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.

Influence of experimental parameters on in vitro human skin permeation of Bisphenol A.

Bisphenol A (BPA) in vitro skin permeation studies have shown inconsistent results, which could be due to experimental conditions. We studied the impact of in vitro parameters on BPA skin permeation using flow-through diffusion cells with ex-vivo human skin (12 donors, 3-12 replicates). We varied skin status (viable or frozen skin) and thickness (200, 400, 800 µm), BPA concentrations (18, 250 mg/l) and vehicle volumes (10, 100 and 1000 µl/cm2). These conditions led to a wide range of BPA absorption (2%-24% after 24 h exposure), peak permeation rates (J = 0.02-1.31 µg/cm2/h), and permeability coefficients (Kp = 1.6-5.2 × 10-3 cm/h). This is the first time steady state conditions were reached for BPA aqueous solutions in vitro (1000 µl/cm2 applied at concentration 250 mg/l). A reduction of the skin thickness from 800 and 400 µm to 200 µm led to a 3-fold increase of J (P < 0.05). A reduction of the vehicle volume from 1000 to 100 led to a 2-fold decrease in J (P > 0.05). Previously frozen skin led to a 3-fold increase in J compared to viable skin (P < 0.001). We found that results from published studies were consistent when adjusting J according to experimental parameters. We propose appropriate J values for different exposure scenarios to calculate BPA internal exposures for use in risk assessment.

Impact of biological and environmental variabilities on biological monitoring--an approach using toxicokinetic models.

Biological monitoring of occupational exposure is characterized by important variability, due both to variability in the environment and to biological differences between workers. A quantitative description and understanding of this variability is important for a dependable application of biological monitoring. This work describes this variability, using a toxicokinetic model, for a large range of chemicals for which reference biological reference values exist. A toxicokinetic compartmental model describing both the parent compound and its metabolites was used. For each chemical, compartments were given physiological meaning. Models were elaborated based on physiological, physicochemical, and biochemical data when available, and on half-lives and central compartment concentrations when not available. Fourteen chemicals were studied (arsenic, cadmium, carbon monoxide, chromium, cobalt, ethylbenzene, ethyleneglycol monomethylether, fluorides, lead, mercury, methyl isobutyl ketone, penthachlorophenol, phenol, and toluene), representing 20 biological indicators. Occupational exposures were simulated using Monte Carlo techniques with realistic distributions of both individual physiological parameters and exposure conditions. Resulting biological indicator levels were then analyzed to identify the contribution of environmental and biological variability to total variability. Comparison of predicted biological indicator levels with biological exposure limits showed a high correlation with the model for 19 out of 20 indicators. Variability associated with changes in exposure levels (GSD of 1.5 and 2.0) is shown to be mainly influenced by the kinetics of the biological indicator. Thus, with regard to variability, we can conclude that, for the 14 chemicals modeled, biological monitoring would be preferable to air monitoring. For short half-lives (less than 7 hr), this is very similar to the environmental variability. However, for longer half-lives, estimated variability decreased.

Dynamin-related protein 1 is required for normal mitochondrial bioenergetic and synaptic function in CA1 hippocampal neurons.

Disrupting particular mitochondrial fission and fusion proteins leads to the death of specific neuronal populations; however, the normal functions of mitochondrial fission in neurons are poorly understood, especially in vivo, which limits the understanding of mitochondrial changes in disease. Altered activity of the central mitochondrial fission protein dynamin-related protein 1 (Drp1) may contribute to the pathophysiology of several neurologic diseases. To study Drp1 in a neuronal population affected by Alzheimer's disease (AD), stroke, and seizure disorders, we postnatally deleted Drp1 from CA1 and other forebrain neurons in mice (CamKII-Cre, Drp1lox/lox (Drp1cKO)). Although most CA1 neurons survived for more than 1 year, their synaptic transmission was impaired, and Drp1cKO mice had impaired memory. In Drp1cKO cell bodies, we observed marked mitochondrial swelling but no change in the number of mitochondria in individual synaptic terminals. Using ATP FRET sensors, we found that cultured neurons lacking Drp1 (Drp1KO) could not maintain normal levels of mitochondrial-derived ATP when energy consumption was increased by neural activity. These deficits occurred specifically at the nerve terminal, but not the cell body, and were sufficient to impair synaptic vesicle cycling. Although Drp1KO increased the distance between axonal mitochondria, mitochondrial-derived ATP still decreased similarly in Drp1KO boutons with and without mitochondria. This indicates that mitochondrial-derived ATP is rapidly dispersed in Drp1KO axons, and that the deficits in axonal bioenergetics and function are not caused by regional energy gradients. Instead, loss of Drp1 compromises the intrinsic bioenergetic function of axonal mitochondria, thus revealing a mechanism by which disrupting mitochondrial dynamics can cause dysfunction of axons.

Skin permeation and metabolism of di(2-ethylhexyl) phthalate (DEHP).

