Document jy7YeqjLJpGVyjjpNm8R6x9g9

Tv* Int. Arch, f/.-cup. Environ, filch. 41, 199-205 (1973) #JSO A-c.*es o <iW!l;)i:ilimil itiK? Mr.vli-jwiit.fiiini iJmillh ...... qr V--IA.I V Determination of Thiodigiycolic Acid in Urine Specimens of Vinyl Chloride Exposed Workers G. Muller*, K. Norpoth1. E. Kusters2. K. Hcrvcg3 and-H. Versin* 1 tnstitut fur Staubhincenfoischun': und Arbcitsmcihin tier VVestf. Withobns-Universitat. >4400 Munster. federal Republic of Germany ! Wcrksarrtlielte Alii.liun-j Ucr BASF. Antwerpen. Belgien * Werksarztiirhc Abteilung dcr Chcmischcn W'erke Hiils AG, .Marl-Hiils. Federal Republic of Germany Summary. A strong correlation was found between vinyl chloride concentrations at working places and the increased excretion of thiodigiycolic acid of 18 exposed workers. The mean air .>ncentrafion of vinyl chloride was calculated referring to personal exposure. The values obtained were in the range of 0.14 - 7.0 ppm. The excretion of thiodigiycolic acid - measured by GC-MS analysis - amounted to 0,3 - 4.0 mg/L. It could be demonstrated that significant increases of the metabolire excretion occur even at V'C-concentratior.s below 5 pom. Key words: Vinyl chloride -- Thiodigiycolic acid - vinvl chloride metabolism in man - Vinyl chloride urine metabolites -- Biological exposure control Today the air concentrations of vinyl chloride a; working places are rigorously reduced mainly due to its carcinogenic [5. 14. 16. I'T 20-23. 34) and mutagenic (6. 8. 9, 14, o. 18.1'h 30] properties. Exposure controls arc commonly based on air analyses by means of highly sensitive, bin no! always strongly specific analytical methods. Therefore, we emphasized the useful.toss of additional biological exposure controls 124. 26], _ By animal experiments we couid demonstrate the possibility of determining the excretion of thiodigiycolic acid and S-carboxy-rtcihyicysteine. two urine metabolites 'f vinyl chloride [ 10. 24. 26. 27, 35] Data of thiodigiycolic acid in the urine ot workers slightly exposed to vinyl chloride and the improved analytical method used for determination arc reported her>.. Materials and Methods Chemicals ihsodiglycoiic acid (9S purity) was purchased from Janssen Pharrr.accutica, Dept. Aldrich ! urope. 142540 IVersc. Belgium. Diazomethane was generated from N-methyl-N'-nitroso-pMuenv-vtifnnaiiiuie acec'd.ny h> do Boer and Backer |3|. Alt other substances were purchased at nnalyi.eii crack from 1 . Merck AG. !>'. 100 Darmstadt. 0304-0131 /7S '004 i/0201/ S 1.40 i j i | j i j ] ] ! | ! j j j t j : j . I j I SPI-04103 N i l -1 L .iB K A W Y ' iqq I'roccdarti Determinations of Individual exposure by Personal Air Sampling G. Muller et al. j ) ! | i j Tluodigly ro'is r . I .-re -i Samplin'! ) Sampling l ube. Traces of orcamc pollutants (anions which vinyl chloride) in the factory air j Mere collected by glass (Li-, x.. ..piers v..;h a length of 12 cm ar.J an internal diameter of a mm. filled with 35-50 rr.esli charcoal 1? |. 'I iiese ciiareoal tubes had a breakthrough velume. for vi:iy 1 i chloride at 20 C, of about 5 liters (33|. i pump. To drate (he air t .'trough the tube samplers we used a small pump named personal sampler manufactured by Anatolc j. Sipi.n Co. The sampled air volume can be calculate-: from thenumber of pistonplays of the pump, registered by a ba.it in counter. As the amount of ci-aircoai and the density of packing varied from, xansplu.r : ate !0 sampling tube, we had :o calibre:.- the pump before each ex peri:: -r.t. The rsliltortvhip between pistonplays and normal gas volume was easily measured with a gasbubbtc meter and a stopwatch. Analysis j 1 j t aon I~ ! A Analytical Conditions. A special injection por: licit allows for direct introduction of the total J t"OA l I sample, cartridge included, was used for the thermal desorption of the trapped vinyl chloride ll, 29|. The vaporized vinyl chloride was routed to a gaschromat.etaphic column. To separate the vinyl chloride from other sampled organic vapor pollutants we used a 25 m wait coated carbowax 20 M capillary a! S0C. The detector used was. of course, a flame ionization detector. j ! i< The surface of the vinyl chloride peak was measured by an integrating computer. 