Organisation For Economic Co-operation And Development Test Guideline Research Articles

Evaluation of Teratogenic Profile of 
Combination of Lumacaftor and Ivacaftor in Sprague–Dawley Rats

Background: The individual teratogenicity profiles of Lumacaftor and Ivacaftor are known, but the combined effects remain unexplored. Purpose: To evaluate the teratogenic profile of a combination of Lumacaftor and Ivacaftor in Sprague-Dawley Rats. Methods: This study adhered to the Committee for Control and Supervision of Experiments on Animals (CCSEA) and Organisation for Economic Co-operation and Development (OECD) guidelines. Sprague–Dawley rats were acclimated and housed individually under controlled conditions. A polygamous breeding scheme was implemented, and pregnant female rats were assigned to control, vehicle control or treatment group following OECD Test Guideline 414. Lumacaftor and Ivacaftor were administered to pregnant rats at various doses, such as MRHD, 5MRHD, 8MRHD and 10MRHD, from gestation days 6 to 17. Clinical observations, body weight and food consumption of pregnant rats were monitored. Postmortem examinations included caesarean section, reproductive performance (the number of corpora lutea, implantation sites, early and late resorptions and live foetuses) and assessment of foetal analysis. The foetal analysis incorporated external malformation evaluation, visceral examination and skeletal examination using alizarin red solution. Results: In pregnant rats exposed to Lumacaftor and Ivacaftor, the external examination was conducted on 206, 209, 199, 184, 198 and 201 foetuses in their respective groups, each consisting of 19 litters. Within this cohort, approximately 33% of foetuses underwent visceral examination. Additionally, across six groups approximately 50% of foetuses underwent skeletal examination. Importantly, no abnormalities were detected in skeletal, visceral or histopathological assessments across the treatment groups encompassing doses of a combination of Lumacaftor and Ivacaftor at MRHD, 5MRHD, 8MRHD and 10MRHD. Additionally, no significant morbidity or mortality related to the treatment was observed. Conclusion: In conclusion, the study indicates that the combination of Lumacaftor and Ivacaftor at doses equivalent to MRHD, 5MRHD, 8MRHD and 10MRHD did not induce significant teratogenic effects, affirming their safety during pregnancy in rats.

A Comparison of In Vitro Metabolic Clearance of Various Regulatory Fish Species Using Hepatic S9 Fractions.

Bioaccumulation predictions can be substantially improved by combining in vitro metabolic rate measurements derived from rainbow trout hepatocytes and/or hepatic S9 fractions with quantitative structure-activity relationship (QSAR) modeling approaches. Compared with in vivo testing guidelines Organisation for Economic Co-operation and Development (OECD) 305 and Office of Chemical Safety and Pollution Prevention (OCSPP; an office of the US Environmental Protection Agency) 850.1730, the recently adopted OECD test guidelines 319A and 319B are in vitro approaches that have the potential to provide a time- and cost-efficient, humane solution, reducing animal use while addressing uncertainties in bioaccumulation across species. The present study compares the hepatic clearance of the S9 subcellular fraction of rainbow trout, bluegill, common carp, fathead minnow, and largemouth bass, discerning potential differences in metabolism between different warm- and cold-water species. With refinements to the in vitro metabolic S9 assay for high-throughput analysis, we measured in vitro clearance rates of seven chemicals crossing multiple classes of chemistry and modes of action. We confirmed that data from rainbow trout liver S9 fraction metabolic rates can be utilized to predict rainbow trout bioconcentration factors using an in vitro to in vivo extrapolation model, as intended in the OECD 319B applicability domain per the bioaccumulation prediction. Also, we determined that OECD 319B can be applied to other species, modified according to their habitat, adaptations to feeding behavior, and environmental conditions (e.g., temperature). Once toxicokinetics for each species is better understood and appropriate models are developed, this method can be an excellent tool to determine hepatic clearance and potential bioaccumulation across species. The present study could be leveraged prior to or in place of initiating in vivo bioconcentration studies, thus optimizing selection of appropriate fish species. Environ Toxicol Chem 2024;00:1-16. © 2024 SETAC.

