Dermal exposure to aqueous solutions of N-methyl pyrrolidone

Toxicology Letters 134 (2002) 265– 269 www.elsevier.com/locate/toxlet

Dermal exposure to aqueous solutions of N-methyl pyrrolidone Peter Akrill a,*, John Cocker a, Steve Dixon b b

a Health and Safety Laboratory, Broad Lane, Sheffield S3 7HQ, UK Health and Safety Executi6e, Stanley Precinct, Bootle, Merseyside, UK

Received 21 September 2001

Abstract N-methyl pyrrolidone (NMP) is a substance widely used for its strong and selective solvent capacity. The strong potential NMP has for skin absorption makes biological monitoring ideal for exposure assessment. This study looked at brief exposures to NMP in aqueous solutions over a range of concentrations. Two volunteers placed one hand in NMP solutions ranging from 5 to 25% for as long as 15 min followed by urine collection for 48 h. The analyte of interest (analysed by GC-MS) was the NMP metabolite 5-hydroxy-N-methyl pyrrolidone (5-HNMP). Excretion of 5-HNMP was plotted against time and this showed that urine concentrations were at a maximum after about 10 h and 5-HNMP excretion continued for 48 h after exposure. The half-life of excretion was found to be approximately 11 h. The mean correlation between exposure (as a measure of exposure duration and solution concentration) and total 5-HNMP excreted was 0.9297. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: N-Methyl pyrrolidone; Dermal exposure; Biological monitoring

1. Introduction N-methyl pyrrolidone (NMP, CAS number 872-50-4) is a clear liquid with a boiling point of 202 °C at 101.3 kPa. The UK Occupational Exposure Standard (OES) is 25 ppm (103 mg m − 3), 8 h Time Weighted Average, and 75 ppm (309 mg m − 3) Short Term Exposure Limit (HSE Books, 2002). The lead health effect used for setting the OES of 25 ppm was upper respiratory tract irri* Corresponding author. Tel.: + 44-114-289-2774; fax: + 44-114-289-2768 E-mail address: [email protected] (P. Akrill).

tancy. In view of the strong potential for skin absorption of NMP a ‘Sk’ notation was also added. Since NMP has strong and selective solvent capacity it is widely used. This is a major factor in its use in the petrochemicals industry. An increasing use for NMP in occupational situations is as a replacement for solvents presenting greater risks e.g. dichloromethane in paint strippers and as a graffiti cleaning agent (Anundi et al., 1993, 2000; Langworth et al., 2001). Many monomers and polymers are soluble in NMP and it is used to catalyse polymerisation reactions. NMP is also used in the microelectronics fabrication industry. In agriculture NMP is used as a

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solvent-carrier in pesticides. Ursin et al. (1995) measured the human skin permeability of NMP as 171 g m − 2 h. The pharmaceutical industry has investigated this extensive percutaneous absorption for enhancing the percutaneous penetration of certain pharmaceuticals involved in transdermal therapy (Barry, 1987; Prı´borsky´ et al., 1988; Sasaki et al., 1988). Human volunteer studies have shown that NMP is efficiently absorbed through the respiratory tract (A, kesson and Paulsson, 1997), and following oral exposure (A, kesson and Jo¨ nsson, 1997). Studies have shown that 0.8% of NMP is excreted in urine unchanged, 44% as 5-hydroxyN-methyl pyrrolidone (5-HNMP), 0.4% as Nmethylsuccinimide (MSI), and 20% as 2-hydroxy-N-methyl succinimide (2-HMSI), and that no conjugation occurs with glucuronic acid or sulphate (A, kesson and Jo¨ nsson, 1997). A proposed major metabolic pathway is that NMP is first hydroxylated to 5-HNMP and then further oxidised to MSI, which is hydroxylated to 2HMSI. A, kesson and Paulsson (1997) report that as much as 2% of inhaled NMP was excreted unchanged and showed that NMP has a half-life in both plasma and urine of around 4 h, and A, kesson and Jo¨ nsson (2000) showed that 5HNMP has a half-life of 6.3 and 7.3 h in plasma and urine, respectively. Very little exposure data exist for NMP. In a study by A, kesson and Paulsson (1997) six male volunteers were exposed to NMP at 0, 10, 25 and 50 mg m − 3 (0, 2.4, 6.1, 12.1 ppm) for 8 h. NMP was measured in plasma and urine. The mean levels of NMP found in plasma after 10, 25 and 50 mg m − 3 exposures were 0.33, 0.99 and 1.6 mg l − 1, respectively, and the levels in urine correlated well with blood (\0.95). Jo¨ nsson and A, kesson (1997) describe a study where an individual was exposed to 25 mg m − 3 (6.1 ppm) NMP for 8 h, and a urine sample collected immediately following exposure contained 94 mg ml − 1 (  1 mmol l − 1) 5-HNMP. A study where six male volunteers were exposed to NMP on four occasions over the range 0–50 mg m − 3 for 8 h found mean urinary and plasma 5-HNMP levels of 0– 117.3 mmol mol − 1 creatinine and 0– 44.4 mmol l − 1, respec-

