Urine Excretion, Organ Distribution, and Placental Transfer of 6PPD and 6PPD-Quinone in Mice and Potential Developmental Toxicity through Nuclear Receptor Pathways.

The rubber antioxidant 6PPD has gained significant attention due to its highly toxic transformation product, 6PPD-quinone (6PPDQ). Despite their detection in urines of pregnant women, the placental transfer and developmental toxicity of 6PPD and 6PPDQ are unknown. Here, we treated C57Bl/6 mice with 4 mg/kg 6PPD or 6PPDQ to investigate their urine excretion and placental transfer. Female and male mice exhibited sex difference in excretion profiles of 6PPD and 6PPDQ. Urine concentrations of 6PPDQ were one order of magnitude lower than those of 6PPD, suggesting lower excretion and higher bioaccumulation of 6PPDQ. In pregnant mice treated with 6PPD or 6PPDQ from embryonic day 11.5 to 15.5, 6PPDQ showed ∼1.5-8 times higher concentrations than 6PPD in placenta, embryo body, and embryo brain, suggesting higher placental transfer of 6PPDQ. Using in vitro dual-luciferase reporter assays, we revealed that 6PPDQ activated the human retinoic acid receptor α (RARα) and retinoid X receptor α (RXRα) at concentrations as low as 0.3 µM, which was ∼10-fold higher than the concentrations detected in human urines. 6PPD activated the RXRα at concentrations as low as 1.2 µM. These results demonstrate the exposure risks of 6PPD and 6PPDQ during pregnancy and emphasize the need for further toxicological and epidemiological investigations.

Application of a LC-MS/MS method developed for the determination of p-phenylenediamine, N-acetyl-p-phenylenediamine and N,N-diacetyl-p-phenylenediamine in human urine specimens.

Cases of poisoning by p-phenylenediamine (PPD) are detected sporadically. Recently an article on the development and validation of an LC-MS/MS method for the detection of PPD and its metabolites, N-acetyl-p-phenylenediamine (MAPPD) and N,N-diacetyl-p-phenylenediamine (DAPPD) in blood was published. In the current study this method for detection of these compounds was validated and applied to urine samples. The analytes were extracted from urine samples with methylene chloride and ammonium hydroxide as alkaline medium. Detection was performed by LC-MS/MS using electrospray positive ionization under multiple reaction-monitoring mode. Calibration curves were linear in the range 5-2000 ng/mL for all analytes. Intra- and inter-assay imprecisions were within 1.58-9.52 and 5.43-9.45%, respectively, for PPD, MAPPD and DAPPD. Inter-assay accuracies were within -7.43 and 7.36 for all compounds. The lower limit of quantification was 5 ng/mL for all analytes. The method, which complies with the validation criteria, was successfully applied to the analysis of PPD, MAPPD and DAPPD in human urine samples collected from clinical and postmortem cases.

Human systemic exposure to [¹4C]-paraphenylenediamine-containing oxidative hair dyes: Absorption, kinetics, metabolism, excretion and safety assessment.

Systemic exposure was measured in humans after hair dyeing with oxidative hair dyes containing 2.0% (A) or 1.0% (B) [(14)C]-p-phenylenediamine (PPD). Hair was dyed, rinsed, dried, clipped and shaved; blood and urine samples were collected for 48 hours after application. [(14)C] was measured in all materials, rinsing water, hair, plasma, urine and skin strips. Plasma and urine were also analysed by HLPC/MS/MS for PPD and its metabolites (B). Total mean recovery of radioactivity was 94.30% (A) or 96.21% (B). Mean plasma Cmax values were 132.6 or 97.4 ng [(14)C]-PPDeq/mL, mean AUC(0-∞) values 1415 or 966 ng [(14)C]-PPDeq/mL*hr in studies A or B, respectively. Urinary excretion of [(14)C] mainly occurred within 24 hrs after hair colouring with a total excretion of 0.72 or 0.88% of applied radioactivity in studies A or B, respectively. Only N,N'-diacetylated-PPD was detected in plasma and the urine. A TK-based human safety assessment estimated margins of safety of 23.3- or 65-fold relative to respective plasma AUC or Cmax values in rats at the NOAEL of a toxicity study. Overall, hair dyes containing PPD are unlikely to pose a health risk since they are used intermittently and systemic exposure is limited to the detoxified metabolite N,N'-diacetyl-PPD.

