Concentration Of PPD Research Articles

Analysis and quantification of selected heavy metals and paraphenylenediamine in commercially available herbal black hair dyes in Sri Lanka

Currently, the use of henna-based herbal black hair dyes is widespread in the community. However, it is important to note that these dyes cannot be classified as completely natural due to the addition of various ingredients to enhance their effectiveness. Most black hair dyes use paraphenylenediamine (PPD) to get a black hue, however, PPD is widely recognized as a contact sensitizer. The toxicity of heavy metals is also prevalent in the cosmetics sector. These metallic elements can permeate the skin and provide widespread exposure throughout the body. Hair dye requires an alkaline pH value because of reactions that occur during the dyeing process, which is incompatible with scalp pH. The varying concentrations of these chemicals and their frequency of usage can result in diverse health issues. The present research implemented High-Performance Liquid Chromatography and Atomic Absorption Spectrometry to quantify the concentrations of PPD and heavy metals (Pb, Cd, and Fe) in seven frequently used brands in Sri Lanka. The lead content in the examined samples exhibited variability, ranging from 0.04 ± 0.01 ppm to 0.28 ± 0.14 ppm. However, all the samples remained below the allowed level of 2 ppm according to the BVL standard. Regarding Cd, certain examined materials surpassed the acceptable thresholds, while others did not and varied between 0 ppm and 2.33 ± 1.92 ppm. Iron was found at a significantly higher concentration compared to the other two heavy metals. A significant quantity of PPD was detected, exceeding the allowable threshold of 6 % according to the European Union legislation. All the samples that were analyzed had a pH value within the alkaline range. The study indicates that herbal dyes generated from natural materials may contain harmful compounds, such as a high concentration of PPD in herbal black hair color potentially inducing sensitization.

Ratiometric electrochemical sensing platform based on N-doped MOF-derived CoNi/C for the determination of p-phenylenediamine in hair dyes.

Astable ratiometric electrochemical sensing platform is introduced for the determination of p-phenylenediamine (PPD). Specifically, the proposed sensing platform employs nitrogen-doped MOF pyrolysis-derived CoNi/C (N-CoNi/C) which was deployed as the sensing agent and methylene blue (MB) as the internal reference, and the MB combined with N-CoNi/C nanomaterials by a simple immersion adsorption process. Full characterization of N-CoNi/C was carried out with respect tomorphology, composition, and electrochemical behavior, and the sensing performance of the ratiometric electrochemical sensing platform was evaluated. Complete separation of the oxidation peaks of PPD and MB was achieved using the MB/N-CoNi/C composite modified glassy carbon electrode (MB/N-CoNi/C/GCE) and their ratio signals were used for quantitative determination of PPD. The electrical signal was linearly related to the concentration of PPD in the concentration range 0.3-100μM, with a fitted correlation coefficient of 0.9987 and a detection limit of 0.091μM (S/N = 3). Additionally, the sensor has been successfully used for the determination of PPD in commercial hair dyes with a recovery rate of over 95%.

Fast In Vitro Release and In Vivo Absorption of an Anti-Schizophrenic Drug Paliperidone from Its Soluplus®/TPGS Mixed Micelles.

The purpose of this study was to develop a drug delivery system for paliperidone (PPD) in order to provide a more effective therapeutic strategy for patients with acute schizophrenia. PPD-loaded Soluplus®/TPGS mixed micelles (PPD-S/T-MM) were prepared using the thin-film hydration method. The critical micelle concentration (CMC) of blank S/T-MM was 4.77 × 10−2 mg/mL. PPD presented much higher solubility in PPD-S/T-MM formulation than that in pure water. The particle size of blank or drug loaded S/T-MM was around 60 nm. The polydispersity index (PDI) was less than 0.1. PPD-S/T-MM presented a nearly spherical shape under transmission electron microscopy. The encapsulation efficiency (EE%) of PPD-S/T-MM was higher than 94%. Based on the analysis of XRD and DSC, it was proved that PPD was incorporated in the core of the mixed micelles as amorphous dispersion or solid solution. PPD-S/T-MM were stable when they were undergoing dilution with water and the change of environmental pH. Although PPD-S/T-MM showed lower rates to release PPD than those from PPD raw material in acidic solution, they provided faster release rates in neutral conditions than those from PPD raw material who only showed modest dissolution in the same neutral condition. This proves that PPD-S/T-MM can release PPD in a more controlled manner. After oral administration of PPD-S/T-MM (dose of PPD, 6 mg/kg) in rats, the plasma concentration of PPD increased rapidly: Tmax was 0.83 ± 0.29 h, and Cmax was 844.33 ± 93.73 ng/mL. Oral administration of PPD suspension resulted in longer Tmax and lower Cmax. The relative oral bioavailability was about 158% for PPD-S/T-MM over PPD suspension. These findings confirm that PPD-S/T-MM can provide faster release in neutral conditions and better oral absorption in rats than those from PPD raw material, which should potentially benefit patients with acute schizophrenia.

