good. These data indicate that the comet assay in EpiDerm™ skin models is a promising model for the safety assessment of compounds with a dermal route of exposure.

The fish embryo test (FET) is a potential animal alternative for the acute fish toxicity (AFT) test. A comprehensive validation program assessed 20 different chemicals to understand intra- and interlaboratory variability for the FET. The FET had sufficient reproducibility across a range of potencies and modes of action. In the present study, the suitability of the FET as an alternative model is reviewed by relating FET and AFT. In total, 985 FET studies and 1531 AFT studies were summarized. The authors performed FET-AFT regressions to understand potential relationships based on physical-chemical properties, species choices, duration of exposure, chemical classes, chemical functional uses, and modes of action. The FET-AFT relationships are very robust (slopes near 1.0, intercepts near 0) across 9 orders of magnitude in potency. A recommendation for the predictive regression relationship is based on 96-h FET and AFT data: log FET median lethal concentration (LC50) = (0.989 × log fish LC50) - 0.195; n = 72 chemicals, r = 0.95, p < 0.001, LC50 in mg/L. A similar, not statistically different regression was developed for the entire data set (n = 144 chemicals, unreliable studies deleted). The FET-AFT regressions were robust for major chemical classes with suitably large data sets. Furthermore, regressions were similar to those for large groups of functional chemical categories such as pesticides, surfactants, and industrial organics. Pharmaceutical regressions (n = 8 studies only) were directionally correct. The FET-AFT relationships were not quantitatively different from acute fish-acute fish toxicity relationships with the following species: fathead minnow, rainbow trout, bluegill sunfish, Japanese medaka, and zebrafish. The FET is scientifically supportable as a rational animal alternative model for ecotoxicological testing of acute toxicity of chemicals to fish., (Copyright © 2013 SETAC.)

Human interleukin 17 (IL-17) is a proinflammatory cytokine derived mainly from activated T cells. Extensive evidence points to a significant role of IL-17 in many autoimmune and infectious diseases, as well as tumorigenesis and transplant rejection, and suggests that targeting IL-17 could be a promising therapeutic strategy. Robust cell-based assays would thus be essential for lead identification and the optimization of therapeutic candidates. Herein, we report a well-characterized two-step assay, consisting of (a) in vitro activation and stimulation of CD4(+) T lymphocytes by a defined complex of antibodies and cytokines, leading to T helper 17 (Th17) cell differentiation and IL-17 production, and (b) IL-17 quantification in cell supernatants using a homogeneous time-resolved fluorescence (HTRF) assay. The system was optimized for and shown to be reliable in high-throughput compatible 96- and 384-well plate formats. The assay is robust (Z' > 0.5) and simple to perform, yields a stable response, and allows for sufficient discrimination of positive (IL-17-producing cells) and negative controls (uninduced cells). The assay was validated by performing dose-response testing of rapamycin and cyclosporine A, which had previously been reported to inhibit IL-17, and determining, for the first time, their in vitro potencies (IC(50)s of 80 ± 23 pM and 223 ± 52 nM, respectively). Also, IKK 16, a selective small-molecule inhibitor of IκB kinase, was found to inhibit IL-17 production, with an IC(50) of 315 ± 79 nM.

In the absence of toxicological data on a chemical, the threshold of toxicological concern (TTC) approach provides a system to estimate a conservative exposure below which there is a low probability of risk for adverse health effects. The original toxicology dataset underlying the TTC was based on NOELs from repeat dose studies. Subsequently there have been several efforts to assess whether or not these limits are also protective for reproductive/developmental effects. This work expands the database of chemicals with reproductive and developmental data, presents these data in a comprehensive and transparent format and groups the chemicals according to the TTC "Cramer Class" rules. Distributions of NOAELs from each of these classes were used to assess whether the previously proposed TTC values based on repeat dose data are protective for reproductive/developmental toxicity endpoints as well. The present analysis indicates that, for each Cramer Class, the reproductive and developmental endpoints would be protected at the corresponding general TTC tiers derived by Munro et al. (1996)., (Copyright © 2011 Elsevier Inc. All rights reserved.)

