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British Journal of Industrial Medicine 1984;41:533-538 n-Hexane metabolism in occupationally exposed workers A MUTTI,' M FALZOI,' S LUCERTINI,' G ARFINI,' MAGDA ZIGNANI,2 S LOMBARDI,2 AND I FRANCHINI' From the Chair ofOccupational Medicine ofthe University ofParma, ' Parma I-43100, and the Occupational Health Service,2 USL 38, Forli 1-47100, Italy ABSTRACr Lung uptake and excretion of n-hexane were studied in ten workers in a shoe factory. Simultaneous samples of inhaled and alveolar air were collected with the aid of a Rhan-Otis valve, personal samplers, and charcoal tubes. Alveolar excretion was monitored during a six hour postexposure period. Uptake was calculated from lung ventilation, the retention coefficient, and environmental concentrations. The amount of exhaled n-hexane was calculated from the decay curve. According to the experimental data, alveolar retention was about 25% of the inhaled n-hexane, corresponding to a lung uptake of about 17%. The postexposure alveolar excretion was about 10% of the total uptake. The main metabolites of n-hexane were identified and measured by capillary GC/MS in spot urine samples collected before, at the end, and 15 hours after the same working shift. Urinary concentrations were low, though related to n-hexane in the air. 2,5-Hexanedione in the end of shift samples gave the best estimate of overall exposure. About 3 mg/g creatinine of 2,5-hexanedione would correspond to about 50 ppm of n-hexane in the air (mean daily exposure). The use of n-hexane is widespread in industry. It is an excellent and inexpensive solvent used especially in glues, varnishes, and paints. Many mixtures are commercially available, and the most common among them contain different proportions of n-hexane and its isomers 2-methyl and 3-methyl pentane, cyclohexane, and methyl ethyl ketone. Occupational exposures have been reported to cause peripheral neuropathies in workers'-3; electrophysiological changes have also been found in subjects exposed to relatively low airborne concentrations.48 The pharmacokinetics of n-hexane has been widely investigated in rats but much less is known for man. Among the published studies there is little agreement about the retention of inhaled n-hexane and about the proportion which is excreted through the lung during the postexposure period. Urinary metabolites of n-hexane in man have been shown by Perbellini et aP after occupational exposure to solvent mixtures containing n-hexane. The present study was undertaken to investigate Received 1 September 1983 Accepted 26 September 1983 the metabolism of n-hexane in man, with the aim of evaluating more specifically: (a) the respiratory uptake and elimination in workers occupationally exposed under constant model ventilation conditions; (b) the possible relation between n-hexane uptake and urinary excretion of its metabolites; and (c) the usefulness of either alveolar concentration or urinary excretion of n-hexane metabolites for biological monitoring purposes. Method The study was performed on 10 young healthy subjects aged 18-30 years who continued their job during the experiment. SAMPLING n-Hexane concentrations in the breathing zone were monitored with personal samplers; inhaled and alveolar air were sampled with the aid of a RhanOtis valve (commercially available from PK Morgan, Chatham, UK). The samples were collected simultaneously for 5 min with two pumps, one for the inhaled air and the other for the last 100 ml of the tidal volume, which was taken as alveolar air. 533 534 Mutti, Falzoi, Lucertini, Arfini, Zignani, Lombardi, and Franchini Exposure period Postexposure period t- Inhaled and alveolar air sampling I: Alveolar air sampling J9 ItI I 0 I J 09v-VTT-. *r07v *- I - -(-v,Y*) (T(v 1* v\) A B-Breathing zone air sampling U1 U2 U3 9 8 10 am . i2 2 pm lbI I I I I 4 68 Time (h) 12 8 am Fig 1 Experimental design: dots = sampling ofalveolar and inhaled air; squares = sampling ofalveolar air during postexposure period; squares in brackets = samples available for only two subjects. Distance between each double arrow indicates sampling time ofenvironmental air in breathing zone. U = spot urine samples collected before, at end, and 15 hours after same working shift. The total sampled volume was about one litre