Paclitaxel-Induced Epidermal Alterations: An In Vitro Preclinical Assessment in Primary Keratinocytes and in a 3D Epidermis Model.

Paclitaxel is a microtubule-stabilizing chemotherapeutic agent approved for the treatment of ovarian, non-small cell lung, head, neck, and breast cancers. Despite its beneficial effects on cancer and widespread use, paclitaxel also damages healthy tissues, including the skin. However, the mechanisms that drive these skin adverse events are not clearly understood. In the present study, we demonstrated, by using both primary epidermal keratinocytes (NHEK) and a 3D epidermis model, that paclitaxel impairs different cellular processes: paclitaxel increased the release of IL-1α, IL-6, and IL-8 inflammatory cytokines, produced reactive oxygen species (ROS) release and apoptosis, and reduced the endothelial tube formation in the dermal microvascular endothelial cells (HDMEC). Some of the mechanisms driving these adverse skin events in vitro are mediated by the activation of toll-like receptor 4 (TLR-4), which phosphorylate transcription of nuclear factor kappa B (NF-κb). This is the first study analyzing paclitaxel effects on healthy human epidermal cells with an epidermis 3D model, and will help in understanding paclitaxel's effects on the skin.

In vitro assessment of the photo(geno)toxicity associated with Lapatinib, a Tyrosine Kinase inhibitor.

The epidermal growth factor receptors EGFR and HER2 are the main targets for tyrosine kinase inhibitors (TKIs). The quinazoline derivative lapatinib (LAP) is used since 2007 as dual TKI in the treatment of metastatic breast cancer and currently, it is used as an oral anticancer drug for the treatment of solid tumors such as breast and lung cancer. Although hepatotoxicity is its main side effect, it makes sense to investigate the ability of LAP to induce photosensitivity reactions bearing in mind that BRAF (serine/threonine-protein kinase B-Raf) inhibitors display a considerable phototoxic potential and that afloqualone, a quinazoline-marketed drug, causes photodermatosis. Metabolic bioactivation of LAP by CYP3A4 and CYP3A5 leads to chemically reactive N-dealkylated (N-LAP) and O-dealkylated (O-LAP) derivatives. In this context, the aim of the present work is to explore whether LAP and its N- and O-dealkylated metabolites can induce photosensitivity disorders by evaluating their photo(geno)toxicity through in vitro studies, including cell viability as well as photosensitized protein and DNA damage. As a matter of fact, our work has demonstrated that not only LAP, but also its metabolite N-LAP have a clear photosensitizing potential. They are both phototoxic and photogenotoxic to cells, as revealed by the 3T3 NRU assay and the comet assay, respectively. By contrast, the O-LAP does not display relevant photobiological properties. Remarkably, the parent drug LAP shows the highest activity in membrane phototoxicity and protein oxidation, whereas N-LAP is associated with the highest photogenotoxicity, through oxidation of purine bases, as revealed by detection of 8-Oxo-dG.

Assessment of novel tobacco heating product THP1.0. Part 7: Comparative in vitro toxicological evaluation.

In vitro studies have been widely used to support the toxicological evaluation of chemicals and complex mixtures including cigarette smoke. In this study, the total particulate matter and whole aerosol from a Kentucky reference 3R4F cigarette and two commercially available tobacco heating products (THPs) were assessed using in vitro mutagenicity, cytotoxicity and tumour-promoting activity assays. The Ames assay assessed mutagenicity using Salmonella typhimurium tester strains TA98, TA100, TA1535, TA1537 and TA102 ± metabolic activation (S9). The mouse lymphoma assay was used with short 3 h and longer 24 h exposures. The Bhas 42 cell transformation assay was incorporated as an in vitro alternative for detecting tumour promoters, and the neutral red uptake cell viability assay provided an acute measure of cytotoxicity. To complement the approach, the Ames assay was also employed with S. typhimurium tester strains TA98, TA100, TA1535, TA97 and TA102 using a scaled down methodology for the assessment of aerosols. All the in vitro techniques employed produced a clear positive response with cigarette smoke and in contrast, a negative response to THPs at doses equivalent to or higher than a cigarette smoke test matrix. The data show little difference between the THPs assessed suggesting parity between products.

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

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

Comparative assessment of local tolerance of alcohols commonly used in alcohol-based hand rubs for hand hygiene.

