Challenges to improve toxicological risk assessment to meet the demands of the EU chemical's legislation, REACH, and the EU 7th Amendment of the Cosmetics Directive have accelerated the development of non-animal based methods. Unfortunately, uncertainties remain surrounding the power of alternative methods such as in vitro assays to predict in vivo dose-response relationships, which impedes their use in regulatory toxicology. One issue reviewed here, is the lack of a well-defined dose metric for use in concentration-effect relationships obtained from in vitro cell assays. Traditionally, the nominal concentration has been used to define in vitro concentration-effect relationships. However, chemicals may differentially and non-specifically bind to medium constituents, well plate plastic and cells. They may also evaporate, degrade or be metabolized over the exposure period at different rates. Studies have shown that these processes may reduce the bioavailable and biologically effective dose of test chemicals in in vitro assays to levels far below their nominal concentration. This subsequently hampers the interpretation of in vitro data to predict and compare the true toxic potency of test chemicals. Therefore, this review discusses a number of dose metrics and their dependency on in vitro assay setup. Recommendations are given on when to consider alternative dose metrics instead of nominal concentrations, in order to reduce effect concentration variability between in vitro assays and between in vitro and in vivo assays in toxicology.
The nominal concentration
is generally used to express concentration–effect
relationships in in vitro toxicity assays. However, the nominal concentration
does not necessarily represent the exposure concentration responsible
for the observed effect. Surfactants accumulate at interphases and
likely sorb to in vitro system components such as serum protein and
well plate plastic. The extent of sorption and the consequences of
this sorption on in vitro readouts is largely unknown for these chemicals.
The aim of this study was to demonstrate the effect of sorption to
in vitro components on the observed cytotoxic potency of benzalkonium
chlorides (BAC) varying in alkyl chain length (6–18 carbon
atoms, C6–18) in a basal cytotoxicity assay with
the rainbow trout gill cell line (RTgill-W1). Cells were exposed for
48 h in 96-well plates to increasing concentration of BACs in exposure
medium containing 0, 60 μM bovine serum albumin (BSA) or 10%
fetal bovine serum (FBS). Before and after exposure, BAC concentrations
in exposure medium were analytically determined. Based on freely dissolved
concentrations at the end of the exposure, median effect concentrations
(EC50) decreased with increasing alkyl chain length up
to 14 carbons. For BAC with alkyl chains of 12 or more carbons, EC50’s based on measured concentrations after exposure
in supplement-free medium were up to 25-times lower than EC50’s calculated using nominal concentrations. When BSA or FBS
was added to the medium, a decrease in cytotoxic potency of up to
22 times was observed for BAC with alkyl chains of eight or more carbons.
The results of this study emphasize the importance of expressing the
in vitro readouts as a function of a dose metric that is least influenced
by assay setup to compare assay sensitivities and chemical potencies.
The 2012 UsedSoft decision of the Court of Justice of the European Union ('ECJ') disrupted the digital distribution of computer programs. Since then, unauthorised sellers of product and game keys often try to utilise the UsedSoft ruling and its principles to justify the resale of key codes. Against this background, we will review the new developments in this area, taking into account recent court decisions in Germany, which are based on European Directives. Furthermore, the impact of the UsedSoft decision on the gaming sector, where game keys are often distributed in digital form only, will be examined. Since the ECJ in its Nintendo decision and the German Federal Supreme Court 'BGH') in its World of Warcraft I decision both considered video games to be hybrid products, we will discuss whether and to what extent the Software Directive 2009/24/EC and the InfoSoc Directive 2001/29/EC are applicable. We then apply the findings to the distribution models used in the gaming sector and examine their legality.
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