SHORT COMMUNICATION

Michael Bader Æ Stephen A. Keener Æ Renate Wrbitzky

Dermal absorption and urinary elimination of N-methyl-2-pyrrolidone

Received: 26 November 2004 / Accepted: 13 April 2005 / Published online: 5 July 2005� Springer-Verlag 2005

Abstract Objectives: The dermal absorption of the solventN-methyl-2-pyrrolidone (NMP) and its elimination inurine was investigated in an experimental study. Meth-ods: Seven volunteers were exposed to 1045 mg of liquidNMP under occlusive conditions for 2 h. Urine wascollected before, during and up to 72 h after the exposureand analysed for NMP by GC/MS after liquid–liquidextraction. Additionally, the remaining NMP in the padswas determined to estimate the total dermal uptake.Results: The concentration of NMP in urine increasedrapidly after beginning of the exposure up to 1 h after theexposure was completed. A peak concentration of1,836±863 lg/l was observed, the half-life in urine was3.2 h. About 0.5% of the absorbed dose was excretedmetabolically unchanged. An average dermal absorptionof 5.5 mg cm�2 h�1 was calculated. Conclusions: Theresults of this study show that the percutaneous absorp-tion of NMP may contribute significantly to the overalluptake of the solvent, e.g. in the workplace. Therefore, abiological monitoring of NMP exposed workers isessential for occupational-medical surveillance.

Keywords N-methyl-2-pyrrolidone Æ Dermalabsorption Æ Urinalysis Æ Biomonitoring

Introduction

N-Methyl-2-pyrrolidone is a heterocyclic ketone thatdemonstrates excellent miscibility with water and mostorganic solvents. In the European Union, about38,000 tons of NMP are produced per year (Germany:20,000 tons) (Bader et al. 2003). The most prominent

applications comprise the production of polymers andresins, hydrocarbon extraction, agrochemistry, and sur-face cleaning e.g. in the microelectronics industry orgraffiti removal (HSE 1997). NMP is a mild irritant to theeyes, the mucous membranes and the skin. Animalexperiments, however, have demonstrated developmentaltoxicity of the compound when administered in high do-ses (Greim and Lehnert 1994; HSE 1997). Due to the highpercutaneous absorption of the compound, an exposureto NMP results in a dermal uptake rather than inhalationof airborne solvent (Anundi et al. 1993, 2000; Langworthet al. 2001). From the occupational medical point of view,this aspect is of particular relevance for the implementa-tion of safety measures and surveillance in the workplace.In Germany, the evaluation of a biological exposure limitvalue for NMP is currently under discussion.

The dermal absorption of NMP has been investigatedpreviously in tissue samples and animal experiments butthe varying approaches make a full comparison betweenthese studies difficult (Ursin et al. 1995; Payan et al.2003). In general, the dermal uptake was mainly influ-enced by skin thickness, desquamation and NMP con-centration. Akesson et al. (2004) reported a four tofivefold higher absorption of undiluted NMP comparedto a 50% aqueous dilution. All studies suggest a relevantcontribution of the dermal exposure route to the overalluptake of NMP that, in the workplace, might well sur-pass the inhaled dose, especially as airborne NMP istypically below current limit values (e.g. German MAK:80 mg/m3). To estimate the relevance of dermal NMPuptake, the absorption through living human skin andthe subsequent elimination pattern in urine were studied.

Materials and methods

Study group

Seven healthy volunteers (four women, three men,average age: 38±6 years) participated in the study. Aphysical examination confirmed a regular skin appear-

M. Bader (&) Æ S. A. Keener Æ R. WrbitzkyDepartment of Occupational Medicine,Hannover Medical School, Carl-Neuberg-Str. 1,D-30625, Hannover, GermanyE-mail: bader.michael@mh-hannover.deTel.: +49-511-5329333Fax: +49-511-5329332

Int Arch Occup Environ Health (2005) 78: 673–676DOI 10.1007/s00420-005-0008-0

ance in every case, no sensitisation towards chemicalswas reported. The study was approved by the EthicsCommittee of Hannover Medical School. All partici-pants gave their informed consent at the beginning ofthe study. They were notified about the irritating prop-erties of NMP and about the observed developmentaltoxicity towards animals.

Experimental exposure and sampling

Regular medical cellulose pads (3.5·5 cm2) were spikedwith 1 ml of liquid NMP (1,045 mg) and attached to theback of a hand of every participant. NMP vaporizationwas avoided by covering the pads with a 40 lm alu-minium foil that was also crimped around the pad edges.This set-up was further covered with a larger aluminiumfoil sheet (4.5·6 cm2) and fixed tightly on the skin withmedical adhesive tape. Prior to the study, this occlusivedesign was tested by attaching aluminium coveredspiked pads onto a small glass frame for 4 h at 37�C. Noweight loss was observed in the pads during this period.Additionally, non-spiked pads attached under occlusiononto human skin for 4 h showed no weight gain. In theexperimental study, the pads were removed after 2 h,transferred into 20 ml gastight crimp vials and stored at�27�C. The exposed skin was washed thoroughly withwater. A urine specimen of every study participant wassampled right before the experiment started, as well asduring the exposure, and as frequently as possible,thereafter. The urine specimens were collected andstored at �27�C. The participants recorded the samplingtime in a standardized protocol.