Hand hygiene plays a key role in nosocomial infection prevention. To achieve users' adherence, products' dermal tolerance is essential. We aimed at making a comparative assessment of skin irritation and phototoxicity of the 3 alcohols commonly used in alcohol-based hand rubs (Ethanol, Propan-2-ol, Propan-1-ol) at 60, 70, 80 or 85% w/w in water or with co-formulates (hydrating, emollient and skin protective agents). In vitro validated OECD methods 439 and 432 were used. For irritation, EpiSkin™ Small Model was the chosen Reconstructed Human Epidermis (RhE). For phototoxicity, co-formulates alone or in mixture with and without alcohol were tested using BALB/c 3T3 cell cultures. Whilst Ethanol and Propan-2-ol could not be differentiated and displayed good skin tolerance profiles, Propan-1-ol based products lead to significant viability impairments of RhE at 60, 70 or 80% and at 60% in the presence of co-formulates. However, these results could not be reproduced in another RhE model. Taking also into account bibliographic data on Propan-1-ol, this suggests that our results are probably related to a lack of specificity of the used RhE. Therefore, it can be relevant in case of significant results to use two different RhE models before performing any classification and/or performing any complementary tests.

In vitro assessment of skin sensitization, photosensitization and phototoxicity potential of commercial glyphosate-containing formulations.

This study evaluated the applicability of a modified Direct Peptide Reactivity Assay (DPRA) (OECD N° 442C, 2015) through the 10-fold reduction of reaction volume (micro-DPRA, mDPRA) for skin sensitization evaluation of six commercial glyphosate-containing formulations. In addition, another modification of DPRA was proposed by adding a UVA (5J/cm2) irradiation step, namely photo-mDPRA, to better characterize (photo)sensitizer materials. The phototoxicity profile of pesticides was also evaluated using the 3T3 Neutral Red Uptake Phototoxicity Test (3T3-NRU-PT) (OECD N° 432, 2004). The mDPRA could represent an environmentally acceptable test approach, since it reduces costs and organic waste. Peptide depletion was greater in photo-mDPRA and changed the reactivity class of each test material, in comparison to mDPRA. Thus, the association of mDPRA with photo-mDPRA was better for correctly characterizing human (photo)sensitizer substances and pesticides. In general, cysteine depletion was greater than that of lysine for all materials tested in both mDPRA and photo-mDPRA. Furthermore, while 3T3-NRU-PT is unable to predict (photo)sensitizers, it was capable of correctly identifying the phototoxic potential of the tested agrochemical formulations. In conclusion, mDPRA plus photo-mDPRA and 3T3-NRU-PT seem to be preliminary non-animal test batteries for skin (photo)sensitization/phototoxicity assessment of chemicals, agrochemical formulations and their ingredients.

A comparative assessment of cigarette smoke aerosols using an in vitro air-liquid interface cytotoxicity test.

This study describes the evaluation of a modified air-liquid interface BALB/c 3T3 cytotoxicity method for the assessment of smoke aerosols in vitro. The functionality and applicability of this modified protocol was assessed by comparing the cytotoxicity profiles from eight different cigarettes. Three reference cigarettes, 1R5F, 3R4F and CORESTA Monitor 7 were used to put the data into perspective and five bespoke experimental products were manufactured, ensuring a balanced and controlled study. Manufactured cigarettes were matched for key variables such as nicotine delivery, puff number, pressure drop, ventilation, carbon monoxide, nicotine free dry particulate matter and blend, but significantly modified for vapor phase delivery, via the addition of two different types and quantities of adsorptive carbon. Specifically manufacturing products ensures comparisons can be made in a consistent manner and allows the research to ask targeted questions, without confounding product variables. The results demonstrate vapor-phase associated cytotoxic effects and clear differences between the products tested and their cytotoxic profiles. This study has further characterized the in vitro vapor phase biological response relationship and confirmed that the biological response is directly proportional to the amount of available vapor phase toxicants in cigarette smoke, when using a Vitrocell® VC 10 exposure system. This study further supports and strengthens the use of aerosol based exposure options for the appropriate analysis of cigarette smoke induced responses in vitro and may be especially beneficial when comparing aerosols generated from alternative tobacco aerosol products.

General Cytotoxicity Assessment by Means of the MTT Assay.

Cytotoxicity assays were among the first in vitro bioassay methods used to predict toxicity of substances to various tissues. In vitro cytotoxicity testing provides a crucial means for safety assessment and screening, and for ranking compounds. The choice of using a particular cytotoxicity assay technology may be influenced by specific research goals. As such, four main classes of assays are used to monitor the response of cultured cells after treatment with potential toxicants. These methods measure viability, cell membrane integrity, cell proliferation, and metabolic activity. In this chapter, we focus on the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazolium reduction colorimetric assay to evaluate detrimental intracellular effects on metabolic activity. This assay is well-characterized, simple to use and remains popular in several laboratories worldwide.

