Multicenter field trial on possible health effects of toluene

Toxicology 168 (2001) 185– 209 www.elsevier.com/locate/toxicol

Multicenter field trial on possible health effects of toluene III. Evaluation of effects after long-term exposure Christine Gericke a,*, Bernd Hanke c, Gudrun Beckmann d, Margret M. Baltes b, Klaus-Peter Ku¨hl b, Diether Neubert a, Toluene Field Study Group 1 a

Institute of Clinical Pharmacology and Toxicology, Benjamin Franklin Medical Center, Free Uni6ersity Berlin, Garystrasse 5, D-14195 Berlin, Germany b Psychiatric Uni6ersity Clinic, Benjamin Franklin Medical Center, Free Uni6ersity Berlin, D-14050 Berlin, Germany c Occupational Medicine Axel-Springer Verlag AG, Hamburg, Germany d Occupational Medicine Broschek Druck GmbH, Hamburg, Germany Received 15 December 2000; received in revised form 25 April 2001; accepted 26 April 2001

Abstract In rotogravure industry, contributing considerably to mass color printing of catalogues and magazines, toluene is still extensively used as paint solvent, and many printers have been exposed to this chemical for several decades. Information on adverse health effects associated with long-term toluene exposure is still controversial. In a multi-center study, adverse health effects possibly associated with long-term toluene exposure were evaluated. In 12 rotogravure factories, 1226 male volunteers were recruited, and sufficient information on exposure and on medical data was compiled for about 1077 of them. Evaluations included: physical examination, standard tests of psychophysiological and psycho-motoric performances, self-report of subjective symptoms, and data on a variety of laboratory blood tests. The medical data were correlated with the length (months) of toluene exposure, and a rough estimate of the extent of exposure (i.e. highly exposed printers and their helpers versus employees working at locations with low or negligible toluene exposure). A small reference group (n = 109) was selected from companies of the paper industry. When linear regression curves were calculated (test results versus duration of exposure), extremely low overall coefficients of determination (r 2 adj.) of a few percent were estimated within the data clouds, with sometimes

* Corresponding author. Tel.: + 49-30-84451795. E-mail address: [email protected] (C. Gericke). 1 Further members of the Toluene Field Study Group are: Dr med. Hermann-Josef Bungert (Axel Springer Verlag AG, Darmstadt); Dr med. Marielis Dorn, (U.E. Sebald Druck + Verlag GmbH, Nu¨rnberg); Dr med. Inge Elster (Schlott Tiefdruck KG, Freudenstadt); Dr med. Rudolf Hoppe (Tiefdruck Schwann-Bagel GmbH, Mo¨nchengladbach); Dr med. Michael Ja¨gers (Maul + Co.-Chr. Belser, Nu¨rnberg); Dr med. Christian Kurth (Melitta, Minden); Dr med. Franz Ott (Burda News Druck GmbH, Darmstadt); Dr med. Gudrun Riedesel (PVG GmbH, Spende); Dr med. Irmgard Rotax (Gruner + Jahr AG & Co., Itzehoe); Dr med. Wolfgang Sieke (Zewawell AG, Minden); Dr med. Fredericke von Stosch (Burda GmbH, Offenburg); Dr med. Arnold Tenbaum (Klambt Druck GmbH, Speyer); Dr med. Christa Wetzel (Herlitz, Berlin); Dr med. Volkmar Wolters (Badenia Verlag +Druckerei GmbH, Karlsruhe). 0300-483X/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 0 - 4 8 3 X ( 0 1 ) 0 0 4 0 8 - 5

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statistically significant P-values. Closer analyses revealed a strong influence of the confounding factor age, no clustering of abnormal values of highly toluene-exposed volunteers, and the vast majority or all values of the highly and long-term toluene-exposed participants staying within the reference ranges. Thus, no medical relevance of P-values B0.05 could be recognized in this evaluation, and there may have been some border-line deviations or results largely occurring by chance in the large trial. In a small cluster of the many rotogravure printers toluene-exposed for more than 20 years, the highest systolic blood pressure values of the study were found, but many possible confounding factors were not taken into account. Data on acute exposure and possible effects are presented in accompanying papers (Neubert et al., 2001a, Neubert et al., 2001b). Restricting the conclusions to the end points evaluated as well as the apparent limitations of the evaluation, no evidence was found that long-term occupational toluene exposure extending over several decades in the rotogravure industry in the Western part of Germany was convincingly associated with chronic adverse health effects or convincingly altered surrogate markers in still working male volunteers. Several peculiarities and pitfalls arising when interpreting medical data associated with such a type of environmental exposure must be considered. Reversibility of alterations previously induced at higher levels of toluene-exposure, as well as a healthy workers effect, cannot be excluded for some of the medical end points evaluated. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Toluene; Chronic effects; Humans; Blood pressure; Clinical chemistry; Psycho-physiological examinations; Rotogravure printers; Occupational medicine

