Penetration of ammonium perfluorooctanoate through rat and human skin in vitro.

Rat and human epidermal membranes were mounted onto in vitro diffusion cells with an exposure area of 0.64 cm2, and skin integrity was confirmed using electrical impedance. Following membrane selection, Fluorad FC-118, a 20% aqueous solution of ammonium perfluorooctanoate (AFPO), was applied to the epidermal surface of each skin replicate at approximately 150 microL/cm2 and the donor chamber opening occluded with Parafilm. Serial receptor fluid samples were collected hourly from 1 to 6 h and at 12, 24, 30, and 48 h and analyzed by liquid chromatography-mass spectrometry (LC-MS) for APFO anion (PFO-). For rat skin, the time to steady-state penetration (6500+/-3000 ng APFO x cm(-2) x h(-1)) occurred in less than 12 h, which was sustained until termination (48 h). Based on the concentration of the applied test material, the permeability coefficient (Kp) for APFO in rat skin was calculated to be 3.25+/-1.51 x 10(-5) cm/h. By end of the 48-h exposure period, only a small portion of the total APFO applied (1.44+/-1.13%) had penetrated through rat skin. For human skin, steady-state penetration of APFO (190+/-57 ng APFO x cm(-2) x h(-1)) was reached by 12 h. Based on the concentration of the applied test material, the permeability coefficient for APFO in human skin was calculated to be 9.49+/-2.86 x 10(-7) cm/h. By the end of the 48-h exposure period, only a negligible amount of the total APFO applied (0.048+/-0.01%) had penetrated through human skin. Thus, under infinite dose and occlusive conditions, the steady-state penetration of APFO from a 20% solution was approximately 34-fold faster through rat skin than human skin.

The Tinsley LCR Databridge Model 6401 and electrical impedance measurements to evaluate skin integrity in vitro.

Electrical impedance is used to confirm skin integrity for in vitro dermal regulatory testing and as a tool to evaluate skin condition to determine the irritation and corrosion potential of various chemicals and personal care products. In this experiment, samples of dermatomed human skin were mounted onto static diffusion cells (0.64 cm2) maintained at 32 degrees C. Following equilibration with 0.9% saline in the donor and receptor chambers, an impedance measurement was taken with a Tinsley LCR Databridge Model 6401 set in the resistance mode (R) and in (a) the serial-equivalent mode (SER) with an alternating current (AC) frequency of 100 hertz (Hz), (b) SER and 1000 Hz, (c) parallel-equivalent mode (PAR) and 100 Hz, and (d) PAR and 1000 Hz. With the databridge set in the SER-equivalent mode and an AC frequency of 1000 Hz, the minimum (7.2 kOmega), maximum (10.0 kOmega), and median (8.6 kOmega) impedance values exhibited a limited response range (2.8 kOmega). However, when the Tinsley 6401 was set in the PAR-equivalent mode at the lower AC frequency of 100 Hz the minimum (16.7 kOmega), maximum (134.6 kOmega), median (83.2 kOmega), and range (117.9 kOmega) of values were the highest obtained. The results confirm that the operator-selected settings on the Tinsley LCR Databridge Model 6401 affect the impedance measurement and the dynamic range of values observed for dermatomed human skin in vitro.

A prevalidation study on the in vitro skin irritation function test (SIFT) for prediction of acute skin irritation in vivo: results and evaluation of ECVAM Phase III.