[Determination of 2, 4-toluenediamine in urine by gas chromatography].

OBJECTIVE: To establish a method for determining the content of 2,4-toluenediamine, a urinary metabolite of toluene diisocyanate, by gas chromatography. METHODS: Urine samples were collected, and acidification, extraction, derivatization, separation with a capillary column, and detection with an electron capture detector were performed. The target compound was qualified by retention time and quantified by peak area. RESULTS: The concentration of 2, 4-toluenediamine showed a linear relationship with peak area within 0.0∼40 ng/ml, with a correlation coefficient 0.9995; the limit of detection was 0.44 ng/ml, the lower limit of quantification was 1.47 ng/ml, the relative standard deviation was 1.85%∼4.05%; the recovery rate was 97.98%∼99.28%. CONCLUSION: The method has the advantages of high sensitivity and high accuracy and can be used for determination of 2, 4-toluenediamine in urine.

Biological monitoring as a valid tool to assess occupational exposure to mixtures of 2,4-:2,6-toluene diisocyanate.

BACKGROUND AND OBJECTIVES: Despite its advantages over environmental monitoring, biological monitoring of exposure to 2,4-:2,6-toluene diisocyanate (TDI) mixtures is still underused. The present study was designed in order to evaluate the feasibility and reliability of biological monitoring in a factory producing polyurethane foam blocks. METHODS: Airborne TDI isomers were sampled by both static and personal pumps and determined by HPLC with fluorimetric detection. Specific metabolites 2,4- and 2,6-toluenediamine (TDA) were determined by gas chromatography-mass spectrometry on hydrolysed urine samples collected from 16 workers at the beginning of the workweek and both before (BS) and at the end (ES) of the 4th workday. Additional samples were collected at the end of the 1st half-shift and at the beginning of the 2nd half-shift in 5 workers. RESULTS: In the foam production shop, TDI values were on average about 20 microg/m3, with higher levels in the 2nd half-shift and peak levels in workers operating along the polymerization tunnel. Average TDI levels were significantly correlated with ES TDA concentrations (p < 0.0001). TDA showed a fast urinary elimination phase leading to progressively higher TDA levels either during the shift (5 workers) and at the end-of-shift. A slower elimination phase with a weekly accumulation was demonstrated by values at the beginning of the workweek (higher than in unexposed subjects) and by their elevation in subsequent BS samples. CONCLUSIONS: The study demonstrates the feasibility and reliability of biological monitoring in workers exposed to 2,4-:2, 6-TDI mixtures. This approach can provide information about both the daily and weekly exposure levels.

Air exposure assessment of TDI and biological monitoring of TDA in urine in workers in polyurethane foam industry.

OBJECTIVES: Toluene diisocyanate (TDI) is used in the manufacturing process of polyurethane (PU) foams and is a potent inducer of occupational asthma. The objective of this study was to evaluate the correlation between the exposure to total TDI (2,4- and 2,6-TDI) in air and the corresponding biomarker concentration of total TDA (2,4- and 2,6-TDA) in hydrolysed urine. The aim was also to propose an appropriate biological exposure limit for total TDA in urine. METHODS: 9 workers from two production lines in a PU foam producing plant were studied. Personal exposure to TDI during four representative production shifts was monitored by an active air sampling method (filter impregnated with 1-(2-methoxyphenyl)piperazine) and quantified by high-performance liquid chromatography and diode array detection (NIOSH n° 2535, 5521). In parallel, pre-shift and post-shift urinary samples were collected from the exposed workers, and TDA concentrations were determined by gas chromatography-mass spectrometry after alkaline hydrolysis. All samples were collected on four measuring days: two Fridays (end of workweek) and two Mondays (start of workweek) separated by a weekend without exposure. RESULTS: Strong correlations between the personal air concentrations of total TDI and the corresponding biomarker levels of total TDA in urine (r=0.816) were observed. An increase of 18.12 µg TDA/l (post-shift minus pre-shift concentration) corresponds to an exposure of 5 ppb (37 µg/m(3), the current American Conference of Governmental Industrial Hygienists threshold limit value) during the shift. CONCLUSIONS: The increase in TDA during the shift is a suitable biomarker for exposure to TDI during the same shift. Further research is needed to evaluate the use of start of week or end of week post-shift TDA in urine as biomarker since TDA was found to accumulate during the working week and thus the moment of sampling will clearly influence the result.