There is an urgent need to validate in vitro human skin models for use in safety testing. An important component of validation is characterizing the metabolizing capacity of these models. We report comparison of the expression of 139 genes encoding xenobiotic metabolizing enzymes in the EpiDerm model and human skin. In microarray analysis, the expression of 87% of the genes was consistent between the EpiDerm model and human skin indicating the presence of similar metabolic pathways suggesting commonality in function. Analysis of EpiDerm models constructed from four donors showed highly comparable expression of xenobiotic metabolizing genes demonstrating reproducibility of the model. Overall, the expression of Phase II enzymes appeared to be more pronounced in human skin and the EpiDerm model than that of Phase I enzymes, consistent with the role of skin in detoxification of xenobiotics. Though the basal expression of CYPs in particular was low in EpiDerm, significant induction of CYP1A1/1B1 activity was observed following treatment with 3-methylcholanthrene. These results indicate that the xenobiotic metabolizing capacity of the EpiDerm model appears to be representative of human skin. Models such as EpiDerm provide a valuable in vitro approach for evaluation of metabolism and toxicity of cutaneous exposures to xenobiotics., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Microbial enzymes have been used in laundry detergent products for several decades. These enzymes have also long been known to have the potential to give rise to occupational type 1 allergic responses. A few cases of allergy among consumers using dusty enzyme detergents were reported in the early 1970s. Encapsulation of the enzymes along with other formula changes were made to ensure that consumer exposure levels were sufficiently low that the likelihood of either the induction of IgE antibody (sensitization) or the elicitation of clinical symptoms be highly improbable. Understanding the consumer exposure to enzymes which are used in laundry and cleaning products is a key step to the risk management process. Validation of the risk assessment conclusions and the risk management process only comes with practical experience and evidence from the marketplace. In the present work, clinical data from a range of sources collected over the past 40 years have been analysed. These include data from peer reviewed literature and enzyme specific IgE antibody test results in detergent manufacturers' employees and from clinical study subjects. In total, enzyme specific IgE antibody data were available on 15,765 individuals. There were 37 individuals with IgE antibody. The majority of these cases were from the 1970s where 23 of 4687 subjects (0.49%) were IgE positive and 15 of the 23 were reported to have symptoms of allergy. The remaining 14 cases were identified post-1977 for a prevalence of 0.126% (14/11,078). No symptoms were reported and no relationship to exposure to laundry and cleaning products was found. There was a significant difference between the pre- and post-1977 cohorts in that the higher rates of sensitization with symptoms were associated with higher exposure to enzyme. The clinical testing revealed that the prevalence of enzyme specific IgE in the population is very rare (0.126% since 1977). This demonstrates that exposure to these strong respiratory allergens via use of laundry and cleaning products does not lead to the development of sensitization and disease. These data confirm that the risk to consumers has been properly assessed and managed and support the concept that thresholds of exposure exist for respiratory allergy. Expansion of enzyme use into new consumer product categories should follow completion of robust risk assessments in order to continue ensuring the safe use of enzymes among consumers.

