British Journal of Industrial Medicine 1993;50:1 111-1118

Biological monitoring of occupational exposure totoluene diisocyanate: measurement oftoluenediamine in hydrolysed urine and plasma bygas chromatography-mass spectrometry

Pemilla Persson, Marianne Dalene, Gunnar Skarping, Mikael Adamsson, Lars Hagmar

AbstractExposure to toluene diisocyanate (TDI) wasstudied during 48 hours and biologicalsamples from nine subjects were taken in afactory producing flexible polyurethane(PUR) foam. Five PUR workers, two whitecollar workers, and two volunteers werestudied. The concentrations of TDI in airwere determined by high performance liquidchromatography with the 9-(N-methyl-aminomethyl)-anthracene reagent. Urine andplasma samples were collected and the TDIrelated amines, 2,4-toluenediamine (2,4-TDA) and 2,6-toluenediamine (2,6-TDA),were determined (after hydrolysis) as penta-fluoropropionic anhydride (PFPA) derivativesby capillary gas chromatography-mass spec-trometry (GC-MS) with selected ion monitor-ing (SIM) in the negative chemical ionisationmode. The concentration of TDI in air was1%/o10% of the Swedish threshold limit value(TLV) of 40 ,ugIm3. The ratio between 2,4-TDIand 2,6-TDI varied in the air samples in therange of 60%:40%/9-5%:95%. Calibration plotsfor human urine spiked with 2,6-TDA and 2,4-TDA in the range of 0-2-12 ugln were producedon eight different occasions during five weeks.The SDS of the calibration plot slopes (n = 8)were less than 4%. Urine and blood sampleswere taken on six occasions for eight of thestudied subjects and on four occasions for onesubject during a two day period. The five malePUR workers showed the highest average uri-nary elimination rate of TDA. Two PURworkers and the two white collar workers hadan elimination rate of 20-70 ng on average for

the sum of 2,6-TDA and 2,4-TDA per hourand three PUR workers had an average of100-300 ng TDA per hour. The eliminationrate curves for all the studied subjects had alinear relation with exposure to TDI.The concentrations of 2,4-TDA and 2,6-

TDA in plasma for the PUR factory employ-ees were virtually stable. No relation betweenthe elimination rates of TDA in urine andplasma concentrations of TDA was found.The five PUR workers showed plasma concen-trations of the sum of 2,4-TDA and 2,6-TDAin the range 1-8 ng per ml. The two whitecollar workers, present only on occasions inthe factory, had 0-2-1 ng TDA per ml plasma.The two volunteers showed an increasing con-centration ofTDA in plasma with time. At theend of the study their plasma concentrationswere 0-6 ng/ml and 0-2 nglml plasma. Threesubjects had the same concentration of thetwo TDA isomers in plasma, two subjects hadabout double, and two subjects had 12 timeshigher concentrations of 2,6-TDA than 2,4-TDA. The presented study indicates that it ispossible to monitor exposure to TDI by moni-toring plasma concentrations ofTDA.

(British Journal of Industrial Medicine 1993;50: 1111-1118)

Toluene diisocyanates (2,4-TDI and 2,6-TDI) areimportant industrial chemicals in the manufactureof polyurethane (PUR) foams, paints, coatings, andelastomers.' They are commercially available as anisomer mixture containing 80:20 or 65:35 of2,4-TDI and 2,6-TDI. A relative enrichment ofthe 2,6-isomer has been found in industrialatmospheres.2

Exposure to TDI may cause irritation of themucous membranes and a progressive impairmentof pulmonary function.3 It has sensitising propertiesand may cause allergic asthma.4 Further, commer-cial grade TDI is carcinogenic to animals.5 Noincreased overall incidence of cancer was, however,found among workers from the Swedish

Department of Occupational and EnvironmentalMedicine, University Hospital S-221 85 LundSwedenP Persson, M Dalene, G Skarping, M Adamsson,L Hagmar