Chemicals

Toluene and potassium hydroxide were of p.a. gradefrom Merck (Darmstadt, Germany). NMP p.a. gradewas purchased from Sigma-Aldrich (Deisenhofen, Ger-many) and ASTM type I water was prepared in-house.Standards for urine analyses were diluted in pooled ur-ine from nonexposed persons in a concentration rangebetween 10 lg/l and 2,000 lg/l. Additionally, NMPstandards in toluene were prepared in the same con-centration range for the analysis of the cellulose pads.

Biomonitoring

NMP in urine was analysed according to Akesson andPaulsson (1997a, b). Two millilitre of each sample wastransferred into a 10 ml screw capped vial and mixedwith 4 ml of 12 M potassium hydroxide. The sampleswere then extracted with 2 ml of toluene for 10 min on alaboratory shaker. After centrifugation to separate thelayers (5 min, 800· g), 1 ml of each toluene extract wastransferred into a sample vial and analysed by single ionmonitoring gas chromatography–mass spectrometry.

GC/MS conditions: injector temperature 230�C, ovenprogram: 80�C initial, 10�/min to 160�C, 15�C/min to250�C, isothermal for 5 min, interface temperature260�C, column: HP InnoWax 30 m·0,25 mm, 0,25 lmfilm, constant flow 1.2 ml/min Helium 5.0, injection vol-ume 1 ll split 1:50, ion source temperature 230�C,quadrupole temperature 150�C, electron energy 70 eV,electronmultiplier 2600 V,mass fragments: 99m/z (NMPbase peak) and 98m/z (hydrogen loss), dwell time 100 ms.The imprecision was 3% within-series and 8% between-series (c = 1,000 lg/l). NMP levels below the limit ofquantification (10 lg/l) were generally adjusted to 5 lg/l.

Pad analyses

The cellulose pads were weighed before and after theexperimental procedure. To analyse the residual NMP,approximately 25 mg of each pad was extracted with10 ml of toluene in a screw capped vial for 20 min on alaboratory shaker. 1 ml of the toluene was then trans-ferred into a sample vial and analysed according to theGC/MS conditions described above. The NMP recoveryranged from 94% to 108%. The total amount of NMPin a pad fragment was calculated from the concentrationof the compound in the toluene extract. The residualNMP in a whole pad was then extrapolated on the basisof the pads total weight, corrected for the pads tareweight.

Results and discussion

The participants were examined by an occupationalphysician during the study, especially with regard toirritative effects on the skin. All volunteers experiencedacute effects during the application such as feelings ofheat, pricking and itchiness. Moderate swellings limitedto the pad area were observed in all cases, one persondeveloped a local erythema (2–3 mm). The symptomsfaded rapidly upon exposure cessation within 24 h. Norelation was observed between the intensity or durationof the effects and the absorbed dose, but it is noteworthythat the participant with the erythema was among thethree persons with the highest NMP uptake.

The occlusive design of the dermal exposure allowedthe calculation of the NMP absorption through the skinfrom the analysis of the pads. Skin contact to NMPunder virtually occlusive conditions is likely to be seene.g. with cleaning workers using NMP resistant protec-tion such as rubber gloves.

All participants were topically exposed to 1 ml ofNMP, corresponding to 1,045 mg of the solvent at 22�Cin the laboratory. The pads were evenly soaked withNMP and the surface was measured (average: 17.5 cm2,range: 16.4–18.2 cm2). After the exposure, the meanresidual amount of NMP in the pads was 851 mg (range:755–905 mg), indicating that an average of (1,045–851 mg) = 194 mg NMP (range: 140–290 mg) had been

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absorbed through the skin during the 2 h exposureinterval. With an exposed skin surface of 17.5 cm2 anabsorption of (194 mg/(17.5 cm2 * 2 h) = 5.5 mgcm�2 h�1 was calculated (range: 4.0–8.3 mg cm�2 h�1)(Table 1). Some possible influence factors, however,could not be controlled with this study design. Althoughthe secretion of sweat was not observed with unspikedpads, this could not be ruled out, given the irritative anddehydrating effects of NMP. An aqueous dilution ofNMP significantly decreases the dermal absorption ofthe solvent (Akesson et al. 2004). Additionally, waterabsorption into the pads would result in a respectiveweight gain and an underestimation of the flux. Incontrast, residual NMP on the skin surface that wasswabbed after the exposure, would increase the calcu-lated absorption.

At this point, the quantitative implications of thesefactors are difficult to estimate. Therefore, a comparisonwas made between the total amount of urinary NMPfound in this study and the data presented by Akessonand Jonsson (1997a, b) after oral administration of100 mg NMP to three human volunteers. The totalamount of NMP excreted in urine in our study was1.04 mg (range: 0.53–1.49 mg), calculated from theNMP concentrations measured and the volume of theunderlying urine samples. Akesson and Jonsson (1997a,b) reported that an average of 0.8% of the NMP dosewas eliminated unchanged in urine. Based on that value,the 1.04 mg NMP found in the dermal study suggest anabsorbed amount of 130 mg NMP and an average fluxof 3.7 mg cm�2 h�1 . The difference of about 32%compared to the pad weight approach might be relatedto the influence factors discussed above. Nevertheless,both calculations point to a considerable dermalabsorption of NMP.

