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.

Impact of physiologically relevant temperatures on dermal absorption of active substances - an ex-vivo study in human skin.

Skin temperature plays a certain role in the dermal absorption of substances, but the extent and mechanisms of skin temperatures-induced modulation in ranges caused by physiological thermoregulation or environmental conditions are largely unknown. The influence of dermal temperature on the absorption of the model lipophilic compound (anisole) and the model hydrophilic compounds (1,4-dioxane, ethanol) through human skin was investigated at three dermal temperatures (25, 32 and 39 °C) in an ex-vivo diffusion cell model. The substances were applied to the skin and transdermal penetration was monitored. All substances showed temperature dependent variations in their penetration behavior (3 h: 25-39 °C: 202-275% increase in cumulative, transdermally penetrated amounts). The relative differences in absorption in relation to temperature were greatest within 45 min after exposure (25-39 °C: 347-653% rise in cumulated penetration), although absolute amounts absorbed were small (45 min vs. 3 h: 4.5-14.5%). Regardless of blood circulation, skin temperature significantly influences the amount and kinetics of dermal absorption. Substance-dependent, temperature-related changes of the lipid layer order or the porous pathway may facilitate penetration. The early-stage modulation of transdermal penetration indicates transappendageal absorption, which may be relevant for short-term exposures. For both, toxicological evaluation and perfusion cell studies, it is important to consider the thermal influence on absorption or to perform the latter at a standardized temperature (32±1 °C).

Neural and endothelial control of the microcirculation in diabetic peripheral neuropathy.

OBJECTIVE: To examine the role played by endothelium-dependent and endothelium-independent vasodilation of the cutaneous microcirculation and their relationship to neural microcirculatory control in type 1 and type 2 diabetic patients. METHODS: Acetylcholine and sodium nitroprusside were iontophoresed using a dose-response technique. Endothelium-dependent, endothelium-independent, and C-fiber-mediated vasodilation were measured with a laser Doppler device. RESULTS: Endothelium-dependent vasodilation of the forearm cutaneous microcirculation was attenuated in diabetic subjects. The response was less in type 2 diabetic subjects than in controls (p < 0.005). In contrast, there was no significant difference between type 1 diabetic subjects and controls. There was no significant abnormality in endothelium-independent vasodilation in either diabetic group. The C-fiber-mediated axon reflex in the forearm was impaired in both type 1 and type 2 diabetics, which is consistent with a small-fiber neuropathy (p < 0.005). The duration of diabetes in type 2 diabetics was a significant predictor of the maximum endothelium-mediated vasodilation. CONCLUSION: Changes in cutaneous blood flow are seen relatively early in the course of diabetic peripheral neuropathy and are characterized by endothelial and neural but not smooth muscle dysfunction. The presence of significant C-fiber impairment in both diabetic groups, together with significantly greater dysfunction in endothelium-dependent vasodilation in type 2 diabetics, suggests that the endothelial function and nitric oxide may play a greater role in the pathogenesis of diabetic peripheral neuropathy in type 2 diabetic patients.

Inflammatory models of cutaneous hyperalgesia are sensitive to effects of ibuprofen in man.

A new experimental procedure was developed to quantify the analgesic actions of non-steroidal anti-inflammatory drugs (NSAIDs) in healthy human subjects. In order to mimic the clinical situation, the drug was 'therapeutically' administered 1 day after induction of inflammation by freezing a small skin area. The procedure was easily tolerated and led to a marked hyperalgesia without ongoing pain which was tested using mechanical impact stimulation and magnitude estimation. For comparison, we used a previously established model of repeated noxious pinching of an interdigital skin web which induces a hyperalgesia to pressure (rated via visual analogue scale), and topical application of capsaicin which leads to quantifiable flare and allodynia responses. The effects of a cumulative drug regime of ibuprofen in 2 different doses (3 x 400 mg and 3 x 800 mg at 2-h intervals) were tested versus placebo using a double-blind cross-over design with 24 volunteers of either gender. Ibuprofen caused a significant suppression of the hyperalgesia to repeated pinching and of the hyperalgesia to impact stimulation following freeze trauma. In contrast, there was no effect on the areas of flare and allodynia induced by capsaicin application and on the impact evoked sensations from untreated skin. The two dosages of ibuprofen, however, appeared to be equally effective in a way that suggests a plateauing of the antihyperalgesic effect. The two models in which hyperalgesia is affected by ibuprofen, i.e., repeated pinching and impact stimulation after freeze trauma, seem to provide comparable sensitivity. The freeze model may in the future have the advantage to allow for a better temporal resolution of the drug's action profile.