Analytical investigations of toxic p-phenylenediamine (PPD) levels in clinical urine samples with special focus on MALDI-MS/MS.

Para-phenylenediamine (PPD) is a common chromophoric ingredient in oxidative hair-dyes. In some African countries like Sudan, Egypt and Morocco but also in India this chemical is used alone or in combination with colouring extracts like Henna for dyeing of the hair or the skin. Excessive dermal exposure to PPD mainly leads to the N-mono- and N,N'-diacetylated products (MAPPD, DAPPD) by N-acetyltransferase 1 and 2 (NAT1 and 2) catalyzed reactions. Metabolites and PPD are mainly excreted via renal clearance. Despite a low risk of intoxication when used in due form, there are numerous cases of acute intoxication in those countries every year. At the ENT Hospital - Khartoum (Sudan) alone more than 300 cases are reported every year (~10% fatal), mostly caused by either an accidental or intended (suicidal) high systemic exposure to pure PPD. Intoxication leads to a severe clinical syndrome including laryngeal edema, rhabdomyolysis and subsequent renal failure, neurotoxicity and acute toxic hepatitis. To date, there is no defined clinical treatment or antidote available and treatment is largely supportive. Herein, we show the development of a quick on-site identification assay to facilitate differential diagnosis in the clinic and, more importantly, the implementation of an advanced analytical platform for future in-depth investigations of PPD intoxication and metabolism is described. The current work shows a sensitive (~25 µM) wet chemistry assay, a validated MALDI-MS/MS and HPLC-UV assay for the determination of PPD and its metabolites in human urine. We show the feasibility of the methods for measuring PPD over a range of 50-1000 µM. The validation criteria included linearity, lower limit of quantification (LLOQ), accuracy and precision, recovery and stability. Finally, PPD concentrations were determined in clinical urine samples of cases of acute intoxication and the applied technique was expanded to identify MAPPD and DAPPD in the identical samples.

Determination of 2,5-toluylenediamine (2,5-TDA) and aromatic amines in urine after personal application of hair dyes: kinetics and doses.

The personal use of hair dye products is currently under discussion due to the potentially increased risk of bladder cancer among long-time users described in epidemiological literature. In order to investigate the dermal absorption of aromatic diamines as well as aromatic amines possibly present as contaminants in hair dye formulations, we conducted a biomonitoring study under real-life conditions and calculated kinetics and doses for the urinary excretion. Urine samples of two female subjects were collected for a time period of 48 h after personal application of a hair dye cream and analysed for aromatic diamines as well as o-toluidine and 4-aminobiphenyl using highly specific GC/MS-methods. 2,5-Toluylenediamine (2,5-TDA) as active ingredient of hair dyes is rapidly absorbed dermally. After a distribution phase of 12 h, 2,5-TDA is excreted with a half-time of 8 h. Excretion was 90% complete within 24 h after application. The doses of 2,5-TDA excreted within 48 h were 700 µg for application of a brown-reddish hair dye cream and 1.5 mg for the application of a brown-black hair dye cream. Urinary 4-aminobiphenyl as well as contaminations with other aromatic diamines were not detectable in our study. Due to the artifactual formation of o-toluidine in the presence of high concentrations of urinary 2,5-TDA, our results could not prove an increased internal exposure of humans to carcinogenic amines after personal application of hair dyes.

Internal exposure of hairdressers to permanent hair dyes: a biomonitoring study using urinary aromatic diamines as biomarkers of exposure.

