Document jBLqq79Je7Dyqp2vKnmBZEVL9

hods for the 3, 173-215. roth and H. I, a natural thylether, a 11. Toxicol., Alirilrfirwr R i w m h , 26 I ( IO9I 12 17-223 y1 l Y Y l Elscvier Science l'til~lislicrs1I.V. All rights resctvcd Ol~~S-l2lX/~~l/$O3.SO A f m v I S 0 16512 I 891001511 PLAlNTIFF'S EXHIBIT 217 G. Nise B. Hogstedt 3, I. Bratt and S. Skerfxing * 'Depurrrneni of Occupuiionul Medicine, Kurolinsku tiospirul, S-104 01 Siockholm, Depuriment of Occupuiional and Ennvironmenrul Medicine, Unii:ersiiy Hospital, S-221 XS Lund und Depurrment of Occupuiionul Medicine, Cenirul fiospitul, S-301 85 Hulmstud (Sweden) (Received 26 February 1991) (Revision received 3 June 1991) (Accepted 1 1 June 1991) Keywords: Blood lymphocytes; Chromosomal aberrations; Micronuclei; Toluene +$., Q05 <@\ q@ci$qc*Y.P@JJP't,Q'\\Q%G\SGQb%J* (pp d' \! ,-;.SP, \'&'-- Summary $0 Qqp' c;o Comparing 21 rotogravure printers exposed to toluene (medians: time-weighted air level 150 mg/m3, blood toluene 1.6 pmole/l) and 21 unexposed controls (median blood toluene I 0.01 pmole/l) there was a significant increase in the frequency of micronuclei (MN) in pokeweed mitogen (PWMI-stimulated peripheral blood lymphocytes in the printers, as compared to the controls (2.8%0 vs. 1.5%; p = 0.03; all p adjusted for age and smoking). The frequency of small MN (size ratio MN/main nucleus I 0.03) in PWM-stimulated lymphocytes was associated with the exposure (1% vs. 0.3960;p = 0.05). Furthermore, among the exposed subjects there was an association between blood toluene and small MN (0.17%0 per pmole/l; p = 0.0005). Small MN in phytohemagglutinin (PHA) cultures displayed no association with any exposure parameter. However, in the printers, an estimated cumulative exposure index was weakly correlated with the frequency of total MN in PHA-stimulated cells (0.00003%0 per mg/m' X year; p = 0.07). Among the printers, chromosomal breaks in I'HA-stimulated cells were irssociated with the duration of earlier benzene exposure (0.03% per year; p=O.Ol). The results of this study strongly indicate that toluene causes a clastogenic effect on the B-cells even at low exposure levels. Further, earlier benzene exposure seems to have caused chromosomal breaks in T-cells. Toluene is a commonly used industrial solvent, In humans, toluene is mainly metabolized into and is generally considered to be a safe substitute benzoic acid, which is further conjugated with for benzene, an established genotoxic agent glycine to hippuric acid (Jelnes, 1989). A minor (Dean, 1985). proportion of the retained toluene (about 1%) undergocs aromatic ring oxidation (Low et al., 1988). This metabolic pathway is mediated by Correspondence: Gun Nise, Dr. Med. Sc., Department of cytochrome P-450 and may initially form arene Occupational Medicine, Karolinska Hospital, S-104 01 Stock- oxides, which rearrange spontaneously to 0-and 5 4 holm (Sweden). p-cresol (Kaubisch et al., 1972; Nise, 1991). Arcne r 218 oxides are often genotoxic (Wislocki et al., 1976). However, studies on the cytogenetic effects of toluene show contradictory results (IARC, 1989). Increases in the frequencies o f micronuclei (MN) and chromosomal aberrations were found i n rat or mouse hotle marrow cclls after intraperitoneal injections of toluene (Mohtashamipur et al., 1985; Roh et ai., 1987). However, neither MN nor chromosomal aberrations were observed in mice after oral doses of toluene (Gad-El-Karim et al., 1984). The discrepancy may depend on the different strains of mice used and/or the varying amounts of toluene administered via different routes. MN have not been studied in toluene-exposed humans. An excess of chromosomal aberrations was reported in the lymphocytes of rotogravure printers (Funes-Cravioto