In vivo genotoxicity testing strategies: Report from the 8th International Workshop on Genotoxicity Testing (IWGT)

AbstractThe in vivo working group (WG) considered three topics: acceptable maximum doses for negative erythrocyte micronucleus (MN) tests, validation status of MN assays in non‐hematopoietic tissues, and nuisance factors in the comet assay. The WG reached agreement on many issues, including: negative erythrocyte MN studies should be acceptable if dosing is conducted to Organisation for Economic Co‐operation and Development (OECD) test guideline (TG) 474 recommendations and if sufficient bone marrow exposure is demonstrated; consensus on the evidence required to demonstrate “sufficient” exposure was not reached. The liver MN test using six‐week‐old rats is sufficiently validated to develop an OECD TG, but the impact of animal age warrants additional study. Ki‐67 is a reliable marker for cellular proliferation in hepatocytes. The gastrointestinal tract MN test is useful for detecting poorly absorbed or rapidly degraded aneugens, and for genotoxic metabolites formed in the colon. Although current validation data are insufficient to support the development of an OECD TG, the methodologies are sufficient to consider as an appendix to OECD TG474. Comparison of comet assay results to laboratory historical control data (HCD) should not be used in data evaluation, unless the HCD distribution is demonstrated to be stable and the predominant source of HCD variation is due to animal, not study, factors. No universally acceptable negative control limit for any tissue was identified. Methodological differences in comet studies can result in variable data interpretations; more data are required before best practice recommendations can be made. Hedgehogs alone are unreliable indicators of cytotoxicity and additional investigations into cytotoxicity markers are required.

Defined approaches to classify agrochemical formulations into EPA hazard categories developed using EpiOcularTM reconstructed human corneal epithelium and bovine corneal opacity and permeability assays

Many sectors have seen complete replacement of the in vivo rabbit eye test with reproducible and relevant in vitro and ex vivo methods to assess the eye corrosion/irritation potential of chemicals. However, the in vivo rabbit eye test remains the standard test used for agrochemical formulations in some countries. Therefore, two defined approaches (DAs) for assessing conventional agrochemical formulations were developed, using the EpiOcularTM Eye Irritation Test (EIT) [Organisation for Economic Co-operation and Development (OECD) test guideline (TG) 492] and the Bovine Corneal Opacity and Permeability (OECD TG 437; BCOP) test with histopathology. Presented here are the results from testing 29 agrochemical formulations, which were evaluated against the United States Environmental Protection Agency’s (EPA) pesticide classification system, and assessed using orthogonal validation, rather than direct concordance analysis with the historical in vivo rabbit eye data. Scientific confidence was established by evaluating the methods and testing results using an established framework that considers fitness for purpose, human biological relevance, technical characterisation, data integrity and transparency, and independent review. The in vitro and ex vivo methods used in the DAs were demonstrated to be as or more fit for purpose, reliable and relevant than the in vivo rabbit eye test. Overall, there is high scientific confidence in the use of these DAs for assessing the eye corrosion/irritation potential of agrochemical formulations.

Exploration of the anti-diabetic potential of hydro-ethanolic leaf extract of Koenigia polystachya L.: an edible wild plant from Northeastern India