1111

Persson, Dalene, Skarping, Adamsson, Hagmar

polyurethane foam manufacturing industry.6 Anon-significant increased risk of non-Hodgkin'slymphoma and rectal cancer was found but it couldnot be conclusively associated with exposure toTDI. Kennedy and Brown have recently reviewedisocyanates found in the work environment andtoxicity and illnesses caused, together with experi-mental approaches, biological mechanisms, and theformation of protein TDI adducts.7

Several methods for the determination of iso-cyanates in air have been described.8-'0 Detectionlimits for these methods are much lower than thethreshold limit values. Short term exposure controlis therefore possible. In Sweden a 5 minute TLVhas been applied since 1984. Air monitoring, whichis the present available method for assessing occu-pational exposure to TDI, has several dis-advantages. In practice, measuring the airconcentration of isocyanates with time during aworkday requires many samples and is costly.Measuring random peak exposure is therefore notfeasible in the work environment. Also the methoddoes not take dermal absorption into consideration.This is a disadvantage regarding the potential riskof cancer and also asthma as it has been shown byKarol et al that dermally absorbed TDI may beresponsible for pulmonary hypersensitivity to TDII."

Diisocyanates are highly reactive with manycompounds containing active hydrogen-for exam-ple, carboxyl and amino groups in proteins.'2 13 It istherefore likely that TDI reacts with the tissue itinitially reaches rather than being absorbed anddistributed throughout the body as unchangedcompounds.' In an inhalation study carried outwith radioactively labelled TDI in guinea pigs,'4 theradioactivity in the blood seemed to be irreversiblyattached to a specific plasma component, a proteinwith a molecular weight of 71 000 Da; this findingindicates the possibility of an extremely specificinteraction between proteins and TDI. Kennedy etal have shown that the rate of uptake of TDI intothe blood of guinea pigs is linear during exposure. 5

Rosenberg et al have studied exposure of workersto toluene diisocyanate monomer in a factory pro-ducing flexible polyurethane.'6The aim of our study was to develop a method

for the biological monitoring of occupational expo-sure to TDI in urine and plasma samples fromPUR workers.

Material and methodsAPPARATUSA VG-Quattro triple quadrupole mass spectrom-eter (Fisons Instruments, VG-Biotech, Altringham,Cheshire, England) connected to a Carlo-ErbaMega gas chromatograph equipped with an A200Sauto sampler (Fisons Instruments, Carlo-Erba,Milan, Italy) was used with a splitless injection

technique. The injector temperature was 290°C,and the starting temperature of the column ovenwas isothermal at 1100C for one minute, thenraised at 15°C/min to 2800C where it was kept fortwo minutes. The split exit valve was kept closedfor one minute after injection. The temperature ofthe ion source was 2300C and the gas chromato-graphy mass-spectrometry (GC-MS) interfacetemperature was 280°C. The capillary inlet pres-sure of helium was 0-8 kg/mi. The solvent delaywas set to 2-9 minutes. The instrument was used inthe chemical ionisation mode with negative ionmonitoring and ammonia as the reagent gas. Whendetermining pentafluoropropionic anhydride deriv-atives of TDA the ions monitored were m/z = 394and 374 corresponding to the molecular weight(M)-20 and the M-40 ions and the m/z = 397 and377 ions of the 3H labelled internal standard. Thedwell time for each of the ions was 0-2 s and theinterscan delay was 0-02 s. The pressure in the ionsource was, in the case of chemical ionisation withammonia and negative ion monitoring, kept atabout 2 x 10-4 mBar. The emission current was200 mV and the electron energy 60 eV. The tuningof the instrument was optimised with nonafluoro-tributylamine as a calibrant.The high performance liquid chromatography

(HPLC) equipment consisted of a Waters 600multisolvent delivery system (Millipore-Waters,Millford, MA, USA), a Waters 712 WISP withvariable injection volume, and a Waters 490 UWdetector. For air sampling, SKC model 224-36pumps (SKC Inc, Eighty Four, Pa, USA) and30 ml midget impingers containing 10 ml absorp-tion solution were used. Temperature and relativehumidity were measured with a Solomat MPM2000 thermohygrometer (Solomat Corp,Rowayton, USA).A Sigma 3E-1 centrifuge (Sigma Hartz,

Germany) was used for phase separation in thework up procedure. The samples were evaporatedin a Speed-Vac 290 centrifuge (Savant,Farmingdale, NY, USA).