A reliable in vitro model to determine the potential estrogenic activity of chemicals of interest is still unavailable. To further investigate the usefulness of a human-derived cell line, we determined the transcriptional changes induced by bisphenol A (BPA) in Ishikawa cells at various doses (1 nM, 100 nM, 10 microM, and 100 microM) and time points (8, 24 and 48 h) by comparing the response of approximately 38,500 human genes and ESTs between treatment groups and controls (vehicle-treated). By trend analysis, we determined that the expression of 2794 genes was modified by BPA in a dose- and time-dependent manner (p< or =0.0001). However, the majority of gene expression changes induced in Ishikawa cells were elicited by the highest doses of BPA evaluated (10-100 microM), while the genomic response of the cells exposed to low doses of BPA was essentially negligible. By comparing the Ishikawa cells' response to BPA vs.17 alpha-ethynyl estradiol we determined that the change in the expression of 307 genes was identical in the direction of the change, although the magnitude of the change for some genes was different. Further, the response of Ishikawa cells to high doses of BPA shared similarities to the estrogenic response of the rat uterus, specifically, 362 genes were regulated in a similar manner in vivo as well as in vitro. Gene ontology analysis indicated that BPA results in changes to multiple molecular pathways affecting various biological processes particularly associated with cell organization and biogenesis, regulation of translation, cell proliferation, and intracellular transport; processes also affected by estrogen exposure in the uterus of the rat. These results indicate that Ishikawa cells are capable of generating a biologically relevant estrogenic response after exposure to chemicals with varied estrogenic activity, and offer an in vitro model to assess this mode of action., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

The fish acute toxicity test is a mandatory component in the base set of data requirements for ecotoxicity testing. The fish acute toxicity test is not compatible with most current animal welfare legislation because mortality is the primary endpoint and it is often hypothesized that fish suffer distress and perhaps pain. Animal alternative considerations have also been incorporated into new European REACH regulations through strong advocacy for the reduction of testing with live animals. One of the most promising alternative approaches to classical acute fish toxicity testing with live fish is the fish embryo toxicity (FET) test. The FET has been a mandatory component in routine whole effluent testing in Germany since 2005 and has already been standardized at the international level. In order to analyze the applicability of the FET also in chemical testing, a comparative re-evaluation of both fish and fish embryo toxicity data was carried out for a total of 143 substances, and statistical approaches were developed to evaluate the correlation between fish and fish embryo toxicity data. Results confirm that fish embryo tests are neither better nor worse than acute fish toxicity tests and provide strong scientific support for the FET as a surrogate for the acute fish toxicity test.

Three new antibiotics, neopyrrolomycins B (1), C (2), and D (3), with potent activity against Gram-positive pathogens were discovered. They exhibited MIC values < 1 microg/mL versus a number of resistant strains. The compounds were obtained from the ethyl acetate extracts of a Streptomyces sp. after purification by column chromatography and RP-HPLC. Their structures were elucidated using X-ray crystallography (1) and NMR spectroscopy (2 and 3).

We have determined the gene expression profile induced by 17 alpha-ethynyl estradiol (EE) in Ishikawa cells, a human uterine-derived estrogen-sensitive cell line, at various doses (1 pM, 100 pM, 10 nM, and 1 microM) and time points (8, 24, and 48 h). The transcript profiles were compared between treatment groups and controls (vehicle-treated) using high-density oligonucleotide arrays to determine the expression level of approximately 38,500 human genes. By trend analysis, we determined that the expression of 2560 genes was modified by exposure to EE in a dose- and time-dependent manner (p

Two new xanthone antibiotics, citreamicin delta (1) and epsilon (2), with potent activity against Gram-positive pathogens including multidrug-resistant Staphylococcus aureus (MDRSA) were discovered. Compounds 1 and 2 exhibited MIC values < 1 microg/mL versus a number of resistant strains. The compounds were obtained from EtOAc extracts of Streptomyces vinaceus and were purified by countercurrent chromatography and reversed-phase HPLC. Their structures were elucidated using primarily NMR and mass spectroscopy.

Resistance to currently available antibiotics has become a widely recognized crisis in the medical community. To address this, many companies and researchers are refocusing their attention towards natural products, which have an excellent track record of producing effective antibacterial drugs. The AMRI natural product library was screened for activity against multi-drug resistant Staphylococcus aureus (MDRSA). The active samples were counter screened for cytotoxicity against the human hepatocellular carcinoma HepG2 cell line to determine an in vitro therapeutic index (in vitro TI). Those samples with a high in vitro TI were selected for fractionation and dereplication. This led to the discovery of a new anthracycline structure. This metabolite, named mutactimycin E (1), exhibited moderate activity against several gram positive organisms. Here we report the isolation, structure elucidation and biological activities of this new compound.

