Assessment of Biological Activities, Acute and Sub-chronic Toxicity of Liang (Gnetum gnemon var. tenerum) Leaves Powder, a Natural Product.

Gnetum gnemon var. tenerum (Gnetaceae) is a shrub plant native to South-East Asia. In Thailand, Liang leaves are commonly consumed in South of Thailand as vegetable. According to literature, they have an antihyperglycemic capacity because of their rich chlorophyll, fiber, and protein. However, there is need to assess the safety since natural food products are not completely devoid of toxicity. This study aimed to assess the biological activities as well as the acute and sub-chronic oral toxicity of Liang leaves powder (LLP). The evaluation of LLP for acute oral toxicity was performed at dose level 2000 mg/kg body weight in Wistar rats while the sub-chronic oral toxicity of LLP was performed at the effective dose (1.47 g/kg) for antihyperglycemic property according to Organisation for Economic Co-operation and Development (OECD)-425. The results showed that LLP demonstrated anti-inflammatory activities. It also showed no clinical signs of toxic effects and mortality in rats throughout 90 d. Thus, LLP could be classified in GHS category 5 which are of relatively low acute toxicity and then the lethal dose, 50% (LD50) cut off at 5000 mg/kg body weight to infinity (∞). Administration of LLP to the experimental rats significantly increased (p < 0.05) the concentration of triglyceride and increased concentration of creatinine as a result of kidney malfunction was also noticed in the experimental rats. Hematological alteration was not noticed in the treated female rats, but red blood cell, hemoglobin and hematocrit concentrations significantly increased in the treated male rats. The study concludes that sub-chronic administration of 1.47 g/kg LLP is relatively safe.

Reflections on the OECD guidelines for in vitro skin absorption studies.

At the 8th conference of Occupational and Environmental Exposure of the Skin to Chemicals (OEESC) (16-18 September 2019) in Dublin, Ireland, several researchers performing skin permeation assays convened to discuss in vitro skin permeability experiments. We, along with other colleagues, all of us hands-on skin permeation researchers, present here the results from our discussions on the available OECD guidelines. The discussions were especially focused on three OECD skin absorption documents, including a recent revision of one: i) OECD Guidance Document 28 (GD28) for the conduct of skin absorption studies (OECD, 2004), ii) Test Guideline 428 (TGD428) for measuring skin absorption of chemical in vitro (OECD, 2004), and iii) OECD Guidance Notes 156 (GN156) on dermal absorption issued in 2011 (OECD, 2011). GN156 (OECD, 2019) is currently under review but not finalized. A mutual concern was that these guidance documents do not comprehensively address methodological issues or the performance of the test, which might be partially due to the years needed to finalize and update OECD documents with new skin research evidence. Here, we summarize the numerous factors that can influence skin permeation and its measurement, and where guidance on several of these are omitted and often not discussed in published articles. We propose several improvements of these guidelines, which would contribute in harmonizing future in vitro skin permeation experiments.

Improved Assessment of Soil Nonextractable Residues of the Pyrethroid Insecticide Cyphenothrin.

The metabolic fate of pyrethroid insecticide cyphenothrin (1) [(RS)-α-cyano-3-phenoxybenzyl (1RS)-cis-trans-2,2-dimethyl-3-(2-methylprop-1-enyl)cyclopropanecarboxylate] in soils was investigated using 14C-labeled (1R)-cis/trans isomers at the cyclopropane ring. Both isomers degraded with half-lives of 19.0-47.4 days, and 48.9-56.0% and 27.5-38.7% of the applied radioactivity (AR) were mineralized to CO2 and incorporated into nonextractable residues (NER), respectively, after 120 days at 20 °C. NER analyses revealed 37.5-42.2% (cis-1) and 44.9-54.1% (trans-1) of each residue at 30/120 days were comprised of 14C-amino acids (AAs) as microbial products. Assuming that 50% of microbial biomass is AAs, it was estimated that 11.3-22.9%AR (cis-1, 75.0-84.4% of NER) and 13.9-30.4%AR (trans-1, 89.8-108.2% of NER) were nonhazardous biogenic NER (bio-NER), while type I/II xenobiotic NER (xeno-NER) characterized by silylation was insignificant at 0.9-1.0%/2.8-3.3%AR (cis-1). Detailed 14C-AA quantitation indicated a high relevance of the tricarboxylic acid cycle and pyruvate pathway during bio-NER formation, offering new insights into the microbial assimilation of the chrysanthemic moiety.