Study of P-Phenylenediamine (PPD) Concentrations after Hair Dye Mixing: A Call for Safety Reassessment

Para-phenylenediamine (PPD) is a chemical that is widely used in hair dyes. Multiple safety and regulatory agencies have categorized PPD as a potent sensitizer. In addition, PPD has carcinogenicity and genotoxicity attributes and, consequently, it is regulated at a maximal concentration of 2.0%. The aim of this study was to test whether the limit for PPD is surplus, and hence whether the consumer may be exposed to unnecessarily PPD levels. Experimentally, the analysis of PPD was performed using HPLC, where method validation and an inter-laboratory comparison test (ILC) were conducted to evaluate method performance. Thirty-three commercial products were analyzed, and five products were chosen to study the unconsumed PPD. Successfully, the implemented method confirmed its suitability and validity for the determination of PPD. For ILC results, PPD levels were 0.97 ± 0.04% and 0.92 ± 0.02%, quantified by our laboratory and an accredited laboratory, respectively. For all products, the initial concentration (T0) of PPD was lower than the regulatory limit. After 45 min, the content of PPD significantly reduced compared to T0. One product showed unconsumed PPD to be as high as 96% following the recommended dyeing time. In conclusion, the existence of high levels of unreacted PPD increases the likelihood of allergic events and elevates the risk of PPD-related chemicals. Collaborative efforts between industries, regulatory bodies, and health-related decision makers are deemed necessary to establish safe concentrations for PPD.

The Potential Effect of Henna (Stone Dye) Extract Paraphenylenediamine on Human Blood Cells, Liver and Renal Function

Some of the dyes that are used for coloring hair and skin contain a chemical compound called paraphenylenediamine (PPD), which is potentially toxic. The study comprised 50 adult Sudanese women who were selected by stratified random sampling and working as henna artist. Urine samples were taken for detection of PPD while blood samples were taken for investigating kidney and liver functions by measuring its relevant hematological indices. In other hand Thin Layer Chromatography (TLC) and High Performance Liquid Chromatography (HPLC) were used to detection of PPD in black henna (stone dye) samples and urine. The PPD was detected in the urine of all women who used the black henna its concentration varied according to duration of exposure being significantly higher in those with >10 years of exposure (p≤0.01). Similarly significant changes in liver function, kidney function and hematological indices were found in the group of women who used natural henna. This study concluded that natural henna is safe to use, while Black dye is potentially hazardous. It contains the high concentration of PPD, is the one most commonly associated with adverse effects.

Cytotoxic effect of paraphenylenediamine (PPD) on Danio rerio (Zebra Fish)

Paraphenylenediamine (PPD) is a synthetic chemical widely used as a substitute for henna as a permanent hair dye because it gives a natural look to the hair and the dyed hair can also be shampooed or permed without losing the colour. However, the intermediate and partially oxidized form of PPD is reported to cause adverse reactions in people. Effects of sublethal concentrations of PPD in the zebra fish were examined for 15 days following exposure to the chemical. On the 7th day and the 15th day following the exposure, blood smears were examined for presence of micronuclei using the Giemsa stain. The micronuclei percentage indicating the cytotoxic effect of PPD was significantly greater in the exposed fishes and the extent of toxicity was directly proportional to the concentration of PPD and to the length of exposure. Therefore, waste water reaching any aquatic ecosystem should be monitored for PPD in order to prevent any adverse impacts on the aquatic ecosystem and to minimize the threat to human beings due to biomagnification.