Unresponsive afferent nerve fibres in the sural nerve of the rat.

1. The proportion of primary afferent nerve fibres in a skin nerve of the rat that responded or failed to respond to mechanical or thermal stimulation of the skin in the noxious and non-noxious range was analysed. 2. Activity of afferent nerve fibres was recorded from the dorsal roots. Units projecting into the sural nerve were selected using supramaximal electrical stimulation of the nerve stem. All other hindleg nerves were cut. 3. The receptive fields were searched by carefully examining the hindleg skin with noxious and innocuous mechanical, cooling and warming stimuli. Probing of the intrinsic foot muscles and manipulation of the ankle and toe joints was employed to recruit units projecting to deeper tissues. 4. In a first series of twenty-two experiments, eighty-nine rapidly conducting myelinated A beta units, thirty slowly conducting myelinated A delta units and 101 unmyelinated C units were investigated. Most units were identified as belonging to one of the established classes of cutaneous sensory units and this was also ascertained by a collision test. 5. Two A beta, eight A delta and forty-six C fibres did not respond to any one of the stimuli. Electrical thresholds and conduction velocities of the unresponsive C fibres were not significantly different from those of the units responding to natural stimulation of their receptive fields. In the A delta group unresponsive and high threshold mechanoreceptive units were preferentially found among the units with the slowest conduction velocities. 6. In a second series of seven experiments, one single nerve filament containing responsive and unresponsive C fibres was tested repetitively at 30 min intervals. Twenty unresponsive units and seven units responding to noxious mechanical and/or heat stimuli were studied. Ten of the twenty initially unresponsive units became activated by mechanical and/or heat stimuli after observation times of 30-150 min. Some of these units had mechanical thresholds as low as 64 mN (tested with calibrated von Frey hairs), or thermal thresholds down to 42 degrees C. 7. Two of the ten C units which became responsive in the course of an experiment later lost their responsiveness again. On the other hand, two of the C units which were initially responsive to noxious heat and/or noxious mechanical stimuli became completely unresponsive after repetitive stimulation, whereas one unit initially only responding to noxious heat became responsive to mechanical stimuli, suggesting that mechanical and heat responsiveness may be separately gained or lost by sensory C fibres.(ABSTRACT TRUNCATED AT 400 WORDS)

Different patterns of hyperalgesia induced by experimental inflammation in human skin.

Different types of hyperalgesia were studied after experimental induction of inflammation in small skin areas of healthy volunteers either by topical application of capsaicin solution (1% in 70% ethanol) or by briefly freezing a skin area of similar size to -28 degrees C. Sensory tests were performed 30 min after capsaicin application and 22 h after freeze lesions. Heat pain thresholds were lowered after both treatments, probably due to nociceptor sensitization. Hyperalgesia to four types of mechanical stimulation was studied. (i) Hyperalgesia to punctate stimuli was encountered at the skin site directly affected by the noxious chemical or freeze stimulus (1 degree zone) and in the surrounding skin (2 degrees zone) in both models though the area of 2 degrees hyperalgesia to punctate stimuli after freezing was smaller than after capsaicin. (ii) Hyperalgesia to gently brushing the skin was prominent after capsaicin in 1 degree and 2 degrees zone, but almost absent after freezing. It was concluded that both hyperalgesia to punctate stimuli and brush-evoked pain are due to central nervous plasticity changes rather than nociceptor sensitization. As revealed by differential nerve blocks, brush-evoked pain is mediated by low threshold mechanosensitive A beta-fibres, whilst hyperalgesia to punctate stimuli can be elicited when only C-fibres conduct. In contrast to hyperalgesia to punctate stimuli it requires continuous background discharges in nociceptor units. (iii) Pressure hyperalgesia to tonic stimulation with a blunt probe was encountered in the 1 degree zone of both types of inflammation and is probably due to recruitment of sensitized nociceptor units. (iv) Impact hyperalgesia was studied by shooting small bullets against the skin at predetermined velocities. It was found in the 1 degree zone after freezing and absent in the capsaicin model. Differential nerve blocks revealed that it is probably mediated by sensitized C-fibres. In conclusion, different types of inflammatory changes may result in characteristic different patterns of hyperalgesia.