Concentration and time-dependent changes in hepatic gene expression were examined in adult, female zebrafish (Danio rerio) exposed to 0, 0.1, 0.7, 4.9 microg/L of a model androgen, 17alpha-methyldihydrotestosterone (MDHT). At 24 and 168 h, fish were sacrificed and liver was extracted for gene expression analysis using custom Affymetrix GeneChip Zebrafish Genome Microarrays. In an effort to link gene expression changes to higher levels of biological organization, blood was collected for measurement of plasma steroid hormones (17beta-estradiol (E2), testosterone (T)) and vitellogenin (VTG) using ELISA. Body and ovary weight were also measured. A significant reduction in E2 occurred at 24h (0.7 and 4.9 microg/L) and 168 h (4.9 microg/L) following MDHT exposure. In contrast, T was significantly increased at 24h (4.9 microg/L) and 168 h (0.1, 0.7, 4.9 microg/L). 171 and 575 genes were significantly affected in a concentration-dependent manner at either 24 or 168 h by MDHT exposure at p

The rat uterus responds to acute estrogen treatment with a series of well-characterized physiological responses; however, the gene expression changes required to elicit these responses have not been fully characterized. In order to understand early events induced by estrogen exposure in vivo, we evaluated the temporal gene expression in the uterus of the immature rat after a single dose of 17 alpha-ethinyl estradiol (EE) by microarray analysis, evaluating the expression of 15,923 genes. Immature 20-day-old rats were exposed to a single dose of EE (10 microg/kg), and the effects on uterine histology, weight, and gene expression were determined after 1, 2, 8, 24, 48, 72, and 96 h. EE induced changes in the expression of 3867 genes, at least at one time point (p < or = 0.0001), and at least 1.5-fold (up- or downregulated). Specifically, the expression of 8, 116, 3030, 2076, 381, 445, and 125 genes was modified at 1, 2, 8, 24, 48, 72, or 96 h after exposure to EE, respectively (p < or = 0.0001, t-test). At the tissue and organ level, a clear uterotrophic response was elicited by EE after only 8 h, reaching a maximum after 24 h and remaining detectable even after 96 h of exposure. The uterine phenotypic changes were induced by sequential changes in the transcriptional status of a large number of genes, in a program that involves multiple molecular pathways. Using the Gene Ontology to better understand the temporal response to estrogen exposure, we determined that the earliest changes were in the expression of genes whose products are involved in transcriptional regulation and signal transduction, followed by genes implicated in protein synthesis, energy utilization, solute transport, cell proliferation and differentiation, tissue remodeling, and immunological responses among other pathways. The compendium of genes here presented represents a comprehensive compilation of estrogen-responsive genes involved in the uterotrophic response.