Hair dye and risk of skin sensitization induction: a product survey and quantitative risk assessment for para-phenylenediamine (PPD)

Background Para-Phenylenediamine (PPD) is a commonly used dye intermediate in permanent hair dye formulations, and exposure to PPD has been associated with allergic contact dermatitis at certain doses. Purpose Determine the concentration of PPD in a survey of self-application permanent hair dye products, and perform a quantitative risk assessment to determine the risk of skin sensitization induction following application of these products. Methods Consumer exposure levels (CELs) to PPD following application of hair dye products were estimated using the maximum amount of hair dye that can adhere to the surface area of the scalp, the measured concentration of PPD in the hair dye product, a retention factor, the dermal absorption of PPD, and the surface area of the scalp. CELs were calculated for various exposure scenarios, and were stratified by hair dye shade. Results All estimated CELs did not exceed the acceptable exposure level. Specifically, margins of safety ranged from 2.3 to 1534 for black dyes, 2.9 to 5031 for brown dyes, and 26 to 5031 for blonde dyes. Conclusions Findings suggest that use of the evaluated permanent hair dyes, under the evaluated exposure scenarios, would not be expected to induce skin sensitization due to PPD exposure at concentrations ≤0.67%.

Ginsenoside Rh3 Inhibits Proliferation and Induces Apoptosis of Colorectal Cancer Cells

Background: Colorectal cancer is a common malignant tumor of the digestive tract, the morbidity rate of which is rising in recent years. Ginsenoside Rh3 was reported to have anticancer activity; however, the underlying mechanism still needs to be explored in depth. Methods: Rabbit blood was used to test hemolytic effects of ginsenoside protopanaxadiol (PPD), Rh2, Rh3, and Rg3. Human colorectal cancer SW1116 cells were treated with different concentration of ginsenoside PPD, Rh2, Rh3, and Rg3 in vitro. MTT and TUNEL assay were used to examine cell proliferation and apoptosis. Semi quantitative RT-PCR, immunocytochemistry assay and flow cytometry assay were used to detect the expression of caspase3. Results: The results showed that the inhibiting effects on SW1116 cells of PPD and Rh2 were stronger than those of Rh3 (p < 0.01), but Rh3 had better solubility and slighter hemolytic effects on blood cells than those ginsenosides. Ginsenoside Rh3 inhibited the proliferation of SW1116 cells at 60 μg/mL (p < 0.01), the inhibition effect was increased sharply when the dose of Rh3 was increased from 60 to 120 μg/mL, the inhibition rate was 62.1% at 120 μg/mL, the inhibition appeared at 9 h, and the peak activity occurred at 12 h and maintained until 48 h (p < 0.01). Compared to the control group, the ratio of apoptotic cells, the expression level of mRNA and protein of caspase3 increased in 120 μg/mL Rh3 treated group. Conclusion: As a potential anticancer medicine, ginsenoside Rh3 could inhibit the proliferation of colorectal cancer cells in a dose- and time-dependent manner and induce cell apoptosis through upregulating the expression of caspase3.

A highly sensitive perovskite oxide sensor for detection of p-phenylenediamine in hair dyes.

Effective regulation of p-phenylenediamine (PPD), a widely used precursor of hair dye that is harmful to human health in large concentration, relies upon an accurate yet simple detection of PPD. In this context, amperometric electrode sensor based on perovskite oxide becomes attractive given its portability, low cost, high sensitivity, and rapid processing time. This work reports the systematic characterization of a series of Sr-doped PrCoO3-δ perovskite oxides with composition of Pr1-xSrxCoO3-δ(x = 0, 0.2, 0.4, 0.6, 0.8, and 1) for PPD detection in an alkaline solution. PSC82 deposited onto glassy carbon electrode (PSC82/GCE) generates the highest redox currents which correlates with the highest hydrogen peroxide intermediates (HO2-) yield and the σ*-orbital (eg) filling of Co that is closest to unity for PSC82. PSC82/GCE provides the highest sensitivities of 655 and 308 μA mM-1 cm-2 in PPD concentration range of 0.5-2,900 and 2,900-10,400 μM, respectively, with a limit of detection of 0.17 μM. PSC82/GCE additionally demonstrates high selectivity to PPD and long term stability during 50 consecutive cyclic voltammetry scans and over 1-month storage period. The potential applicability of PSC82/GCE was also demonstrated by confirming the presence of very low concentration of PPD of below 0.5% in real hair dyes.