at a flow rate of 200 mVmin. Vapours were absorbed on NIOSH approved charcoal tubes interposed between the valve and the pumps. Urinary samples were taken before, at the end, and 15 hours after exposure. The experimental design is summarised in fig 1. During each alveolar air sampling, pulmonary ventilation was measured by means of a volumetric counter (SIM Brunt AB) connected to the Rhan-Otis valve. ANALYSIS After desorption with CS2, gas chromatographic analysis was performed using a Perkin Elmer Sigma 3B gas chromatograph equipped with an FID and a silicone OV-101 Column 50 m x 0-25 mm (id). Helium (1 mlmin) was used as the carrier gas. The column temperature was 40C and the injector and detector temperatures were 160C and 200C respectively. Urinary metabolites of n-hexane were measured using a slightly modified version of the method of Perbellini et al.'0 A silica capillary column (25 m x 0-25 mm id) was used, coated with Carbowax 20M. The column temperature was programmed after 2 min isothermal operation from 40C to 150C at a rate of 20C per min and the final temperature was maintained for 5 min. The injector temperature was 200C, the detector temperature 2500C, and the flow rate 1 ml helium per minute. Particular interest was focused on the following metabolites: 2 and 3-hexanol, 2,5-hexanediol, methyl n-butyl ketone, 2,5 hexanedione, y-valerolactone, and 2,5-dimethylfuran. Authentic samples of each substance were obtained from Fluka AG (Buchs, Switzerland). The urinary metabolites were identified on the basis of gas chromatographic retention time, and their mass spectra were com- pared with those of authentic samples, using a Finnigan MAT 1020 mass spectrometer (Finnigan MAT, Cincinnati, OH, USA). EQUATIONS Intake, uptake, and alveolar excretion were calculated according to the following equations: R I1 C_Ca (1) I= k'(-rwA)Vet D = kCi(WA)VaRt (2) (3) E = KVa Ctdt to (4) where Ca = alveolar concentration of n-hexane (ppm); C = inhaled concentration of n-hexane (ppm); (A) = time weighted average of breathing zone levels of n-hexane (ppm); R = pulmonary retention coefficient; Ve = respiratory minute volume (1/min); Va = alveolar ventilation, calculated according to Astrand" (1/min); t = time (min); I = total respiratory intake (mg); D = alveolar uptake or dose (mg); E = alveolar excretion (mg); and k = factor converting ppm to mg/l (3.5 x 10-3). Results Technical grade hexane is a mixture containing variable proportions of n-hexane, of its isomers, and of n-Hexane metabolism in occupationally exposed workers 535 Table 1 Concentration oforganic solvents in the air (8h- TWA) Solvent 8h-TWA* air concentration (mg/M3) Hygienict effect Median Range Median Range n-Hexane 2-Methyl pentane 3-Methyl pentane Cyclohexane n-Heptane 243 133 100 18 65 8-1143 5-685 4-514 0-892 0-136 1-34 0-07 0 05 0-02 0-04 0-04-637 0-0-38 0-0-28 0-085 0-008 *8h-TWA = Time weighted average. tHygienic effect is the ratio between the measured concentration of the compound and its threshold limit value. When specific information about synergism does not exist, the sum of the ratio does not exceed 1-0, which is the threshold limit value for the mixture. Annn -. ._c 2 QD 1500- 0 EJ Intake = 661 mg m Uptake =112mg .Alveolar excretion from to to t., =12 6mg ._ la c. 0 500 Intake Intake __-- ~ a 10 12 2 14 6 8 10 am pm Time (h) Fig 2 Time course of total respiratory intake and absorption ofn-hexane during eight hours of exposure (left) and ofalveolar excretion during the postexposure period (right) in a worker exposed to 243 mg/m3 ofn-hexane in air (mean daily exposure). other solvents. The main components of the mixture used by our study subjects and their 8 h time weighted average (TWA) concentrations in the air during the study are summarised in table 1. The solvent concentrations showed a wide scatter, the exposure to n-hexane ranging from a few to more than 1000 mg/iM3. Figure 2 shows the time course of n-hexane intake and uptake during exposure in a study subject and that of alveolar excretion during the postexposure period. From the difference between alveolar uptake and excretion it seems reasonable to con- clude that a considerable amount of absorbed n-hexane was either retained or metabolised. In fig 3 the time course of n-hexane alveolar excretion is shown in the same subject as in fig 2. During the postexposure period the alveolar excretion of unchanged n-hexane accounted for about 10% of the total uptake. Elimination from the lungs was biphasic, the median half lives of the fast and slow phases being 11 min and 99 min, respectively. The results from the individual experiments are 536 Mutti, Falzoi, Lucertini, Arfini, Zignani, Lombardi, and Franchini Io. 3I I I 9x Cnv=30-12eO006't, 0-82eO00007t y = -0-481. 