A prevalidation study sponsored by the European Centre for the Validation of Alternative Methods (ECVAM) on in vitro tests for acute skin irritation is aimed at identifying non-animal tests capable of discriminating irritants (I) from non-irritants (NI), as defined according to European Union and OECD. This paper reports on Phase III for one of the methods, the skin integrity function test (SIFT), assessing the protocol performance of the SIFT, in terms of reproducibility and predictive ability, in three laboratories. The barrier function properties of excised mouse skin were determined using a set of 20 coded chemicals (10 I, 10 NI), using the endpoints of trans-epidermal water loss (TEWL) and electrical resistance (ER). The basis of the SIFT prediction model is if the ratios of the pre- and post-application values for either TEWL or ER are greater than five-fold, then the test chemical is deemed irritant (I). If the ratio of both parameters is less than five-fold then the chemical is deemed non-irritant (NI). Analysis of variance (ANOVA) indicated that the intra-lab reproducibility was acceptable but that the inter-lab reproducibility was not. Overall, the SIFT test under-predicted the irritancy of the test chemicals chosen for Phase III with an overall accuracy of only 55%. The sensitivity value (ability to correctly predict I) was only 30%. The specificity (ability to predict NI) of the test was better at 80%. A retrospective examination of the SIFT results was undertaken using Student's t-test and a significance level of P<0.05 to predict an irritant based on changes in the TEWL ratio values. This improved the predictivity of the SIFT test, giving a specificity of 60%, a sensitivity of 80% and an overall accuracy of 70%. Appropriate modifications to the prediction model have now been made and the SIFT will be re-examined in a new validation exercise to investigate the potential of this non-animal method to predict acute skin irritation potential.

Rapid integrity assessment of rat and human epidermal membranes for in vitro dermal regulatory testing: correlation of electrical resistance with tritiated water permeability.

An approach is presented that allows for rapid selection of robust rat and human epidermal membranes for use on in vitro dermal regulatory studies. Tritiated water (THO) permeability was correlated with electrical resistance (ER) and the results used to propose ER values to judge membrane integrity. Rat and human epidermal membranes were prepared and mounted onto in vitro glass static diffusion cells (0.64 cm(2)) maintained at 32 degrees C. THO permeability coefficients (Kp) were determined and compared with ER measurements. Electrical resistance was also determined for various in vitro cell exposure areas from 0.64 cm(2) to 2.54 cm(2). Our results show that rat epidermal membrane THO Kp values exhibited a lognormal distribution with a median value of 2.76 x 10(-3) cm/h. Human epidermal membrane THO Kp values were best described by a Weibull distribution with a median value of 1.13 x 10(-3) cm/h. The corresponding median electrical resistance measurements were 5.59 kOmega for rat and 23 kOmega for human epidermal membranes. Based on the widely used and accepted single point THO Kp thresholds of /=5.87 kOmega and >/=17.1 kOmega were calculated and proposed as acceptable benchmarks for pre-qualifying membranes. In our research exploring the relationship between ER and exposure area we report that an inverse relationship exists between ER and in vitro cell exposure area; as cell area increased, ER decreased. The use of electrical resistance provides a rapid and reliable method for evaluating the integrity of rat and human epidermal membranes for in vitro dermal kinetic testing.

Early cell proliferative and cytotoxic effects of phenacetin on rat nasal mucosa.

The oral analgesic drug phenacetin is known to cause nasal and renal tumors in rats when fed in the diet for 2 years at 1.25 or 2.5%. Long-term exposure to chemicals at cytotoxic concentrations may lead to tumor formation secondary to chronic restorative cell proliferation. In the present experiments, cell proliferative and cytotoxic effects on the nasal mucosa were examined after short-term phenacetin administration. One week of daily gavage treatment of rats with phenacetin at doses comparable to those used in oncogenicity studies resulted in dose-related increases in DNA synthesis in both respiratory and olfactory mucosa. The increase in respiratory mucosa was due to inflammatory cells in the lamina propria and not proliferation of the respiratory epithelial cells. This observation demonstrates a potentially serious artifact in analytical approaches to DNA synthesis using tissue homogenates. One or two weeks of daily phenacetin treatment resulted in degenerative changes in the olfactory epithelium and necrosis of Bowman's glands. These changes were associated with increases in cell proliferation only in the olfactory epithelium. Two-week daily gavage treatment of rats with phenacetin at 100, 625, or 1250 mg/kg/day increased olfactory epithelial cell replication 62.4, 174, or 763%, respectively. The dose-response relationship for cell proliferation was similar to that of nasal tumor formation. These data suggest that the primary site of phenacetin toxicity within the nose is the olfactory mucosa, with restorative cell proliferation being confined to the epithelial cell layer. The data indicated that early cell proliferative responses may be important in the genesis of nasal tumors and that cell proliferation data may be useful for setting dose levels for oncogenicity studies.