et at., 1977). Further, Bauchinger et at. (1982) found increases in the frequency of sistcr-chromatid exchanges (SCE), chromatid breaks, chromatid exchanges, and gaps in workers exposed to toluene. However, Forni and co-workers (1971) reported no significant difference in the frequency of lymphocyte chromosomal breaks in rotogravure printers. Neither was there any increase in the frequencies of chromosomal aberrations or SCE in rotogravure printers (Maki-Paakkanen et al., 1980) or in paint-industry workers (Haglund et al., 1980). All studies employed phytohemagglutinin (PHA)-stimulated cultures. One possible explanation for the varying results in these studies is differences in exposure, with regard to intensity, duration, and benzene impurities in the toluene. The information on this matter is scanty in some of the reports. Using a cohort with well-defined exposure, the objective of the present study was to investigate whether toluene exposure causes cytogenetic effects in peripheral lymphocytes. We also investigated whether the type of mitogen used, PHA or pokeweed (PWM), is of importance for tolueneinduced MN frequencies and size ratios. \ Subjects and methods Subjects Twenty-one male rotogravure printers exposed to toluene, and 21 workers from a margarine factory, with no solvent exposure, were studied in 1987. Ten of the printers were smokers, with a median consumption of 14 cigarettes/day (range 3-30). Thirteen of the controls were smokers (median 20 cigarettes/day, range 10-20). Each printer's exposure had been thoroughly followed with a personal sampler for the entire workshift during a whole work week in 1986 (Nise and (arbaek, 1988). The median individual week exposure was 150 mg/m3 (range 30-420). Toluene in blood was measured before and after shift on Monday, Wednesday, and Friday in 1986. The post-shift toluene in blood has been found to reflect the exposure during the preceding shift (Nise and Orb~ek,1988). Mid-shift blood samples, obtained in connection with the sampling for cytogenetic assays, were also analyzed for toluene (see below). Methods Blood sampling Venous blood samples were obtained in heparinized tubes from all the examined subjects between 11 a.m. and 2 p.m. on a day in the middle of a work week. The samples were taken during 3 consecutive weeks, since samples from all subjects could not be cultured simultaneously. At each sampling time, about half of the subjects were controls. On the day of sampling, 1 blood sample from each subject was coded and immediately sent to the laboratory for lymphocyte cultivation. Blood toluene For both printers and controls, 1 blood sample was analyzed for toluene on the day after sampling, with the head-space gas chromatography technique (Nise and 0 r b a k , 1988). The detection limit was 0.01 pmoIe/l. The precision of the method was 16% for blood toluene levels at 0.01 pmole/l (coefficient of variation of duplicate samples). Micronuclei in lymphocytes Buffy coat leukocytes from the blood were cultured for 80 h in RPMI 1640 medium with 15% fetal calf serum according to Hogstedt (1984). Parallel cultures were set up with either PHA (Gibco; 1 m1/100 ml) or PWM (Gibco; 1 ml/l( were ! Frc were MN. were I was e: the h (Hogs blind Cltl Blc RPMl ment (0.075 were 1 chrom Giemi were tern r for HI result! latter break! centric His AI forme1 printir were condit ing co ing ex from 250 h Tht during curren 30 h curren mg/m mated measu pation mated and a - uitli a ,range iokers wghly entire (Nise week luene ift on The id to shift iples, 5 for uene hepjects the iken rom usly. ects ood ediulti- iple .imPhY ion the 1.01 ate :re ith :dt ier ,I m1/100 ml) as a mitogen. The M N preparations were stained with Giemsa. From each subject, 4000 