COLUMNSFused silica capillary columns 30 m x 0-24 mminternal diameter with chemically bound stationaryphases (DB-5 J and W Scientific, Folsom, CA,USA) with a film thickness of 0-25,m were usedfor the GC-MS determinations. Columns forHPLC were of stainless steel (200 mm, 5 mm ID),packed with 5,im Nucleosil C,6 (Macherey-Nageland Co, Duren, Germany).

CHEMICALS2,4-TDA and 2,6-TDA was obtained from Fluka(Buchs, Switzerland); 2,4-TDI and 2,6-TDI(80:20), triethylamine, HCl, NaOH, and K,HPO4,

1112

Biological monitoring of occupational exposure to toluene diisocyanate

from E Merck (Darmstadt, Germany); pentafluoro-propionic anhydride (PFPA), from Pierce(Rockford, II, USA); acetonitrile and toluene, fromLab-Scan (Dublin, Ireland); and trideuterated-2,4-TDA and 2,6-TDA (CD,C6H,(NH2)2) and 9-(N-methylaminomethyl)-anthracene hydrochloride(MAMA) from Synthelec (Lund, Sweden).

PROCEDUREPreparation of standard solutionsStandard solutions of the 2,4-TDA and 2,6-TDAisomers were prepared by dissolving accuratelyweighed amounts in 0-1 M hydrochloric acid. The

solutions were further diluted to the appropriateconcentrations.

Sampling and analysis of TDI in airFor sampling of TDI, we used 30 ml midgetimpinger flasks containing 10 ml of 1 x 104 Mamine reagent 9-(N-methylaminomethyl)-anthra-cene (MAMA) in toluene. The TDI reacted duringsampling with the amine reagent in the impingerflask to form urea derivatives. Different volumes ofair were drawn through the absorption reagents at a

speed of 1 1/min. The samples were kept in a

refrigerator until analysis. The toluene solution wasevaporated to dryness with an evaporation cen-

trifuge. The urea derivatives were dissolved in 1 mlof acetonitrile-water (70:30, v/v) and analysed byHPLC with UV detection at 254 nm. The eluentacetonitrile-water was in the ratio 70:30, v/v, andthe water phase contained 3% (v/v) triethylaminephosphate (pH 3).

Sampling, handling, and storing of biological samplesAll urine produced during the study by the ninesubjects was collected in polyethylene bottles. Thesamples were acidified with 5 ml of 6 M HC1 solu-tion per 100 ml of urine and stored in a refrigeratoruntil analysis. The density, creatinine concentra-tion, pH, and total volume of the urine sampleswere determined.The urine samples were analysed after acid

hydrolysis. Urinary 2,4-TDA and 2,6-TDA excre-tion was monitored for a 48 hour period.Ten ml venous blood samples were taken on six

occasions over a two day period for eight of thesubjects and on four occasions for one subject.Plasma was separated within eight hours, trans-ferred to a new tube and kept at - 20°C untilanalysis.

All analyses were done after acid hydrolysis ofthe plasma samples. The amounts given as 2,4-TDA and 2,6-TDA therefore reflect the sum offree and hydrolysable compounds.

Samples of plasma and urine were taken on the

first morning before the 48 hours started, for analy-sis of the baseline values.

Work up procedure for biological samplesTo a 1 ml urine or plasma sample 1-5 ml of 6 MHC1, containing D, 2,6-TDA and 2,4-TDA asinternal standards each at a concentration of0-5 pg/l was added. The sample was then hydro-lysed at 1 00°C overnight. A 5 ml volume ofsaturated NaOH and 2 ml of toluene were addedand the mixture was shaken for about 10 minutesand centrifuged at 1500 g for 10 minutes. A 1-5 mlvolume of the organic layer was transferred to anew test tube and 10 ,ul of the anhydride derivatisa-tion reagent was added. The mixture was immedi-ately shaken vigorously for about 10 minutes. Theexcess of the reagent and acid formed was removedby extraction with 2 ml of 1M phosphate buffersolution (pH 7 5). A lml volume of the toluenelayer containing the amide derivative and the inter-nal standard was transferred to a 1-5 ml autosampler vial and injected into the GC-MS system.Two aliquots from each sample were worked upand two GC-MS determinations were performedfor each aliquot. The average of the four GC-MSmeasurements was used as an estimate of theconcentration.