1. Introduction Toluene has been extensively used in rotogravure printing but risk assessment on chronic adverse health effects after many years of toluene exposure has remained difficult because the published data are contradictory. Toluene concentrations at the work-areas have decreased considerably during the past decades, but most previous investigations are from the 1980s. Data of the largest field study on toluene exposure performed so far were used to also reveal possible chronic adverse health effects after longterm toluene exposure over several decades. Information on more than 1000 toluene-exposed male individuals from 12 German rotogravure companies, and an external reference group of about 100 not toluene-exposed male volunteers of the paper industry, formed the basis for these evaluations. Intentions as well as predictive value of this evaluation of possible chronic effects of long-term and comparatively high toluene exposure are quite different from those of the accompanying assessment of acute exposure (Neubert et al., 2001a,b): dose– response relationships or a lack of acute effects at defined concurrent body burdens may be revealed from the cross-sectional trial. Quantitatively assessing the exact extent of individual chronic exposure to variable daily doses is

virtually impossible for substances with rather short elimination half-lives, such as toluene. However, individuals with presumed high toluene exposure may be compared with a group of participants exposed to very small doses of toluene, if any. Furthermore, the duration of exposure may be taken into account as a semi-quantitative measure of the extent of long-term exposure, required for the development of chronic adverse effects. These approaches were used in our trial. The evaluations were aimed at testing the following hypotheses, which were based on contradictory information from previously published studies: 1. Long-term exposure to high concentrations of toluene may be associated with deficits to be revealed in special psycho-physiological and psycho-motoric tests (Iregren, 1982; AnttiPoika et al., 1985; Juntunen et al., 1985; Mørck et al., 1988; Ørbæk and Nise, 1989; Foo et al., 1990, 1993). 2. Some subjective complaints, such as headache, may be mentioned more frequently (Mørck et al., 1988) by highly and long-term toluene-exposed persons. 3. Serum transaminases may deviate from the reference range (Mørck et al., 1988) in highly and long-term toluene-exposed persons.

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4. Renal functions may be altered (Askergren, 1982) in highly and long-term toluene-exposed persons. 5. FSH and testosterone levels may deviate from the reference range (Mørck et al., 1988; Svensson et al., 1992a,b) in highly and longterm toluene-exposed workers. 6. Systolic blood pressure may be increased (Mørck et al., 1988) in highly and long-term toluene-exposed volunteers. A few additional routinely measured medical endpoints were compiled in order to possibly generate further hypotheses.

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pate. An additional small external reference group was recruited from four companies of the paper industry (REF-EXT, with 109 men). These served especially as independent references for the psycho-physiological tests and for confirming possible age-dependent effects. The incidence of subtle neurological deficits could not be revealed in this study, since such examinations should be performed by a single experienced neurologist, a design not possible in this large study. End points evaluated for possible effects of long-term exposure are compiled in Table 1.

2.2. Exposure assessment 2. Study design and methods

2.1. Study design Data were evaluated from the multi-center, controlled toluene field study. For the evaluations on possible adverse health effects of long-term toluene exposure results of the psycho-physiological and psycho-motoric tests were used, as well as data of additional relevant medical end points. Different exposure scenarios (duration of exposure as well as type of work-area [gross extent of exposure]) were taken into account. The study was restricted to male participants still employed at the factories. An evaluation of all the employees of the participating rotogravure factories, as may have been desirable, was impossible because the assessment of psycho-physiological capabilities should be included, and only a limited number of such tests could be carried out. Furthermore, we could not recruit retired printers who had been toluene-exposed during extended periods. For practical reasons, the evaluation was performed on the participating 1225 employees from 12 rotogravure printing companies in the Western part of Germany. Volunteers were selected randomly by independent members of the Federal Professional Association of the Printing and Paper Processing Industry. However, the selection was not truly random, because workers initially chosen could refuse to take part in the study, and the next randomly selected person was asked to partici-