Effects of p-Phenylene diamine on liver and kidney functions of occupational exposed workers

Objective: To study the effect of p-phenylenediamine (PPD) on liver and kidney function in occupational exposed workers. Methods: Workers in a hair dye production enterprise which used p-phenylenediamine as a raw material for production were selected as the main research population. Then we conducted a questionnaire survey on the basic conditions of workers and conducted occupational health checkups on general health status, liver and kidney function. Occupational health examination assessment results were tested in Taizhou Cancer Hospital. All data was built using EpiData 3.1 software, and statistical analysis was performed using software SPSS 20.0. Results: The liver function indicators including direct bilirubin, prealbumin, total protein, and white protein, globulin, aspartate aminotransferase, glutamyl transpeptidase, and total bilirubin in the workers exposed to high concentration of PPD were at high normal values, and these indicators were significantly different from low PPD concentration group (P<0.05) . The serum creatinine and serum uric acid in the renal function index were significantly higher in workers exposed to PPD than in workers exposed to low concentrations and in the control group (P<0.05) . Conclusion: Occupational exposed to PPD may have a hazard to the workers' liver and kidney function. Long-term occupational exposure to PPD may lead to increased cumulative exposure of workers, which may cause potential chronic liver and kidney damage in occupationally exposed populations.

A highly sensitive HPLC–MS/MS method for quantification of 20( S )-protopanaxadiol in human plasma and its application in phase IIa clinical trial of a novel antidepressant agent

A highly sensitive HPLC-MS/MS assay method was established to quantify 20(S)-protopanaxadiol (PPD) in human plasma with dexamethasone as an internal standard. The electrospray ion mass spectrometry (ESI-MS) was operated under the multiple reactions monitoring mode (MRM) using positive ion mode. PPD was extracted from 500μL plasma samples by liquid-liquid extraction then separated by a C18 analytical column with gradient elution. The concentration of PPD could be determined by this HPLC-MS/MS method over the range of 0.05-20ng/mL with the lower limit of quantification (LLOQ) of 0.05ng/mL. The method was successfully applied to phase IIa clinical trial of Yuxintine (PPD capsule) in which plasma samples of 87 subjects were analyzed following 6 weeks of oral administration of placebo or PPD capsules in 5 different doses. In this study, the measured concentration was linearly related to the oral dosage with R=0.9901. The minimum and maximum values of measured concentration were 0.06 and 11.60ng/mL, respectively. In addition, plasma concentrations of PPD in depression patients were reported for the first time in our study.

Production of the dammarene sapogenin (protopanaxadiol) in transgenic tobacco plants and cultured cells by heterologous expression of PgDDS and CYP716A47.

Protopanaxadiol (PPD) is an aglycone of dammarene-type ginsenoside and has high medicinal values. In this work, we reported the PPD production in transgenic tobacco co-overexpressing PgDDS and CYP716A47. PPD is an aglycone of ginsenosides produced by Panax species and has a wide range of pharmacological activities. PPD is synthesized via the hydroxylation of dammarenediol-II (DD) by CYP716A47 enzyme. Here, we established a PPD production system via cell suspension culture of transgenic tobacco co-overexpressing the genes for PgDDS and CYP716A47. The concentration of PPD in transgenic tobacco leaves was 2.3-5.7 µg/g dry weight (DW), depending on the transgenic line. Leaf segments were cultured on medium with various types of hormones to induce callus. Auxin treatment, particularly 2,4-D, strongly enhanced the production of DD (783.8 µg g(-1) DW) and PPD (125.9 µg g(-1) DW). Treatment with 2,4-D enhanced the transcription of the HMG-Co reductase (HMGR) and squalene epoxidase genes. PPD production reached 166.9 and 980.9 µg g(-1) DW in a 250-ml shake flask culture and in 5-l airlift bioreactor culture, respectively.

Photovoltammetric behavior and photoelectrochemical determination of p-phenylenediamine on CdS quantum dots and graphene hybrid film

A photoelectroactive film composed of CdS quantum dots and graphene sheets (GS) was coated on F-doped SnO2 (FTO) conducting glass for studying the electrochemical response of p-phenylenediamine (PPD) under photoirradiation. The result indicated that the cyclic voltammogram of PPD on CdS–GS hybrid film became sigmoidal in shape after exposed under visible light, due to the photoelectrocatalytic reaction. Such a photovoltammetric response was used to rapidly optimize the photoelectrocatalytic activity of hybrid films composed of different ratios of CdS to GS toward PPD. The influences of scan rate and pH on the photovoltammetric behavior of PPD on CdS–GS film revealed that although the controlled step for electrochemical process was not changed under photoirradiation, more electrons than protons might participate the photoelectrocatalytic process. Furthermore, the photoelectroactive CdS–GS hybrid film was explored for PPD determination based on the photocurrent response of film toward PPD. Under optimal conditions, the photocurrent signal on CdS–GS film was linearly proportional to the concentration of PPD ranging from 1.0×10−7 to 3.0×10−6molL−1, with a detection limit (3S/N) of 4.3×10−8molL−1. Our work based on CdS–GS hybrid film not only demonstrated a new facile photovoltammetric way to study the photoinduced electron transfer process of PPD, but also developed a sensitive photoelectrochemical strategy for PPD determination.

