For years, the strive for in vitro methods for toxicological assessment suitable to replace animal studies gained progressive importance. OECD Test Guideline (TG) 431 was implemented in 2004, ...allowing to circumvent animal testing according to OECD TG 404 while reliably predicting skin corrosion potential of many substances and products. However, non-animal assays often show protocol-dependent limitations, that complicate or even prevent the testing of several groups of substances. In this study, the suitability of the OECD TG 431 for assessment of the skin corrosion potential of known acidic, thus often skin corrosive or irritating acrylic and methacrylic acid-based adhesives and monomers, was investigated. The commercially available Phenion® Open Source Reconstructed Epidermis (OS-REp) model, developed at Henkel & Co. KGaA, was used. The EpiDerm™ prediction model was considered most applicable to the Phenion® OS-REp model. All Proficiency Substances listed in OECD TG 431, amongst them six acids, were correctly classified and subcategorized as Skin Corr. 1 A or 1B/C corrosives. The OS-REp model was shown to be suitable for the assessment of skin corrosion potential in accordance with OECD TG 431. However, our results also indicate that acrylic and methacrylic monomer-based adhesives might fall outside the applicability domain of this guideline.
The purpose of this study was to develop a defined approach (DA) for eye hazard identification according to the three UN GHS categories for surfactants (DASF). The DASF is based on a combination of ...Reconstructed human Cornea-like Epithelium test methods (OECD TG 492; EpiOcular™ EIT and SkinEthic™ HCE EIT) and the modified Short Time Exposure (STE) test method (0.5% concentration of the test substance after a 5-min exposure).
DASF performance was assessed by comparing the prediction results with the historical in vivo data classification and against the criteria established by the OECD expert group on eye/skin.
The DASF yielded a balanced accuracy of 80.5% and 90.9% of Cat. 1 (N = 22), 75.0% of Cat. 2 (N = 8), and 75.5% of No Cat. (N = 17) surfactants were correctly predicted. The percentage of mispredictions was below the established maximum values except for in vivo No Cat. surfactants that were over-predicted as Cat. 1 (5.6%, N = 17), with a maximum value set at 5%. The percentage of correct predictions did meet the minimum performance values of 75% Cat. 1, 50% Cat. 2, and 70% No Cat. established by the OECD experts. The DASF has shown to be successful for eye hazard identification of surfactants.
•develop defined approach (DA) for eye hazard assessment of surfactants (DASF).•proposed DA: combination of OECD TG 492 with the modified STE test method.•The DASF can distinguish between the three UN GHS categories.
In recent years many efforts have been made to replace dermal toxicity testing of chemicals in the animal by in vitro assays. As a member of a German research consortium, we have previously ...contributed to the validation of an in vitro test protocol for percutaneous absorption studies on the basis of reconstructed human epidermis and both human and pig skin ex vivo. Aiming to assess the barrier properties of a newly developed reconstructed skin model, this protocol has now been transferred to the Phenion Full-Thickness Skin Model (FT model). The permeation of testosterone and caffeine was quantified in parallel to that of pig skin using Franz-type diffusion cells. In addition, the permeation of benzoic acid and nicotine was studied. As expected, the FT model is more permeable than pig skin, yet its barrier properties are well in accordance with those of reconstructed human epidermis when compared to previous data. In fact, the FT model most efficiently retards testosterone as the compound of highest lipophilicity, which can be explained by an additional uptake by a reservoir formed by the dermis equivalent. Thus, the structure closely parallels human skin. In consequence, the Phenion FT model appears to be suitable for percutaneous absorption studies in hazard analysis and should be subjected to a catch-up validation study.
To replace the Draize skin irritation assay (OECD guideline 404) several test methods based on reconstructed human epidermis (RHE) have been developed and were adopted in the OECD test guideline 439. ...However, all validated test methods in the guideline are linked to RHE provided by only three companies. Thus, the availability of these test models is dependent on the commercial interest of the producer. To overcome this limitation and thus to increase the accessibility of in vitro skin irritation testing, an open source reconstructed epidermis (OS-REp) was introduced. To demonstrate the capacity of the OS-REp in regulatory risk assessment, a catch-up validation study was performed. The participating laboratories used in-house generated OS-REp to assess the set of 20 reference substances according to the performance standards amending the OECD test guideline 439. Testing was performed under blinded conditions. The within-laboratory reproducibility of 87% and the inter-laboratory reproducibility of 85% prove a high reliability of irritancy testing using the OS-REp protocol. In addition, the prediction capacity was with an accuracy of 80% comparable to previous published RHE based test protocols. Taken together the results indicate that the OS-REp test method can be used as a standalone alternative skin irritation test replacing the OECD test guideline 404.