Genomic, proteomic, and metabolomic technologies continue to receive increasing interest from environmental toxicologists. This interest is due to the great potential of these technologies to identify detailed modes of action and to provide assistance in the evaluation of a contaminant's risk to aquatic organisms. Our experimental model is the zebrafish (Danio rerio) exposed to reference endocrine disrupting compounds in order to investigate compound-induced changes in gene transcript profiles. Adult, female zebrafish were exposed to 0, 15, 40, and 100ng/L of 17alpha-ethynylestradiol (EE2) and concentration and time-dependent changes in hepatic gene expression were examined using Affymetrix GeneChip Zebrafish Genome Microarrays. At 24, 48, and 168h, fish were sacrificed and liver mRNA was extracted for gene expression analysis (24 and 168h only). In an effort to link gene expression changes to effects on higher levels of biological organization, body and ovary weights were measured and blood was collected for measurement of plasma steroid hormones (17beta-estradiol (E2), testosterone (T)) and vitellogenin (VTG) using ELISA. EE2 exposure significantly affected gene expression, GSI, E2, T, and VTG. We observed 1622 genes that were significantly affected (p< or =0.001) in a concentration-dependent manner by EE2 exposure at either 24 or 168h. Gene ontology (GO) analysis revealed that EE2 exposure affected genes involved in hormone metabolism, vitamin A metabolism, steroid binding, sterol metabolism, and cell growth. Plasma VTG was significantly increased at 24, 48, and 168h (p< or =0.05) at 40 and 100ng/L and at 15ng/L at 168h. E2 and T were significantly reduced following EE2 exposure at 48 and 168h. GSI was decreased in a concentration-dependent manner at 168h. In this study, we identified genes involved in a variety of biological processes that have the potential to be used as markers of exposure to estrogenic substances. Future work will evaluate the use of these genes in zebrafish exposed to weak estrogens to determine if these genes are indicative of exposure to estrogens with varying potencies.

The multi-lab International Life Sciences Institute (ILSI) project on the application of genomics to risk assessment offered the unique opportunity to investigate the influence of variability within and between laboratories on identifying biologically relevant gene expression changes. We assessed the gene expression profiles of mouse lymphoma L5178Y cells treated with hydroxyurea (HU) in three independent studies from two different laboratories, Sanofi Aventis and Procter and Gamble. Cells were dosed for 4 hr and harvested immediately at the end of the treatment or after a 20-hr recovery period. Cytotoxicity and genotoxicity were evaluated by standard assays. Statistical analysis of these data revealed that, while gene expression responses to HU treatment were markedly different at 4 hr vs. 24 hr, there was otherwise a consistent pattern of dose-response across the three studies. Therefore, the studies were merged and each time point was evaluated separately. At 4 hr, we identified 173 (P < 0.0001) dose-responsive genes with a common trend in all three studies. These were mainly associated with the cell cycle, DNA repair and DNA metabolism, and in particular, the intra-S and G2/M phase checkpoints. At 24 hr, we identified 434 dose-responsive genes common across studies. These genes were involved in lymphocyte-specific activities and the activation of apoptosis via the caspase cascade. Our results show that despite inter-laboratory variability, combining the three studies in a single statistical analysis identifies more significantly-modulated genes than in any of the individual studies, due to improved statistical sensitivity. The genes identified in our study provide information that is relevant to HU biology.

Recently gene expression studies have been multiplied at an accelerated rate by the use of high-density microarrays. By assaying thousands of transcripts at a time, microarrays have led to the discovery of dozens of genes involved in particular biochemical processes, for example, the response of a tissue/organ to a given chemical with therapeutic or toxic properties. The next step in these studies is to focus on the response of a subset of relevant genes to verify or refine potential therapeutic or toxic properties. We have developed a sensitive, high-throughput gene expression assay for this purpose. In this assay, based on the Luminex xMAP system, carefully selected oligonucleotides were covalently linked to fluorescently coded microspheres that are hybridized to biotinylated cRNA followed by amplification of the signal, which results in a rapid, sensitive, multiplexed assay platform. Using this system, we have developed an RNA expression profiling assay specific for 17 estrogen-responsive transcripts and three controls. This assay can evaluate up to 100 distinct analytes simultaneously in a single sample, in a 96-well plate format. This system has improved sensitivity versus existing microsphere-based assays and has sensitivity and precision comparable with or better than microarray technology. We have achieved detection levels down to 1 amol, detecting rare messages in complex cRNA samples, using as little as 2.5 microg starting cRNA. This assay offers increased throughput with decreased costs compared with existing microarray technologies, with the trade-off being in the total number of transcripts that can be analyzed.