activated lymphocytes were analyzed for the presence of intracellular MN. The sizes of the cell nuclei and the MN were measured in 3000 lymphocytes, and the size was expressed as the ratio between the surface of the MN and the corresponding main nucleus (Hogstedt et al., 1985). All scoring was performed blind by 1 person. Chromosomal aberrations in lymphocytes Blood was incubated at 37C with PHA in the RPMI 1640 medium for 48 h. Hypotonic treatment was performed with potassium chloride (0.075 mole/l) at room temperature. The cells were fixed in methanol/acetic acid ( 3 :I ) and the chromosome preparations were stained with Giemsa. For each individual, 100 metaphases were scored, according to the classification system recommended by the lnternational System for Human Cytogenetic Nomenclature (1985). The results are presented as `gaps' and `breaks', the latter comprising chromatid and isochromatid breaks, pericentric inversions, rings, and dicentrics. History of tooluene and benzene exposure A retrospective exposure assessment was performed back to 1948, the first year of rotogravure printing in the present plant. All the printers were interviewed in detail about their working conditions, past and present. The printers' working conditions are also described, both concerning exposure levels and durations, in 6 reports from the 1970s and early 191(0s, totaling about 250 h of air toluene measurements. The evaluation of the toluene exposure level during different time periods started with the current exposure (each printer was followed for 30 h by personal air sampling in 1986). The current time-weighted exposure level was 150 mg/m3. Then, by working backwards, the estimated exposure level was revised whenever any measure of importance was taken affecting occupational hygiene, The toluene exposure was estimated to be about 800 mg/m3 during the 1970s, and around 1500 mg/m3 during the 1950s and 210 I960s, with variation from year to year (Svensson et a]., 1990). For each printer, a cumulative exposure index (CEI) was calculated as the sum of the exposure time (in years) multiplied by the corresponding exposure estimates (in mg/m3). The exposure estimates used for the calculation of the CEls probably deviate by less than a factor of 2 from the true levels, as judged by air measurements in other printing plants with corresponding production in the studied time periods. Benzene was not measured in the present plant. However, benzene was a component of the toluene used in rotogravure printing before the mid-1950s. The benzene content could be up to about 10% (Svensson et al., 1990). After the late 1950s, the solvent used only occasionally contained more than 0.5% benzene. Today the impurity of benzene in the toluene used is less than 0.01%. Further, benzene was not found in any air sample during our measurements. Statistical methods For intra-individual comparisons of the fre- quencies and size ratios of MN, between PHA and PWM cultures, Wilcoxon matched-pairs signed-rank test and Spearman's rank correlation (I ;) were used. The effects of exposure on the cytogenetic parameters were also evaluated in a multiple linear regression analysis, with allowance for age and smoking habits. As the frequencies of MN and chromosomal aberrations were low, the aver- (fi+age square root transformation 43)/2 was applied to the individual measurements of these endpoints, to stabilize the variance (Whor- ton, 1985). All comparisons between quantitative expo- sure variables and the cytogenetic endpoints were performed within the exposed group. All p-val- ues are 2-tailed. Results The printers' toluene concentrations in blood ranged from 1.0 to 6.6 pmole/l (median 1.6; Table 1). These levels are in agreement with the