A prospective study of the Bovine Corneal Opacity and Permeability (BCOP) Laser Light-Based Opacitometer (LLBO) test method was conducted to evaluate its usefulness to identify chemicals as inducing ...serious eye damage (Cat. 1) or chemicals not requiring classification for eye irritation (No Cat.) applying United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS).
The aim was to demonstrate the reproducibility of the BCOP LLBO protocol for liquids and solids and define its predictive capacity. Briefly, 145 chemicals were simultaneously tested with BCOP LLBO and OP-KIT (OECD TG 437), one to two times in one laboratory. When used to identify Cat. 1, the BCOP LLBO has a false negative rate (FNR) of 24.1% (N = 56) compared to 34.8% (N = 56) for the BCOP OP-KIT, with a comparable false positive rate (FPR, N = 89) of 18.5% and 20.8%, respectively. When used to identify chemicals not requiring classification (No Cat.) the BCOP LLBO and BCOP OP-KIT had a FNR (N = 104) of 6.2% and 7.2% and a FPR (N = 41) of 45.1% and 42.7%, respectively.
The OP-KIT and LLBO devices are interchangeable at no cost to data quality and reliability. The OP-KIT and LLBO devices are interchangeable at no cost to data quality and reliability. The performance of the LLBO is at least as good as the OP-KIT, both methods can be used to identify UN GHS Cat. 1 and UN GHS No Cat. chemicals.
•Performance of the BCOP Laser Light-Based Opacitometer (LLBO) test method•Test Method for Identifying Chemicals Inducing Serious Eye Damage•Test Method for Identifying Chemicals Not Requiring Classification for Eye Irritation/Serious Eye Damage
We have developed a new in vitro skin irritation test based on an open source reconstructed epidermis (OS-REp) with openly accessible protocols for tissue production and test performance. Due to ...structural, mechanistic and procedural similarity, a blinded catch-up validation study for skin irritation according to OECD Performance Standards (PS) was conducted in three laboratories to promote regulatory acceptance, with OS-REp models produced at a single production site only. While overall sensitivity and predictive capacity met the PS requirements, overall specificity was only 57%. A thorough analysis of the test results led to the assumption that some of the false-positive classifications could have been evoked by volatile skin-irritating chemicals tested in the same culture plate as the non-irritants falsely predicted as irritants. With GC/MS and biological approaches the cross-contamination effect was confirmed and the experimental set-up adapted accordingly. Retesting of the affected chemicals with the improved experimental set-up and otherwise identical protocol resulted in correct classifications as non-irritants. Taking these re-test results into account, 93% overall sensitivity, 70% specificity and 82% accuracy was achieved, which is in accordance with the OECD PS. A sufficient reliability of the method was indicated by a within-laboratory-reproducibility of 85-95% and a between-laboratory-reproducibility of 90%.
We model "soft" error rates for writing (WSER) and for reading (RSER) for spin-torque memory devices that have a free layer with easy axis perpendicular to the film plane by solving the Fokker-Planck ...equation for the probability distribution of the angle that the free layer magnetization makes with the normal to the plane of the film. We obtain: 1) an exact, closed form, analytical expression for the zero-temperature switching time as a function of initial angle; 2) an approximate analytical expression for the distribution function of the direction of the magnetization and the exponential decay of the WSER as a function of the time the current is applied; 3) comparison of the approximate analytical expressions for the distribution function and WSER to numerical solutions of the Fokker-Planck equation; 4) an approximate analytical expression for the distribution function and WSER for the case in which the pinned layer is not collinear with the perpendicular free layer; 5) an approximate analytical expression for the linear increase in RSER with current applied for reading; 6) comparison of the approximate analytical formula for the RSER to the numerical solution of the Fokker-Planck equation; and 7) confirmation of the accuracy of the Fokker-Planck solutions by comparison with results of direct simulation using the single-macrospin Landau-Lifshitz-Gilbert equations with a random fluctuating field in the short-time regime for which the latter is practical. We find that the WSER decays at long times as exp-2( i -1)τ where the reduced time τ is related to the switching time, Gilbert damping and precession frequency through τ = αω 0 t , and the reduced current i is the ratio of the applied current to the critical current density for switching i = I̅ / I 0 . This exponentially decaying tail in WSER is not easily reduced by tilting the pinned layer magnetization.