It is impossible to assess the exact individual toluene exposure experienced occupationally, i.e. quite variably over decades. We have refrained from assessing special quantitative ‘exposure scores’, because this cannot be achieved with any reasonable accuracy. For the analyses of possible associations of chronic adverse health effects with long-term exposure to toluene, clinical and laboratory data were correlated in our trial with the duration of toluene exposure (months) at the typical work-area. Additionally, workers assumed to have been heavily exposed (printers and their helpers) were compared with those having experienced only slight exposure (e.g. via processing of the printed material) in the same companies, and additionally in some instances with the external reference group (REF-EXT, with no occupational toluene exposure). If a printer or his helper was typically and simultaneously heavily exposed to another substance or factor over the years, this would be a strong confounder. We are not aware of such a specific agent, but possible effects of poly-exposure, e.g. to toluene and noise, cannot be excluded. The residual body burdens of the volunteers (mg toluene/l blood) at the time when the psychophysiological tests were given before the workshift are compiled in Table 2. Residual body burdens were higher in ‘printers’ than in ‘nonprinters’, but only a small percentage exhibited toluene concentrations \ 80 mg/l blood. Body burdens achieved during the same work-shift (i.e. measured after the work-shift) are published in

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Table 1 Selection of variables evaluated in this study Variable

Psycho-physiological tests Immediate visual memory (visual reproduction) Self-rating of feeling (scales of self-feeling) Verbal memory span, forward Verbal memory span, backward Visuo-motoric performance (digit symbol) Objecti6e tests Bisensory vigilance test Critical flicker fusion frequency test

Alterations to be expected after exposure Acute

Chronic

x

X

x

X

x

X

x

X

x

X

x x

X X

Self assessment of minor complaints Burning tongue Diarrhea Dry mucous membranes Fever Headache Insomnia Loss of appetite Muscle cramps Nausea Skin itching

X X X X X X X X X X

Physical examinations Blood pressure, diastolic Blood pressure, systolic Color vision Lung function Neurological symptoms Skin alterations Visus

X X (X) X X X X

Clinical laboratory 6alues (serum) GOT GPT gGT Uric acid Creatinine Cholesterol (total) Blood glucose (fasted)

X X X X X X X

Hormone concentrations (serum) FSH LH Testosterone TSH

X X X X

Confounders considered Alcohol (acute) Body mass index Age Gender

x x x

X X X

detail elsewhere for comparison (Neubert et al., 2001a). This provides an overview on the toluene exposure within the participating German rotogravure factories during the period (1993–1995) the trial was performed. As a group, printers were much higher toluene-exposed than ‘non-printers’. It is more difficult to assess the possible previous toluene exposure of the volunteers during the past decades at various work-areas. There is no doubt that printers (including proof printers) and their helpers also were the workers with the highest toluene exposure within the factories, and that they have been exposed to higher levels during the past decades. The initially tolerable maximal toluene concentration at the work-place (MAK-value) of 200 ppm was reduced in 1985 in the Federal Republic of Germany to 100 ppm, and to 50 ppm in 1993.

2.3. Number of 6olunteers e6aluated The initial number of male volunteers participating in this part of the study was 1225 of the rotogravure factories, and 109 references from factories of the paper industry (sum: 1334). However, of only 1077 participants reliable information on toluene exposure and on the duration spent at a toluene-exposed work-place could be obtained. Interchanging of jobs with different types of exposure is one example of an exclusion criterion. Included in the final selection of participants were 768 printers and their helpers (286 of these toluene-exposed for ] 240 months), and 309 ‘non-printers’ (117 of these employed for ] 240 months). Furthermore, the numbers of workers to be evaluated was somewhat reduced by the fact that not all individual data sets were complete, and single individual data could not be reliably considered. In compliance with the German ‘data protection’ law, anonymous data were passed on by the examining physicians, and it was difficult to correct missing information later on.

2.4. Psycho-physiological tests and analyses of psycho-motoric functions The tests selected allowed to evaluate various aspects of psycho-physiological capabilities and

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Table 2 Toluene body burden measured at the time of the psycho-physiological and psycho-motoric testing (i.e. before the work-shift) Exposure group

Median

Q1

Q3

Min

Max

1.3 14 39

0.9 6 20

1.8 32 82

0.3 0.7 1.7

12 876 911

Typical exposure during the work-shift (mg toluene/m 3 air) References (paper industry) 109 0.5 17 ‘Non-printers’ 455 Printers and their helpers 770 91