BackgroundGlobally, medicinal plants are used to treat diseases like diabetes. The present study evaluates the possible antioxidant, acute oral toxicity, the in-vitro and in-vivo antidiabetic potential of the hydro-ethanolic leaf extract of Koenigia polystachya (HELeKP) against beta-cell damage in experimentally induced diabetes mellitus. The DPPH (2,2-diphenyl-1-picrylhydrazine), ABTS [2,2′-azino bis-(3-ethylbenzothiazoline-6-sulfonic acid)], H2O2 (Hydrogen peroxide), superoxide radical scavenging activity and NO (Nitric oxide) assay estimated the in-vitro antioxidant assay of HELeKP. The acute oral toxicity study was evaluated per the OECD (Organization for Economic Cooperation and Development) test guidelines 425. Diabetes was stimulated in rats with a single dose of Streptozotocin (STZ), and after confirmation of diabetes, HELeKP was given orally for 21 days. Blood/serum samples were gathered and examined for biochemical changes, while tissue samples were evaluated for histopathological alterations.ResultsThe IC50 value of the HELeKP for all the anti-oxidant assays confirms the free radical scavenging activity. The data on acute oral toxicity revealed that the HELeKP used in the study was comparatively very safe. The outcomes of the in-vivo study suggested that the extract significantly reduced (p < 0.001) the fasting glucose level in STZ-induced diabetic rats. Furthermore, the lipid profile level was significantly normalized (p < 0.01, p < 0.001) in diabetic rats. The histopathological observation of the pancreas in HELeKP-treated rats showed significant beta-cell restoration.ConclusionsBased on the outcomes of this study, the HELeKP-treated rats have significant free radical scavenging and anti-diabetic potential. Therefore, it can be recommended as a beneficial functional vegetable for consumption.

Assessing the Quality and Making Appropriate Use of Historical Negative Control Data: A Report of the International Workshop on Genotoxicity Testing (IWGT).

Historical negative control data (HCD) have played an increasingly important role in interpreting the results of genotoxicity tests. In particular, Organisation for Economic Co-operation and Development (OECD) genetic toxicology Test Guidelines recommend comparing responses produced by exposure to test substances with the distribution of HCD as one of three criteria for evaluating and interpreting study results (referred to herein as "Criterion C"). Because of the potential for inconsistency in how HCD are acquired, maintained, described, and used to interpret genotoxicity testing results, a workgroup of the International Workshops for Genotoxicity Testing was convened to provide recommendations on this crucial topic. The Workgroup used example data sets from four in vivo tests, the Pig-a gene mutation assay, the erythrocyte-based micronucleus test, the transgenic rodent gene mutation assay, and the in vivo alkaline comet assay to illustrate how the quality of HCD can be evaluated. In addition, recommendations are offered on appropriate methods for evaluating HCD distributions. Recommendations of the Workgroup are: When concurrent negative control data fulfill study acceptability criteria, they represent the most important comparator for judging whether a particular test substance induced a genotoxic effect. HCD can provide useful context for interpreting study results, but this requires supporting evidence that i) HCD were generated appropriately, and ii) their quality has been assessed and deemed sufficiently high for this purpose. HCD should be visualized before any study comparisons take place; graph(s) that show the degree to which HCD are stable over time are particularly useful. Qualitative and semi-quantitative assessments of HCD should also be supplemented with quantitative evaluations. Key factors in the assessment of HCD include: i) the stability of HCD over time, and ii) the degree to which inter-study variation explains the total variability observed. When animal-to-animal variation is the predominant source of variability, the relationship between responses in the study and an HCD-derived interval or upper bounds value (i.e., OECD Criterion C) can be used with a strong degree of confidence in contextualizing a particular study's results. When inter-study variation is the major source of variability, comparisons between study data and the HCD bounds are less useful, and consequentially, less emphasis should be placed on using HCD to contextualize a particular study's results. The Workgroup findings add additional support for the use of HCD for data interpretation; but relative to most current OECD Test Guidelines, we recommend a more flexible application that takes into consideration HCD quality. The Workgroup considered only commonly used in vivo tests, but it anticipates that the same principles will apply to other genotoxicity tests, including many in vitro tests.