Neuronal nicotinic receptor expression in sensory neurons of the rat trigeminal ganglion: demonstration of alpha3beta4, a novel subtype in the mammalian nervous system.

The identification of a family of neuronal nicotinic receptor subunit genes establishes the potential for multiple subtypes with diverse physiological functions. Virtually all of the high affinity nicotinic receptors measured to date in the rodent CNS are composed of alpha4 and beta2 subunits only. However, the demonstration of other subunit transcripts in a variety of central and peripheral nervous tissues suggests a greater degree of receptor subtype heterogeneity than so far has been elucidated. The purpose of the present studies was to determine at the mRNA and protein levels which neuronal nicotinic receptor subunits are expressed by sensory neurons of the rat trigeminal ganglion and in what combinations these gene products associate to form neuronal nicotinic receptor subtypes in this tissue. Radioreceptor binding analysis indicated that in the adult rat trigeminal ganglion there exist at least two nicotinic receptor binding sites with differing affinities for [3H]-epibatidine. In situ hybridization histochemical studies revealed the existence of mRNA encoding the alpha3, alpha4, alpha5, beta2, and beta4 subunits, but not the alpha2 subunit. Immunoprecipitation with subunit-specific antisera demonstrated that each of the subunits present in the ganglion at the mRNA level is a constituent of nicotinic receptors capable of binding 3H-epibatidine. Various applications of these approaches yielded strong evidence that, in addition to alpha4beta2, which is thought to be the predominant neuronal nicotinic receptor subtype in the rodent CNS, trigeminal sensory neurons express as the principal subtype alpha3beta4, which has not been demonstrated previously in mammalian nervous tissue.

Capsaicin-evoked CGRP release from rat buccal mucosa: development of a model system for studying trigeminal mechanisms of neurogenic inflammation.

Many of the physiological hallmarks associated with neurogenic inflammatory processes in cutaneous tissues are similarly present within orofacial structures. Such attributes include the dependence upon capsaicin-sensitive sensory neurons and the involvement of certain inflammatory mediators derived therein, including calcitonin gene-related peptide (CGRP). However, there are also important differences between the trigeminal and spinal nervous systems, and the potential contributions of neurogenic processes to inflammatory disease within the trigeminal system have yet to be fully elucidated. We present here a model system that affords the ability to study mechanisms regulating the efferent functions of peptidergic terminals that may subserve neurogenic inflammation within the oral cavity. Freshly dissected buccal mucosa tissue from adult, male, Sprague-Dawley rats was placed into chambers and superfused with oxygenated, Krebs buffer. Serial aliquots of the egressing superfusate were acquired and analysed by radioimmunoassay for immunoreactive CGRP (iCGRP). Addition of the selective excitotoxin, capsaicin (10-300 microm), to the superfusion buffer resulted in a significant, concentration-dependent increase in superfusate levels of iCGRP. Similarly, release of iCGRP from the buccal mucosa could also be evoked by a depolarizing concentration of potassium chloride (50 mm) or by the calcium ionophore A23187 (1 microm). The specific, capsaicin receptor antagonist, capsazepine (300 microm), completely abolished the capsaicin-evoked release of iCGRP while having no effect whatsoever on the potassium-evoked release. Moreover, capsaicin-evoked release was dependent upon the presence of extracellular calcium ions and was significantly, though incompletely, attenuated by neonatal capsaicin denervation. Collectively, these data indicate that the evoked neurosecretion of iCGRP in response to capsaicin occurs via a vanilloid receptor-mediated, exocytotic mechanism. The model system described here should greatly facilitate future investigations designed to identify and characterize the stimuli that regulate the release of CGRP or other neurosecretory substances in isolated tissues. This system may also be used to elucidate the role of these mediators in the aetiology of inflammatory processes within the trigeminal field of innervation.