concentrations found in their blood during the 231 -rAuLLi I TI1E FREQUENCY OF T O T A L A N D SMAI,I. (SIZE RATIO I 0.03) MICRONUCLEI (MN) IN P E R I P H E R A L LYMPHOCYTES STIMULATED WITII PI I A O K PWM MITOGENS, A N D THE CIIROMOSOMAL ABERRATIONS IN LYMPHOCYTES IN 21 TOLUENE-EXPOSED AND ? I C'ONTROL SUBJECTS Age (years) Smokers ( n ) Exposure (years) Blood toluene ( p m u l e / l ) CEI (rng/m3 x years) Exposed M c i i~i ti 48 IO 2s 1.6 I O 200 Riirigc 30-03 - 0.5-37 I.o-6.6 75-37500 C'otitrol Mediat i 33 13 0 0.0 I 0 Range 25-63 - < 0.01-0.01 - 'I - - The significance level\ ( p ) are adjusted for age and smoking Iiahils. 1.0-4.0 1).3-2.7 0.2-3.8 0- 1.7 0-3 0-6 0.4 > 0.5 0.03 0.05 0 . I3 0.008 study in 1986. The blood toluene levels in the controls were at or below the detection limit of 0.01 pmole/l. With PHA-stimulated lymphocytes, the median freqirency of MN, for all examined subjects, was 2.5%0, compared to 2%0 with PWM stimulation ( p = 0.06; Wilcoxon test). Furthermore, there was a significant correlation between the 2 scores (r\ = 0.36, p = 0.02). For lymphocytes stimulated with PWM, there was a significant difference in the frequency of MN between the printers and the controls, when allowance was made for age and smoking (medians 2.8%0 vs. 1.5%0; p = 0.03; Table I). These frequencies are in agreement with those found earlier in piperazine-exposed workers (Hogstedt et a]., 1988b). No difference was found for the frequency of MN in lymphocytes stiniulated with PHA. I n the niultiple regression analy-' sis, age and smoking habits displayed no statistically significant effect, either with PHA- or with PWM-stimulated cultures. The estimated CEJ displayed a weak association with MN frequency in PHA-stimulated lymphocytes ( r , = 0.40.p = 0.08).The association was still present after allowing for age and smoking (0.00003%0 per mg/mg3 X year, p = 0.07). PWM-stimulated MN showed no association with cumulative exposure. The MN frequencies (PHA and PWM).displayed no association with the printers' toluene levels in blood at the time of cytogenetic sampling or with the benzene exposure during employment in the 1950s. No difference was found in individual geometric mean MN size ratios between the printers and the controls, irrespective of the mitogen used (PHA: 0.027 vs. 0.024, p 2 0.5; PWM: 0.031 vs. (1.033, p 2 0.5, respectively). The median frequency of small MN (size ratio I0.03) in PWM-stimulated lymphocytes was 1%0 (range 0-3.7) in the toluene-exposed printers, compared with 0.3%0 (range 0-1,7) in the controls after allowing for age and smoking ( p = 0.05, Fig. 1, Table 1). The medians were 1.3%0 (range 0.3-3.0) and 1%O (range 0.3-2.71, respectively, in PHA-stimulated cultures ( p > 0.5, Fig. 1 , Table 1). The low frequency of small MN in PWMstimulated lymphocytes probably depends on the baseline level. Since the sample preparations were done for exposed and non-exposed subjects simultaneously and the analyses were performed Number! 901 ...... .. :. ..,: : I :. ......... -.. ... Fig. I . 'llii ratio > O.( blind, th small 1'1 blood to ( r , = 0.5: tistically smoking There in chron, the conti age and nifican tl) the conti No a: quencies rotogravi toluene both chrl ated wit} ing with I p = 0.004 when all1 habits th was still : Discussio The pi with an i small onc the small were assc tive tolue .YMI'I 10.YMI'I 10- 0.07). n with I) dishene * samg emomet-s and used 3 1 vs. ratio 1%0 liters, con0.05, xnge ly, in 'able WM1 the were s sinied PHA PWM 60 30 0- - 50.03 >0.03 SO.03 Size ratioof MN __ >0.03 Controls Printers Fig. I . Tlic riuniber ol'sin;rll' bizc r i i l i o 5 0.03)aiid .l)ig'( s i x r o l i o > 0 . 