A step-by-step approach for assessing acute oral toxicity without animal testing for additives of quasi-drugs and cosmetic ingredients.

Animal testing of cosmetic ingredients and products has been banned in the European Union since 2013. However, in Japan, the application of new quasi-drugs requires the generation of data on acute oral toxicity through animal testing. A weight of evidence approach for assessing oral toxicity was challenged. This approach used a combination of safety data, including a neutral red uptake cytotoxicity assay using BALB/c3T3 cells (3T3-NRU cytotoxicity assay), which can assess the acute oral toxicity of quasi-drugs or cosmetic ingredients. We conclude that the step-by-step approach can be used to assess test substances that cause low acute oral toxicity, such as the median lethal dose (LD 50) > 2000 mg/kg, thereby avoiding animal testing.

The effect of on-site application density on the UV protection efficacy of sunscreens.

BACKGROUND: Sun protection factor (SPF) and UVA protection factor (UVAPF) are performance indicators consumers recognize for UV protective cosmetics such as sunscreens. However, on-site application density affects actual UV protection, despite these indicators. To understand actual UV protection better, a more reliable manner is needed to verify application density for further discussion of photoprotection efficacy regarding public health. OBJECTIVES: To estimate the UV protective efficacy of sunscreen in actual use based on the application density of UV protective cosmetics and the analysis of UV protective effect modulated by application density. METHOD: The subjects applied the SPF-labeled sunscreens as usual. We measured the application amount and area including any amount on their hands to calculate the average application density on the face. Also, sunscreens were applied at densities of 0.5, 1.0, and 2.0 mg/cm2 . The SPF values were measured at each application site to evaluate the effect of application density on photoprotection efficacy. RESULT: We established a method of measuring application density utilizing three-dimensional photograph analysis. The median application density of the sunscreen applied in actual use was 1.33 mg/cm2 . The measured SPF values decreased in association with the decreased application density of sunscreens. Based on the estimate assuming the first-order correlation, the SPF value required to get the protective effect equivalent to a sunscreen with SPF 15, 30, or 50 at 2 mg/cm2 was calculated to be 23.8, 47.5, and 79.2, respectively, with the application density of 1.33 mg/cm2 . CONCLUSION: We demonstrated a reasonable procedure for estimating the photoprotection efficacy of sunscreens on the face. A suggestion was made to consider the application density for further discussion of photoprotection among consumers, especially for the long term with respect to public health.

Light source spectrum strongly influences the in vitro estimation of sun protection factor.

Irradiation from an artificial solar simulator that matches the relative cumulative erythema effectiveness (RCEE) of sunlight is used for in vivo sun protection factor (SPF) testing, whereas irradiation that matches the spectrum of natural sunlight (NS) is generally used for in vitro SPF testing. This study was designed to clarify whether this difference is important. Eight sunscreens spread on artificial substrates and several optical filters as sunscreen mimics were used. Their in vitro SPF values were evaluated using RCEE-compliant and NS spectrum-matching light sources in calculation. The calculated in vitro SPF values obtained using NS light (SPF [NS]) were lower than those obtained using RCEE light (SPF [RCEE]). The in vitro SPF (RCEE) values showed a better correlation and better agreement with in vivo SPF values, as compared with the in vitro SPF (NS) values. A marked difference between in vitro SPF values obtained with the two light sources in calculation was found for sunscreens showing low transmittance in the ultraviolet B region. To obtain in vitro SPF values that correspond well to in vivo SPF values measured with currently accepted methodology, it is important to use an RCEE-compliant light source.

Influence of application amount on sunscreen photodegradation in in vitro sun protection factor evaluation: proposal of a skin-mimicking substrate.

Widely used polymethylmethacrylate substrates for in vitro sun protection factor (SPF) testing of sunscreens do not mimic the rough surface structure of skin, and in addition, sample loading is less than that used in in vivo SPF testing (2.00 mg cm(-2)). We have developed a skin-mimicking substrate (SMS), which has furrows and ridges on its surface, like human skin. A comparison of the photodegradation profiles of sunscreens on commercially available substrates (including SMS) at the recommended application amounts, and on SMS at various application amounts showed that the photodegradation rate of photounstable sunscreen was dependent on the application amount being higher at lower application amounts. SMS at the recommended application amount of 2.00 mg cm(-2) provided in vitro SPF values that were comparable with in vivo SPF values. Our results confirm that, in order to develop a reliable in vitro SPF method, which is consistent with in vivo SPF determination, it is important to use the same application amount of sample as in the in vivo method, in order to take proper account of sunscreen photostability.