Enhancing the sensitivity of the thymidine kinase assay by using DNA repair-deficient human TK6 cells.

The OECD guidelines define the bioassays of identifying mutagenic chemicals, including the thymidine kinase (TK) assay, which specifically detects the mutations that inactivate the TK gene in the human TK6 lymphoid line. However, the sensitivity of this assay is limited because it detects mutations occurring only in the TK gene but not any other genes. Moreover, the limited sensitivity of the conventional TK assay is caused by the usage of DNA repair-proficient wild-type cells, which are capable of accurately repairing DNA damage induced by chemicals. Mutagenic chemicals produce a variety of DNA lesions, including base lesions, sugar damage, crosslinks, and strand breaks. Base damage causes point mutations and is repaired by the base excision repair (BER) and nucleotide excision repair (NER) pathways. To increase the sensitivity of TK assay, we simultaneously disrupted two genes encoding XRCC1, an important BER factor, and XPA, which is essential for NER, generating XRCC1 -/- /XPA -/- cells from TK6 cells. We measured the mutation frequency induced by four typical mutagenic agents, methyl methane sulfonate (MMS), cis-diamminedichloro-platinum(II) (cisplatin, CDDP), mitomycin-C (MMC), and cyclophosphamide (CP) by the conventional TK assay using wild-type TK6 cells and also by the TK assay using XRCC1 -/- /XPA -/- cells. The usage of XRCC1 -/- /XPA -/- cells increased the sensitivity of detecting the mutagenicity by 8.6 times for MMC, 8.5 times for CDDP, and 2.6 times for MMS in comparison with the conventional TK assay. In conclusion, the usage of XRCC1 -/- /XPA -/- cells will significantly improve TK assay.

Is a Water Content of 60% Maximum Water Holding Capacity Suitable for Folsomia candida Reproduction Tests? A Study with Silver Nanoparticles and AgNO3.

Silver nanoparticles (AgNP) are increasingly emitted to the environment due to a rise in application in various products; therefore, assessment of their potential risks for biota is important. In this study the effects of AgNP at environmentally relevant concentrations (0.6-375 µg kg-1 soil) on the soil invertebrate Folsomia candida in OECD (Organisation for Economic Co-operation and Development) soil was examined at different soil water contents. Animals were retrieved by heat extraction, which had an efficiency of about 90% compared with the floatation method. The tested water content range is set by OECD Guideline 232 (40-60% of the maximum water holding capacity, WHC), and we detected significant differences in toxicity due to these. With AgNO3, used as an ionic control, the number of juveniles significantly decreased only at 40% WHC, which might be due to dilution of the toxicant at higher soil water content. In turn, at 60% WHC, the reproduction of F. candida significantly increased in the presence of AgNP compared with in the control. However, at this water content, the required number of juveniles in the control treatment was not reached in three independent tests. The fact that the OECD validity criterion is not met indicates that the soil conditions are not suitable for reproduction at 60% WHC.

Incorporation of 14C-cholesterol in human adrenal corticocarcinoma H295R cell line and online-radiodetection of produced 14C-steroid hormone metabolites.