1 3 3 Gilibration. A calibration gas was dynamically prepared by passing a known gasstream over a teflon permeation tube filled with liquid vinyl chloride 123, 311. the concentration of vinyl ; ; chloride was calculated by the loss of weight of the permeation tube over several days. A I 3 known volume of this gas was drawn through a charcoal tube and this tube was analyzed as ` + described above. The gnsehro.-autographic results were reported as the amount of pg absorbed by the cliarcoa! tube sampler. j Evaluation of the Personal Exposure Data. The mean concentration of vinyl chloride in the air during the experiment was calculated from the amount of absorbed vinyl chloride analyzed by gasdtromatography and from the sampled air volume calculated from the number of pistonplay-s of the pump during sampling. ! j VC-Determination in the Air of Working Places. The measurements were performed using multipoint flame ionisation detectors (HD), type RADF1SH, spread in the working area. Total hydrocarbons in the air of each sampling point were analyzed once a minute. It has been shown by CC-mcasurements that approximately 2 ppm of the total hydrocarbons in this working area are not VCM. Considering this amount of 2 ppm not - VCM - hydrocarbons the true air concentration of vinyl chloride monomer was calculated. The personal exposure of each worker was evaluated. Thus, from registrations based on the duration of stay at different working places, all values of VCM air concentrations given in this paper, which are not obtained by personal air sampling, refer also to personal exposure. Determination of Thiodiglycolic Acid by CC-MS-Analysis. In a 10 ml glass tube 1 ml of urine and 0,3 ml of cone. HC1 were evaporated to dry ness in vacuo at room temperature. The residue was suspended in metharoi and. the suspension was completely methylated by means of diazoincthane in ethyl ether ?3 J. After 15 minutes 500 mg dry sa'in exchange resign (Dov.ex 50 WX 8; M were added. i'hiodiglycoMc acid dimethyl ester was determine : by guschrorr.aiographic separation and sir.rde ion detection of "he mass fragment M 146. It i> necessary to use the standard addition lechr.ajuc to obtain exact measurements. / > ' I Conditions Cascliromatogrr 0,3 cm y.-r OV 130 C. Injectw Results The analytical small anunints two groups cl AS dC.MIOi (group A) anc 1 V- signified! SPI-04104 Muller et .if rir,<'di"lyeo!ic Add in ihc Urine or' Vinyl Chloride Expos'd Horse.-. 201 ernry air t hi rr.:n. : re. lor vinyl : ..Tin! sampler ram i lie number ..he to 1 hip between '.I a stopwalch. the total l chloride in separate ,;l coated Hum detector. ..at over a v< of vinjl lays. A ais red as e alisotbed 1 ale m the air . inaly/e.l hv iii pistonplays vil ns:: v; area e U has r'on- '.a l;> druearhotis i,' "S's'Mire or' . at diMetent . not obtained * till il urine and I he residue wins of 'egn (Dowex - jtasehromato- vary lo me ' t AB -c * Fig. I. Urine itorccntrations of tlitodigivcr-li: acid fTDGAV Group A: 20 nor-exposed male-. Group B: workers exposed to. 0.1 d - J.35 ppm VCM mea-ured by FH) aj analysis. Group C: 8 workers exposed to 1.02 - 7,0 ppm VCM measured by use or' persona! a samplers Fig. 2. Urine eoneeniralions of IhioJielycolic acid (TDGA) after classification of 37 data according to four collectives w his It are related to four different VCM exposure rat'jct: A * n.O ppri, B * 0.01 2..'0 ppm. C- 2.51 - 5.00 pern. D * 5.01 - 7,50 ppm Conditions i si<chromatograph 9500 combined with mass spoettometcr 3200 (Finmian). Column 1.5 m x cm 327 OV I on f.il/hO