tively. Close correlation between both urinary and plasma 5-HNMP and NMP exposure was reported. (A, kesson and Jo¨ nsson, 2000). No data appear to have been published from human volunteer studies involving dermal exposure to NMP. An occupational study of 38 Swedish graffiti cleaners using cleaning agents containing NMP among other solvents found urinary 5HNMP levels ranging from 0.03 to 18.3 mmol mol − 1 creatinine) and suggested that dermal exposure played a significant part in the exposure of the workers (Anundi et al., 2000). The work published here looks at short-term dermal exposure to aqueous solutions of NMP. This approach was used to investigate the feasibility of using a relatively low toxicity substance, such as NMP, which is readily absorbed through the skin, as a safe model substance, to assess the potential dermal absorption of more hazardous substances during a variety of work-tasks. The volunteer study reported here was approved by the Research Ethics Committee of the Health and Safety Executive (ETHCOM/REG/99/09) and was sponsored by the Health and Safety Executive.

2. Method Methanol and ethyl acetate (HPLC Grade) were provided by Rathburn Chemicals, Walkerburn, UK, and NMP and bis(trimethylsilyl)trifluoroacetamide) with 1% trimethylchlorosilane (BSTFA+ 1% TMCS) by Aldrich Chemical Company, Gillingham, UK. 5-HNMP was synthesised in two batches by Ultrafine Chemicals, Manchester, UK and Key Organics, Camelford, UK and the internal standard deuterated 5-hydroxy-N-methyl pyrrolidone (d4-5HNMP) (94%) was kindly given by Dr Bengt Akesson, University Hospital, Lund, Sweden. Double distilled water was prepared in house using a Milli-Q system (Waters). The end-capped C8 Isolute extraction cartridges (3 ml, 500 mg) were provided by IST, Hengoed, UK and the GC column (HP-5, 30 m×0.32 mm, 1 mm film thickness) by Hewlett Packard.

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2.1. Exposure study Two volunteers were exposed to a 5% solution of NMP for 5, 10 and 15 min, then to 10, 15, 20 and 25% solutions for 15 min. This involved them placing one hand up to the wrist into a beaker containing the NMP solution for the prescribed time. The volunteers wore half-face organic vapour cartridge masks and were situated in a room maintained at negative pressure to remove any possibility of inhalation exposure to the NMP. Following exposure the volunteers patted dry their hands with a paper towel.

2.2. Urine collection and analysis The volunteers gave a urine sample immediately prior to the exposure and then collected complete voidings at regular intervals for up to 48 h following the start of exposure. The analysis of urine samples from the study followed a method reported by Jo¨ nsson and A, kesson (1997), which briefly comprised: conditioning of the C8 solid phase extraction cartridges with methanol and distilled water; addition of 1 ml of sample urine containing d4-5-HNMP as internal standard; washing with distilled water and elution with an 80:20 mixture of ethyl acetate and methanol. The published method was modified to use a Gilson ASPEC XL automated solid phase extraction apparatus (Anachem, Luton, UK) for the extraction process. The eluent was then evaporated to dryness under a stream of nitrogen before the addition of BSTFA +1% TMCS for derivatisation at 100 °C for 1 h. After ethyl acetate had been added to the derivatising reagent, which was then transferred to a GC vial, the sample was injected into a HP 5890 GC with MS detection (Hewlett Packard, UK). The GC oven (which contained a 30 m ×0.32 mm HP-5 column with a film thickness of 1 mm) was set at 70 °C for 1 min, then the temperature was increased by 15 °C min − 1 to 200 °C where it remained for a further minute. The injection port temperature was 250 °C; solvent delay set at 4 min; transfer line temperature 280 °C; ionisation mode, EI+ at 70 eV; source temperature 200 °C; quad temperature 100 °C; column head pressure 20 kPa. The ions monitored

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with dwell time 100 ms were m/z 98 for 5-HNMP and 102 for d4-5-HNMP.