0.5 7 44

1.5 39 161

0.5 0.3 1.6

48 200a 800b

n

Before the work-shift (vg toluene/liter blood) References (paper industry) 109 455 ‘Non-printers’ Printers and their helpers 770

a b

+1 value: 550. +1 value: 1715 (contaminated?), 10 mg toluene/m3 = 2.7 ppm.

psycho-motoric performances. The well-established tests had been validated on a number of psychopathological conditions and diseases, including drug actions. Tests were given on a single day before the work-shift, in order to avoid interference with higher acute toluene exposures. The test repertoire used by us included: (1) verbal memory span (digit span test (Wechsler, 1958)), consisting of two parts— digit span forward and digit span backward; (2) visuomotoric performance (digit symbol test (Wechsler, 1958)); (3) immediate visual memory (visual reproduction test, (Wechsler and Stone, 1983)); (4) bisensory (auditory and visual) vigilance test (Wiener reaction), using the special testing equipment; (5) critical flicker fusion frequency test, using the special test apparatus. The tests are described in more detail in an accompanying paper (Neubert et al., 2001b).

2.5. Subjecti6e complaints and self-rating of feeling The volunteers were asked to mention subjective complaints, such as: headache, insomnia, nausea, loss of appetite, diarrhea/constipation, etc. As further subjective data they provided information on self-rating of feeling (using the established scales of self-feeling, Zerssen, 1976; Zerssen et al., 1979).

2.6. Anamnestic information and results of physical examinations The medical examinations were performed once on each of the participants. They included routine physical examination and simple neurological tests.

2.7. Clinical chemistry The laboratory tests were performed once on each of the participants. Reference ranges for the individual laboratory tests were established by the different laboratories of clinical chemistry.

2.8. Statistical analyses The hypothesis was tested that abnormal reactions and adverse health effects may be associated with the long-term toluene exposure, and with semi-quantitative measures of the extent of exposure (printers vs. ‘non-printers’). The duration of working at a typical work-area with known toluene exposure was taken as a basis for evaluation, a semi-quantitative measure of the extent of exposure served as another criterion. Usually, data of the cross-sectional part of the study are presented, but the evidence was never changed when the 45–46 volunteers of the longitudinal part (all were printers and their helpers) are included. Standard

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Fig. 1. Association between duration of toluene exposure (months) and age (years) of male volunteers of the printing industry (n=1077). Regression equation: Duration of exposure = − 178 +9age, coefficient of determination (r 2 [adj.]): 60%, PB 0.0001.

statistical tests and analyses were applied, using the SAS® software. Of all evaluations individual data were supplied, mostly in the form of scatter plots. Individual data deviating from the reference range were especially analyzed. Because of the extensive data set and the numerous end points, statistical associations by chance and nonsense associations are bound to occur.

2.9. Confounding factors Recognizing and controlling for confounding factors is a crucial aspect in any epidemiological evaluation. With respect to the psycho-physiological tests, concurrent alcohol consumption was considered as confounding factor, and controlled by measuring blood alcohol levels when the tests were given. However, the volunteers were informed about these tests and warned not to consume any alcohol during this work-shift. It was not possible to effectively control for effects of chronic alcohol consumption as a confounding factor. With regard to some of the psycho-physiological tests as well as with respect to the duration of long-term toluene exposure, age was the strongest confounding factor, and the age-dependency was always taken into account. Our data were not controlled for noise as a confounder.

3. Results In previous studies, long-term occupational toluene exposure has been associated with some manifestations of chronic diseases or altered surrogate markers of chronic diseases (e.g. Tha¨ ti et al., 1981; Antti-Poika et al., 1985; Cherry et al., 1985; Juntunen et al., 1985; Chia et al., 1987; Ha¨ nninen et al., 1987; Mørck et al., 1988; Ørbæk and Nise, 1989; Svensson et al., 1992a,b). However, the results of these studies are contradictory, and several previous reports suffer from a number of inadequacies. In particular, a small number of volunteers were not allowed the control for important confounding factors. As to be expected, the duration of exposure (period employed at a given work-place with known toluene exposure) was highly correlated in our study with age (Fig. 1), and this was a strong modifying factor for several of the end points evaluated.