Cosmetics Europe recently established HPLC/UPLC-spectrophotometry as a suitable alternative endpoint detection system for measurement of formazan in the MTT-reduction assay of reconstructed human ...tissue test methods irrespective of the test system involved. This addressed a known limitation for such test methods that use optical density for measurement of formazan and may be incompatible for evaluation of strong MTT reducer and/or coloured chemicals. To build on the original project, Cosmetics Europe has undertaken a second study that focuses on evaluation of chemicals with functionalities relevant to cosmetic products. Such chemicals were primarily identified from the Scientific Committee on Consumer Safety (SCCS) 2010 memorandum (addendum) on the in vitro test EpiSkin™ for skin irritation testing. Fifty test items were evaluated in which both standard photometry and HPLC/UPLC-spectrophotometry were used for endpoint detection. The results obtained in this study: 1) provide further support for Within Laboratory Reproducibility of HPLC–UPLC-spectrophotometry for measurement of formazan; 2) demonstrate, through use a case study with Basazol C Blue pr. 8056, that HPLC/UPLC-spectrophotometry enables determination of an in vitro classification even when this is not possible using standard photometry and 3) addresses the question raised by SCCS in their 2010 memorandum (addendum) to consider an endpoint detection system not involving optical density quantification in in vitro reconstructed human epidermis skin irritation test methods.
•HPLC/UPLC-spectrophotometry for formazan measurement in in vitro reconstructed human tissue test methods.•Evaluation of 50 test items in EpiSkin™ test system.•Reproducibility and concordance between OD and HPLC-UPLC-spectrophotometry demonstrated.•Applicable for highly coloured chemicals interfering with standard detection method.•Applicability to evaluation of chemicals with functionalities relevant to cosmetic products demonstrated.
•Key qualification parameters defined for HPLC/UPLC-spectrophotometry systems.•HPLC/UPLC-spectrophotometry for formazan detection is highly reproducible.•High concordance of classification using OD ...and HPLC/UPLC-spectrophotometry.•Applicable for highly coloured chemicals interfering with standard detection method.•Applicable to in vitro RhT test methods for eye/skin irritation and skin corrosion.
A number of in vitro test methods using Reconstructed human Tissues (RhT) are regulatory accepted for evaluation of skin corrosion/irritation. In such methods, test chemical corrosion/irritation potential is determined by measuring tissue viability using the photometric MTT-reduction assay. A known limitation of this assay is possible interference of strongly coloured test chemicals with measurement of formazan by absorbance (OD). To address this, Cosmetics Europe evaluated use of HPLC/UPLC-spectrophotometry as an alternative formazan measurement system. Using the approach recommended by the FDA guidance for validation of bio-analytical methods, three independent laboratories established and qualified their HPLC/UPLC-spectrophotometry systems to reproducibly measure formazan from tissue extracts. Up to 26 chemicals were then tested in RhT test systems for eye/skin irritation and skin corrosion. Results support that: (1) HPLC/UPLC-spectrophotometry formazan measurement is highly reproducible; (2) formazan measurement by HPLC/UPLC-spectrophotometry and OD gave almost identical tissue viabilities for test chemicals not exhibiting colour interference nor direct MTT reduction; (3) independent of the test system used, HPLC/UPLC-spectrophotometry can measure formazan for strongly coloured test chemicals when this is not possible by absorbance only. It is therefore recommended that HPLC/UPLC-spectrophotometry to measure formazan be included in the procedures of in vitro RhT-based test methods, irrespective of the test system used and the toxicity endpoint evaluated to extend the applicability of these test methods to strongly coloured chemicals.
► We present the construction of a tissue engineered hemi-cornea model based on human cell lines. ► We show the reproducible construction of this model in independent laboratories. ► We have ...developed an in vitro test system for eye irritation testing based on this model. ► We present the predictive capacity of this system and the inter-laboratory reproducibility.
We have developed a 3-dimensional human hemi-cornea which comprises an immortalized epithelial cell line and keratocytes embedded in a collagen stroma. In the present study, we have used MTT reduction of the whole tissue to clarify whether the production of this complex 3-D-model is transferable into other laboratories and whether these tissues can be constructed reproducibly. Our results demonstrate the reproducible production of the hemi-cornea model according to standard operation procedures using 15 independent batches of reconstructed hemi-cornea models in two independent laboratories each.
Furthermore, the hemi-cornea tissues have been treated with 20 chemicals of different eye-irritating potential under blind conditions to assess the performance and limitations of our test system comparing three different prediction models. The most suitable prediction model revealed an overall in vitro–in vivo concordance of 80% and 70% in the participating laboratories, respectively, and an inter-laboratory concordance of 80%. Sensitivity of the test was 77% and specificity was between 57% and 86% to discriminate classified from non-classified chemicals. We conclude that additional physiologically relevant endpoints in both epithelium and stroma have to be developed for the reliable prediction of all GHS classes of eye irritation in one stand alone test system.
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