New Photosafety Assessment Strategy Based on the Photochemical and Pharmacokinetic Properties of Both Parent Chemicals and Metabolites.

Photoreactivity and dermal/ocular deposition of compounds have been recognized as key considerations for evaluating the phototoxic risk of compounds. Because some drugs are known to cause phototoxic reactions via generation of potent phototoxic metabolites, photosafety assessments on parent drugs alone may lead to false predictions about their photosafety. This study aimed to establish a new photosafety assessment strategy for evaluating the in vivo phototoxic potential of both a parent substance and its metabolites. The in vivo phototoxic risk of fenofibrate (FF) and its metabolites, fenofibric acid (FA) and reduced fenofibric acid, were evaluated based on photochemical and pharmacokinetic analyses. FF and FA exhibited intensive UV absorption, with molar extinction coefficient values of 17,000 (290 nm) and 14,000 M(-1)cm(-1) (295 nm), respectively. Superoxide generation from FA was significantly higher than from FF, and a marked increase in superoxide generation from FF was observed after incubation with rat hepatic S9 fractions, suggesting enhanced photoreactivity of FF after metabolism. FA showed high dermal/ocular deposition after oral administration (5 mg/kg, p.o.) although the concentration of FF was negligible, suggesting high exposure risk from FA. On the basis of these findings, FA was deduced to be a major contributor to phototoxicity induced by FF taken orally, and this prediction was in accordance with the results from in vitro/in vivo phototoxicity tests. Results from this study suggest that this new screening strategy for parent substances and their metabolites provides reliable photosafety information on drug candidates and would be useful for drug development with wide safety margins.

Non-animal photosafety screening for complex cosmetic ingredients with photochemical and photobiochemical assessment tools.

Previously, a non-animal screening approach was proposed for evaluating photosafety of cosmetic ingredients by means of in vitro photochemical and photobiochemical assays; however, complex cosmetic ingredients, such as plant extracts and polymers, could not be evaluated because their molecular weight is often poorly defined and so their molar concentration cannot be calculated. The aim of the present investigation was to establish a photosafety screen for complex cosmetic ingredients by using appropriately modified in vitro photosafety assays. Twenty plant extracts were selected as model materials on the basis of photosafety information, and their phototoxic potentials were assessed by means of ultraviolet (UV)/visible light (VIS) spectral analysis, reactive oxygen species (ROS)/micellar ROS (mROS) assays, and 3T3 neutral red uptake phototoxicity testing (3T3 NRU PT). The maximum UV/VIS absorption value was employed as a judgment factor for evaluating photoexcitability of samples, and the value of 1.0 was adopted as a tentative criterion for photosafety identification. The ROS/mROS assays were conducted at 50 µg/mL, and no false negative prediction was obtained. Furthermore, the ROS/mROS assays at 50 µg/mL had a similar predictive capacity to the ROS/mROS assays in the previous study. A systematic tiered approach for simple and rapid non-animal photosafety evaluation of complex cosmetic ingredients can be constructed using these modified in vitro photochemical assays.

Toxicological assessment of kretek cigarettes Part 6: the impact of ingredients added to kretek cigarettes on smoke chemistry and in vitro toxicity.

Mainstream smoke (MS) from experimental kretek cigarettes with three ingredient mixes at low (typical use level) and high (2.5 or 3 times that level) inclusion rates was compared to a control kretek cigarette of identical construction (cloves and humectants), but without the addition of ingredients. 350 ingredients, commonly used in various combinations and in a limited number in a given brand in the manufacture of marketed kretek cigarettes were assessed. The MS composition of the kretek cigarettes was characterized by a comprehensive set of analytes (55 smoke constituents). Furthermore, the smoke was assessed in vitro for its cytotoxicity in the Neutral Red Uptake assay (particle phase and gas/vapor phase separately) in mouse embryo BALB/c 3T3 cells, and for mutagenicity/genotoxicity in the Salmonella typhimurium reverse mutation assay and the mammalian cell mouse lymphoma TK assay in L5178Y cells, the latter with and without metabolic activation. There were some statistically significant differences in the yield of smoke constituents (increases as well as decreases, nearly all of them less than ± 20%) as a result of the addition of the ingredient mixes. However, the addition of the three different mixes of ingredients to the experimental kreteks did not change the in vitro cytotoxicity and mutagenicity/genotoxicity of the smoke, when compared to the control kretek cigarette.