Potential oestrogenic effects (following the OECD test guideline 440) and thyroid dysfunction induced by pure cyanotoxins (microcystin-LR, cylindrospermopsin) in rats

Potential endocrine-disrupting properties of cyanotoxins, such as microcystin-LR (MC-LR) and cylindrospermopsin (CYN) are of concern due to their increasing occurrence, the scarcity of reports on the topic (particularly for CYN) and the impact of human's health at different levels. Thus, this work performed for the first time the uterotrophic bioassay in rats, following the Organization for Economic Cooperation and Development (OECD) Test Guideline 440, to explore the oestrogenic properties of CYN and MC-LR (75, 150, 300 μg/kg b.w./day) in ovariectomized (OVX) rats. Results revealed neither changes in the wet and blotted uterus weights nor in the morphometric study of uteri. Moreover, among the steroid hormones analysed in serum, the most remarkable effect was the dose-dependent increase in progesterone (P) levels in rats exposed to MC-LR. Additionally, a histopathology study of thyroids and serum levels of thyroids hormones were determined. Tissue affectation (follicular hypertrophy, exfoliated epithelium, hyperplasia) was observed, as well as increased T3 and T4 levels in rats exposed to both toxins. Taken together, these results point out that CYN and MC-LR are not oestrogenic compounds at the conditions tested in the uterotrophic assay in OVX rats, but, however, thyroid disruption effects cannot be discarded.

Applicability of OECD TG 201, 202, 203 for the aquatic toxicity testing and assessment of 2D Graphene material nanoforms to meet regulatory needs

Tests using algae and/or cyanobacteria, invertebrates (crustaceans) and fish form the basic elements of an ecotoxicological assessment in a number of regulations, in particular for classification of a substance as hazardous or not to the aquatic environment according to the Globally Harmonised System of Classification and Labelling of Chemicals (GHS-CLP) (GHS, 2022) and the REACH regulation (Registration, Evaluation, Authorisation and Restriction of Chemicals, EC, 2006). Standardised test guidelines (TGs) of the Organisation for Economic Co-operation and Development (OECD) are available to address the regulatory relevant endpoints of growth inhibition in algae and cyanobacteria (TG 201), acute toxicity to invertebrates (TG 202), and acute toxicity in fish (TG 203). Applying these existing OECD TGs for testing two dimensional (2D) graphene nanoforms may require more attention, additional considerations and/or adaptations of the protocols, because graphene materials are often problematic to test due to their unique attributes. In this review a critical analysis of all existing studies and approaches to testing used has been performed in order to comment on the current state of the science on testing and the overall ecotoxicity of 2D graphene materials. Focusing on the specific tests and available guidance's, a complete evaluation of aquatic toxicity testing for hazard classification of 2D graphene materials, as well as the use of alternative tests in an integrated approach to testing and assessment, has been made. This information is essential to ensure future assessments generate meaningful data that will fulfil regulatory requirements for the safe use of this “wonder” material.

Validation of a method to determine transformation of chemicals in anaerobic liquid pig and cattle manure for the OECD test guideline programme

Manure is widely used as a fertilizer and applied to agricultural land. It may contain highly active chemicals like veterinary medicinal products or biocides, which enter into the environment by this pathway. This is recognized by several regulatory frameworks, however, a detailed method for examining the transformation of chemicals in manure was lacking. This article describes the validation of a method for studying the anaerobic transformation of chemicals in pig and cattle liquid manure. Different steps are covered with an emphasis on the validation ring test and the OECD (Organisation for Economic Cooperation and Development) process that led to the recent adoption of the method as OECD Test Guideline (TG) 320.