Figure 1 shows a combined NMP elimination curveof all seven participants during the first 26 h (exposuretime + 24 h). The NMP concentration in urine in-creased rapidly upon exposure and reached a maximumabout 1 h after cessation. An interindividual variation of30–60 min was observed, probably not only affected byinterindividual differences in NMP metabolism but alsoby imprecise time records and the relatively low numberof samples. Nevertheless, this result coincides with datapresented by Akesson and Paulsson (1997a, b) after

exposure to airborne NMP. The average NMP concen-tration in urine 1 hour after the end of exposure was1,836±863 lg/l with a broad interindividual variation(687–3,190 lg/l).

Table 1 Calculation of the dermal absorption rate of NMP and the percentage of metabolically unchanged urinary NMP

Study subject Residual amountof NMP inthe pad [mg]

Absorbed amountof NMP [mg]

Total dermalabsorption[mg cm�2 h�1 ]

Total amountof NMP inurine [mg]

Percentage ofabsorbeddose [%]

1 905 140 4.0 0.526 0.382 900 145 4.1 0.739 0.513 794 251 7.2 1.040 0.414 901 144 4.1 0.986 0.685 846 199 5.7 1.217 0.616 853 192 5.5 1.491 0.787 755 290 8.3 1.247 0.43Average 851 194 5.5 1.035 0.54

Fig. 1 NMP concentrations in urine samples of seven volunteersbefore, during and after 2 h of topical exposure to 1,045 mg NMP

Fig. 2 Semi-logarithmic plot of NMP-(U) versus time. Theexponential regression analysis is limited to the 3–24 h postexpo-sure interval

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The NMP concentrations in urine declined rapidlyfrom the maximum value and in most cases, dropped tobelow the limit of quantification 12 h after cessation ofexposure. Three participants showed a second, lesspronounced increase of NMP in urine in the 8–18 hinterval after the end of exposure. In one case, a thirdelimination interval 26–43 h after the end of exposurewas observed (data not shown). The pattern did notchange when NMP concentrations were corrected forurinary creatinine. A possible explanation for the de-layed elimination is the storage effect of the skin, but thishas not been observed in other studies on dermal NMPuptake, e.g. Akesson and Jonsson (1997a, b), Akesson etal. (2004). The route of exposure seems to influence thetoxikokinetic of NMP and its metabolites (Akrill et al.2002; Akesson et al. 2004) and it is likely that urinaryNMP in urine is also affected.

The half-life of NMP in urine was calculated from asemi-logarithmic concentration versus time curve(Fig. 2) according to the equation t1/2 = ln2/k, with k= slope of the regression curve. A calculation based onthe data from the first 24 h yielded a t1/2 for NMP inurine of 3.2 h (r = 0.936), a value well in line with theresults of Akesson et al. (2004) from dermal exposureexperiments (2.9–3.7 h). Akesson and Paulsson (1997a,b) reported a mean half-life of NMP in urine of 3.9–5.2 h after inhalation of the compound. The dermalapplication results in a short high peak exposure and agreat part of the absorbed NMP is then rapidly elim-inated with the urine. In contrast, the uptake of air-borne NMP corresponds to a relatively low dose over alonger period of time and the distribution of NMP inthe body compartments probably decelerates the elim-ination. A closer examination of the time-dependentNMP elimination (Fig. 2) suggests a biphasic processwith an initial biological half-life of 2.2 h (r = 0.921)during the first 12 h, and a slower elimination with t1/2= 4.4 h (r = 0.693) afterwards. The significance of thisinterpretation is, however, limited due to the lownumber of samples and NMP concentrations close tothe limit of quantification.

In conclusion, the study points to a significant roleof dermal exposure in the overall uptake of NMP. Atypical workplace exposure to airborne NMP rangesbelow 10 mg/m3 as reported by Anundi et al. (2000)and Langworth et al. (2001). Thus, about 100 mg ofNMP might be incorporated by inhalation alone duringan 8 h shift under the assumption of a standardbreathing volume of 10 m3 and 100% pulmonalretention. The same dose would be absorbed from asmall skin area of 10 cm2 during 2 h with a dermal

absorption rate of 5.5 mg cm�2 h�1 . A relatively shortand topically limited exposure to undiluted NMP onthe skin is likely to result in a significant uptake of thesolvent. This is all the more important as NMP irritatesthe skin and thereby might enhance its own absorption.As the dermal exposure route is not covered by airsampling, a biological monitoring of NMP exposedworkers, e.g. based on the NMP metabolites 5-hydro-xy-NMP and 2-hydroxy-N-methylsuccinimide, isstrongly advisable (Akesson and Paulsson 1997a, b;Akesson et al. 2004).

Acknowledgements The authors thank Mr Wolfgang Rosenbergerfor his excellent technical assistance.

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