Persistent Pigment Darkening Research Articles

Evaluating hyperpigmentation: is invasion of the skin necessary?

BackgroundThe combination of long wavelength ultraviolet A1 radiation (LWUVA1) and visible light (VL) has been shown to produce photodamage and the majority of organic sunscreens lack protection against this spectrum. Currently, established testing protocols for VL photoprotection are lacking.ObjectiveTo compare pigmentation assessment methods, including Investigator’s Global Assessment (IGA) scoring, Diffuse Reflectance Spectroscopy (DRS), and immunohistochemistry, to assess the utility of each in evaluating VL + LWUVA1 induced pigmentation.MethodsAnonymized IGA, DRS, and immunohistochemistry data for 37 subjects (Fitzpatrick skin phototypes IV-VI) was retrospectively analyzed for pigment evaluation. Pigmentation was induced on the subjects’ backs with VL + LWUVA1 (0%–0.5% UVA1) irradiation at a dose of 480 J/cm2. Comparisons were made for all three assessment techniques between non-irradiated skin and VL + LWUVA1 irradiated skin, as well as between pure VL (0% UVA1) and VL + LWUVA1 (0.5% UVA1) irradiated sites. All comparisons were made for data collected approximately 24 h after irradiation to evaluate persistent pigment darkening (PPD).ResultsAmong all 37 subjects, both IGA scores and DRS detected a statistically significant difference in PPD between irradiated and non-irradiated sites, as well as between VL + LWUVA1 and pure VL irradiated sites. However, MART-1/Melan-A did not indicate a statistically significant difference in PPD between irradiated and non-irradiated sites or between VL + LWUVA1 and pure VL irradiated sites.ConclusionSubjective and objective noninvasive assessments were more sensitive in detecting VL + LWUVA1 induced pigmentation and should be preferred over invasive methods. Researchers are advised to initiate assessments with IGA and subsequently incorporate DRS for more objective and comprehensive insights in pigment evaluation.

Oxidative stress in the skin: Impact and related protection.

Skin, our first interface to the external environment, is subjected to oxidative stress caused by a variety of factors such as solar ultraviolet, infrared and visible light, environmental pollution, including ozone and particulate matters, and psychological stress. Excessive reactive species, including reactive oxygen species and reactive nitrogen species, exacerbate skin pigmentation and aging, which further lead to skin tone unevenness, pigmentary disorder, skin roughness and wrinkles. Besides these, skin microbiota are also a very important factor ensuring the proper functions of skin. While environmental factors such as UV and pollutants impact skin microbiota compositions, skin dysbiosis results in various skin conditions. In this review, we summarize the generation of oxidative stress from exogenous and endogenous sources. We further introduce current knowledge on the possible roles of oxidative stress in skin pigmentation and aging, specifically with emphasis on oxidative stress and skin pigmentation. Meanwhile, we summarize the science and rationale of using three well-known antioxidants, namely vitamin C, resveratrol and ferulic acid, in the treatment of hyperpigmentation. Finally, we discuss the strategy for preventing oxidative stress-induced skin pigmentation and aging.

French maritime pine bark extract (pycnogenol) in association with triple combination cream for the treatment of facial melasma in women: a double-blind, randomized, placebo-controlled trial.

Melasma can be recalcitrant to treatment, and relapses are common. Pycnogenol has been reported to be effective in treating melasma. To compare the efficacy, safety and tolerability of 75mg pycnogenol taken orally twice a day vs. a placebo, in association with the triple combination and broad-spectrum sunscreen for the treatment of facial melasma. A randomized, double-blind, parallel, placebo-controlled study was conducted on 44 women with facial melasma in a single centre from May 2019 through November 2019. Patients with melasma were randomly assigned to orally take 75mg pycnogenol (PYC) or a placebo (PLAC) twice a day for 60days. Both groups also received tinted sunscreen [Sun Protection Factor (SPF) 50; Persistent Pigment Darkening (PPD) 17] for daytime use and a topical triple combination at bedtime. The primary outcome was a change from the baseline Modified Melasma Area Severity Index (mMASI) score. Secondary outcomes were improvements in the patients' quality of life (MELASQoL), colorimetric indices and Global Aesthetic Improvement Scale (GAIS). All participants completed the trial. The mean (SD) age of the participants was 39 (7) years, and 91% were phototypes III-IV. Both groups exhibited a reduction in mMASI scores, MELASQoL scores and colour contrast (P<0.01). The mean (CI 95%) reductions of the mMASI scores were 49% (36-61%) for PYC and 34% (16-47%) for PLAC. The reductions in mMASI scores and colorimetric contrast were superior for the PYC group (P<0.05). The analysis of GAIS resulted in an improvement of 86% (CI 95%: 68-96%) for the participants in the PYC group and 55% (CI 95%: 32-73%) for those in the PLAC group. There were no adverse effects related to oral treatment. Pycnogenol is well-tolerated and increases the effectiveness of broad-spectrum sunscreen and the triple combination in the treatment of facial melasma in women.