PROCESS AND EXPOSUREThe factory produced flexible polyurethane foam ina continuous foam block. The generated foam hada cross section of 1 m x 2 m. The plant used an80:20 mixture of 2,4-TDI and 2,6-TDI. The TDImonomer, polyol, catalyst, and blowing agentswere pumped from tanks and mixed in a foamingnozzle. The material was poured on to movingkraft paper, which passed into a ventilated curingtunnel. The foam had reached its final size withinone minute; the paper lining was stripped off andthe PUR foam was cut into pieces and moved to astorage room for cooling before further handling.The production was generally based on curing oneblock in the morning and one block in the after-noon. The TDI foam blocks could be severalhundred metres long.The first day of the study was a Monday. In the

morning the workers mainly prepared the machinefor the week. In the afternoon a 330 metre blockwas produced in 75 minutes. The next morning a90 metre block was produced in about 20 minutesand in afternoon a 180 metre block was producedin 40 minutes. In the first two foam blocks pro-duced, water was used as the blowing agent andcarbon dioxide was liberated. In the production ofthe third block formic acid was used as a blowingagent and carbon monoxide was produced. Bothstationary and personal air samples were collected

1113

Persson, Dalene, Skarping, Adamsson, Hagmar

at the pouring, paper stripping, and cutting workstations during active production.

SUBJECTSBlood and urinary samples for analyses of TDAwere taken from seven employees from the plant(mean age 35 (range 24 48) years) and twovolunteers (H, I), 40 and 28 years of age. All weremen with the exception of a female laboratory tech-nician (F) working regularly 4-5 hours a day. Ofthe remaining employees five (A to E) were processoperators and one the plant manager (G). Thework was organised in one eight hour shift a day forfive days a week. Subjects H and I were mainlyinvolved in collecting air samples, taking notes,photos etc. They were present in the productionarea for about 90% of the study period and also inplaces where exposure could be expected to beconsiderable. Only subjects B and E used protec-tive devices (fresh air masks). These were used dur-ing the short period of work in the curing tunnel.

HEALTH EXAMINATIONAll nine subjects were interviewed by a physician(LH) for occupational history, smoking habits, andhealth history. Pulmonary function tests for vitalcapacity (VC) and forced expiratory volume in onesecond (FEV,) were carried out with a Vitalographspirometer, both immediately before and immedi-ately after the first work day. A skin prick test with15 common allergens (Allergologisk Laboratorium,Copenhagen, Denmark) and with a conjugate ofTDI and human serum albumin was performed onall subjects. Venous blood samples and urinarysamples were taken in the morning. A dipstix test(Labstix) was used for testing for the presence ofglucose, albumin, blood, and ketone bodies inurine. Analyses of haemoglobin in blopd and whiteblood cells were performed by routne methods.Antibodies against TDI in plasma were measuredwith techniques described in detail elsewhere.'7

ResultsEXPOSUREThe workers regularly wore personal respiratoryprotection devices only on occasions when highexposure to TDI was expected. During the study,the air concentration of TDI varied greatly. Thegeneral TDI concentration around the processmachine was 1%-10% of the Swedish TLV. Veryhigh concentrations were found in the tunnel (50times the TLV). Further exposure to TDI occurredduring infrequent work in a TDI tank room andduring the filling of a tank from a TDI transporta-tion vehicle. During the study subject A wasinvolved in this work. The TDI used had an iso-meric composition of 2,4-TDI and 2,6-TDI in aratio of 80%:20%. The ratio between 2,4-TDI and

2,6-TDI varied in the air samples in the range of60%:40%-5%:95%. The air temperature wasabout 21°C and the relative humidity was about40%. Under normal conditions, dermal exposureto TDI was probably infrequent due to the closedsystem technique. Dermal exposure to freshlymade foam and possibly partly unreacted mixtureofTDI and polyol was, however, noted.