DMCS Chromusorh \V. Cameras 30 tnl/mm. Column temperature I )(f` C. Injection port temperature 20O' C. Separator tetnpei.iturc 200" C. Results The analytical procedure which we developed for sufficiently precise determination of small amounts of thiodigivcolic acid in n 'ir.e samples allowed us to compare slain n: two groups of VC-workers with those of;; non-exposes! group. As demonstrated by l:ig. I. it is possible to distinguish between normal values (group A) and such obtained from slightly exposed workers (group B .uni C) at the I '.if significance level. As we calculated from our normal values, in all cases concern I j i ! ti I ! SPI-04105 ,S*.-T<VC.tTdy'.. >' b.\;*'s''**. .- . - : cci >* X3 r<: 202 TOGi ! (h1 CZ] too II 3CO C. MulJer ct al. VClppmln 3 2 lIi Fig. 3. Results of a comparison in.VNfi^uiion on VCM expuv.irc levels and the excretion of thiodiglycolic acid (TDCA)/hour in the case of a single worker, who was observed during a three<hy period 0 trations of thiodiglycolic acid exceeding 2,1 /rg/m! urine can be distinguished from norma! concentrations at the 1 % significance level (99 % probability of confidence). Those values were found twice in group B and five times in group C. The mean values of the three groups suggest a correlation between the extent of exposure and the amount of thiodiglycolic acid excreted. This correlation could be proved by classifi cation of the analytical data according to four classes of different exposure. As shown in Fig. 2, an increase of metabolite concentrations in the urine is found with in creasing exposure. The correlation was tested by a distribution free statistical proce dure f 17] and could be confirmed at the level of p < 0,01. Since individual exposure control by means of personal air sampling seems to provide more precise data than global air analysis, we started exact examinations of single workers controlling the personal exposure during time periods of two or three days. In this period all urine portions were collected separately and in each of them the concentration of thiodiglycolic acid was measured. The results of one investigation are shown in Fig. 3. It can be seen that the increase of metabolite excretion began shortly after starting the exposure each day and that the highest concentrations were found in the first or second urine sample obtained after the highest possible VC-uptake. Furthermore, it was a result of this investigation that the metabolite excretion exceeded the normal range significantly even after a mean exposure level of about 1,5 ppru but not after an individual exposure of less than 1,0 ppm. Thio 1 ij'ycu Discussion h uieta annuals art [11, 24, 2< Previo S-carboxygical expo: experirnen thiodiglycr higher tha: working p! performed IS worker: of thiodigl; alternative the expo: me reap tu thiodigly, more prei previous!) in man th a naturall; method tin urine of wc 1.5 and 5 g level. Negiec daily amou fo ng e The explan VSh = lung Cvc - !".ec KVC = rcs0 = relaliv. mclabMGtdaa MGvCM = The th rates in the values as sh in rat and r more in aci analyses us hydrocarbc t f 11 G 'taller ct al. VClp*vnlU 3 7 1 *. fclion of i during a I . ushed from >f confidence), file mean values ire and the .'d by classifisure. As shown d with in' stical proce- c seems to ..initiations of ' two or three each of them me investigation etion began ^rations were 'iblc VC-upiie excretion . -`-of about Thiodiglycolic Acid in the I'rine of Vinyl Chloride Exposed Workers Discussion 203 Main metabolites of vinyl chloride which are excreted in the urine of laboratory animals are thiodiglycolic acid [ 10,24. 26, 27. 35], S-carboxy-methylcvsteine [ 11. 24. 26. 27] and hydroxyethyl mercapturic acid f 11. 35]. Previously, we proposed quantitative determinations of thiodiglvcoiic acid and S-carboxy-methyl cysteine in the urine of vinyl chloride exposed workers for biolo gical exposure control [24. 