The purpose of this study was to determine (1) the transcriptional program elicited by exposure to three estrogen receptor (ER) agonists: 17 alpha-ethynyl estradiol (EE), genistein (Ges), and bisphenol A (BPA) during fetal development of the rat testis and epididymis; and (2) whether very low dosages of estrogens (evaluated over five orders of magnitude of dosage) produce unexpected changes in gene expression (i.e., a non-monotonic dose-response curve). In three independently conducted experiments, Sprague-Dawley rats were dosed (sc) with 0.001-10 microg EE/kg/day, 0.001-100 mg Ges/kg/day, or 0.002-400 mg BPA/kg/day. While morphological changes in the developing reproductive system were not observed, the gene expression profile of target tissues were modified in a dose-responsive manner. Independent dose-response analyses of the three studies identified 59 genes that are significantly modified by EE, 23 genes by Ges, and 15 genes by BPA (out of 8740), by at least 1.5 fold (up- or down-regulated). Even more genes were observed to be significantly changed when only the high dose is compared with all lower doses: 141, 46, and 67 genes, respectively. Global analyses aimed at detecting genes consistently modified by all of the chemicals identified 50 genes whose expression changed in the same direction across the three chemicals. The dose-response curve for gene expression changes was monotonic for each chemical, with both the number of genes significantly changed and the magnitude of change, for each gene, decreasing with decreasing dose. Using the available annotation of the gene expression changes induced by ER-agonist, our data suggest that a variety of cellular pathways are affected by estrogen exposure. These results indicate that gene expression data are diagnostic of mode of action and, if they are evaluated in the context of traditional toxicological end-points, can be used to elucidate dose-response characteristics.

Background: In a previous study, we determined the effects of 17-alpha-ethynyl estradiol (EE) on gene expression using microarrays that represented approximately 9,000 genes, which was the state of-the-art. Higher content arrays with almost double the number of genes have since become available. In order to better determine whether common sets of gene expression changes can be predictive of estrogenic activity, we have replicated the previous experiment using the more comprehensive microarray., Methods: Immature 20-day-old Sprague-Dawley rats were exposed to 0.1, 1, and 10 microg EE/kg/day ( subcutaneously [s.c.]), for four days, dosing from postnatal day (PND) 20-23). Changes in a more comprehensive expression level of 15,923 rat annotated genes and expressed sequence tags were evaluated on PND 24., Results: By comparing the response of the treatment groups versus controls using various statistical parameters, we determined that the expression of 1,394 genes showed a significant change with respect to control (p< or =0.0001), to at least one of the EE dosages. The tissues from animals exposed to 0.1 microg EE/kg/day showed changes in the expression of only 33 genes, whereas when they were exposed to 1 or 10 microg EE/kg/day, the expression of 409 and 548 genes was modified, respectively. A dose-dependent analysis indicated that 592 genes showed a robust and significant response to EE exposure (increased or decreased). Our analysis confirmed the regulation of previously identified estrogen-sensitive genes, and clearly identified novel mediators of estrogen actions, both in the uterus as well as in the ovary., Conclusions: This compendium of genes represents the largest compilation of estrogen-responsive genes that has ever been identified for the immature uterus and ovary of any species, and can be used to generate testable hypothesis to improve the understanding of the molecular pathways associated with physiological and pathophysiological responses to exposure to chemicals with estrogenic properties.