Development a high-throughput zebrafish embryo acute toxicity testing method based on OECD TG 236

The Organization for Economic Co-operation and Development (OECD）Test Guideline (TG) 236 for zebrafish embryo acute toxicity testing was adopted for chemical toxicity assessment in 2013. Due to the increasing demand for prediction and evaluation of the acute toxicity using zebrafish embryos, we developed a method based on OECD 236 test guideline with the aim to improve the testing efficiency. We used 4–128 cell stage zebrafish embryos and performed an exposure assay in a 96-well microtiter plate, observing the lethality endpoints of embryos at 48-h postexposure. A total of 32 chemicals (two batches) were used in the comparison study. Our results indicated that the logarithmic LC50 (half lethal concentration) obtained by the modified method exhibited good correlation with that obtained by the OECD 236 testing method, and the R2 of the linear regression analysis was 0.9717 (0.9621 and 0.9936 for the two batches, respectively). Additionally, the intra- and inter-laboratory coefficient of variation (CVs) for the LC50 from the testing chemicals (17 chemicals in second batch) was less than 30%, except for CuSO4. Therefore, the developed method was less time-consuming and demonstrated a higher throughput for toxicity testing compared to the prior method. We argue the developed method could be used as an additional choice for high-throughput zebrafish embryo acute toxicity test.

Scientific concepts and methods for moving persistence assessments into the 21st century.

The evaluation of a chemical substance's persistence is key to understanding its environmental fate, exposure concentration, and, ultimately, environmental risk. Traditional biodegradation test methods were developed many years ago for soluble, nonvolatile, single-constituent test substances, which do not represent the wide range of manufactured chemical substances. In addition, the Organisation for Economic Co-operation and Development (OECD) screening and simulation test methods do not fully reflect the environmental conditions into which substances are released and, therefore, estimates of chemical degradation half-lives can be very uncertain and may misrepresent real environmental processes. In this paper, we address the challenges and limitations facing current test methods and the scientific advances that are helping to both understand and provide solutions to them. Some of these advancements include the following: (1) robust methods that provide a deeper understanding of microbial composition, diversity, and abundance to ensure consistency and/or interpret variability between tests; (2) benchmarking tools and reference substances that aid in persistence evaluations through comparison against substances with well-quantified degradation profiles; (3) analytical methods that allow quantification for parent and metabolites at environmentally relevant concentrations, and inform on test substance bioavailability, biochemical pathways, rates of primary versus overall degradation, and rates of metabolite formation and decay; (4) modeling tools that predict the likelihood of microbial biotransformation, as well as biochemical pathways; and (5) modeling approaches that allow for derivation of more generally applicable biotransformation rate constants, by accounting for physical and/or chemical processes and test system design when evaluating test data. We also identify that, while such advancements could improve the certainty and accuracy of persistence assessments, the mechanisms and processes by which they are translated into regulatory practice and development of new OECD test guidelines need improving and accelerating. Where uncertainty remains, holistic weight of evidence approaches may be required to accurately assess the persistence of chemicals. Integr Environ Assess Manag 2022;18:1454-1487. © 2022 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Evaluating the Ready Biodegradability of Biodegradable Plastics.

Reducing the environmental burden and assessing the safety of plastics are huge global challenges. However, standard test data on the ready biodegradability of plastics are limited. We evaluated the ready biodegradability of 8 biodegradable plastics using Organisation for Economic Co-operation and Development (OECD) test guideline 301F with nonspecific bacteria and examined the effects of prolonging the test duration to a maximum of 90 d. Cellulose used as a potential reference material for plastics was not comparable to the reference material of OECD test guideline 301, but it may be improved by using a test concentration lower than the typical test concentration (100 mg/L). Of the 8 plastics examined, polyamide 4, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), polycaprolactone, and poly(butylene succinate adipate; PBSA) were biodegraded by >60% by day 28 and considered to show ready biodegradability. Poly(3-hydroxybutyrate; PHB), poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid; PHBV), and poly(butylene succinate; PBS) were biodegraded but did not fulfill the ready biodegradability criteria. Because the typical test concentration is considered to have negative effects on biodegradation and calculation of biodegradation percentage, using a lower concentration may result in PHB, PHBV, and PBS fulfilling the ready biodegradability criteria. Poly(d,l-lactide; PLA) was not biodegraded. The biodegradation of PBS and PBSA was noted to vary depending on the used inoculum and/or particle size. For the 7 plastics except PLA, the percentage biodegradation on day 60 was larger than that on day 28, indicating that a longer test period could be useful for evaluating the environmental persistence of plastics. In tests in which the plastics were not biodegraded by day 60, no marked biodegradation was observed by day 90. Environ Toxicol Chem 2021;40:2443-2449. © 2021 SETAC.