HEALTH EXAMINATIONTwo of the process operators (A, C) and one of thetwo volunteers (I) were current smokers. Two ofthe employees wheezed when they had colds, butno work related airway symptoms. Due to an acci-dent in production one of them had been exposedto very high momentary concentrations of TDI 10years previously. This caused an episode of acuteobstructive bronchitis which lasted for severalweeks after the accident.The mean FEV1 for the seven employees as a

percentage of the expected (considering age andheight'8) was 104-4% before the work shift and105'0% after the work shift. The correspondingfigures for VC were 99 9% and 100-0%. None ofthem had any significant decrease in pulmonaryfunction by the end of the shift. None of theemployees or the two chemists had any IgE or IgGantibodies against a TDI-HSA conjugate. Noabnormalities in haemoglobin, sedimentation rate,white blood cell counts, and Labstix test werefound, except for a diabetic subject (B) who hadslight glucosuria.

PROCEDUREChromatography and mass spectrometryThe m/z = M-20 and the m/z = M-40 fragmentswere monitored by GC-MS. Urine from all nineexposed persons was analysed. For the m/z = M-20 measurements urine samples from one of theworkers showed unresolved separation of the 2,6-TDA isomer relative to the matrix. The valley tothe baseline between the peaks was about 30% ofthe peak height of the 2,6-TDA peak. This resultedin uncertain evaluation of peak areas. Whenanalysing plasma samples the chromatograms weremuch cleaner and when plasma was analysed fromthe same worker no interfering peaks were seen.When monitoring the m/z = M-40 fragment nointerfering peaks were seen for either urine orplasma samples. It is therefore recommended thatboth the m/z = M-20 and the m/z = M-40 frag-ments be monitored when performing quantitativedeterminations.

Quantification.Coefficients of variation (CVs) defined as (8(5)2 X(2n)-')1/2 x x-I (6 = the difference between theresult of two analysed aliquots, n = number of

1114

Biological monitoring of occupational exposure to toluene diisosyanate

samples, x = average of the determinations) were

calculated for the TDA measurements. The CVwas 6%-7% for both 2,6-TDA and 2,4-TDA, atlow concentrations of plasma and urine (table 1).At higher concentrations the CV decreased tolower values. There was a slightly higher CV forurinary 2,4-TDA. In the procedure two aliquotsfrom each sample were worked up and two GC-MS determinations were performed for eachaliquot and the average of the four GC-MS mea-

surements was used as an estimate of the concen-

tration. Therefore when performing the actualmethod, lower CVs than those given in the tablewould be expected. Calibration plots for humanurine spiked with 2,6-and 2,4-TDA in the range of0-2-12 jug/l were produced on eight different occa-sions over a period of five weeks. The SDs of thecalibration plot slopes (n = 8) were less than 4%.When the average from the work up procedure ofthe two aliquots from the same sample differed bymore than 40% the result was disregarded and theprocedure was repeated. This was the case in about5% of the sample determinations. The overall pre-

cision for human urine spiked to a concentration of1 pg/l was 1-6% for 2,6-TDA and 3-5% for 2,4-TDA (n = 10).

2,4-TDA and 2,6-TDA in urineIt was not possible to investigate the individualurinary elimination of TDA relative to the con-

centration of TDI in the inhalation air. This was

due to the limited number of air samples taken(n = 46). The average TDI concentration was esti-mated to be within the range 0O4-4 ,ug TDI m3.Five of the subjects (A to E) were male workersmainly involved with the TDI foam process. Theywere considered to get the highest TDI dose duringthe study. It was not possible to estimate the indi-vidual dose in detail. The figure shows that theyalso had the highest average elimination rate of uri-nary TDA. The between individual differences inelimination rate of TDA were, however, significant.Two PUR workers (B, C) and two white collarworkers (F,G) had similar elimination rate patternsof about 20-70 ng TDA per hour (TDA = the

sum of 2,4-TDA and 2,6-TDA). Two subjects (A,D) had an elimination rate of about 100-300 ng

TDA per hour. This discrepancy may have beendue to different working habits, which we couldnot identify.