Melanopsin mediates UVA-dependent modulation of proliferation, pigmentation, apoptosis, and molecular clock in normal and malignant melanocytes

Cutaneous melanocytes and melanoma cells express several opsins, of which melanopsin (OPN4) detects temperature and UVA radiation. To evaluate the interaction between OPN4 and UVA radiation, normal and malignant Opn4WT and Opn4KO melanocytes were exposed to three daily low doses (total 13.2 kJ/m2) of UVA radiation. UVA radiation led to a reduction of proliferation in both Opn4WT cell lines; however, only in melanoma cells this effect was associated with increased cell death by apoptosis. Daily UVA stimuli induced persistent pigment darkening (PPD) in both Opn4WT cell lines. Upon Opn4 knockout, all UVA-induced effects were lost in three independent clones of Opn4KO melanocytes and melanoma cells. Per1 bioluminescence was reduced after 1st and 2nd UVA radiations in Opn4WT cells. In Opn4KO melanocytes and melanoma cells, an acute increase of Per1 expression was seen immediately after each stimulus. We also found that OPN4 expression is downregulated in human melanoma compared to normal skin, and it decreases with disease progression. Interestingly, metastatic melanomas with low expression of OPN4 present increased expression of BMAL1 and longer overall survival. Collectively, our findings reinforce the functionality of the photosensitive system of melanocytes that may subsidize advancements in the understanding of skin related diseases, including cancer.

Comparison of UVA Protection Factor Measurement Protocols.

BackgroundIn the past, it was taught that UVA wavelengths (320- 400nm) only plays a major role in skin aging but recently the scientific researches also show that UVA cause cancerous keratinocyte cells in deep layer of the epidermis. Therefore, the protective ability of the product against UVA is important in addition to protection against UVB rays. The UVA protective factor (UVA-PF) is used to evaluate the effectiveness of sunscreen products against UVA rays. This study aims to review and compare all outstanding protocols in the field of UVA-PF measurement and finally the introduction of the best method of measuring UVA-PF based on the further benefits.Materials and MethodsFour standards including ISO 24443 (AS/NZS 2604: 2012 recommended approach), CEN 2006, FDA 2007 and FDA 2011 are selected.ResultsIn order to measure UVA-PF with in vivo method, two standards of CEN 2006 and FDA 2007 recommended persistent pigment darkening (PPD) method. Although the general principle of both is similar, there are some differences in detail. For in vitro measurement of UVA-PF, CEN and FDA 2011 standards use critical wavelengths. FDA 2007 introduces the modified Diffey fraction, and ISO 24443 standard meets the UVA-PF measurement in a manner that is consistent with PPD.ConclusionFinally, this review discussed the comparison of all in vitro and in vivo UVA-PF measurement standards and provided information in the form of texts and tables to move towards the creation of an integrated standard. Since in vitro methods of UVA-PF measurement are not reproducible due to differences in test conditions, it may be concluded that the in vivo PPD method is a more suitable option.

The dynamics of pigment reactions of human skin to ultraviolet A radiation.

The pigment responses of human skin to broadband UVA radiation (320-400nm) occur in three distinct phases. The first phase includes immediate pigment darkening (IPD), the pigment that appears immediately after irradiation. The second phase involves an intermediate step, termed persistent pigment darkening (PPD), which leads to the third phase of neomelanogenesis or delayed tanning (DT). Since DT results from synthesis of new melanin, it persists beyond 5-7days. We conducted studies on human subjects to investigate the dynamic responses of the IPD and PPD reactions to broadband UVA radiation at threshold and superthreshold doses. The threshold doses for IPD, PPD, and DT were found to be approximately 1, 11, and 18J/cm2 , respectively. The colorimetry ΔL* value corresponding to minimal clinically perceptible pigmentation was found to be 0.8±0.1. IPD appeared immediately and had an associated decay constant of approximately 1.4minutes. At doses greater than PPD threshold, IPD reaction decayed while PPD developed indicating toward IPD being used as a substrate in the formation of PPD.