Assessment of the different skin sensitization potentials of irritants and allergens as single substances and in combination using the KeratinoSens assay.

People are exposed to mixtures containing allergens and irritants often causing contact dermatitis. Therefore, regulatory authorities require systematic information on the effects of mixtures on the sensitization threshold. In this study a moderate (cinnamal) and a weak (ethylene glycol dimethacrylate) allergen were combined with irritants covering different mechanisms of action (sodium dodecyl sulfate, salicylic acid, and α-pinene). For a systematic approach, the single substances were initially tested using the KeratinoSens assay. Thereafter, each allergen was combined with noncytotoxic concentrations of the irritants. The KeratinoSens assay was applied for the single substances according to OECD (Organisation for Economic Co-operation and Development) Test Guideline 442D. Based on these results, three noncytotoxic concentrations of the irritants were selected and applied simultaneously with 12 concentrations of the allergens to the KeratinoSens cells. Sensitization threshold and cytotoxicity were measured and compared with the individual testing. The combinations of allergens and irritants differed from the effects of the single substances and lowered the sensitization threshold. The quantitative approach allowed a clear description of the changes which varied by factors between 1.1 and 10.3. Overall, the allergen was the prominent compound in the mixture and its nature appeared to determine the degree of the response.

Summary of reference chemicals evaluated by the fish short-term reproduction assay, OECD TG229, using Japanese Medaka, Oryzias latipes.

Under the Organisation for Economic Co‐operation and Development (OECD), the Ministry of the Environment of Japan (MOE) added Japanese medaka (Oryzias latipes) to the test guideline fish short‐term reproduction assay (FSTRA) developed by the United States Environmental Protection Agency (US EPA) using fathead minnow (Pimephales promelas). The FSTRA was designed to detect endocrine disrupting effects of chemicals interacting with the hypothalamic–pituitary–gonadal axis (HPG axis) such as agonists or antagonists on the estrogen receptor (Esr) and/or the androgen receptor (AR) and steroidogenesis inhibitors. We conducted the FSTRA with Japanese medaka, in accordance with OECD test guideline number 229 (TG229), for 16 chemicals including four Esr agonists, two Esr antagonists, three AR agonists, two AR antagonists, two steroidogenesis inhibitors, two progesterone receptor agonists, and a negative substance, and evaluated the usability and the validity of the FSTRA (TG229) protocol. In addition, in vitro reporter gene assays (RGAs) using Esr1 and ARβ of Japanese medaka were performed for the 16 chemicals, to support the interpretation of the in vivo effects observed in the FSTRA. In the present study, all the test chemicals, except an antiandrogenic chemical and a weak Esr agonist, significantly reduced the reproductive status of the test fish, that is, fecundity or fertility, at concentrations where no overt toxicity was observed. Moreover, vitellogenin (VTG) induction in males and formation of secondary sex characteristics (SSC), papillary processes on the anal fin, in females was sensitive endpoints to Esr and AR agonistic effects, respectively, and might be indicators of the effect concentrations in long‐term exposure. Overall, it is suggested that the in vivo FSTRA supported by in vitro RGA data can adequately detect effects on the test fish, O. latipes, and probably identify the mode of action (MOA) of the chemicals tested.

[Current Issues in Photosafety Evaluation].