Subject F was a female laboratory technicianmainly working in a separate room and she visitedthe production area only for very short periods.Subject G was a supervisor present in the produc-tion area about 10% of the time during the study.They showed the same elimination rate pattern as

subjects B,C, and E. Subjects F and G did not useany protective devices during the study, which mayexplain why they were exposed to TDI.The two volunteers, subjects H and I, were pre-

sent close to the production area and also at placeswhere exposure was expected to be considerable.This explains the high urinary TDA eliminationrates during the study.The elimination rate curves with time (figure)

for the workers A to E and the volunteer I showedhigher elimination rates during and soon after theTDI foam production. The elimination rates inurine showed a peak about one hour after the TDIfoaming in the afternoon of day 2 (about 35hours). This corresponds to the use of formic acidas the blowing agent. Even subjects F and G whowere not present in the production area at that timeshowed a peak in the elimination rate curves. Theirexposure may have been due to some unforeseenproblem with, for example, ventilation.The relation between the 2,4-TDA and 2,6-

TDA isomers was not constant in the eliminationrate curves. The subjects A to C and G showed thesame elimination rate of the two isomers. Subject Eshowed a more than double elimination rate of 2,6-TDA compared with that of 2,4-TDA and subjectF showed a more than double elimination rate of2,4-TDA compared with 2,6-TDA. The relationbetween the isomers changed with time for subjectsD, H, and I. This may be explained by exposure todifferent ratios of the TDI isomers in air at differ-ent places in the factory.

Urinary TDA was related to density and creati-nine over time in essentially the same pattern.

Table 1 Coefficients of variation (CVs) for measurements of TDA

Plasma Unine2,6-TDA 2,4-TDA 2,6-TDA 2,4-TDA

Concentration of TDA (pugll) CV(%/.) CV(%) CV(%/.) CV(%)

0-1 7-1 (n = 15) 6-4 (n = 22) 7-3 (n = 64) 16-3 (n = 62)1-5 2-1 (n = 15) 7-9 (n = 30) 6-7 (n = 54) 12-4 (n = 63)5-16 2-1 (n = 21) 3 0 (n = 12) 8-5 (n = 5)

Each TDA value was calculated from two gas chromatography mass spectrometry measurements.

1115

Persson, Dalene, Skarping, Adamsson, Hagmar

81

64

44

24

DO A

Do

00 10 20 30 40 5C

200

160

120

80

40

v)

B

D 101 -C

20 30 40 50

0 10 20 30 40 50

D

.-

100

80

60

4

21

G

10.

10 20 30 40 50

E 200Eo 2-6 TDA* 2-4TDA 160

120

080

40'

00

2001

10

H

0 10 20 30 40 50 0 10 20 30Time (h) Time (h) Time (h)

Concentrations of 2,4-TDA and 2-6-TDA (toluenediamine) in hydrolysed urinefrom workers A, B, C, D, E; white collar work-ers F, G, and volunteers H, I. Urinary excretion is shown at the midpoint of each observation period. The start time was 0600 onthe first morning.

2,4-TDA and 2,6-TDA in plasmaThe concentrations of 2,4-TDA and 2,6-TDA inplasma (table 2) for the subjects A to G were virtu-ally stable. No noticeable correlation between theelimination rate of TDA in urine and plasma con-centration of TDA was seen. The subjects A to Eshowed plasma concentrations of total TDA in therange of 1-8 ng/ml. The laboratory technician (F)had 0-2 ng TDA/ml plasma and the supervisor (G)had about 1 ng TDA/ml plasma. The volunteers(H, I) showed an increasing concentration of TDAin plasma with time. At the end of the study theplasma concentrations were 0-6 ng/ml and 0-2ng/ml plasma.