PURPOSE: To determine whether the occupational exposure of hairdressers to permanent hair dyes can be quantified by the use of biological monitoring of urinary aromatic diamines as one of the main constituents and to compare these levels to those recently determined in persons after personal application of hair dyes. METHODS: Fifty-two hairdressers (40 female and 12 male) from 16 hairdresser salons in and around the city of Aachen took part in this field study. Subjects were asked to document all operations associated with possible exposure to permanent hair dyes like mixing colour, application of colour, washing after dyeing, and cutting of freshly coloured hair. Excretion of aromatic diamines 2,5-toluylene diamine (2,5-TDA) and p-phenylene diamine (p-PDA) as main constituents of commercially available hair dyes was measured in urine samples using a highly specific and accurate GC/MS-method. Urine samples were taken at 5 points of time during the work week: pre-shift before the start of the work week, pre- and post-shift on the third day of the work week and finally pre- and post-shift on the last day of a work week in order to meet different workloads and possible accumulative effects over the week. Nineteen persons matched for age served as a control group and gave spot urine samples. RESULTS: Although the levels were generally low, we could determine a significantly higher internal exposure to 2,5-TDA in hairdressers (medians ranged from <0.2 µg/g creatinine up to 1.7 µg/g creatinine at various sampling times, with a maximum of 155.8 µg/g creatinine) compared to the control group (median <0.2 µg/g creatinine, maximum 3.33 µg/g creatinine). At the same time, p-PDA was detectable only in selected cases in the group of hairdressers but not in the control group. Overall, there was neither an intra-shift effect seen nor an effect across the work week. There was also no significant difference in urinary excretion of participants who reported wearing protective gloves compared to those who reported not wearing protective gloves. CONCLUSION: The internal exposure to aromatic diamines in hairdressers using permanent hair dyes can be determined using biological monitoring. The extent of exposure is low compared to subjects after personal application of hair dyes, who excreted more than 200 times higher amounts of aromatic diamines. This slight work-related exposure might be reduced by the strict adherence to the use of suitable gloves as well as long-sleeved clothing.

The GSTP1 Ile105 Val polymorphism modifies the metabolism of toluene di-isocyanate.

BACKGROUND: Toluene di-isocyanate (TDI) is widely used in the production of polyurethane foams and paints. As TDI causes respiratory disease in only a fraction of exposed workers, genetic factors may play a key role in disease susceptibility. Polymorphisms in TDI metabolising genes may affect elimination kinetics, resulting in differences in body retention, and in its turn differences in adverse effects. OBJECTIVES: To analyze how genotype modifies the associations between (i) TDI in air (2,4-TDI and 2,6-TDI) and its metabolites toluene diamine (TDA; 2,4-TDA and 2,6-TDA) in hydrolyzed urine; and (ii) 2,4-TDA and 2,6-TDA in hydrolyzed plasma and 2,4-TDA and 2,6-TDA in urine. METHODS: Workers exposed to TDI were analyzed for 2,4-TDI and 2,6-TDI in air (N=70), 2,4-TDA and 2,6-TDA in hydrolyzed urine (N=124) and in plasma (N=128), and genotype: CYP1A1*2A, CYP1A1*2B, GSTA1-52, GSTM1O, GSTM3B, GSTP1 I105V, GSTP1 A114V, GSTT1O, MPO-463, NAT1*3, *4, *10, *11, *14, *15, NAT2*5, *6, *7, and SULT1A1 R213H. RESULTS: GSTP1 105 strongly modified the relationship between 2,4-TDA in plasma and in urine: ValVal carriers had about twice as steep regression slope than IleIle carriers. A similar pattern was found for 2,6-TDA. CYP1A1*2A, GSTM1, GSTP1, GSTT1, and MPO possibly influenced the relationship between TDA in plasma and urine. CONCLUSION: Our results show, for the first time, genetic modification on the human TDI metabolism. The findings suggest that GSTP1 genotype should be considered when evaluating biomarkers of TDI exposure in urine and plasma. Moreover, the results support earlier findings of GSTP1 105 Val as protective against TDI-related asthma.