Phototoxicity is a toxic response elicited by topically applied or systemically administered photoreactive chemicals after exposure to light and can be broadly categorized into photoirritation, photoallergy, photogenotoxicity, and photocarcinogenicity. The need in the 21st century for accurate evaluation of photosafety has led to the publication of a number of guidelines from government agencies in Europe and the U.S.A. as well as the Organisation for Economic Co-operation and Development (OECD). In this review, we first discuss the mechanisms of phototoxicity and how they can be evaluated. We then discuss the state of the art and challenges now faced in photosafety evaluation of pharmaceuticals and cosmetics. Additionally, we describe the latest developments in OECD test guidelines (TG) for assessing photosafety, including revisions to the in vitro 3T3 neutral red uptake (NRU) phototoxicity test (TG 432) and the newly adopted reactive oxigen species (ROS) assay (TG 495). We will emphasize the importance of selecting the most appropriate means of evaluation with reference to the latest guidelines and other legal criteria for conducting photosafety evaluation.

Intra- and inter-laboratory reproducibility of the rat blood Pig-a gene mutation assay.

The in vivo Pig-a assay is being used in safety studies to evaluate the potential of chemicals to induce somatic cell gene mutations. Ongoing work is aimed at developing an Organization for Economic Cooperation and Development (OECD) test guideline to support routine use for regulatory purposes (OECD project number 4.93). Among the requirements for OECD approval are demonstrations of assay reliability, including reproducibility within and among laboratories. Experiments reported herein address the reproducibility of the rat blood Pig-a assay using the reference mutagens chlorambucil and melphalan. These agents were evaluated for their ability to induce Pig-a mutant erythrocytes in three separate studies conducted across two laboratories. Each of the studies utilized a common treatment schedule: 28 consecutive days of exposure via oral gavage. Whereas one laboratory studied Crl:CD(SD) rats, the other laboratory used Wistar Han rats. One or two days after cessation of treatment blood samples were collected for mutant reticulocyte and mutant erythrocyte measurements that were accomplished with the same analytical technique whereby samples were depleted of wildtype erythrocytes via immunomagnetic separation followed by flow cytometric enumeration of mutant phenotype cells (MutaFlow®). Dunnett's test results showed similar qualitative outcomes within and between laboratories, that is, each chemical and each study demonstrated statistically significant, dose-related increases in mutant reticulocyte and erythrocyte frequencies. Benchmark dose analysis (PROAST software) provided a means to quantitatively analyze the results, and the relatively tight, overlapping benchmark dose confidence intervals observed for each of the two chemicals indicate that within and between laboratory reproducibility of the Pig-a assay are high, adding further support for the development of an OECD test guideline.

Alkali Comet Assay in Genotoxicity Tests.

DNA damage detected by the in vivo comet assay is an initial factor for Clastogenicity and gene mutation, and it is considered that the potential for carcinogenesis can be evaluated. However, there is a problem that the test results were not stable because the results fluctuated largely depending on the test execution conditions. Therefore, the Organisation for Economic Co-operation and Development (OECD) test guideline have described the conditions under which tests should be conducted in order to obtain stable data. Herein, I describe an in vivo comet assay that is based on recently approved the OECD test guideline (TG 489).

Laser Light-Based Opacitometer ‘Peira LLBO 180’: A new and validated opacitometer for use in the Bovine Corneal Opacity and Permeability (BCOP) eye irritation test method

The “Peira LLBO 180” is a Laser Light-Based Opacitometer that can be used as an alternative for the standard OP-KIT device in the Bovine Corneal Opacity and Permeability (BCOP) test Organisation for Economic Co-operation and Development (OECD) Test Guideline (TG) 437 to identify chemicals inducing serious eye damage as defined by United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS), i.e. chemicals to be classified as UN GHS Category 1 and chemicals not requiring classification for eye irritation or serious eye damage under the UN GHS classification system (No Category).• The Peira LLBO 180 offers the advantage of analysing the complete corneal surface and is therefore able to detect more efficiently opaque spots located around the periphery of the excised corneas.• This new device will allow not only a more accurate definition of the eye irritating potential of compounds, but also a more precise ranking of moderate to mild and non-irritating compounds.• The value of Peira LLBO 180 is confirmed during in-house and multi-laboratory evaluation studies and is now included in the updated OECD TG 437, dated 26th of June 2020.The results demonstrate that the presented methodology is an improved new approach methodology (NAM) for ocular irritation testing of liquids and solids.