3. Results Fig. 1 shows the urine concentration against time for both volunteers for exposures to 5–25% NMP for 15 min. The absolute urine concentration is shown in graph A and the creatinine corrected concentration is shown in graph B. Urine concentrations were at a maximum about 10 h following exposure and 5-HNMP excretion continued for 48 h following exposure. From Fig. 1 it can be clearly seen that creatinine correction gives a far smoother excretion profile, and clearer

Fig. 1. Mean urine concentration of 5-HNMP for two volunteers, against time following start of exposure. Each point is the mean concentration for two volunteers of duplicate determinations and plotted at the mid point of the sample collection period. Graph A shows absolute concentration values and graph B shows creatinine corrected concentration values.

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Table 1 Total excretion of 5-HNMP (mmol) for each exposure Exposure indexa

2 3 6 9 12 15

Total excretion (mmoles) Vol 01

Vol 02

44 53 129 225 286 383

– 58 126 342 270 526

a The exposure index (EI) is expressed as a product of the NMP concentration and exposure duration, where a 5 min exposure to 5% NMP solution has an EI of 1, 10 min at 5% EI is 2, etc.

differences in 5-HNMP urine concentration between exposures at different NMP concentrations. The total cumulative excretion of 5-HNMP for both volunteers can be seen in Table 1. No 5HNMP was detected for volunteers following exposure to 5% NMP for 5 min. A regular increase in excretion can be seen as the exposure increases. The correlation is good between total excretion and exposure particularly for volunteer 2. (R = 0.9940). The overall correlation between total excretion and exposure has a coefficient of R = 0.9297.

4. Discussion and conclusion The results exhibited in Fig. 1 show that correc-

Table 2 Determination coefficient (R 2) of urinary 5-HNMP as a function of exposure index Time following exposure (h)

R2

2 4 6 8 10 12 20

0.93 0.95 0.98 0.97 0.90 0.94 0.95

Fig. 2. 5-HNMP excretion profiles following 8 h inhalation exposure to 10 mg m − 3 NMP and 15 min dermal exposure of one hand to 15% NMP (0 h is start of exposure). Data points for the inhalation are the mean of six volunteers (A, kesson and Jo¨ nsson, 2000) and for the dermal exposure the mean of two volunteers.

tion of the concentration results for creatinine is necessary to obtain results, which can be more easily related to exposure dose, creatinine correction of the results also gave better agreement between the results of both volunteers. Table 2 gives a summary of correlations between creatinine corrected urinary 5-HNMP concentrations and exposure dose for different time points following exposure. From Table 2 it can be seen that urinary levels and dose correlate well over the entire sampling time. The continued increase in urinary 5-HNMP concentration in the urine for 10 h following the start of exposure is surprising. This could be caused by a slow release of NMP from the skin into the body, or a slow metabolism of NMP to its 5-HNMP metabolite. The similarity between the excretion profiles obtained after a 15 min dermal exposure to 15% NMP in this study and during and after a study of an 8 h inhalation exposure at 10 mg m − 3 conducted by A, kesson and Jo¨ nsson (2000) would suggest the former to be the case (see Fig. 2), with the apparent 15 min dermal exposure being like an 8 h inhalation exposure. Probably because NMP absorbed into the skin during the immersion was being released slowly into the body over the following 8 h. The estimated half-life from the data obtained from the two volunteers for the exposures of 15

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min is 10.6 h this compares to 7.3 h as reported by A, kesson and Jo¨ nsson (2000) for an 8 h inhalation exposure. This together with the delayed maximum urine concentration would suggest a biological monitoring strategy for dermal exposure to NMP solutions based on a preshift next day urine collection. The results also raise the issue of the effects of multiple or repeated dermal exposure and whether they may be additive. A good dose-response relationship for long NMP inhalation exposures has been demonstrated previously by A, kesson and Jo¨ nsson (2000). The work reported here shows a good dose-response relationship for very short dermal exposures to NMP solutions, reinforcing the fact that NMP is very easily absorbed through the skin. The main purpose of this pilot study was to investigate the feasibility of using a relatively low toxicity substance, such as NMP, which is readily absorbed through the skin, as a safe model substance, to assess the potential dermal absorption of more hazardous substances during a variety of work-tasks. This will be done by using the NMP solution whilst performing various cleaning type tasks and then measuring urinary levels of NMP metabolite to assess dermal uptake of NMP during these tasks. The results show that NMP would be suitable for such a purpose since the NMP absorbed from the solutions used has been easily detected following immersion for 15 min of a full hand. The good dose-response relationship obtained in this pilot study suggests that analysing urinary 5-HNMP concentrations following the use of a 25% NMP solution during a variety of tasks would be a valid way of ranking the tasks in order of exposure.

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