This study demonstrates the addition of 14C-cholesterol to the human cell line H295R will in-situ form radiolabeled steroid hormones allowing for new mechanistic and metabolic insights. The aim of the present study was to in-situ radiolabel steroid hormones from cell line-incorporated 14C-cholesterol using the OECD guideline 456, H295R steroidogenesis in-vitro assay. Radiodetection of the steroid metabolites of the steroidogenic pathway allows for an improved understanding of the various enzymatic mechanisms involved without necessarily being dependent on quantification. Generated radiolabeled steroids were analyzed using HPLC hyphenated with a Flow Scintillation Analyzer (FSA). H295R cells were incubated with radiolabeled cholesterol and cell media were collected and prepared by solid phase extraction and analyzed with HPLC-FSA. For successful radiolabeling of the steroids in the steroidogenesis of H295R cells, radioactive cholesterol may potentially only need to be added just before the cells are incubated for 72h in well plates. Based on the obtained HPLC-FSA chromatograms, and confirmation of the observations by studies in the literature, a qualitative time profile for the production of steroid hormones was estimated. Multiple radiolabeled steroid hormones were identified by means of analytical standards and UV (ultraviolet) co-chromatography, though the elucidation of multiple metabolites remains unresolved. Although online radiodetection proved to suffer from suboptimal sensitivity, the concept of radiolabeling the steroidogenesis in H295R cells with 14C-cholesterol and detecting the radiolabeled steroid hormones online was proved and may assist in further toxicological studies.

Alternative methods for the replacement of eye irritation testing.

In the last decades significant regulatory attempts were made to replace, refine and reduce animal testing to assess the risk of consumer products for the human eye. As the original in vivo Draize eye test has been criticized for limited predictivity, costs and ethical issues, several animal-free test methods have been developed to categorize substances according to the global harmonized system (GHS) for eye irritation.This review summarizes the progress of alternative test methods for the assessment of eye irritation. Based on the corneal anatomy and the current knowledge of the mechanisms causing eye irritation, different ex vivo and in vitro methods will be presented and discussed in regard of possible limitations and their status of regulatory acceptance. In addition to established in vitro models, this review will also highlight emerging, full thickness cornea models that might be applicable to predict all GHS categories.

The fish embryo test (FET): origin, applications, and future.

Originally designed as an alternative for the acute fish toxicity test according to, e.g., OECD TG 203, the fish embryo test (FET) with the zebrafish (Danio rerio) has been optimized, standardized, and validated during an OECD validation study and adopted as OECD TG 236 as a test to assess toxicity of embryonic forms of fish. Given its excellent correlation with the acute fish toxicity test and the fact that non-feeding developmental stages of fish are not categorized as protected stages according to the new European Directive 2010/63/EU on the protection of animals used for scientific purposes, the FET is ready for use not only for range-finding but also as a true alternative for the acute fish toxicity test, as required for a multitude of national and international regulations. If-for ethical reasons-not accepted as a full alternative, the FET represents at least a refinement in the sense of the 3Rs principle. Objections to the use of the FET have mainly been based on the putative lack of biotransformation capacity and the assumption that highly lipophilic and/or high molecular weight substances might not have access to the embryo due to the protective role of the chorion. With respect to bioactivation, the only substance identified so far as not being activated in the zebrafish embryo is allyl alcohol; all other biotransformation processes that have been studied in more detail so far were found to be present, albeit, in some cases, at lower levels than in adult fish. With respect to larger molecules, the extension of the test duration to 96 h (i.e., beyond hatch) has-at least for the substances tested so far-compensated for the reduced access to the embryo; however, more research is necessary to fully explore the applicability of the FET to substances with a molecular weight >3 kDa as well as substances with a neurotoxic mode of action. An extension of the endpoints to also cover sublethal endpoints makes the FET a powerful tool for the detection of teratogenicity, dioxin-like activity, genotoxicity and mutagenicity, neurotoxicity, as well as various forms of endocrine disruption.

Homologue specific analysis of a polyether trisiloxane surfactant in German surface waters and study on its hydrolysis.

The occurrence of a polyether trisiloxane surfactant in the ng L(-1) range in German surface waters is reported for the first time. The studied surfactant does not ubiquitously occur in the aquatic environment but can reach surface waters on a local scale. As a first step towards the understanding of the environmental fate, the hydrolysis was studied according to the OECD guideline 111. It confirmed that the trisiloxane surfactant is sensitive to hydrolysis and that the hydrolysis rate strongly depends on the pH and the temperature. If one takes only into account the hydrolysis, the trisiloxane surfactant could persist several weeks in river water (the half-life in water is approximately 50 days at pH 7, 25 °C, and an initial concentration of 2 mg L(-1)). A degradation product, more polar than the initial trisiloxane surfactant, was identified by high resolution mass spectrometry.