Photoprotection against visible light-induced pigmentation.

This paper presents invivo an invitro studies demonstrating the induction of pigmentation in human skin by visible light which can be blocked by using formulation containing the correct amount of yellow iron oxide (YIO). An invitro absorption method was developed to determine the protection provided by a test formulation containing 4.5% YIO using an IPD UVA-VIS action spectrum. Following the development of the invitro method and invivo study with 10 normal healthy volunteers with Fitzpatrick skin phototypes IV to VI was conducted to verify if the predictive model. The invitro model for visible light protection provided a protection factor of 2.5 using the invitro absorption spectrum of 4.5% of YIO with a very similar result from the invivo study with a protection factor of 3.0. Multiple daily exposures of visible light have shown increase in skin pigmentation and the application of YIO provide less development of pigmentation when compared to unprotected skin. In vitro testing of the absorbance of the pigmented formulation using a proposed action spectrum for immediate pigment darkening (IPD) response in the visible light range supports the invivo protection observations for persistent pigment darkening (PPD) and can be used as predictor for skin pigmentation induced by visible light.

Homeostasis in Topical Photoprotection: Getting the Spectral Balance Right.

The solar radiation range has harmful and beneficial effects. Sunscreens, which selectively block specific spectral regions, may potentially interfere with skin homeostasis. For instance, the ultraviolet (UV) B waveband produces erythema and DNA damage; simultaneously, it induces pre-vitamin D3 synthesis. UVA1 and visible light can both induce pigmentation in skin phototypes IV–VI, and act in synergy to induce erythema and persistent pigment darkening. In contrast, UVA may contribute to blood pressure control and cardioprotection by inducing release of nitric oxide from intracutaneous photolabile nitric oxide derivatives. Finally, infrared A radiation alters the collagen equilibrium of the dermal extracellular matrix but is involved in the regulation of body temperature and in nitric oxide release, with a potential beneficial impact on blood pressure regulation. Ideally, photoprotection should thus be performed with a neutral density filter, mitigating all radiation ranges homogeneously, to maintain solar spectrum homeostasis. Natural compounds such as mycosporine-like amino acids are promising natural UV radiation-filtering compounds for an improved homeostasis with our environment. Lastly, we should not forget individual characteristics and behavior, as homeostasis differs according to individual phototypes and skin exposure behaviors.

ESTUDO COMPARATIVO DE FORMULAÇÕES DE FATOR DE PROTEÇÃO SOLAR DE FARMÁCIAS MAGISTRAIS UTILIZANDO SIMULADORES

A importância do uso de protetor solar tem sido amplamente divulgada na mídia. Há muitas opções de formulações de protetor solar disponíveis no mercado magistral, incluindo uma gama de fatores proteção solar e preços. No momento da compra, dúvidas e controvérsias podem surgir em relação ao fator de proteção ideal e suficiente para minimizar os efeitos da radiação ultravioleta. Portanto, o objetivo deste estudo foi avaliar formulações de fotoprotetores disponíveis em farmácias de manipulação. Seis formulações foram testadas com fator de proteção solar atribuído por farmácias de manipulação no Estado de São Paulo. Foram observadas discrepâncias entre fator rotulado e eficácia em alguns protetores solares testados. Os resultados mostraram que algumas formulações não atingiram o fator de proteção solar descrito na embalagem nem o grau de proteção contra raios UVA, utilizando como base o método Persistent Pigment Darkening (PPD), em relação ao valor mínimo exigido na RDC 30, de 1 de Junho de 2012. Tendo em vista que a fotoproteção é de extrema importância para evitar os efeitos da radiação ultravioleta, há necessidade de um controle mais rigoroso de qualidade na produção de protetores solares manipulados.

Sun Protection Factors in Evaluation of Sunscreen Potency: The Persistent Pigment Darkening Method

Sun Protection Factors in Evaluation of Sunscreen Potency: The Persistent Pigment Darkening Method The Persistent Pigment Darkening (PPD) method has been modified by Bissonette et al. as an in vivo assay for UVA exposure. This is the gold standard with which to compare many other methods that assay the potency of sunscreens that block UVA damage. In view of the current interest in evaluation of sun protection factors (SPF), it is important to ensure the validity of current methodology used to evaluate UVB and UVA exposure before meaningful evaluation of sun protection factors and potency of sunscreens against UVinduced damage can be made.

