In Vitro Biotransformation Assays Using Liver S9 Fractions and Hepatocytes from Rainbow Trout (Oncorhynchus mykiss): Overcoming Challenges with Difficult to Test Fragrance Chemicals.

In vitro metabolic stability assays using rainbow trout (Oncorhynchus mykiss) isolated hepatocytes (RT-HEP) or hepatic S9 fractions (RT-S9) were introduced to provide biotransformation rate data for the assessment of chemical bioaccumulation in fish. The present study explored the suitability of the RT-HEP and RT-S9 assays for difficult test chemicals, and the in vitro-based predictions were compared to in silico-based predictions and in vivo-measured bioconcentration factors (BCFs). The results show that volatile or reactive chemicals can be tested with minor modifications of the in vitro protocols. For hydrophobic chemicals, a passive dosing technique was developed. Finally, a design-of-experiment approach was used to identify optimal in vitro assay conditions. The modified assay protocols were applied to 10 fragrances with diverse physicochemical properties. The in vitro intrinsic clearance rates were higher in the S9 than in the hepatocyte assay, but the in vitro-in vivo (IVIV) predictions were comparable between the 2 assays. The IVIV predictions classified the test chemicals as nonbioaccumulative (BCF < 2000), which was in agreement with the in vivo data but in contrast to the in silico-based predictions. The findings from the present study provide strong evidence that the RT-HEP and RT-S9 assays can provide reliable estimates of in vivo biotransformation rates for test chemicals with difficult physicochemical properties. Environ Toxicol Chem 2020;39:2396-2408. © 2020 SETAC.

Extending the concept of predicting fish acute toxicity in vitro to the intestinal cell line RTgutGC.

Testing chemicals for fish acute toxicity is a legal requirement in many countries as part of environmental risk assessment. To reduce the numbers of fish used, substantial efforts have been focussed on alternative approaches. Prominently, the cell viability assay with the rainbow trout (Oncorhynchus mykiss) gill cell line, RTgill-W1, has been recognized, owing to its high predictive power and robustness. Like gills, the intestine is considered a major site of chemical uptake and biotransformation but, in contrast to gills, is expected to be exposed to rather hydrophobic chemicals, which enter the fish via food. In the present study, we therefore aimed to extend the cell bioassay to the rainbow trout epithelial cell line from intestine, RTgutGC. Using 16 hydrophobic and volatile chemicals from the fragrance palette, we showed that also the RTgutGC cell line can be used to predict fish acute toxicity of chemicals and yields intra-laboratory variability in line with other bioassays. By comparing the RTgutGC toxicity to a study employing the RTgill-W1 assay on the same group of chemicals, a fragrance specific relationship was established which reflects an almost perfect 1:1 relationship between in vitro and in vivo toxicity results. Thus, both cell lines can be used to predict fish acute toxicity, either by using the obtained in vivo-in vitro relationship or by taking the in vitro results at face value. We moreover demonstrate the derivation of non-toxic concentrations for downstream applications which rely on a healthy cell state, such as the assessment of biotransformation or chemical transfer.

Intestinal Fish Cell Barrier Model to Assess Transfer of Organic Chemicals in Vitro: An Experimental and Computational Study.

We studied the role of the fish intestine as a barrier for organic chemicals using the epithelial barrier model built on the rainbow trout (Oncorhynchus mykiss) intestinal cell line, RTgutGC and the newly developed exposure chamber, TransFEr, specifically designed to work with hydrophobic and volatile chemicals. Testing 11 chemicals with a range of physicochemical properties (logKOW: 2.2 to 6.3, logHLC: 6.1 to 2.3) and combining the data with a mechanistic kinetic model enabled the determination of dominant processes underlying the transfer experiments and the derivation of robust transfer rates. Against the current assumption in chemical uptake modeling, chemical transfer did not strictly depend on the logKOW but resulted from chemical-specific intracellular accumulation and biotransformation combined with paracellular and active transport. Modeling also identified that conducting elaborate measurements of the plastic parts, including the polystyrene insert and the PET filter, is unnecessary and that stirring in the TransFEr chamber reduced the stagnant water layers compared to theoretical predictions. Aside from providing insights into chemical uptake via the intestinal epithelium, this system can easily be transferred to other cell-based barrier systems, such as the fish gill or mammalian intestinal models and may improve in vitro-in vivo extrapolation and prediction of chemical bioaccumulation into organisms.