0 3 ) rnicroiiuclci in PI IA- aiid I'WM-sliniulalcd lyni- phocytes among printers mil controls. blind, this will not affect the results. Further, the sniall PWM-stimulated MN were associated with blood toluene at the time of cytogenetic sampling (rh= 0.55, p = 0.01); the association was still statistically significant after allowing for age and smoking habits (0.17%0 per pmole/l, p = 0.000S). There was no statistically significant difference i n clwomosomul breaks between the exposed and the control groups, when allowance was made for age and smoking habits (Table 1). However, significantly more gup.s ( y = 0.008) were observed i n the controls. N o association was found between the frequencies of chromosomal breaks and gaps in the rotogravure printers and either their blood toluene concentrations, or their CEls. However, both chromosomal breaks and gaps were associated with earlier benzene exposure (ycars working with benzene-contaminated toluenc; r, = 0.64, p = 0.004 and Y, = 0.39, p = 0.07, respectively); when allowance was made for age and smoking habits the association with chroniosoma I breaks was still significant (0.03% per year, p = 0.01). Discussion The printing work environment was associated with an increased frequency of MN (particularly small ones) in PWM-stimulated cultures. Further, the small MN in PWM-stimulated lymphocytes were associated with blood toluene. The cumulative toluene exposure showed a weak association with the frequency o f M N in PHA-stimulated cultures. Also, chromosomal breaks were associated with earlier benzene exposure. Printers are exposed both to toluene and to different organic dyestuffs in the printing ink (Svensson et al., 1990). `The exposure to toluene is the predominant one; the contact with the dyeituffs is less intense. A causal effect of toluene ib in accordance with the dose-dependent increase of MN in mice injected with toluene (Mohtashamipur et al., 198s). This is strongly supportcd by thc association bctwcen blood toluene and (small) MN, although there is the possibility that the blood toluene concentrations affected thc lymphocytes in vitro during culturing. However, it seems more likely that it is an in vivo effect, as the blood toluene was diluted (about S O times) by culture medium. Exposure was particularly associated with an increase in smull PWM-stimulated MN. This indicates ;in effect o n the chromosomes (fragments) rather than on the spincllc apparatus (Yamamoto et al., 1980; Hogstedt et al., 1985). In this study, we have used 2 mitogens PHA, which activates the T-cells, and PWM, which also stimulates B-cells. In lymphocytes cultured with mitomycin C and ionizing radiation in vitro, the frequency of MN was higher when using PWM than with PHA (H6gstcdt et at., 1988a). This has been confirmed by Slavutsky and Knuutila (l989), who also found that the B-cells showed a higher frequency of MN than did the Tlymphocytes, when the genetic damage was induced with peplomycin. In the present study, an effect of toluene on MN was observed in the PWM cultures. In accordance with the present findings in studies of gasoline-pump mechanics (Hiigstedt et al., 199I) anti chemical industry workers (Hijgstedt et al., 1988b), there were increases in MN frequencies in PWM cultures. Thus, there are several indications that Blymphocytes are more sensitive to mutagens than are T-cells. However, the increased sensitivity of the 13-lymphocytesthat is shown in vitro (Hogstedt et al., 1988a; Slavutsky and Knuutila, 1989) is not so easily displayed i n vivo. Still, Hagmar et al. (1989) found styrene exposure to be associated neither with MN frequency nor size ratios, irrespective of the mitogen used. -? -7 -? I t is interesting t h ;It , w11 i 1c crri'rrnf cxposti re was ;issociated with M N in I'WM-stimulated cells, the