The effect of the dietary background of phytoestrogens on the outcome of rodent bioassays used to identify and assess the reproductive hazard of endocrine-disrupting chemicals is controversial. Phytoestrogens, including genistein, daidzein, and coumestrol, are fairly abundant in soybeans and alfalfa, common ingredients of laboratory animal diets. These compounds are weak agonists for the estrogen receptor (ER) and, when administered at sufficient doses, elicit an estrogenic response in vivo. In this study, we assessed the potential estrogenic effects of dietary phytoestrogens at the gene expression level, together with traditional biologic end points, using estrogen-responsive tissues of the immature female rat. We compared the gene expression profile of the uterus and ovaries, as a pool, obtained using a uterotrophic assay protocol, from intact prepubertal rats fed a casein-based diet (free from soy and alfalfa) or a regular rodent diet (Purina 5001) containing soy and alfalfa. Estrogenic potency of the phytoestrogen-containing diet was determined by analyzing uterine wet weight gain, luminal epithelial cell height, and gene expression profile in the uterus and ovaries. These were compared with the same parameters evaluated in animals exposed to a low dose of a potent ER agonist [0.1 microg/kg/day 17alpha-ethynyl estradiol (EE) for 4 days]. Exposure to dietary phytoestrogens or to a low dose of EE did not advance vaginal opening, increase uterine wet weight, or increase luminal epithelial cell height in animals fed either diet. Although there are genes whose expression differs in animals fed the soy/alfalfa-based diet versus the casein diet, those genes are not associated with estrogenic stimulation. The expression of genes well known to be estrogen regulated, such as progesterone receptor, intestinal calcium-binding protein, and complement component 3, is not affected by consumption of the soy/alfalfa-based diet when assessed by microarray or quantitative reverse transcriptase-polymerase chain reaction analysis. Our results indicate that although diet composition has an impact on gene expression in uterus and ovaries, it does not contribute to the effects of an ER agonist.

During the safety evaluation process of new drugs and chemicals, a battery of genotoxicity tests is conducted starting with in vitro genotoxicity assays. Obtaining positive results in in vitro genotoxicity tests is not uncommon. Follow-up studies to determine the biological relevance of positive genotoxicity results are costly, time consuming, and utilize animals. More efficient methods, especially for identifying a putative mode of action like an indirect mechanism of genotoxicity (where DNA molecules are not the initial primary targets), would greatly improve the risk assessment for genotoxins. To this end, we are participating in an International Life Sciences Institute (ILSI) project involving studies of gene expression changes caused by model genotoxins. The purpose of the work is to evaluate gene expression tools in general, and specifically for discriminating genotoxins that are direct-acting from indirect-acting. Our lab has evaluated gene expression changes as well as micronuclei (MN) in L5178Y TK(+/-) mouse lymphoma cells treated with six compounds. Direct-acting genotoxins (where DNA is the initial primary target) that were evaluated included the DNA crosslinking agents, mitomycin C (MMC) and cisplatin (CIS), and an alkylating agent, methyl methanesulfonate (MMS). Indirect-acting genotoxins included hydroxyurea (HU), a ribonucleotide reductase inhibitor, taxol (TXL), a microtubule inhibitor, and etoposide (ETOP), a DNA topoisomerase II inhibitor. Microarray gene expression analysis was conducted using Affymetrix mouse oligonucleotide arrays on RNA samples derived from cells which were harvested immediately after the 4 h chemical treatment, and 20 h after the 4 h chemical treatment. The evaluation of these experimental results yields evidence of differentially regulated genes at both 4 and 24 h time points that appear to have discriminating power for direct versus indirect genotoxins, and therefore may serve as a fingerprint for classifying chemicals when their mechanism of action is unknown.

Background: Enzymes have been safely used in laundry products for many years. The risk of developing adverse responses to enzymes in laundry detergents among consumers in countries where hand laundry predominates is expected to be low., Objectives: To understand how consumers in hand laundry markets used detergent products; to show that use of enzyme-containing detergents did not lead to sensitization in an atopic population with compromised skin; and to show that enzyme detergents did not have an adverse effect on skin condition., Methods: Women in the rural Philippines were chosen since they do hand laundry for several hours a day, every day. The skin prick test (SPT) tested for the presence of IgE antibody to common aeroallergens and to enzymes in detergent product. Atopic women used enzyme-containing laundry bars for hand laundry and personal cleansing. They also used enzyme-containing laundry granules for hand laundry. All subjects were evaluated by SPT with enzymes over 2 years. Hand and body skin conditions were also evaluated., Results: None of the 1,980 subjects screened for eligibility into the 2-year study were SPT positive to enzymes, including 655 women who used enzyme-containing detergent for up to 1 year. None of the subjects in the study developed IgE to the enzymes. Enzymes had no adverse effect on skin condition or on the development of erosions on the hands., Conclusions: The 2-year study confirms that enzymes are safe for use in laundry products at or below levels tested in the study even when used by atopic consumers under extremely harsh conditions.