In vitro model systems for studying the impact of organic chemicals on the skin barrier lipids.

This paper describes two synthetic lipid models designed to replace human stratum corneum (SC) in studies of the impact of volatile organic chemicals on the molecular organization of the skin barrier lipids. The models built upon previously developed self-assembled lipid membranes which have composition and 3D organization similar to those of the lipid matrix in SC. In one model the target chemicals were incorporated in the lipids before their self-assembly, and in the other one they were applied on top of a preformed lipid membrane. The chemicals could be incorporated within the model membranes in quantities close to those reached within human SC upon heavy surface loading. The dose-dependent effects of the chemicals on the lateral molecular organization in the models were qualitatively identical to those observed by infrared spectroscopy in human SC. The models facilitated the interpretation of X-ray diffraction profiles used to determine the nature of the interactions between the chemicals and the lipid lamellae and the position of the exogenous molecules within the unit cell of the lipid phases. These model systems are suitable for in vitro studies in the areas of skin biophysics, dermatology, transdermal drug delivery, and risk assessment.

The impact of surface loading and dosing scheme on the skin uptake of fragrances.

This study compared the skin uptake of γ-undecalactone, decanol, and dodecyl acetate in an in vitro, un-occluded penetration assay in which they were applied to porcine skin at different finite loadings and application schemes. The pattern of fractional uptake differed between the chemicals and did not show the often assumed inverse correlation with surface loading. Furthermore, the mass uptake of identical cumulative amounts of the chemicals was not always additive. These results show that the uptake of fragrances in absence of occlusion and at finite loadings is chemical-specific and depends on the surface loading, the application scheme, and most probably, on the effects of the chemicals on the skin barrier efficiency. The observed lack of additivity might explain some of the differences in the responses observed in patch and repeated open application tests, and the boosting of the allergic state in sensitized individuals by sub-clinical exposures.

In vitro skin penetration of fragrances: trapping the evaporated material can enhance the dermal absorption of volatile chemicals.

This study compared the evaporation and skin absorption profiles of four fragrance chemicals in in vitro skin penetration studies performed in conditions of airflows of low velocity with and without trapping of the evaporated volatiles. The presence of a trapping chamber above the skin surface slowed down the evaporation of the chemicals, possibly due to formation of a gaseous stagnant layer of greater thickness than the one existing at the skin surface in the real-life conditions of multidirectional and/or turbulent flows. In addition, the use of a trapping chamber considerably influenced the distribution of the fragrance chemicals in the skin layers and resulted in 2- to 8-fold increase of the doses available for systemic absorption. Such unrealistic overestimation of the percutaneous absorption can significantly impact the risk assessment of topically applied volatile chemicals and can lead to defining unrealistic margins of safety.

Correlation between the properties of the lipid matrix and the degrees of integrity and cohesion in healthy human Stratum corneum.

The correlation between the degrees of integrity and cohesion in healthy human Stratum corneum (SC) and the properties of the SC lipid matrix could be examined non-invasively in vivo using ATR-FTIR spectroscopy and measurements of pH, conductance, and transepidermal water loss (TEWL) taken in the course of tape-stripping. The change of TEWL following the removal of a SC layer with a predefined thickness served as a measure for the SC integrity, and the amount of protein removed by predefined number of tapes - as a measure for the SC cohesion. The extent of lipids organized in orthorhombic lattices and the pH in the inner SC emerged as the main factors that determine the degree of integrity. The amounts and molecular organization of the SC lipids did not correlate with the degree of cohesion, while the pH and the hydration of SC correlated well with the degree of cohesion in the superficial but not in the inner SC layers. This study evidenced the variability of SC integrity and cohesion existing in healthy human skin, demonstrated the importance of the lipid molecular organization for the SC integrity, and illustrated the limitations in the determination the degree of corneodesmolysis in SC based only on the protein content of tape-strips.