Phthalates are suspected to be endocrine disruptors. Di(2-ethylhexyl) phthalate (DEHP) is assumed to have low dermal absorption; however, previous in vitro skin permeation studies have shown large permeation differences. Our aims were to determine DEHP permeation parameters and assess extent of skin DEHP metabolism among workers highly exposed to these lipophilic, low volatile substances. Surgically removed skin from patients undergoing abdominoplasty was immediately dermatomed (800 µm) and mounted on flow-through diffusion cells (1.77 cm(2)) operating at 32°C with cell culture media (aqueous solution) as the reservoir liquid. The cells were dosed either with neat DEHP or emulsified in aqueous solution (166 µg/ml). Samples were analysed by HPLC-MS/MS. DEHP permeated human viable skin only as the metabolite MEHP (100%) after 8h of exposure. Human skin was able to further oxidize MEHP to 5-oxo-MEHP. Neat DEHP applied to the skin hardly permeated skin while the aqueous solution readily permeated skin measured in both cases as concentration of MEHP in the receptor liquid. DEHP pass through human skin, detected as MEHP only when emulsified in aqueous solution, and to a far lesser degree when applied neat to the skin. Using results from older in vitro skin permeation studies with non-viable skin may underestimate skin exposures. Our results are in overall agreement with newer phthalate skin permeation studies.

A new alternative method for testing skin irritation using a human skin model: a pilot study.

BACKGROUND: Studies assessing skin irritation to chemicals have traditionally used laboratory animals; however, such methods are questionable regarding their relevance for humans. New in vitro methods have been validated, such as the reconstructed human epidermis (RHE) model (Episkin®, Epiderm®). The comparison (accuracy) with in vivo results such as the 4-h human patch test (HPT) is 76% at best (Epiderm®). There is a need to develop an in vitro method that better simulates the anatomo-pathological changes encountered in vivo. OBJECTIVES: To develop an in vitro method to determine skin irritation using human viable skin through histopathology, and compare the results of 4 tested substances to the main in vitro methods and in vivo animal method (Draize test). METHODOLOGY: Human skin removed during surgery was dermatomed and mounted on an in vitro flow-through diffusion cell system. Ten chemicals with known non-irritant (heptylbutyrate, hexylsalicylate, butylmethacrylate, isoproturon, bentazon, DEHP and methylisothiazolinone (MI)) and irritant properties (folpet, 1-bromohexane and methylchloroisothiazolinone (MCI/MI)), a negative control (sodiumchloride) and a positive control (sodiumlaurylsulphate) were applied. The skin was exposed at least for 4h. Histopathology was performed to investigate irritation signs (spongiosis, necrosis, vacuolization). RESULTS: We obtained 100% accuracy with the HPT model; 75% with the RHE models and 50% with the Draize test for 4 tested substances. The coefficients of variation (CV) between our three test batches were <0.1, showing good reproducibility. Furthermore, we reported objectively histopathological irritation signs (irritation scale): strong (folpet), significant (1-bromohexane), slight (MCI/MI at 750/250ppm) and none (isoproturon, bentazon, DEHP and MI). CONCLUSIONS: This new in vitro test method presented effective results for the tested chemicals. It should be further validated using a greater number of substances; and tested in different laboratories in order to suitably evaluate reproducibility.

[From the mixed dentition to the permanent dentition: how to manage space while guiding eruption?]. / De la denture mixte à la denture permanente: comment gérer l'espace en guidant l'éruption?

In the mixed dentition, when there is a discrepancy between the bony structures and the teeth, in certain cases, serial extractions are performed in order to guide the eruption of permanent teeth and obtain a correct occlusal function. The treatment consists in the extraction of primary teeth and then of first bicuspids. The choice of the first tooth to be extracted is based upon the position of the crowns of the permanent teeth and their degree of root maturation. Then the first bicuspid is extracted as soon as or prior to its eruption. This interceptive treatment has limited indications which need to be respected in order to preserve the child's future dental health. This treatment is indicated for Class I malocclusions with severe crowding or moderate crowding associated with bi-maxillary protrusion.

[Perforation of the cervical esophagus during tracheal intubation. Apropos of a personal case]. / Perforation de l'oesophage cervical au cours de l'intubation trachéale. A propos d'une observation personnelle

On the occasion of a personal case of perforation of the cervical oesophagus by laborious tracheal intubation which had necessitated the use of a rigid stilet, we found seven similar cases in the literature since 1971 which can be added to the 16 collected by DUBOST in 1971. We analyse the etiological circumstances, the clinical picture the course and action taken in these eight cases. From this we draw practical conclusions which are imperative during anesthesia.

[The deciduous tooth: therapeutic choices]. / La dent temporaire: les choix thérapeutiques.

Deciduous tooth diagnostic and treatment plan put a specific problem down. Therapeutical choice depends, not only of the child general health, but also of the deciduous tooth physiological period, its pathology, and the young child cooperation. Complexity of deciduous tooth biological cycle gives necessary, for the pedodontist, accuracy and a good clinical sense.

[Synergism of the combination of enzymes or surfactants and a phenolic disinfectant on a bacterial biofilm]. / Synergie de l'association d'enzymes ou de surfactants et d'un désinfectant phénolique sur un biofilm bactérien.

Disrupting bacterial biofilms is necessary for a wide application domains such as reusable medical devices, or systems of pipes for water or fluids in cosmetics, food and chemicals industry. Bacterial cells embedded in a biofilm are less susceptible to disinfectants than suspended cells. This property is referable to the structure of the biofilm itself. The gangue of exopolymers and the thickness of a 5-day-old biofilm of Escherichia coli (more than 200 layers of bacteria), contribute to this decrease of susceptibility. The present work deals with the release of an Escherichia coli biofilm by the sequential action of enzymes and a phenolic disinfectant on the one hand, and by the sequential or simultaneous action of surfactants and the previous disinfectant on the other hand. The decrease of bacteria count per mm2 and the Scanning Electron Microscope observations exhibited a synergic action in every case. Nevertheless, Escherichia coli biofilms quickly reconstructed even after exposition to the previous treatment.