Toxicological assessment of kretek cigarettes Part 4: mechanistic investigations, smoke chemistry and in vitro toxicity.

The smoke chemistry and in vitro toxicity of mainstream smoke (MS) was investigated in American-blended cigarettes with or without the addition of 2.5%, 5% or 10% eugenol to the tobacco and in Indonesian-blended cigarettes with and without the addition of cloves, cloves extracted with hot ethanol, and extracted cloves replenished with eugenol or clove oil. The addition of eugenol reduced the concentration of nearly all toxicants measured in MS as well as the in vitro cytotoxicity of the gas/vapor phase. Reductions were also seen in bacterial mutagenicity of the total particulate matter (TPM) assessed by the Ames Assay. The addition of extracted cloves led to increases and decreases of toxicant concentrations in MS. Replenishment with eugenol or clove oil decreased the toxicant concentrations; with most smoke constituent concentrations reduced below the concentration found in tobacco-only cigarettes. Cytotoxicity of the TPM was not affected by the clove preparations. However, GVP cytotoxicity was reduced (untreated cloves showing the highest reductions). Mutagenicity of TPM was decreased by the clove preparations. Mechanisms for the reductions, (up to 40%), are most likely due to dilution effects by eugenol, changed burning characteristics of the tobacco, and free radical scavenging by eugenol.

A comprehensive evaluation of the toxicology of the "Deli" cast sheet process used in experimental cigarettes.

CONTEXT: Manufacture of cigarettes results in tobacco by-products, some of which can be processed and added back to cigarettes. Such additions (known as reconstituted tobacco or reconstituted leaf) have been shown to reduce tar yields. A new process (termed "Deli" cast sheet) is a potential refinement of the reconstitution process. OBJECTIVE: Compare toxicity of smoke from experimental cigarettes made with reconstituted leaf with that from cigarettes made with Deli cast sheet. MATERIALS AND METHODS: Analytical chemistry, Salmonella mutagenicity and cytotoxicity assays were used to evaluate the composition biological activity of mainstream smoke from experimental cigarettes made with Deli cast sheet or with reconstituted leaf. The effect of different amounts of guar and propylene glycol in Deli cast sheet was also evaluated. RESULTS: Small increases in the amount of nitrogen oxides were found as a result of inclusion of the Deli cast sheet when compared with reconstituted leaf; no differences in cytotoxicity or mutagenicity were found. CONCLUSION: The Deli process neither significantly modified chemical composition of smoke nor affected its biological activity, as measured by the mutagenicity and cytotoxicity assays used here.

Non-animal photosafety assessment approaches for cosmetics based on the photochemical and photobiochemical properties.

The main purpose of the present study was to establish a non-animal photosafety assessment approach for cosmetics using in vitro photochemical and photobiochemical screening systems. Fifty-one cosmetics, pharmaceutics and other chemicals were selected as model chemicals on the basis of animal and/or clinical photosafety information. The model chemicals were assessed in terms of photochemical properties by UV/VIS spectral analysis, reactive oxygen species (ROS) assay and 3T3 neutral red uptake phototoxicity testing (3T3 NRU PT). Most phototoxins exhibited potent UV/VIS absorption with molar extinction coefficients of over 1000M(-1)cm(-1), although false-negative prediction occurred for 2 cosmetic phototoxins owing to weak UV/VIS absorption. Among all the cosmetic ingredients, ca. 42% of tested chemicals were non-testable in the ROS assay because of low water solubility; thereby, micellar ROS (mROS) assay using a solubilizing surfactant was employed for follow-up screening. Upon combination use of ROS and mROS assays, the individual specificity was 88.2%, and the positive and negative predictivities were estimated to be 94.4% and 100%, respectively. In the 3T3 NRU PT, 3 cosmetics and 4 drugs were incorrectly predicted not to be phototoxic, although some of them were typical photoallergens. Thus, these in vitro screening systems individually provide false predictions; however, a systematic tiered approach using these assays could provide reliable photosafety assessment without any false-negatives. The combined use of in vitro assays might enable simple and fast non-animal photosafety evaluation of cosmetic ingredients.

Individual and combined developmental toxicity assessment of bisphenol A and genistein using the embryonic stem cell test in vitro.