All subjects showed equal or higher concentra-tions of the 2,6-TDA isomer compared with the2,4-TDA isomer. The ratio between the isomers,however, varied. The subjects A, C, D, F, and Ghad about the same or double the ratio of 2,6-TDAcompared with 2,4-TDA. Twelve times higherratios were found in plasma from subjects B and E.

The two volunteers (H, I) showed increasing con-centration of both 2,6-TDA and 2,4-TDA inplasma with time.

DiscussionTo measure the average exposure during a work-shift several air samples must be taken as thesolvent in the impinger flasks may evaporate to dry-ness within one hour, depending on the ambienttemperature, which makes prolonged samplingcomplicated. Sampling with the chemosorptiontechnique could have great advantages in thesecases but the technique is not yet completelyevaluated.When personal measurements of air sampling

exposure are performed to assess the dose, differ-ences between individual subjects in work loadaffecting the respiratory volume makes the evalua-tion difficult. One of the greatest disadvantageswith air sampling is the necessity to plan the iso-

a)

co0._

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20 30 40 50a) 100

c 800

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C)= ' 80'

C 40'._-C

40 50

0I, .

1116

'J

Biological monitoring of occupational exposure to toluene diisocyanate

Table 2 Concentrations of2,4-TDA and 2,6-TDA in hydrolysed plasma (nglml)

Time (h)Isomer

Subject of TDA 0-5 9-5 24-5 30 5 33-5 48-5

2,4- 1-8 1-6 1-4 2-2 2-2 2-3A 2,6- 2-4 2-2 2-0 2-5 2-4 2-5B 2,4- 0-54 0-54 0-58 0-56 0-57 0-52

2,6- 5-3 5-8 5-4 5-6 5-7 5-32,4- 0-59 0-60 0.55 0 53 0-58 054

C 2,6- 1-3 1-2 1-2 1-2 1-2 1.1D 2,4- 1-8 1-7 1-5 1 9 2-2 1-8

2,6- 2-4 2-4 2-1 2-4 2-6 2-32,4- 0-55 0-66 0-46 0-46 0-48 047

E 2,6- 7-6 6-5 6-2 6-1 6-4 6-6F 2,4- 0 09 0 07 0 10 0-08

2,6- 0-14 0-12 0-14 0-142,4- 0 43 0-34 0 34 0-41 0-44 0 39

G 2,6- 0 53 0-52 0-46 0 50 0-57 0 53H 2,4- 0 07 0-06 0-12 0-16 0-21 0-25

2,6- 0-05 0-06 0-08 0-15 0-27 0-352,4- 0 07 0-12 0-13 0 10 0.11 0 112,6- 0 03 0-06 0-06 0-08 0.10 0 11

*Start time was 0600 the first day, and the first blood samples (0 5) were taken at 0630. For details of subjects see figure.

cyanate measurements beforehand and therebypossibly influence the environment at the work-place. Recently a more detailed understanding ofisocyanate chemistry in the work environment hasbeen obtained. Workers may not only be exposedto airborne gaseous isocyanate monomers, but alsoto aerosols containing other compounds necessaryfor the build up of the polymer.'0 Furthermore, inan aerosol particle the polymer continues to growwith time and various reaction grades of the iso-cyanates must be present. Only the isocyanatemonomer, however, is determined in air measure-ments. Theoretically other compounds present inthe air and the aerosol particles could influence theuptake of the isocyanate. When exposed to thermaldegradation products from a TDI polymer,compounds such as tolueneaminoisocyanates arereported to be present in the air'9. These types ofcompounds are not generally measured by the mostoften used isocyanate methods.As isocyanates are hazardous, personal protec-

tive devices are widely used nowadays. The effi-ciency of these varies and may also decrease overtime if they are handled improperly, as is often thecase. Personal exposure measurements are in suchsituations meaningless. Furthermore, dermal expo-sure to isocyanates can be expected to happenoccasionally even if protective clothes are used. Amethod for biological monitoring may thus give amore relevant estimate of exposure. Earlier we havereported a GC-MS and electron impact ionisation(EI) method for the determination of TDA inhydrolysed human urine and plasma after exposureto TDI. This method is essentially the same as wehave used here. The method20s22 (and unpublisheddata from our laboratory) has been applied to thestudy of TDA in hydrolysed plasma and urine after