3.1. Psycho-physiological tests and tests of psycho-motoric functions Results of digit span forward and backward tests are compiled in Fig. 2A and B. Only a very small significant trend (P=0.003, r 2 [adj.]: B 1%) can be seen with respect to the digit span forward

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Fig. 2. Association between the scores of the psycho-physiological test and duration of toluene exposure (months) of male volunteers of the printing industry (n= 1077). (A) Digit span forward test: regression equation: score of the test = 6.5 −0.001duration of exposure, coefficient of determination (r 2 [adj.]): 0.7%, P=0.003. (B) Digit span backward test: regression equation: score of the test =4.9 −0.0015duration of exposure, coefficient of determination (r 2 [adj.]): 2%, P =0.0001.

test, and also the tiny trend in the backward test (r 2 [adj.]: 2%) really is not convincing, despite the ‘statistical significance’ (P =0.0001). Without considering confounding factors, there seemed to be an overall trend of an association (Fig. 3A) between the duration of toluene exposure and a reduced performance in the digit-symbol test (analysis of variance: r 2 [adj.]: 6%, PB0.0001). However, this apparent trend is clearly confounded by the age-dependency in the outcome of this test (Fig. 3B). Multiple regression analysis shows a statistically significant dependency on age (P=0.0001), but no decrease in the performance capability with respect to toluene exposure (even a slightly opposite trend is calculated).2 An analogous slight age-dependency (r 2 [adj.]: 18%) is obvious in the independent reference population of the paper industry (Fig. 3C). The second performance for which a very slight but statistically significant overall trend (P = 0.0001) was calculated in the linear regression analysis was the visual reproduction test (Fig. 3D). The coefficient of determination also was negligible (r 2 [adj.]: 5%). Multiple regression analysis again revealed an influence of age (P = 0.0001), but there was no reduced performance which could be attributed to the duration of

toluene exposure, in fact a slightly opposite effect occurred (P= 0.01).3 It was also analyzed whether a higher percentage of performances in the lower range (i.e. below the overall mean value) occurred in volunteers with an especially high (as printers or their helpers) and long (\240 months) toluene exposure when compared with the ‘non-printers’ (Table 3). No special susceptibility of the highly and long-term toluene-exposed workers could be recognized with respect to the four psycho-physiological tests evaluated. At the first glance, the duration of toluene exposure at the work-area also might be associated with a very small trend to a longer decision as well as motoric time in the bisensory (auditory and visual) vigilance (Wiener reaction) test (Fig. 4A and B). However, again an influence of age in the same direction is detectable (Fig. 4C), verified independently by the reference group of the paper industry (Fig. 4D). This fully explains any apparent trend with respect to exposure. It is difficult to recognize a biologically significant trend within the broad data cloud of for example Fig. 4A and B. The independent group of volunteers of the paper industry is of interest, because for most of the psycho-physiological functions tested there is a correlation with respect to age in these participants without any toluene exposure (Table 4).

2 Multi-regression analysis: 0.016duration of exposure.

3 Multi-regression analysis: 0.003duration of exposure.

score = 62− 0.58age+

score = 95 − 0.13age+

192 C. Gericke et al. / Toxicology 168 (2001) 185–209 Fig. 3. Association between the scores of the psycho-physiological test and duration of toluene exposure (months) or age (years) of male volunteers of the printing industry (n=1073). (A) Digit symbol test vs. exposure: regression equation: score of the test =45− 0.02duration of exposure, regression coefficient (r 2 [adj.]): 6%, P=0.0001. (B) Digit symbol test vs. age: regression equation: score of the test = 59 −0.4age (printing industry), coefficient of determination (r 2 [adj.]): 17%, P=0.0001. (C) Digit symbol test vs. age: regression equation: score of the test = 60 − 0.4age (paper industry), coefficient of determination (r 2 [adj.]): 18%, P =0.0001. (D) Visual reproduction test vs. exposure: regression equation: score of the test =12 − 0.006duration of exposure, coefficient of determination (r 2 [adj.]): 4.5%, P=0.0001.

C. Gericke et al. / Toxicology 168 (2001) 185–209 Fig. 4. Association between the scores of the psycho-physiological test and duration of toluene exposure (months) or age (years) of male volunteers of the printing industry (n=880). (A) Bisensory vigilance test (decision time) vs. exposure: regression equation: score of the test =0.4 +0.00004duration of exposure, coefficient of determination (r 2 [adj.]): 0.3%, P= 0.052. (B) Bisensory vigilance test (motoric time) vs. exposure: regression equation: score of the test = 0.1+ 0.00002duration of exposure, coefficient of determination (r 2 [adj.]): 0.2%, P =0.09. (C) Bisensory vigilance test (decision time) vs. age: regression equation: score of the test = 0.3+ 0.002age (printing industry), coefficient of determination (r 2 [adj.]): 5.6%, P =0.0001. (D) Bisensory vigilance test (decision) vs. age: regression equation: score of the test= 0.3− 0.002age (paper industry), coefficient of determination (r 2 [adj.]): 4.6%, P B0.045. 193