The potential developmental toxicity of environmental estrogenic endocrine disruptors have become a great concern in recent years. In this study, two typical environmental oestrogen, namely, bisphenol A (BPA) and genistein (GEN) were investigated for potential embryotoxicity using the embryonic stem cell test model. Afterwards, a 4×4 full factorial design and the estimated marginal means plot were performed to assess the combined effects of these two compounds. According to the linear discriminant functions and classification criteria, bisphenol A and genistein were classified as weakly embryotoxic and strongly embryotoxic respectively. As for combined effects, the overall interaction between BPA and GEN on embryonic stem cells (ESCs) differentiation was synergistic at low dosages, however, on ESCs and 3T3 cell proliferation, the predominate action was additive. Considering the actual daily intake of these chemicals, it is concluded that BPA alone might not have adverse reproductive or developmental effects on human being. However, given that BPA and GEN do have synergistic effect at low concentration, they may disturb normal embryo development together, which could result in birth defect and behavioral alterations later in life.

Chemical-based risk assessment and in vitro models of human health effects induced by organic pollutants in soils from the Olona Valley.

Risk assessment of soils is usually based on chemical measurements and assuming accidental soil ingestion and evaluating induced toxic and carcinogenic effects. Recently biological tools have been coupled to chemical-based risk assessment since they integrate the biological effects of all xenobiotics in soils. We employed integrated monitoring of soils based on chemical analyses, risk assessment and in vitro models in the highly urbanized semirural area of the Olona Valley in northern Italy. Chemical characterization of the soils indicated low levels of toxic and carcinogenic pollutants such as PAHs, PCDD/Fs, PCBs and HCB and human risk assessment did not give any significant alerts. HepG2 and BALB/c 3T3 cells were used as a model for the human liver and as a tool for the evaluation of carcinogenic potential. Cells were treated with soil extractable organic matters (EOMs) and the MTS assay, LDH release and morphological transformation were selected as endpoints for toxicity and carcinogenicity. Soil EOMs induced dose-dependent inhibition of cell growth at low doses and cytotoxicity after exposure to higher doses. This might be the result of block of cell cycle progression to repair DNA damage caused by oxidative stress; if this DNA damage cannot be repaired, cells die. No significant inductions of foci were recorded after exposure to EOMs. These results indicate that, although the extracts contain compounds with proven carcinogenic potential, the levels of these pollutants in the analyzed soils were too low to induce carcinogenesis in our experimental conditions. In this proposed case study, HepG2 cells were found an appropriate tool to assess the potential harm caused by the ingestion of contaminated soil as they were able to detect differences in the toxicity of soil EOMs. Moreover, the cell transformation assay strengthened the combined approach giving useful information on carcinogenic potential of mixtures.

Assessment of the predictive capacity of the 3T3 Neutral Red Uptake cytotoxicity test method to identify substances not classified for acute oral toxicity (LD50>2000 mg/kg): results of an ECVAM validation study.

Assessing chemicals for acute oral toxicity is a standard information requirement of regulatory testing. However, animal testing is now prohibited in the cosmetics sector in Europe, and strongly discouraged for industrial chemicals. Building on the results of a previous international validation study, a follow up study was organised to assess if the 3T3 Neutral Red Uptake cytotoxicity assay could identify substances not requiring classification as acute oral toxicants under the EU regulations. Fifty-six coded industrial chemicals were tested in three laboratories, each using one of the following protocols: the previously validated protocol, an abbreviated version of the protocol and the protocol adapted for an automation platform. Predictions were very similar among the three laboratories. The assay exhibited high sensitivity (92-96%) but relatively low specificity (40-44%). Three chemicals were under predicted. Assuming that most industrial chemicals are not likely to be acutely toxic, this test method could prove a valuable component of an integrated testing strategy, a read-across argument, or weight-of-evidence approach to identify non toxic chemicals (LD50>2000 mg/kg). However, it is likely to under predict chemicals acting via specific mechanisms of action not captured by the 3T3 test system, or which first require biotransformation in vivo.

Prevalidation study of the BALB/c 3T3 cell transformation assay for assessment of carcinogenic potential of chemicals.