earlier exposure (CEI) was related t o 1'1-iAstimulated ones. This is in ;iccoi-d;ince with ;in association between cumti lat ive exposure to i norganic mercury and the frequency o f M N in PIiAstimulated cells (Barregird et al.. I O O I ), `rile cxplanation may be that different subgroups o f Iymphocytes have varying turnover rates, and that ;I fraction of lyniphocytes lives for decades. Thus a cytogenetic effect accumu latcd over many years is not illogical. I n accordance with this. persisting clironiosonial abe rrat ions have been recorcicd many years after radiation cxposurc (Buckton et ill., 1002). Ch roniosoni;i I hrea ks i n I' H A-st i ni ti I it t ed cc I Is were statistically significantly associated with work during decades whcn benzene wiIs present. `I`his finding is in ;IccOt-dilt1ce with rcsults by I-orni a n d co-workers ( 107I 1 in benzene-exposed workers. Fti rt tic r, an i ticre asc cl 1111ni be r of chroni o wti1;i I abeintions has recently been limnd in subjects with low occupatiot i a1 bcnzenc cxposu re (YardIcy-Jones et al., 1090). The discrepancy f o u n d i n earlier stiidics of printcrs (Funcs-Cr;ivioto et ill., 1977: Miiki-l'akkancn et al.. 1980)Ini1y be ;I rcsult o f the combination of studying PI-IA cultures ;tnd lack of consideration o f benzene exposure. I n the present study, the controls had signiricantly more gaps than the exposed workers. I lowever. ;IS all the scores were within the `nornial' limits o f the laboratory, the discrepancy in the number of gaps was probably a spurious finding. What do these cytogenetic findings mean in terms of health risk? There are indications o f ;in association between the ratc o f chromosomal aberrations in peripheral lymphocytes and subscquent cancer niorbidity (Nordic Study Group, 1990). Analogously. Hagmar et al. ( 1 086) found an increased risk o f Hodgkin's lymphoma and myeloma in workers at a chemical plant, where the workers also had increased M N freqiiencies in PWM cultures (H6gstedt et al., 198817). Sevcral other studies show an association between these and other lymphatic tumors and occupational cxposure to solvents ( O h , 1978; Olsson et al., 1980; Hardell et al., 1981). Most such tumors are derived froni the B-lymphocytes. For most of the current printcrs, thc toluene exposure fell considerably during the 1970s and has been low during the last 10 years, under 200 nig/ni' (the present Swedish TLV). This is probably why the effect o f toluene on the lymphocytes w a s rather slight; it might have been more pronounced earlier. Nevertheless, in a cohort study o f itbout 1000 rotogravure printers, half o f whom were exposed in the 19SOs, there was no increased cancer risk, including lymphomas (Svensson et al., 1990). Acknowledgements `I'hc study was supported by grants from the Swctli sli Work Envi ron men t F u 11d and t he Swedish Cancer Society. We thank Ms A. Nilsson for hcr collection of the blood samples, and Mr. R.G. Svensson for the medical examinations. The excellent technical assistance of Ms G. Nilsson is gratefully acknowledged. MI-. Bradley Evanof is :ickiiowlcdgctl for kindly revising the English text. References .13~. I. .I I,.L ~ ~ I~<..I I1%- .~I l.iigsletlt. A. Schiitz. A. Kitrlsson. G . Siillsicii ;ind (i. Tliiringci- ( IOYI ) lil'tcct 01 occupation.:il cxposure to niercuiy vapour on lymphocyte micronuclei, Scand. J. Work Environ. ile;ilth, 17, 2h3-2hX. Iktuchinger. M., E. Sctimid, .I. Dresp, J. Kolin-Gerrescheim, R . 1 I ; i u I and E. Suhr (1082) Chromosome changes in lyniphocytes after occupational exposure to toluene, Mutation lies.. 102, 339-445. I3uckton. K.E.. P.A. .lacobs, W.M. Court Brown and R. Doll (1002) A sludy of chromosome damage persisting after X-ray therapy for ankylosing spondylitis, Lancet, ii, 676682. Dean. 13.J. (1085) Recent findings on the genetic toxicology o f benzene. toluene, xylenes and phenols, Mutation Res., 154. 