0.014x r = 0.967 P<0.001 8 I _. iC !5C 0.1 0 250 500 750 1000 1250 I n- Hexane in air, 8h-TWA, mg/m3 Fig 4 Relation between TWA concentration ofn-hexane in air and 2 5 hexanedione in end ofshift urine samples. 0 360 Postexposure time (min) Fig 3 Time course ofalveolar concentrations (ppm) of n-hexane during postexposure period in same subject as in fig 2. Measured values were plotted versus time both on lineaj!r (continuous line) and on lin-log scale (dashed line). Experimental curve was obtained by extrapolation to zero time. Fast phase (first hour) was calculated by subtracting slower component at each sampling period. 3 a .4',, ~~~~~~145 2 t.~~~~~~~~~~~~~~~L summarised in table 2. The difference in the inhaled and alveolar concentrations indicates that about 25% of alveolar n-hexane is taken up by the blood. Absorption in relation to the total respiratory intake is only 17%, taking into account both the retention coefficient and alveolar ventilation. It should be emphasised that the physical load while working was Table 2 Absorption and alveolar excretion ofn-hexane assuming a mean ventilation of8 llmin Parameters Intake (mg) Retention (%) Uptake (mg) Excretion fmg) Half lives min): Fast phase Slow phase Median 972 252 166 14-7 11 99 Range (32-4572) (221-28-7) (9-1146) (1-106) Fig 5 Typical gas chromatogram ofone end ofshift spot urine sample. 1: 2,5 dimethylfuran, 2: 2-hexanol, 3: cyclohexanone internal standard, 4: 2,5 hexanedione, 5: y-valerolactone. slight, the average ventilation values being close to typical "at rest" values, 7-2 /min. Our results, therefore, do not take into account the possible effect of heavy workload levels. Despite the large amount of n-hexane that was either retained or metabolised during exposure, the recovery of metabolites in urine was low (table 3), even if an accurate estimation is not possible, since spot samples only were available. Assuming a urinary excretion of about 2 g of creatinine a day, however, only about 20-25 mg of n-hexane metabolites were excreted when the Table 3 Urinary excretion on n-hexane metabolites (mmollmol creatinine) n-Hexane metabolites 2,5-Dimethylfuran 2-Hexanol 2,5-Hexanedione y-Valerolactone Preshift Median 0-4 0-2 0-6 0-8 Range <0-01-5-2 <0-1-2-4 <0-1-10-7 <0-1-7-5 End ofshift Median 2-6 0-5 35 2-9 Range <0-1-15-1 <0 1-5-3 <0-1-17-9 0-1-16-4 Next morning Median 1-8 0-3 2-4 2-0 Range <0-1-8-1 <0-1-4-3 <0-1-15-0 <0-1-10-5 n-Hexane metabolism in occupationally exposed workers 537 median uptake was 166 mg during the whole working shift. The main urinary metabolite appeared to be 2,5hexanedione, particularly in the end of shift sample. A good relation was found between exposure to n-hexane, as measured by its TWA concentration in the air, and the urinary excretion of 2,5hexanedione at the end of the working day (fig 4). Nevertheless the scattergram suggests that the measurement of 2,5-hexanedione in the urine is not sensitive enough to detect exposures below 50 mg/ mi3. Figure 5 shows a typical chromatogram of one end of shift urine spot sample. It can be seen that the identification of each metabolite and the interpretation of analytical results is a difficult task. Discussion formation of a given toxic chemical. Nevertheless, practical reasons should also be considered when implementing a biological monitoring programme. When evaluating the usefulness of a biological indicator for monitoring purposes these reasons might represent a serious drawback in terms of analytical difficulties and data interpretation. As a result, a great difference may occur between statistically significant and biologically significant relationships between exposure and biological indicators. We are very grateful to Mr Elio Rosa, Centro Misure UniversitA di Parma, for his help in mass spectra analysis. This work was supported by Consiglio Nazionale delle Ricerche, Progetto Finalizzato