controlled exposure of human volunteers at threeconcentrations in the range of 6-21,ug/m3 of2,4-TDI and 14-50 Pug/m3 2,6-TDI. In urine thecumulated excreted amount over 24 hours was for2,4-TDA 15%-19% and for 2,6-TDA 17%-23%of the estimated dose. The mean ti,, for the con-centrations in urine were 1.9 hours for 2,4-TDAand 1.6 hours for 2,6-TDA. There was a linearrelation between urinary exeretion and dose found.An association between the dose of TDI and theconcentration of TDA in plasma was also noted.The GC-EI method required great care of the massspectrometer and the detection limit of the methodwas set by the instrument. The determination ofTDA in urine and plasma was made close to theultimate detection limit. The instrument could onlyoperate in the electron impact and chemical ionisa-tion modes monitoring positive ions, and it gave anacceptable detection limit for only about 10% ofthe time. Far better sensitivity and selectivity havebeen achieved in this study with GC-MS in thechemical ionisation mode with negative ion moni-toring and ammonia as the reagent gas. The detec-tion limit achieved was 10 fg for 2,4- and 2,6-TDAinjected, corresponding to about 0.01 jug TDAIl ofhuman urine or plasma. To estimate exposure toTDI in workers by analysing urine samples thevariation of the TDA concentration in urine withtime from exposure must be considered. For con-trol of exposure for individual subjects this mightbe complicated. When estimating exposure for agroup this may, however, be a good alternativecompared with air measurements. When exposureto TDI was estimated by the monitoring of plasmaconcentrations of TDA little variation with timeafter exposure was found. As the plasma concentra-tions of TDA are stable it is not critical to take the

1117

Persson, Dalene, Skarping, Adamsson, Hagmar

blood sample immediately after exposure. Plasmasamples can give a measure of long term exposure.

The nature of the TDI-protein bonding in humansis not clear at present, but from polyurethanechemistry, it is known that isocyanate bonding toamine and hydroxyl groups is strong with goodresistance to hydrolysis. This indicates that more

TDI metabolites may be present in the body,which are not determined by the method, based onacid hydrolysis, described in this paper.As we have reported earlier only about 8%-18%

of the inhaled dose of TDI is eliminated in urine.21In another study we found decreasing concentra-tions of 2,4-TDA and 2,6-TDA in plasma withtime22 (t 12 = about 250 hours). How TDI ismetabolised in humans is not known in detail.Kennedy et al have described a TDI/TDA plasmaprotein adduct in rats. In our study the virtuallyconstant plasma concentrations indicate the forma-tion of TDI/TDA plasma protein adducts inhumans. In this study it was not possible to analysethe TDA in plasma from workers during a pro-longed period free of exposure. It is not feasible toexpose volunteers to high doses of TDI necessary

for the build up of high plasma concentrations ofTDA due to ethical considerations. These mattersare presently under study.

It is interesting that the urinary concentrations ofthe five TDI workers at the beginning of the studywere in some cases the same as those of the twovolunteers at the end. Taking into considerationthe half life ofTDA in urine (t1,2 = 1 6-1 9 hours),after TDI exposure several compartments may beinvolved in the biotransformation of TDI. Therelatively high concentration of TDA in urine fromthe TDI workers may only partly be explained bythe degradation of TDI/TDA plasma proteins.

This study indicates that it would be possible tomonitor exposure to TDI by monitoring plasmaconcentrations of TDA. More detailed understand-ing, however, of the biotransformation of TDI anddifferences between subjects is necessary.

This work was supported by grants from the Swedish WorkEnvironment Fund and the Medical Faculty, Lund University.We thank Dr Carsten Sango and Per Brunmark, MSc for valuablehelp and support and Dr Hans Welinder for performing theRAST and ELISA analyses of TDI antibodies. Valuable help was

given by Ms Pia Aprea.

World Health Organisation, Environmental health criteria 75,toluene diisocyanates. Geneva:WHO, 1987.

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Accepted 10 February 1993

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