Are there mechanistic differences between ultraviolet and visible radiation induced skin pigmentation?

Most of the studies on sunlight-induced pigmentation of skin are mainly focused on ultraviolet (UV) radiation-induced pigmentation and ways to prevent it. Recent studies have shown that the visible component of sunlight can also cause significant skin pigmentation. In the current study, the extent of pigmentation induced by UV and visible regions of sunlight in subjects with Fitzpatrick skin type IV-V was measured and compared with pigmentation induced by total sunlight. The immediate pigment darkening (IPD) induced by the visible fraction of sunlight is not significantly different from that induced by the UV fraction. However, the persistent pigment darkening (PPD) induced by visible fraction of sunlight in significantly lower than that induced by the UV fraction. The dose responses of IPD induced by UV, visible light and total sunlight suggest that both UV and visible light interact with the same precursor although UV is 25 times more efficient in inducing pigmentation per J cm(-2) of irradiation compared to visible radiation. The measured diffused reflection spectra and decay kinetics of UV and visible radiation-induced pigmentation are very similar, indicating that the nature of the transient and persistent species involved in both the processes are also likely to be same.

Effects of sunscreens on delayed type hypersensitivity in mice

Objective To study the influence of sunscreens with different efficacy on delayed type hypersensitivity (DTH) and their immunoprotective effect in mice.Methods A cohort of mice were randomly divided into 5 groups with 10 mice in each group:group 1 as the positive control without irradiation,group 2 receiving solar-simulated radiation (SSR) only,group 3 receiving SSR and protected by sunscreen l with sun protection factor 15(SPF15)and persistent pigment darkening(PPD)12,group 4 receiving SSR and protected by sunscreen 2 with SPF 50 and PPD 28,and group 5 as the negative contml receiving SSR only.SSR was carried out on the back of mice with the UVA dose being 1.4 J/cm2 and UVB dose being 100 mJ/cm2 for 10 days.After a 5-day irradiation,the groups 1 to 4 were immunized by intraperitoneal injection with 100 μl(107 cells/ml) of Candida albicans suspension.On the 10th day both sides of the posterior foot pad were measured;then the foot pads were injected with additional 50 μl of the Candida albicans suspension.Twenty-four hours after the injection,the thickness of each foot pad was measured,and immunosuppression rate was calculated.Finally,the mice were sacrificed and skin samples were obtained from the back of these mice followed by the examination of CDla, CD80 and CD86 expression by Western blot.Resets The thickness of edema in foot pads was 0.41±0.38 mm,0.21±0.23 mm and 0.30 ± 0.25 mm in group 1,3 and 4,respectively,significantly higher than in group 5 and 2(0.04±0.03 mm,0.14±0.12 mm,respectively,all P 0.05).Significant differences were observed in the immunosuppression rate between group 2,3 and 4(73.0%±11.3%,54.1%±6.4%,29.7%±7.5%,respectively,all P 0.05).Conclusions The exposure to sub-erythema dose of UV can induce DTH,and sunscreens have an immunoprotective effect in this process.Epidermal Langerhans cells are not essential for UV-induced immunosuppression. Key words: Ultraviolet rays; Sunscreening agents; Dermatitis,photoallergic; Langerhans cells; Disease models,animal

Immediate pigment darkening and persistent pigment darkening as means of measuring the ultraviolet A protection factor in vivo: a comparative study

Persistent pigment darkening (PPD) is a widely used in vivo method for measurement of ultraviolet (UV) A protection factor (UVAPF). However, with increased emphasis on UVA protection and sunscreen products with higher UVAPF gaining popularity, the immediate pigment darkening (IPD) method is drawing attention again. Furthermore, only about a quarter of the recommended quantity of sunscreen is used during daily activities. However, there is as yet no clearly defined relationship between the UVAPF and the amount of sunscreen applied. To analyse the differences between the IPD and PPD methods, and to establish a relationship between the quantity of sunscreen application and the UVAPF. Different doses of sunscreen were applied on the back of 15 healthy volunteers, and the UVAPF was measured using both the IPD and the PPD methods. Both methods proved to be effective for measuring the UVAPF. However, all the UVAPF values determined by the PPD method were lower than those determined by the IPD method. Additionally, an exponential relationship between the amount of sunscreen applied and the UVAPF was observed. The IPD method can also be used as an appropriate endpoint in the determination of UVA protection. It is time saving, and thus considerably lowers the risk of UV exposure, particularly when testing sunscreen products with higher UVAPF. We further state that in order to achieve the desired protective effect of the sunscreen, the quantity of application is also very important.