Algorithm for in vitro Sun Protection Factor based on transmission spectrum measurement with concomitant evaluation of photostability.

In the in vitro evaluation of Sun Protection Factor (SPF), the photostability of the ultraviolet (UV) filters can have a major impact, especially for high-SPF formulations, but is generally not taken into consideration. In this study, we present a UV transmission spectrum measurement system utilizing a high-sensitivity UV photomultiplier tube with concomitant evaluation of photostability. We have developed an algorithm to estimate SPF in vitro by converting the amount of UV light transmission through the sunscreen layer into cumulative relative erythema effectiveness to obtain one minimal erythema dose. Thus, the algorithm uses the same endpoint as in vivo SPF methods, but with a photomultiplier tube as the detector instead of skin. The values obtained showed an excellent correlation with in vivo SPF values, even for high-SPF sunscreens exceeding SPF 50. This method should be suitable as an in vitro SPF testing method for regulatory purposes.

Involvement of photooxidation of melanogenic precursors in prolonged pigmentation induced by ultraviolet A.

Melanin is usually produced in melanocytes; however, our histologic research indicates that brownish black persistent pigmentation occurs at the basal layer and, partially, at the suprabasal layer of the epidermis, outside of melanocytes, in skin exposed to high doses of ultraviolet (UV) A radiation. This pigmentation remains for several weeks. We examined whether colorless melanogenic precursors participate in this UVA-induced persistent pigmentation. Among them, 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and its O-methyl metabolite, 6-hydroxy-5-methoxyindole-2-carboxylic acid (6H5MICA), can change into dark-colored pigment upon exposure to UVA, but not UVB, radiation, producing irreversible brownish black pigmentation. These hydroxyindole derivatives readily changed into dark-colored pigment upon UVA irradiation in the presence of hydrogen peroxide in vitro. Histochemical and biochemical studies demonstrated that 6H5MICA accumulates in excised skin specimens and supernatant of cultured human melanocytes. These results suggest that dark-colored pigment is generated directly, outside of melanocytes, in response to UVA exposure, by photooxidation of O-methyl metabolites of DHICA, such as 6H5MICA, at least. We propose that the pigmentation derived from this compound at the basal and suprabasal layers of the epidermis is involved in the mechanism of skin persistent pigmentation without reddening, observed after exposure to sunlight.

Objective measurement of minimal erythema and melanogenic doses using natural and solar-simulated light.

Very little information exists on the amount of natural and artificial UV light required to cause sunburn and tanning in individuals with very pale skin who are at the greatest risk of developing skin cancer. We have investigated minimal erythema dose (MED) and minimal melanogenic dose (MMD) in a group of 31 volunteers with Fitzpatrick skin types I and II using an Oriel 1000 W xenon arc solar simulator and natural sunlight in Sydney, Australia. We measured the erythemal and melanogenic responses using conventional visual scoring, a chromameter and an erythema meter. We found that the average MED measured visually using the artificial UV source was 68.7 +/- 3.3 mJ/cm2 (3.4 +/- 0.2 standard erythema doses [SED]), which was significantly different from the MED of sunlight, which was 93.6 +/- 5.6 mJ/cm2 (P < 0.001) (11.7 +/- 0.7 SED). We also found significant correlations between the solar-simulated MED values, the melanin index (erythema meter) and the L* function (chromameter). The average MMD (obtained in 16 volunteers only) using solar-simulated light was 85.6 +/- 4.9 mJ/cm2, which was significantly less than that measured with natural sunlight (118.3 +/- 8.6 mJ/cm2; P < 0.05). We mathematically modeled the data for both the chromameter and the erythema meter to see if we were able to obtain a more objective measure of MED and differentiation between skin types. Using this model, we were able to detect erythemal responses using the erythema index function of the erythema meter and the a* function of the chromameter at lower UV doses than either the standard visual or COLIPA methods.