153-1x1. Forni, A., E. Pacafico antl A. Limonta (1971) Chromosome studies in workers exposed to benzene or toluene or both, Arch. Ilnviron. flealth. 22, 373-378. Funes-(`r;iviolo, F., 13. Kolmodin-I It.dniitn, J. Lindsten. M. Nortlcnsk.iiild, C. Zapata-Gayon, B. Lambert, E. Norberg, K. Olin and A. Swensson (1977) Chromosome aberrations antl sister-chromatid exchanges in workers in chemical laboratories and a rotoprinting factory and in children of women laboratory workers, Lancet, ii, 322-325. (3x1-El-Karim. M.M.. B.L. I-larper and M.S. Legator (1984) Modifications in the myeloclastogenic effect of benzene in mice with toluene, phenobarbital, 3-methylcholanthrene, Aroclor 1254 a n d SK12-S25A, Mutation Res., 135,225-24.3. I laglund. [I., I . 1,iindberg and I-. Zech (IOXO) Chromosome ahcrr:itions and sister chrom;itiil exchanges in Swedish paint inth 2`) I -2YX. I lagmar. I... At (eweI I niorhitlity Work Pin I kigmiir. I>.. Riissricr ( plastic wo ron. I lenll f lardell. L... lymphoni;i organic vi control st1 I liigstctlt. t3. scrvctl CVt' cl;ini;igc iii I liigstctlt, 13.. in human used. Mut 1liigstedt. 13., of micron st inirr htcd mitogen, 1 1lijgstedt. I).. ing ( IOXHII in lympho pokeweed 1 leretlil;is, I liigstcdl. 13.. ;ind K. V increased by pokewc lARC (1980) genic Risk ganic Sol\( Pigments : ture and F Cancer, L) J n ter na t ion;I I (19x5) Birt dation, Ne Jelnes. J.E. (I P. Lirntlbe Expert Circ Karibisch. N.. its intcriiic con1polllid! ides. I3ioch Low. L.K., J. . - --- 70s ilild rlcr 200 s probhocytes I'c pr'o1 study . whom no in- [ SVCllS- ,111 1hc d the 4 ilsson id Mr. s. 'I'he isoi1 is mot' is 11 text. 'iiillsten )sure to I. Work diciiii, iges i n M U I;I- <. 1)Oll 5 after il hX1- logy Of Res., osoI11e .I~OIh, 11, M . irbe rg, ations miciil Kll of :l')x4) :ne in IICIIC, -243. )some ulisli piiinl iiidustiy workci'a. Sc;iiitl. J. Woi-h I<nvircin.I Icaltli. 0, 201 -2ox. ll:igm;ir, I-., '1'. l3cllandcr. V. Iiiigliiiidcr. J . l<:ins[iiin, I<. Attewell arid S. Skcrlving ( lO8f)) Mortality ;ind ~;IIIC~Iiiior1)idity among workcrs i n ;I clicniicirl f;ictory, Sciiiitl. J . Work Environ. Ilealth, 12, 545-551. 1l;igniar. L., 13. Iliigstcdt, 1I . Welindcr, A. Karlsson ;ind f:. I<;iswer ( 1080) ('ytogenctic ;ind hcin;itologic;il cflcct> iii plastic workcIs cx~~iisetol styrcric. Sciiiiil. I. Work linviion. l l e ~ l l t l ~IS,, I . l ( I - - l 4 l . lliirdell, I..,M. liriksson and I>.L e i i n c r (10x1 1 M;ilign;int lyniphonias antl exposurc LO chcniic;rl agents, espcciiilly to organic solvents, chloroplicnols and phenoxy acids. A case control study, 131. J. C'anccr, 43, 109- 176. Iliigstedt, U. ( 10x4) Microniiclci i n lynipliocytcs with prcserved cytoplasm. A method lor assessnient o f cytogenetic doniiige in miin, Mutation Res., 130, 63-72. Iliigstedt, l3., and A. Karlssoii (1985) 'Ihc size of micronuclei iii liumiin lyniphocytes varies iiccortlirig to iiitlucing agent used. Mutiition Res., 150, 229-232. Iliigstedt, 13.. A. K;irlsson and A. Ilolm&n ( lO8X;i) 1:requcncy of micr-oiiuclci i n human periplicr;il I)loocl lyriiphocytcs ~tiniulatcdi n v i t r o by pliytolicrii;iygliitiiiin and pokewetid iiiitogeri, Ilereditiis, 109. 53- Ilijgstedt, U., I. Bratt, A. l-lolni&n,L. Ilagmur m d S. Skcrlving ( IYXXb) Frequency and size distribution ol' micronuclei in lyniphocytes stiniul;itetl with pliytolicmagglutiiiin :ind pokcwced mitogen in workers cxposctl to pipcrazinc, Ilcreditas, 109, 139- 142. Iliigsledt, B., A. liolnith, A. Karlsson, Ci. Raihlc, K. Nillius ant1 K. Vestlund (1991) Gasoline piimp mechanics had increased frequencies mid sizes i n lymphocytes stimulatctl by pokeweed mitogen, Mutation Ilcs., 203, 5 1-55. IAKC' (19x0) Monographs on the Evduation of the C'arcinogenic Risk o f Chemicnls t o Iluiii;ins, Vol. 47, Some Organic Solvents, Resin Monomers and Related (.'ompounds, Pigmen ts ii ncl Occupati o n ;iI Exposures i n Paint Manu fuctuw and Painting, Intern;ition:il Agency lor l<cse;irch on ('anccr, Lyon, pp. 70- 123. 