Effects of sunscreen on human skin’s ultraviolet radiation tolerance

To observe the alteration ultraviolet radiation (UVR) of skin's tolerance after its exposure to the small dose of UVR under the protection of sunscreen. Eleven subjects who applied sunscreen were exposed to 0.75 dose minimal persistent pigment darkening (MPPD) and minimal erythema dose (MED) by the Phototherapy Unit for 4 weeks. Each week their MPPDs and MEDs were measured by solar simulator. Meanwhile, SPECTCOLOMETER® and VISIOSCAN VC98® were used to detect the test areas and control areas. The values of MPPD and MED increased significantly after the exposure to UVR. But there were no visible changes on the surface of skin's texture. With the protection of sunscreen, the UVR tolerance of skin was greatly increased after the skin's exposure to the small dose UV.

Assessment of the Photoprotection Properties of Sunscreens by Chromatographic Measurement of DNA Damage in Skin Explants

Evaluation of the photoprotection provided by sunscreens is performed either through the induction of erythema and expressed as the sun protection factor (SPF), or by the UVA-mediated persistent pigment darkening (PPD). None of these two endpoints has a link with skin cancer, the most deleterious consequence of excess exposure to solar UV radiation. We thus set up a complementary approach to evaluate the protection provided by sunscreens to the genome of human skin. This is based on the quantification of the thymine cyclobutane dimer (TT-CPD), the main DNA lesion induced by both UVB and UVA radiations. Irradiations were performed ex vivo on human skin explants and the level of TT-CPD in DNA was determined by HPLC associated with tandem mass spectrometry. The technique was first optimized and validated with three standard sunscreens. The study was then extended to the evaluation of a commercial high SPF sunscreen exhibiting efficient UVA photoprotection. The DNA protecting factor was found to reflect the ratio between UVB and UVA photoprotection, although the absolute values of the genomic protection were, as a general trend, lower than either SPF or PPD. These data show the usefulness of the proposed approach for the evaluation of the genoprotection afforded by sunscreens.

Changes of skin complexion after ultraviolet A (UVA) and ultraviolet B (UVB) exposure.

Objective To observe the alteration of skin complexion after UVA and UVB exposure.Methods The back skin of ten females with skin type Ⅲ was subjected to single exposure to solar-simulated UVA of double minimal persistent pigment darkening (MPPD) or UVB of double minimal erythema dose (MED). Skin reflectance was assessed with clinical grading, spectcolometer and Mexameter MX 18 before irra-diation, 6 hours, 1, 7 and 14 days after the irradiation. Results After UVB irradiation, a~* value and erythema index (EI) abruptly increased at 6 hours and peaked on day 2; L~* value sharply declined on day 1; ITA° markedly decreased on day 7; melanin index (MI) declined within the first 2 days, but notably increased on day 7. After UVA irradiation, a~* and El value experienced no apparent changes; L~* value obviously declined at 6 hours; ITA° reached its lowest value on day 14; MI increased only on day 1. Conclusions There is a significant difference in the kinetics and extent of skin complexion changes after UVA and UVB irradiation. EI and a~* value are sensitive and accurate indices for evaluating sunburn, and MI and ITA ° for analyzing tanning. Key words: Ultraviolet rays; Skin complexion; L~* value; a~* value; Melanin index; Erythema index

The COLIPA in vitro UVA method: a standard and reproducible measure of sunscreen UVA protection