A Multiparametric Approach to Cerium Oxide Nanoparticle Toxicity Assessment in Non-Biting Midges.

Cerium oxide nanoparticles (CeO2 NPs) are included in the Organisation for Economic Co-operation and Development (OECD) priority list of engineered nanomaterials for assessment of their environmental impact. The present study was carried out to assess the CeO2 NP toxicity to the freshwater midge Chironomus riparius larvae at concentrations of 2.5, 25, 250, and 2500 mg of CeO2 NP/kg of sediment. Experiments were designed to assess the prolonged exposure of midges to CeO2 NPs while adhering to OECD test guideline 218. The following parameters were investigated: CeO2 NP uptake by larvae, oxidative stress parameters, in vivo genotoxic effects, and life trait parameters. Inductively coupled plasma-mass spectrometry analysis showed a significant positive correlation between the concentration of CeO2 NPs in the sediment and its uptake by the larvae. No significant mortality was observed in C. riparius, and oxidative stress was not detected. The only significantly induced sublethal effect was genotoxicity, which began to manifest at a lowest-observed-effect concentration of 25 mg kg-1 of sediment and progressively increased at higher concentrations. Our results indicate that exposure to CeO2 NP-contaminated freshwater sediments does not pose a risk to chironomids at environmentally realistic concentrations. However, the significant accumulation of CeO2 NPs by chironomid larvae may pose a risk through trophic transfer to organisms further up the food chain. Environ Toxicol Chem 2019;39:131-140. © 2019 SETAC.

Skin sensitizing effects of sulfur mustard and other alkylating agents in accordance to OECD guidelines

Vesicants cause a multitude of cutaneous reactions like erythema, blisters and ulcerations. After exposure to sulfur mustard (SM) and related compounds, patients present dermal symptoms typically known for chemicals categorized as skin sensitizer (e.g. hypersensitivity and flare-up phenomena). However, although some case reports led to the assumption that SM and other alkylating compounds represent sensitizers, a comprehensive investigation of SM-triggered immunological responses has not been conducted so far. Based on a well-structured system of in chemico and in vitro test methods, the Organization for Economic Co-operation and Development (OECD) established procedures to categorize agents on their skin sensitizing abilities. In this study, the skin sensitizing potential of SM and three related alkylating agents (AAs) was assessed following the OECD test guidelines. Besides SM, investigated AAs were chlorambucil (CHL), nitrogen mustard (HN3) and 2-chloroethyl ethyl sulfide (CEES). The methods are described in detail in the EURL ECVAM DataBase service on ALternative Methods to animal experimentation (DB-ALM). In accordance to OECD recommendations, skin sensitization is a pathophysiological process starting with a molecular initiating step and ending with the in vivo outcome of an allergic contact dermatitis. This concept is called adverse outcome pathway (AOP). An AOP links an adverse outcome to various key events which can be assayed by established in chemico and in vitro test methods. Positive outcome in two out of three key events indicates that the chemical can be categorized as a skin sensitizer. In this study, key event 1 “haptenation” (covalent modification of epidermal proteins), key event 2 “activation of epidermal keratinocytes” and key event 3 “activation of dendritic cells” were investigated. Covalent modification of epidermal proteins measured by using the DPRA-assay provided distinct positive results for all tested substances. Same outcome was seen in the KeratinoSens assay, investigating the activation of epidermal keratinocytes. The h-CLAT assay performed to determine the activation of dendritic cells provided positive results for SM and CEES but not for CHL and HN3. Altogether, following OECD requirements, our results suggest the classification of all investigated substances as skin sensitizers. Finally, a tentative AOP for SM-induced skin sensitization is suggested.