Biological monitoring of TDI-derived amines in polyurethane foam production.

BACKGROUND: Toluene diisocyanate (TDI) is used in industry in the production of flexible polyurethane foam, commonly a mixture of the 2,4- and 2,6- isomers. The production process may lead to exposure to diisocyanates which are associated with respiratory disease. A method has been available for the determination of TDI biomarkers in urine for some years. AIMS: To explore the usefulness of urinary toluenediamine (uTDA) in assessing whether dermal absorption of diisocyanates makes a significant contribution to a worker's total exposure. METHODS: Twenty-six workers took part in the study. Thirteen workers whose duties brought them into physical contact with uncured polyurethane foam during their shift (handlers) were compared to a control group of 13 workers in the same block plant environment had no physical contact with uncured foam on the day that sampling took place (non-handlers). Creatinine-adjusted uTDA levels in the two groups were compared across a work shift. RESULTS: Both groups of workers were exposed to similar levels of airborne TDI. Ten handlers were found to have TDA in post-shift urine samples above detection limits compared with two non-handlers (P < 0.05). No clear relationship was found between the level of airborne TDI exposure and post-shift uTDA. CONCLUSIONS: uTDA provides a useful indication of the contribution which skin absorption makes to total TDI exposure. The results suggest that skin protection when handling uncured polyurethane foam may not receive sufficient consideration.

N-Glucuronidation of the antiepileptic drug retigabine: results from studies with human volunteers, heterologously expressed human UGTs, human liver, kidney, and liver microsomal membranes of Crigler-Najjar type II.

Retigabine (D-23129), an N-2-amino-4-(4-fluorobenzylamino)phenylcarbamine acid ethyl ester, is a novel antiepileptic drug which is currently in phase II clinical development. This drug undergoes N-glucuronidation. We aimed to identify the principal enzymes involved in the N-glucuronidation pathway of retigabine and compared our findings with those obtained from human liver (a pool of 30 donors) and kidney microsomes (a pool of 3 donors) and with results from a human absorption, distribution, metabolism, and excretion study upon administration of 200 microCi of [(14)C]-D-23129. Essentially, microsomal assays with UGT1A1 produced only one of the 2 N-glucuronides, whereas UGT1A9 is capable of forming both N-glucuronides. The rates of metabolism for UGT1A9, human liver microsomes, and UGT1A1 were 200, 100, and 100 pmol N-glucuronide per minute per milligram of protein, respectively. At the 50 micromol/L uridine diphosphate glucoronic acid (UDPGA) concentration, UGT1A4 also catalyzed the N-glucuronidation of retigabine, the rates being approximately 5 and 6 pmol/(min.mg protein). With UGT1A9, the production of metabolites 1 and 2 proceeded at a K(m) of 38+/-25 and 45+/-15 micromol/L, whereas the K(m) for retigabine N-glucuronidation by human liver microsomal fractions was 145+/-39 micromol/L. Furthermore, a V(max) of 1.2+/-0.3 (nmol/[min.mg protein]) was estimated for human liver microsomes (4 individual donors). We investigated the potential for drug-drug interaction using the antiepileptic drugs valproic acid, lamotrigine, the tricyclic antidepressant imipramine, and the anesthetic propofol. These are commonly used medications and are extensively glucuronidated. No potential for drug-drug interactions was found at clinically relevant concentrations (when assayed with human liver microsomes or UGT1A9 enzyme preparations). Notably, the biosynthesis of retigabine-N-glucuronides was not inhibited in human liver microsomal assays in the presence of 330 micromol/L bilirubin, and glucuronidation of retigabine was also observed with microsomal preparations from human kidney and Crigler-Najjar type II liver. This suggests that lack of a particular UDP-glucuronosyltransferase (UGT) isoform (eg, UGT1A1 in kidney) or functional loss of an entire UGT1A gene does not completely abolish disposal of the drug. Finally, chromatographic separations of extracts from microsomal assays and human urine of volunteers receiving a single dose of (14)C-retigabine provided clear evidence for the presence of the 2 N-glucuronides known to be produced by UGT1A9. We therefore suggest N-glucuronidation of retigabine to be of importance in the metabolic clearance of this drug.