The profound effects of 17beta-estradiol on cell growth, differentiation, and general homeostasis of the reproductive and other systems, are mediated mostly by regulation of temporal and cell type-specific expression of different genes. In order to understand better the molecular events associated with the activation of the estrogen receptor (ER), we have used microarray technology to determine the transcriptional program and dose-response characteristics of exposure to a potent synthetic estrogen, 17 alpha-ethynyl estradiol (EE), during prepubertal development. Changes in patterns of gene expression were determined in the immature uterus and ovaries of Sprague-Dawley rats on postnatal day (PND) 24, 24 h after exposure to EE, at 0.001, 0.01, 0.1, 1 and 10 micro g EE/kg/day (sc), for four days (dosing from PND 20 to 23). The transcript profiles were compared between treatment groups and controls using oligonucleotide arrays to determine the expression level of approximately 7000 annotated rat genes and over 1740 expressed sequence tags (ESTs). Quantification of the number of genes whose expression was modified by the treatment, for each of the various doses of EE tested, showed clear evidence of a dose-dependent treatment effect that follows a monotonic response, concordant with the dose-response pattern of uterine wet-weight gain and luminal epithelial cell height. The number of genes whose expression is affected by EE exposure increases according to dose. At the highest dose tested of EE, we determined that the expression level of over 300 genes was modified significantly (p < or = 0.0001). A dose-dependent analysis of the transcript profile revealed a set of 88 genes whose expression is significantly and reproducibly modified (increased or decreased) by EE exposure (p < or = 0.0001). The results of this study demonstrate that, exposure to a potent estrogenic chemical during prepubertal maturation changes the gene expression profile of estrogen-sensitive tissues. Furthermore, the products of the EE-regulated genes identified in these tissues have a physiological role in different intracellular pathways, information that will be valuable to determine the mechanism of action of estrogens. Moreover, those genes could be used as biomarkers to identify chemicals with estrogenic activity.

Exposure to some compounds with estrogenic activity, during fetal development, has been shown to alter development of reproductive organs, leading to abnormal function and disease either after birth or during adulthood. In order to understand the molecular events associated with the estrogenicity of different chemicals and to determine whether common sets of gene expression changes can be predictive of estrogenic activity, we have used microarray technology to determine the transcriptional program influenced by exposure to this class of compounds during organogenesis and development. Changes in patterns of gene expression were determined in the developing uterus and ovaries of Sprague-Dawley rats on GD 20, exposed to graded dosages (sc) of 17alpha-ethynyl estradiol (EE), genistein, or bisphenol A (BPA) from GD 11 to GD 20. Dose levels were roughly equipotent in estrogenic activity. We compared the transcript profiles between treatment groups and controls, using oligonucleotide arrays to determine the expression level of approximately 7000 rat genes and over 1000 expressed squence tags (ESTs). At the highest tested doses of EE, BPA, or genistein, we determined that less than 2% of the mRNA detected by the array showed a 2-fold or greater change in their expression level (increase or decrease). A dose-dependent analysis of the transcript profile revealed a common set of genes whose expression is significantly and reproducibly modified in the same way by each of the 3 chemicals tested. Additionally, each compound induces changes in the expression of other transcripts that are not in common with the others, which indicated not all compounds with estrogenic activity act alike. The results of this study demonstrate that transplacental exposure to chemicals with estrogenic activity changes the gene expression profile of estrogen-sensitive tissues, and that the analysis of the transcript profile of these tissues could be a valuable approach to determining the estrogenicity of different compounds.