1iilcriiation;il Systeni lor llum;rn ('ytogenetic Noniencl:rtirre (19x5) Birth defects, March ol Dimes Birth Delects F h n dation, New York, Original article series, vol. 21, no. I . Jelnes, J.E. (1989) Toluene, in: G. I lcinil>iirgcr. 13. Ikijc ;in11 1'. LII ntllie rg ( Etls.1. ('rile ria Ihicu iiic 11Is I ' t ~ i i i i I tic Noi.tl ic Ikqwt Group IOXO, Arl)ctc I liilsii, 37. 7 - 57. K.;iubisch, N., J.W. Daly a n d I1.M. Jerina (1972) Arene oxides a s intermed iates in the o xitInt ivr met ii hol ism of ;iromiit ic compounds. Isomerization o f methyl-substituted arene oxides, Biochemistry, 1 1, 3080-30XX. I.ow, L.K., J.lL Meeks and C'.R. M x k e r c r (19x8) I le;ilth cl~lccts of the ;ilkyll~cri~ciicaI.. 'l'oluciic, 'l'oxicol. Iiitl. 1 lcillth, 4, J'k75. M:iki-l):ikk:inen, J., K. I ltisg~ilvcl-l~tirsi~iiiicI).i-iI.,. Kiillioniiiki, J. 'l'uomincii antl M. Sorsu ( 1980) ~l'olucnc-cxposctlworkers ;ind chroiiiosonic :il)err:itions, I . 'l'oxicol. Inviroii. IIciilth, 0, 775-7131. Moht;ishaniipur, I-.,K. Norpotli, U. Woclkc and I). I luhcr (108.5) Iffccls of ctllyll~cll/.cllc.Iolucllc. i11111 xylcllc 0 1 1 llic inductioii 01. iiiicroiiuclci iii I)onc n i ~ i i ~ ~pocw~lycliroin;itic erythrocytes ol mice, Arcli. Toxicol.. 5S, 100- 100. Nisc, G. ( IWI 1 Urinary cxcrction of' o-cresol ;ind hippuric acid alter toluene exposure i n rotogrovure printing, lnt. Arch. Occup. Environ. I ledth. i n press. Nise, G., and P. Urtxek ( I W X ) Toluene i n venous I)lood during ;ind ;il'ter work i n rotogr~rvureprinting, I n t . Arch. Occup. Eiiviron. IHealth, 60, 3 1-35, Nordic Study Group (1990) An inter-Nordic prospective study on cytogenetic endpoints antl ciincer risk, Cancer Genet. ('ytogenct.. 45, xs-92. Olin, I<.( 1078) The h;rz:irds of' ;I cheniicd laboratory environment. A study o l the mortality i n two cohorts of Swedish clicmists, Ani. Intl. I lyg. Ass. J., 3 0 , 557-507. Olsson, 1I., iiiitl I,. I~i-;iiitlt( 19x0) Occupiitional exposure to organic solvents and I lodgkin's disease i n man. A casecontrol study, Scand. J. Work Environ. I Ic;ilth, 6, 302-305. Roh. J.. Y.11. Moon and K.Y. Kim (10x7) The cytogenetic effects o l Ixxwcnc iiiitl tolucnc o n boric miirrow cells i n r:its. Yonsci Mctl. J., 2X, 707-300. Slavutsky, I.,and S. Knuutila (1989) Micronucleus formation i n tli fle rent lymphocyte subpopti Litions in pe plomyci ntreated a n d control ciilturcs, Mutation Res., 219- 257-261. Svcnsson, 13.(;., C;. N i x , V. L3iglander, I<. Attcwell, S. Skerlviiig mil '1'. Miiller ( IWO) I>eaths mtl tumoiirs aniong rotogravure printers exposed to toluene, 131. J . I d . Med., 47, 372-370. Whorton, E.B. (19x5) Some experimental design antl analysis considerations for cytogenetics studies, Environ. Mutagen., 7. 0- IS. Wislocki, I).(;., A.W. Wood, R.1,. ('Iiiiiig et al. ( I O T i ) Mutirgcnicity and cytotoxicity o f I>cnzo[n]pyrrnc arcne oxides, phenols, quinones, and dihyilrodiols in bacterial and nianimaliari cells, C'ancer Rcb., 36, 3350. Yaiii;imoto, K.I., ;tiid Y. Kikuchi ( 1080) A comp;irisori of tliiiiiictcrs til' iiiici-oniiclci iiitlucctl by cliist~igciis;ind by spiiiillc piisoiis, Mut;ition I<cs., 71, 127- 131. Yardlry-Jones, A,, I). Anderson, D.P. Lovell and P.C. Jenkinsoli ( 1000) Analysis of chromosomal aberrations i n workers exposed t o low levcl benzene, 131.. J . Ind. Med., 47, 4s-5 I ,