Analysis of paraphenylenediamine.

The toxic Paraphenylenediamine is characterized by infrared spectrophotometry and nuclear magnetic resonance spectroscopy, whereas, high performance liquid chromatography with a refractive index detector was used to determine its purity in the suspect samples using external standardisation. An analytical method for determination of lower traces of paraphenylenediamine in post-mortem biological fluids was developed. This procedure involves deproteneization or hydrolysis followed by liquid-liquid extraction and derivatization with trifluoroacetic anhydride. 1 microL of the extract was then analysed by gas chromatography/iontrap mass spectrometry. Benzidine used as the internal standard for quantification and the extraction recovery test was evaluated to 85%. This method was validated in cases with paraphenylenediamine poisoning.

Improvement in the GC-MS method for determining urinary toluene-diamine and its application to the biological monitoring of workers exposed to toluene-diisocyanate.

OBJECTIVES: To develop a simple and sensitive GC-MS method for determining toluene-diamine (TDA) in urine and to apply the method for biological monitoring of workers exposed to toluene-diisocyanate (TDI). METHODS: After acid hydrolysis of 0.1 ml of urine, diluted tenfold with water, for 1.5 h, the free TDA formed was extracted with dichloromethane, and the heptafluorobutyric anhydride derivative was determined by GC-MS. We applied the method to the biological monitoring of 18 workers who were using an 80:20 mixture of 2,4-TDI and 2,6-TDI. RESULTS: 2,6-TDA and 2,4-TDA were simply determined in 7 min by GC-MS. TDA levels in post-shift urine were well correlated with personal exposure levels of TDI. The correlation was improved by correction with creatinine or specific gravity in the 2,6-isomer, but not in the 2,4-isomer because of low exposure levels. From the correlation equation, the 2,6-TDA level (corrected with creatinine), corresponding to the TDI level of 5 ppb, was calculated to be 31.6 mug/g Cre. TDAs in pre-shift urine also correlated significantly with the personal exposure levels of TDIs, although the slope of the correlations for pre-shift samples was 60%-70% of those for post-shift samples. The correlation between 2,4-TDA and 2,6-TDA levels was significant, although the levels of the 2,4-isomer were less than one-tenth of the 2,6-isomers in both air (personal exposure) and urine. CONCLUSION: The present method is simple and practicable and can be useful for biological monitoring of TDI workers.

Urinary acetylated metabolites and N-acetyltransferase-2 genotype in human subjects treated with a para-phenylenediamine-containing oxidative hair dye.

In the organism of mammals, important detoxification pathways of arylamines are catalysed by N-acetyltransferase 2 (NAT2). A recent case-control epidemiology study suggested that human NAT2 slow acetylators exposed to oxidative hair dyes may be at greater risk to develop bladder cancer. We therefore profiled urinary [(14)C]-metabolites and NAT2 genotype in eight human subjects following treatment with a dark-shade oxidative hair dye containing [(14)C]-para-phenylenediamine (PPD). Genotyping identified three subjects as slow, and five subjects as intermediate NAT2 acetylators. Within 24 h after treatment, the study subjects excreted a mean total of 0.43+/-0.24% of the applied [(14)C] in the urine, where five different metabolites were found. The major urinary metabolites were concluded to be N-mono-acetylated and N,N'-diacetylated PPD. They were present in all urine samples and amounted to 80-95% of the total urinary [(14)C]. Another metabolite, possibly a glucuronic acid conjugate, was found in 6/8 urine samples at 5-13% of the total urinary [(14)C]. All metabolites appeared to be related to PPD, no evidence of the presence of high-molecular weight dye-intermediates or corresponding metabolites was found. The metabolite profile in the study subjects showed no significant differences between the NAT2 intermediate and NAT2 slow acetylator subgroups. Urine of NAT2 slow acetylators contained N-mono-acetylated-PPD at 42.2+/-10.2% and N,N'-di-acetylated-PPD at 54.1+/-7.6% of total urinary radioactivity, while the corresponding values of intermediate acetylators were 46.0+/-8.9% and 45.7+/-9.9%, respectively. Overall, our results suggest that the human acetylation rate of PPD after topical application is independent of the NAT2 genotype status, most likely due to metabolism by epidermal NAT1 prior to systemic absorption.