The cell transformation assays (CTAs) have attracted attention within the field of alternative methods due to their potential to reduce the number of animal experiments in the field of carcinogenicity. The CTA using BALB/c 3T3 cells has proved to be able to respond to chemical carcinogens by inducing morphologically transformed foci. Although a considerable amount of data on the performance of the assay has been collected, a formal evaluation focusing particularly on reproducibility, and a standardised protocol were considered important. Therefore the European Centre for the Validation of Alternative Methods (ECVAM) decided to coordinate a prevalidation study of the BALB/c 3T3 CTA. Three different laboratories from Japan and Europe participated. In the study the following modules were assessed stepwise: test definition (Module 1) consisted of the standardisation of the protocol, the selection of the cell lineage, and the preparation of a photo catalogue on the transformed foci. The within-laboratory reproducibility (Module 2) and the transferability (Module 3) were assessed using non-coded and coded 3-methylcholanthrene. Then, five coded chemicals were tested for the assessment of between-laboratory reproducibility (Module 4). All three laboratories obtained positive results with benzo[a]pyrene, phenanthrene and o-toluidine HCl. 2-Acetylaminofluorene was positive in two laboratories and equivocal in one laboratory. Anthracene was negative in all three laboratories. The chemicals except phenanthrene, which is classified by IARC (http://monographs.iarc.fr) as group 3 "not classifiable as to its carcinogenicity to human", were correctly predicted as carcinogens. Further studies on phenanthrene will clarify this discrepancy. Thus, although only a few chemicals were tested, it can be seen that the predictive capacity of the BALB/c 3T3 CTA is satisfactory. On the basis of the outcome of this study, an improved protocol, incorporating some changes related to data interpretation, has been developed. It is recommended that this protocol be used in the future to provide more data that may confirm the robustness of this protocol and the performance of the assay itself. During the study it became clear that selecting the most appropriate concentrations for the transformation assay is crucial.

Embryotoxicity assessment of developmental neurotoxicants using a neuronal endpoint in the embryonic stem cell test.

The embryonic stem cell test (EST) is a validated in vitro embryotoxicity test; however, as the inhibition of cardiac differentiation alone is used as a differentiation endpoint in the EST, it may not be a useful test to screen embryotoxic chemicals that affect the differentiation of noncardiac tissues. Previously, methylmercury (MeHg), cadmium and arsenic compounds, which are heavy metals that induce developmental neurotoxicity in vivo, were misclassified as nonembryotoxic with the EST. The aim of this study was to improve the EST to correctly screen such developmental neurotoxicants. We developed a neuronal endpoint (Tuj-1 ID50) using flow cytometry analysis of Tuj-1-positive cells to screen developmental neurotoxicants (MeHg, valproic acid, sodium arsenate and sodium arsenite) correctly using an adherent monoculture differentiation method. Using Tuj-1 ID50 in the EST instead of cardiac ID50, all of the tested chemicals were classified as embryotoxic, while the negative controls were correctly classified as nonembryotoxic. To support the validity of Tuj-1 ID50) , we compared the results from two experimenters who independently tested MeHg using our modified EST. An additional neuronal endpoint (MAP2 ID50), obtained by analyzing the relative quantity of MAP2 mRNA, was used to classify the same chemicals. There were no significant differences in the three endpoint values of the two experimenters or in the classification results, except for isoniazid. In conclusion, our results indicate that Tuj-1 ID50 can be used as a surrogate endpoint of the traditional EST to screen developmental neurotoxicants correctly and it can also be applied to other chemicals.

Review of the performance of the 3T3 NRU in vitro phototoxicity assay in the pharmaceutical industry.

Based on current regulatory guidelines a substantial proportion of drug development portfolios within the pharmaceutical industry trigger the requirements for photosafety testing i.e. absorb light in the range 290-700 nm, and are either applied locally/topically, or 'reach' (EMEA)/'significantly partition to' (FDA) the skin or eyes. There has been growing concern within the pharmaceutical industry over recent years regarding the performance of the in vitro photosafety tests with respect to in vivo predictivity (in animals and in humans). Therefore, the Safety Ad hoc Group (SAHG) of the European Federation of Pharmaceutical Industries and Associations (EFPIA) commissioned a survey of member companies to better understand the triggers for photosafety testing and how in vitro hazard characterisation translated to in vivo risk (both in animal models and in humans). Data were collated for 361 compounds from 10 EFPIA member companies and the results of the phototoxicity survey are reported. Based on these results, it appears that the in vitro photosafety assays are substantially over predicting animal photosafety hazard in vivo and also human photosafety risk in the clinic. This raises concern regarding the use of in vitro photosafety assays for the assessment of chemical photosafety of pharmaceuticals for regulatory purposes. At the very least, a review of the current guidance documents for the photosafety evaluation of pharmaceuticals should be undertaken urgently.