26]. In the meantime, we could demonstrate by animal experiments that S-carboxy-methyl cysteine, a precursor of the final metabolite thiodiglycolic acid, accumulates only in the case of vinyl chloride air concentrations higher than 500 ppm in the exposure chamber [25], which are unusual today at working places. Concerning hydroxyethyl mercapturic acid measuremonts where performed using an amino acid analyzer. No amounts where found in the urine of IS workers slightly exposed to vinyl chloride which exceeded the corresponding values of thiodiglycolic acid. It may be taken into consideration that the contribution of alternative metabolic routes to the biotransformation of vinyl chloride depends on the exposure level [13.25.36]. Thus it is not yet clear, whether hydroxyethyl mercapturic acid is the main urine metabolite of VCM in man or whether it is thiodiglycolic acid. Investigating the real concentration of the latter metabolite, a more precise procedure was needed than in our animal experiments performed previously [18, 20] because its increase after VCM exposure is apparently less marked in man than in the rat. Furthermore, it has been shown that thiodiglycolic acid is also a naturally occurring urine metabolite in man [25]. Using an improved analytical method then, we could clearly demonstrate that the metabolite concentrations in the urine of workers exceed the normal level significantly even after VC-exposures between 1.5 and 5 ppm. This increase of urine concentrations depends clearly on the exposure level. Neglecting other elimination routes than urinary excretion theoretical values of the daily amounts (E) of excreted thiodiglycolic acid may be calculated according to the following concerning: E _ V8h X Cyc x Rye x Q x MGtdga MGvc The explanation of the symbols is: Vgh = lung ventilation during an 8 hour interval! CvC='rnean a'r concentration of VC RVC - resorption value, which has been found in animals as about 0,4 (4) Q = relative amount of thiodiglycolic acid as compared to the total amount of VC- metabolitcs. which has been found between 25 > (35) and 50 % (10). MGtdaa = molecular weight of thiodiglycolic acid MGy'CM = molecular weight of VCM. The thus determined theoretical values correspond well to the measured excretion rates in the rat. They are. however, between 2 and 10 times higher than the excretion values as shown in Figs. 1 and 2. This may be due to different biotrar.sformation rates in rat and man. On the other hand, the values obtained by personal air sampling are more in accordance with the theoretical values than those obtained by direct air analyses using FID-detectors. In the latter ease, as mentioned previously, other hydrocarbons are measured together with VCM amounting to an average level of i ti SPI-04107 204 G. Muller el al. about 2,0 ppm. This average may have been exceeded during some of our measurements Thus, more evidence is needed for the true slope of the function between VCM exposure and excretion of thiodigiycoiic acid. Therefore, we started a new study to reinvestigate this slope by measuring the persona; VCM exposure using the personal air sampling tech nique in direct comparis. with the use of FID-detectors for air analyses. As shown in Fig. e-.er; a slight VCM exposure can cause characteristic fluctuations of thicdiglycolic acid excretion and this can be revealed by continuous urine analysis. Considering the excreted amounts of the VCM metabolite per hour, it seems possible to prove a periodical uptake of very low amounts of VCM by single workers. Such measurements could also provide some informations about inter- and mtraitidividuj! differences of VCM metabolism in man. Acknowledgement, This study was supported by a gran: cl the Berul'sgenossenschat't der Chertiisehen Industrie. D-6900 Heidelberg I. BR13. 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