Medicina Preventiva e Riabilitativa, 82.02175.56/ 115.13134. A direct comparison with other field and experimen- tal studies is not possible because of the differences in the methods used. There is, however, a reason- able agreement among the alveolar retention coefficients reported by Brugnone et al,2 by Veulemans et al,3 and that found in the present study. In contrast to the retention data ranging from 0*164 to 02522 reported above a value of 0056 was found by Nomiyama et al.'4 In agreement with the experimental study of Veulemans et al,'3 we found that the postexposure alveo- lar excretion was biphasic and the half lives were similar to those of Veulemans despite the different exposure levels. According to the same authors, the short half life of the fast phase would cause a high variability in the measurements of the concentration of n-hexane in breath samples taken at specified time intervals. As a result, the alveolar concentration does not give better information about the absorbed dose during the whole exposure period than the measurement of the environmental con- centration. As shown earlier by Iwata et al'5 and Perbellini et al'6 a close relation was found between the mean daily exposure to n-hexane and the uri- nary excretion of 2,5-hexanedione in the end of shift sample. In the present study individual exposure was accurately defined and covered a wide range (2317 ppm); regression was linear over this range. Nevertheless, at least three false negative urines were found at exposure levels lower than 50 mg/m3, or about one third of the current TLV. Furthermore, for practical reasons such an indicator cannot be recommended for the routine biological monitoring of occupationally exposed workers. Biological monitoring permits an accurate estimation of the absorbed dose, whatever the absorption route, the physical work load, and the changes in the biotrans- References 'Hersekovitz A, Ishii N, Schaumburg HH. n-Hexane neuropathy. A syndrome occurring as a result of industrial exposure. N Engl J Med 1971;285:82-5. Sobue I, Mitsuo I, Yamamura Y, Takanagui T. n-Hexane polyneuropathy. Int J Neurol 1978; 11:317-30. 3 Cianchetti C, Abbritti G, Perticoni G, Siracusa A, Curradi A. Toxic polyneuropathy of shoe industry workers. A study of 122 cases. J Neurol Neurosurg Psychiatry 1976;39 1151-61. 4Buiatti E, Cecchini S, Ronchi 0, Dolara P, Bulgareili G. Relationship between clinical and electromyographic findings and exposure to solvents in shoe and leather workers. Br J Ind Med 1978;35: 168-73. sMutti A, Cavatorta A, Lucertini S, Arfini G, Falzoi M, Franchini I. Neurophysiological changes in workers exposed to organic solvents in a shoe factory. Scand J Work Environ Health 1982;8(suppl 1):136-41. 6 Mutti A, Ferri F, Lommi G, Lotta S, Lucertini 5, Franchini I. n-Hexane induced changes in nerve conduction velocities and somatosensory evoked potentials. Int Arch Occup Environ Health 1982;51:45-54. Sanagi S, Seki Y, Sugimoto K, Hirata M. Peripheral nervous system functions of workers exposed to n-hexane at a low level. Int Arch Occup Environ Health 1978;47:69-79. 8Seppailinen AM, Raitta C, Hunskonen MS. n-Hexane induced changes in visual evoked potentials and electroretinograms of industrial workers. Electroencephalogr Clin Neurophysiol 1979;47:492-8. 9 Perbellini L, Brugnone F, Pavan I. Identification of the metabolites of n-hexane, cyclohexane and their isomers in man's urine. Toxicol Appl Pharmacol 1980;53: 220-9. Perbeilini L, Brugnone F, Pastorello G, Grigolini L. Urinary excretion of n-hexane metabolites in rats and humans. Int Arch Occup Environ Health 1979;42:349-54. " Astrand I, Engstrom J, Ovrum P. Exposure to xylene and ethylbenzene. I Uptake, distribution and elimination in man. Scand J Work Environ Health 1978;4: 185-94. 12 Brugnone F, Perbellini L, Grigolini L, Apostoli P. Solvent exposure in a shoe upper factory. I n-Hexane and acetone concentration in alveolar and environmental air and in blood. Int Arch Occup Environ Health 1978;42:51-62. 13 Veulemans H, Van Vlem E, Janssen H, Masschelein R, Leplat A. Experimental human exposure to n-Hexane. Study of the 538 Mutti, Falzoi, Lucertini, Arfini, Zignani, Lombardi, and Franchini respiratory uptake and elimination, and of n-hexane concentrations in peripheral venous blood. Int Arch Occup Environ Health 1982;49:251-63. 