Human systemic exposure to a [14C]-para-phenylenediamine-containing oxidative hair dye and correlation with in vitro percutaneous absorption in human or pig skin.

We investigated the absorption of a commercial [14C]-PPD-containing oxidative dark-shade hair dye in human volunteers as well as in vitro using human or pig ear skin. The hair of eight male volunteers was cut to a standard length, dyed, washed, dried, clipped and collected. Hair, washing water, materials used in the study and a 24-h scalp wash were collected for determination of radioactivity. Blood, urine and faeces were analysed up to 120 h after hair dyeing. An identical [14C]-PPD-containing hair dye formulation was applied in vitro for 0.5 h to human and pig ear skin, and radioactivity was determined in skin compartments after 24 h. In humans, the recovery rate was 95.7+/-1.5% of the applied radioactivity. Washing water, cut hair, gloves, paper towels, caps or scalp wash contained a total of 95.16+/-1.46% of the applied [14C]. Absorbed radioactivity amounted to 0.50+/-0.24% in the urine and 0.04+/-0.04% in the faeces, corresponding to a mean of 7.0+/-3.4 mg [14C]-PPD-equivalents absorbed. Within 24 h after application, most of the radioactivity was eliminated. The Cmax of [14C]-PPD-equivalents in the plasma was 0.087 microgeq/ml, the Tmax was approximately 2 h, and the mean the AUC(0-12h) was 0.67 microgeq h/ml. In vitro tests in human or pig skin found total absorbed amounts of 2.4+/-1.6% (10.6+/-6.7 microgeq/cm2) or 3.4+/-1.7% (14.6+/-6.9 microgeq/cm2), respectively. Percentage-based in vitro results were considerably higher than corresponding in vivo data, whereas, in units of microg/cm2, they corresponded to a total absorbed amount of 7.40 or 10.22 mgeq for human or pig skin, respectively. All results suggested that hair dyeing with oxidative hair dyes produces minimal systemic exposure that is unlikely to pose a risk to human health.

Pharmacokinetic interaction between retigabine and lamotrigine in healthy subjects.

PURPOSE: The antiepileptic drugs (AEDs) retigabine (RGB) and lamotrigine (LTG) undergo predominantly N-glucuronidation and renal excretion. This study was performed to evaluate potential pharmacokinetic interactions between both AEDs. METHODS: Twenty-nine healthy male subjects participated in the study. Group A ( n=14) received single oral 200-mg RGB doses on day 1 and day 7, and 25 mg o.i.d. LTG on days 3-8. Group B ( n=15) received single oral 200-mg LTG doses on day 1 and day 17, and was up-titrated to 300 mg RGB b.i.d. on days 6-20. Blood samples were collected to compare the pharmacokinetics of both AEDs and the N-acetyl metabolite of RGB (AWD21-360) after single and concomitant treatments. RESULTS: RGB was rapidly absorbed and eliminated with a mean half-life (t(1/2)) of 6.3+/-1.1 h and an apparent clearance (CL/F) of 0.69+/-1.4 l/h/kg. Under co-administration of LTG, mean RGB t(1/2) and area under the plasma concentration-time curve (AUC) were increased by 7.5% ( P=0.045) and 15% ( P=0.006), respectively, while CL/F was decreased by 13% ( P=0.06). Consistent results were obtained for AWD21-360. LTG was moderately rapidly absorbed, eliminated with a mean t(1/2) of 37+/-10.4 h and a CL/F of 0.028+/-0.007 l/h/kg. Under co-administration of RGB, mean LTG t(1/2) and AUC decreased by 15% and 18%, respectively, while CL/F increased by 22% (all parameters, P=0.001). CONCLUSIONS: RGB and LTG exhibit a modest pharmacokinetic interaction on each other. The slight decline in RGB clearance due to LTG is believed to result from competition for renal elimination rather than competition for glucuronidation. The induction of LTG clearance due to retigabine was unexpected since RGB did not show enzyme induction in various other drug-drug interaction studies. Further studies in patients are needed to assess the clinical relevance of these findings for concomitant treatment with both drugs in the upper recommended dose range.