Hematopathology in Sprague–Dawley Rats Following Sub-Chronic Topical Application of Para-Phenylenediamine

The aim of the present study was to analyze the hematological profile of male SD rats treated topically with aqueous solution of para-phenylenediamine (PPD), a component of almost all hair dye formulations. The rats were painted with different concentration of PPD (0, 1, 2 and 3 mg Kg(-1) Day(-1)) for 90 days and then sacrificed. The hematological profile indicated severe anemia characterized by significant (p < 0.05, 0.001) reduction of total RBC count (59%), packed cell volume (PCV, 50%) and haemoglobin level (70%) in the peripheral blood of PPD treated animals when compared to control group. The leucocytes profile exhibited an overall elevation of around twofold as compared to the control group with significant lymphocytosis (44.4%) and a higher percentage of blast cells (8.5%) as well as smudge (10.3%) and hairy cells (6.2%) in the peripheral blood of treated animals. Histopathological examination of spleen from treated rat's exhibit red pulp congestion, expansion of the germinal centre, hyperplasia of the membrane capsule and extensive accumulation of hemosidderin pigments in the red pulp of the spleen. Overall this study indicated an abnormal pathophysiological condition indicating adverse effect of PPD in the treated animal groups. The risk assessment of hair dye formulation needs to be reviewed in view of widespread usage of paraphenylenediamine in almost all hair dye formulation.

Severe allergic reactions to para‐phenylenediamine in children and adolescents: should the patch test concentration of PPD be changed?

Semipermanent henna tattoos containing para-phenylenediamine (PPD) are a well-known cause of severe contact dermatitis, mainly in children. Another relevant exposure source to PPD are hair dyes, which are increasingly used by children and have also been reported to cause intense facial and scalp dermatitis. German patch test guidelines for children recommend that PPD should only be tested in patients who have had a henna tattoo, and then at a reduced concentration of PPD 0.5 % for 24 hours. We report on patch test results in four patients, three children and one adolescent, with a history of contact dermatitis to henna tattoos or hair dye. We used the recommended or even lower patch test concentrations of PPD with 24-hours exposure in all patients. All patients showed very strong patch test reactions to PPD and cross-reactions to related compounds even after dilution of PPD to as low as 0.05 %. Therefore, we suggest that in children with a history of allergic reactions to this compound, a titration test should be performed beginning at a concentration of maximal 0.05 %. This procedure has also been proposed previously based on a larger cases series in adults.

Extreme patch test reactivity to p-phenylenediamine but not to other allergens in children

According to clinical impression, extreme patch test reactions (+++) to p-phenylenediamine (PPD) are not uncommon in children. To investigate the patch test reactivity in children (aged 1-14 years) in comparison with other age groups and other allergens. A retrospective analysis was performed of data from the German Information Network of Departments of Dermatology, including all patients consecutively patch tested between 1994 and 2004 with PPD, and, for comparison, nickel, fragrance mix I, and methylchloroisothiazolinone (MCI)/methylisothiazolinone (MI). The distribution of +, ++ and +++ grades of positive reactions among those with a positive reaction were analysed in five age strata. We found a strikingly higher proportion of +++ reactions to PPD in children than in all other age groups (p < 0.001). No such difference was observed for the other allergens. The main suspected exposures associated with extreme reactions to PPD in children were hair dyes and 'henna tattoos'. The high proportion of extreme patch test reactions to PPD, but not to other important allergens, in children reflects intense sensitization, probably because of a high induction dose from PPD-containing 'henna tattoos'. In children with a history of contact allergy to 'henna tattoos' or hair dyes, the standard patch test concentration of PPD 1% should be drastically reduced.

Determination of p-phenylenediamine by perturbation of a non-equilibrium stationary state in the B-Z reaction

A convenient method for determination of p-phenylenediamine (PPD) in wastewater has proposed, based on the perturbation of a non-equilibrium stationary state near a bifurcation for the Belousov-Zhabotinskii (B-Z) reaction, due to this point being very sensitive to the surrounding change. Results indicate that the change of the potential (ΔE) is linearly proportional to the negative logarithm concentration of PPD (-logC) in the range of 4.68 × 10-7 - 1.95 × 10-5 mol L-1 (r = 0.9981) with a limit of detection (LOD) of 9.50 × 10-8 mol L-1. A good recovery was obtained at ca. 95.1 to 102.3%.