14Nomyama K, Nomyama H. Respiratory retention, uptake and excretion of organic solvent in man. Benzene, toluene, n-hexane, trichloroethylene, acetone, ethylacetate and ethylalcohol. Internationale Archiv fPr Arbeitzmedizin 1974;32: 75-83. 5 Iwata M, Takeuchi Y, Hisanaga N, Ono Y. A study on biological monitoring of n-hexane exposure. Int Arch Occup Environ Health 1983;51:253-60. 16 Perbellini L, Brugnone.F, Faggionato G. Urinary excretion of the metabolites of n-hexane and its isomers during occupational exposure. Br J Ind Med 1981;38:20-6. Notices International Symposium on Hexachlorobenzene (HCB), Lyon, France, 24-28 June 1985 5th International Conference on Heavy Metals in the Environment, Athens, 10-13 September 1985 Themes for the Athens conference are environmental pathways, fate and effects, exposure and health effects, disposal, control, and legislation with emphasis on: marine and estuarine environments; fate of heavy metals in sludge treatment/disposal and problems with dredged materials; aquatic chemistry; atmospheric particulates and dispersion; microbial interactions and availability; speciation-in soil, water, and organisms; health effects-case studies, specific effects-neurotoxic, immunological, renal, human studies of combined exposures, remobilisation, and excretion; metal cycling and effects on forest ecosystemsevolution and mechanisms of metal tolerance, ecosystems as sinks for heavy metals; detoxification of soils and water by plants; control strategies and legislation; and clean technology in metal processing industries. Papers for the conference will be welcome, and authors should submit an abstract of 200-300 words to the secretariat before 1 February 1985. Further information is available from Heavy Metals Secretariat, CEP Consultants Ltd, 26 Albany Street, Edinburgh EH1 3QH, UK. (Tel: 031 557 2478). The symposium is jointly sponsored by the International Agency for Research on Cancer, World Health Organisation, International Programme on Chemical Safety WHO-UNEP-ILO, US Environmental Protection Agency, US National Cancer Institute, and Commission of the European Communities, Health and Safety Directorate, Luxembourg. The sponsoring organisations are concerned about the hazards associated with exposures to HCB. The major aim of the symposium is to provide an up to date evaluation of both known exposures and biological effects of HCB. In particular, the symposium will focus on the following objectives: presentation of available data on HCB and its impact on human health and the environment-sources, distribution, exposures, and effects; comparability of HCB toxicity to that of other environmental chemicals of similar structure and toxicity; and recommended future research and risk management options. For further details contact the symposium secretariat: Dr JRP Cabral, IARC, 150 cours Albert Thomas, 69372 Lyon Cedex 08, France. Tel: (7)875-81-81, telex: 380023 F. 1st Intemational Conference on Environmental Contamination, London, 10-13 July 1984 Impact of Biotechnlogy on Diagnstics, Rome, 16-18 April 1985 At this international symposium the list of topics will be: production and genetic manipulation of monoclonal antibodies, tumor detection by immunocytochemical and radioimmunometric methods, recombinant DNA and hybridoma technology, DNA probes in diagnostics, biotechnology in animal and human vaccines, and antibodies to synthetic antigenic determinants. Further details available from the organising secretariat at Fondazione Giovanni Lorenzini, Via Monte Napoleone, 23-20121 Milan, Italy. Tel: (02) 783868-702267, Cable: Lorenzfound. The published proceedings of the first conference are now available. The proceedings contain over 150 papers from 23 countries covering: environmental asbestos; organic and inorganic contaminants in agriculture; contaminants in drinking water, river waters, stormwaters and sediments; contaminants in wastewaters and sludges; hazardous and toxic wastes; airborne contaminants; land contamination and reclamation; marine contaminants; and radioactive wastes. Published by CEP Consultants Ltd, 26 Albany Street, Edinburgh EH1 3QH, UK. Price (inc p&p) UK: 50-00, Americas: US$75.00, Elsewhere: 55-00. Available direct from the publishers CEP Consultants Ltd. Payment is required with order.