Simultaneous determination of oxidative hair dye p-phenylenediamine and its metabolites in human and rabbit biological fluids.

A liquid chromatography with an electrochemical detector method has been developed for the quantitative measurement for three diamine derivatives (p-phenylenediamine, N,N(')-p-phenylenebisacetamide, and 4-aminoacetanilide) in human urine and rabbit blood, urine, and feces. The detection cell consisted of a glassy carbon electrochemical signal obtained with a supporting electrolyte containing 20% methanol-5mM octylammonium orthophosphate (pH 6.30) as the mobile phase. A comparison of the results obtained from HPLC-UV shows agreement.

LC-MS determination of urinary toluenediamine in workers exposed to toluenediisocyanate.

To improve the biological monitoring method for 2,6- and 2,4-toluenediisocyanate (TDI) exposure, we developed a simple and rapid method for analysis of the corresponding urinary metabolites, 2,6- and 2,4-toluenediamine (TDA) using liquid chromatograph-mass spectrometry (LC-MS). One ml of urine was hydrolyzed at 100 degrees C for 1.5 h with H(2)SO(4). Alkalinized hydrolysate was extracted with dichloromethane (DCM) and analyzed by atmospheric pressure chemical ionization (APCI) LC-MS, in positive-ion mode. The mass spectra of TDA isomers showed the protonated molecule [M+H](+), at m/z 123 as the base peak. Calibration curves of 2,6-TDA were linear up to 400 microg/l. TDA isomers in urine of exposed workers as determined by LC-MS correlated well with those obtained by gas chromatography-mass spectrometry. 2,6- and 2,4-TDA were not detected in non-exposed subjects, whereas exposed workers showed urinary levels up to 250 and 63 microg/l, respectively.

Exposure to 2,4- and 2,6-toluene diisocyanate (TDI) during production of flexible foam: determination of airborne TDI and urinary 2,4- and 2,6-toluenediamine (TDA).

Occupational exposure to 2,4- and 2,6-toluene diisocyanate (2,4- and 2,6-TDI) was measured during the production of flexible foam. The usefulness of urinalysis of the TDI-derived amines, 2,4- and 2,6-toluenediamine (2,4- and 2,6-TDA), for exposure assessment was compared with air monitoring. Urine samples were collected from 17 employees at two plants. The workers' personal exposure was measured using 1-(2-methoxyphenyl)-piperazine (2MP)-impregnated glass fibre filters for sampling and high-performance liquid chromatography (HPLC) with ultraviolet (UV) and electrochemical (EC) detection for quantification. The limit of detection (LOD) of 2,4- and 2,6-TDI was 0.01 microtg ml(-1) for a 20 microl injection. The precision of sample preparation, expressed as the relative standard deviation (RSD), was 0.6% with UV detection and 0.8% with EC detection at a 2,4-TDI concentration of 0.2 microg ml(-1) (n = 6). For 2,6-TDI, the corresponding RSDs were 0.5% and 0.8%. The urinary 2,4- and 2,6-TDA metabolites were determined after acid hydrolysis as heptafluorobutyric anhydride derivatives by gas chromatography-mass spectrometry. The LOD in urine was 0.35 nmol l(-1) for 2,4-TDA and 0.04 nmol l(-1) for 2,6-TDA. The precision (RSD) of six analyses of human urine spiked to a concentration of 100 nmol l(-1) was 3.7% for 2,4-TDA and 3.6% for 2,6-TDA. There was a trend for linear correlation between urinary TDA concentration and the product of airborne TDI concentration and sampling time. Urinalysis of TDA is proposed as a practical method for assessing personal exposures in workers exposed intermittently to TDI.