Development of a UPLC-ESI-MS/MS Assay for 20(S)-Protopanaxadiol and Pharmacokinetic Application of Its Two Formulations in Rats

An ultra-performance liquid chromatography-electrospray tandem mass spectrometry (UPLC-ESI-MS/MS) method was developed to investigate 20(S)-protopanaxadiol (PPD) pharmacokinetics in rats. Rat plasma samples were treated using a solid-phase extraction with satisfactory recovery (> 81%). The method showed an excellent sensitivity that the limit of detection (LOD) and the lower limit of quantitation (LLOQ) of PPD were 0.5 and 2 ng/mL, respectively. The method was applied to the evaluation of pharmacokinetics from two types of PPD formulations. The PPD emulsion showed more rapid and efficient drug absorption, and higher and more persistent plateau concentration of PPD in plasma than PPD oil solution. PPD emulsion was demonstrated to be a promising dosage form. In spite of lower plateau plasma drug concentration, PPD oil solution was characterized by the easiness in preparation and the persistent, durative plateau plasma concentration of PPD, there is room to further improve its bioavailability.

Determination of para-Phenylenediamine (PPD) in Henna in the United Arab Emirates

Henna is very popular in the United Arab Emirates (UAE); it is part of the culture and traditions. Allergy to natural henna is not usual; however the addition of para-phenylenediamine (PPD) to the natural henna increases the risk of allergic contact dermatitis. The objectives of the study were to identify the presence and concentration of PPD in henna available in UAE. Fifteen henna salons were selected randomly from three cities in UAE. Twenty five henna samples were acquired from these selected salons. The presence of PPD in henna samples was determined qualitatively and quantitatively using High Performance Liquid Chromatography (HPLC). The study showed that PPD was present in all of the black henna samples at concentrations ranging between 0.4% and 29.5% and higher than that recommended for hair dyes in most of the black henna samples. The presence of PPD in the black henna increases the risk of allergic contact dermatitis among users of black henna and a number of cases have already been reported in UAE.

Late reactions to the patch-test preparations para-phenylenediamine and epoxy resin: a prospective multicentre investigation of the German Contact Dermatitis Research Group

Late patch-test reactions, developing at day (D) 7 or later have been described for several allergens. Late reactions may reflect patch-test sensitization. Para-phenylenediamine (PPD) and epoxy resins (ER) are potent allergens and therefore may potentially induce patch-test sensitization. Up to now, there has been no prospective study on the frequency of late reactions in routine patch testing with these allergens. To assess the frequency of late reactions to PPD and ER. In 1748 patients PPD (PPD-base, 1% pet.) and ER [based on diglycidylether of bisphenol A (DGEBA, 1% pet.)], and in 812 patients, nickel sulphate (5% pet.) were removed from the test panel of the standard series and applied on the medial side of the upper arm. Patch-test occlusion time was 24 h in 588 (PPD and ER) and 241 patients (nickel sulphate), respectively, and 48 h in 1160 (PPD and ER) and 571 (nickel sulphate) patients, respectively. Patch tests were read on D1-3 and D2-3, respectively; additional late readings were performed on D7, D14 and D21 after patch-test application. Patients who were not able to return for all scheduled late readings were telephoned on D7, D14 or D21, and questioned about a reaction at the test sites. Patients were instructed to perform daily self-examination from D4 onwards and to return immediately to the clinic if a reaction at the upper arm became visible. Data of 1428 patients (ER and PPD) and 638 patients (nickel) were evaluable. In 25 patients (1.8%), patch tests became positive not before D7, among them 21 reactions to PPD (1.5%) and four reactions to ER (0.3%). In five of seven patients, repeated patch tests with PPD disclosed patch-test sensitization as the cause of the late reaction. All late reactions, except for one, occurred in patients in whom patch tests were applied for 48 h. No late reactions were seen with nickel sulphate. PPD (1% pet.) elicited late reactions in 1.5% of routine patch tests, the majority of them probably being caused by patch-test sensitization. Therefore, the German Contact Dermatitis Research Group decided to remove PPD 1% pet. from the German standard series and to take efforts to optimize the patch-test conditions of PPD. One way to optimize PPD testing could be to reduce the exposure of PPD 1% to 24 h. Alternatively the patch-test concentration of PPD might be reduced.