There is a continuing need to measure and communicate reliably the UVA protection offered by commercial sunscreens. To that end, the COLIPA (European Cosmetics Trade Association) 'In Vitro Sun Protection Methods' group has developed a new in vitro method for measuring UVA protection in a standardized, reproducible manner. The method is based on in vitro UV substrate spectrophotometry and convolution of resulting absorbance data with the action spectrum for the in vivo Persistent Pigment Darkening (PPD) endpoint to provide an in vitro UVA protection factor (UVAPF) which is correlated with an in vivo measure. This method has been published as a COLIPA guideline, used currently in European geographies for testing and labelling sunscreen products. This article summarizes two 'ring' studies, involving eight separate testing laboratories, which both defined critical parameters for the method and validated it. In Ring Study 1, eight laboratories tested the in vitro UV transmission of a total of 24 sunscreens and, from the data, a unit dose of UVA (D(0) of 1.2 J cm(-2)) was defined to provide a single irradiation step which, by taking into account potential sunscreen photo-instability, gave the closest agreement with in vivo UVAPF values. In Ring Study 2, eight laboratories tested the in vitro UV transmission of a total of 13 sunscreens using this single irradiation step and established a very good correlation (r(2) = 0.83; slope = 0.84, P < 0.0001) between resulting in vitro UVAPF values and corresponding values derived from the in vivo PPD method. This new method, therefore, can be used to provide a reliable in vitro metric to describe and label UVA efficacy in sunscreen products, in line with the EU Commission recommendation 2006/247/EC.

Diffuse reflectance spectroscopy for ultraviolet A protection factor measurement: correlation studies between in vitro and in vivo measurements

Assessing the ultraviolet (UVA) protection factor of sunscreen formulations has been discussed for the past 20 years. The purpose of this study is to correlate the measurements of the UVA protection factor value (PFA value) via in vivo diffuse reflectance spectroscopy (DRS) and to compare this method with the in vitro method of measuring the PFA value, as well as with the in vivo persistent pigment darkening (PPD) and PFA methodologies. The UVA protection factor via DRS technique was assessed in two clinical studies. The first study was performed in 12 subjects and the second one consisting of 10 subjects. All subjects in these studies had Fitzpatrick skin phototypes II-IV. DRS measurements were performed using a SkinScan spectrofluorimeter (Spex SkinScan, Yvon Horiba). The in vitro PFA measurements were performed using Labsphere UV-1000s UV Transmission Analyzer. The results obtained from the non-invasive DRS studies were used to correlate with the in vitro testing and with the in vivo PFA/PPD multicenter study. A positive relationship (regression coefficient r(2)=0.90) of PFA values was found between in vitro PFA testing and the in vivo DRS testing. There was also a very good correlation (regression coefficient r(2)=0.99) between the in vivo PFA/PPD values and UVA protection factor obtained from the DRS method. This paper presents studies using the DRS technique to assess the UVA protection factor in different sunscreen formulae correlating with in vitro and in vivo PFA/PPD values. It is a fast method, non-invasive and does not involve any subject irradiation. The technique is a good estimator for the in vivo UVA protection factor as well as a way to assess, in vivo, the photostability of sunscreen formulation in the UVA.

Long‐lasting pigmentation of human skin, a new look at an overlooked response to UV

Long‐lasting pigmentation of human skin, a new look at an overlooked response to UV

In vivo persistent pigment darkening method: proposal of a new standard product for UVA protection factor determination

The European Commission (EC) has recommended assessing the level of ultraviolet A (UVA) protection afforded by sunscreen products using the in vivo persistent pigment darkening (PPD) method or other methods giving equivalent results. In this context, the reproducibility of the in vivo PPD method is of importance. To check the validity of the UVA protection factor (UVAPF) tests, the Japanese Cosmetic Industry Association (JCIA) recommends using a standard product (JCIA standard) with an expected UVAPF 3.75 (SD 1.01). However, considering the increase in UVA efficacy of the new sunscreen products available in the market, with UVAPF up to 30, it seemed useful to develop a new standard product to be used when testing products with expected UVAPF > or =10. The PPD method was used in six centres to determine the UVAPF of the two products. Reproducibility of results was also studied by testing two batches of the new product at two different times. There was no statistical difference between the six centres with regard to the JCIA standard. The ring study showed that the mean value of UVAPF (4.3) was higher than that given by JCIA (3.75). These data enable the proposal of a new acceptance range for the JCIA standard product (3.4-5.2) derived from actual results from European laboratories. Whereas this range is different from that proposed by JCIA (2.74-4.76), there is an overlapping of the values. Data on the new standard product show that reproducibility is not influenced by the batches of this product. The mean UVAPF value obtained is 12.1. An acceptance range (9.6-14.6) is proposed for the new standard. Data presented here demonstrate that if an identical protocol is used, reproducible results can be expected and that the PPD method is reproducible and reliable.