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Table 3 Number and percentage of male volunteers with very high and long toluene exposure, showing scores below average in some of the psycho-physiological tests ¡ Test

Verbal memory span, forward Verbal memory span, backward Visuomotoric performance Immediate visual memory

Number of all volunteers

Toluene-exposure ]240 months

‘Non-printers’

‘Non-printers’

Printers and helpers

Printers and helpers

All

With low valuesa

All

With low valuesa

304

758

117

24

20.5%

286

67

23.4%

304

758

117

64

54.7%

286

153

53.5%

308 304

754 758

117 117

64 36

54.7% 30.8%

285 286

157 94

55.1% 32.9%

There is no statistical significance between printers and their helpers and ‘no-printers’. a With values below average (these are not pathological values).

This may be taken as a ‘positive control’, indicating the responsiveness of the test. Some results of the critical flicker fusion frequency test (as evaluated before the work-shift) were discussed in an accompanying publication (Neubert et al., 2001b). The data suggesting that highly toluene-exposed printers and their helpers might recognize shorter flicker frequencies as continuous (compared with the combined references) are not very convincing.

3.3. Anamnestic information and objecti6e results of the physical examinations Anamnestic information did not reveal any increased incidence of cardiovascular, lung or kidTable 4 Coefficients of determination (r 2 [adj.]) for some of the tests vs. duration of toluene exposure (months) or age (years) of the male volunteers Printing industry

Paper industrya

Regression for: “

Exposure

Age

¡ Test

r 2 [adjusted] (%)

Digit span, forward Digit span, backward Immediate visual memory Visuomotoric performance

2.0

4.8

5.2

0.2

1.8

2.6

3.2

9.6

10.9

10.7

22.3

19.8

8.3 4.7

12.7 7.4

3.2. Self-assessment of minor complaints Impairment of subjective well-being was not mentioned more often in volunteers clearly longterm and highly toluene-exposed in the rotogravure printing industry (printers and their helpers). The frequency of overall complaints such as: headache, insomnia, nausea, loss of appetite, diarrhea/constipation, etc. was not correlated with the duration or the extent of toluene exposure. When all the volunteers of the printing industry were compared with those of the paper industry, a significantly higher percentage of the former ones ( 2, P B0.05) mentioned (Table 5) sleeping disorders (43 vs. 22%), ‘dry mucous membranes’ (18 vs. 9%), and the occurrence of allergies (19 vs. 8%). Thus, the difference is apparently due to some unknown confounding factors, related to the type of factories, and not to toluene.

Audio-6isual 6igilance test Decision time 6.2 Motoric time 3.2

Age

Bold: coefficient of determination for age at least double of that of exposure period. All r 2-values are so small that the medical relevance is questionable. a Volunteers from the paper industry are considered as references.

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Table 5 Subjective symptoms in male volunteers toluene exposed in the printing factories compared with participants of the paper industry Symptom

Printing industry (with toluene exposure)

Paper Industry (without toluene exposure)

Total

With symptoms

Total

With symptoms

n

n

%

n

n

%

Loss of appetite Diarrhea Constipation Nausea Headache Insomnia Muscle cramps Neuralgia Fever Dry mucous membranes Itchy skin Burning tongue

1226 1226 1226 1225 1225 1226 1225 1226 1224 1223 1223 1222

105 93 62 197 402 525 148 96 9 214 234 21

8.6 7.6 5.1 16.1 32.8 42.8a 12.1 7.8 0.7 17.5a 19.1 1.7

107 107 107 107 108 106 106 107 107 107 107 107

5 5 1 7 28 23 9 5 0 10 15 3

4.7 4.7 0.9 6.5 25.9 21.7 8.5 4.7 0 9.4 14.0 2.8

Allergies

1209

231

19.1a

107

9

8.4

The number and percentage of volunteers subjectively indicating the symptoms listed (self-assessment) is shown for all male volunteers from the printing industry (possibly toluene exposed) and participants from the paper industry (references). a PB0.05;  2-test.

ney diseases in the highly and long-term toluene-exposed volunteers. The percentage of dermatological diseases was also not increased in printers and their helpers. Routine neurological examinations revealed no indication for toluene-induced effects among the highly and long-term toluene-exposed printers and their helpers (Table 6). There were no increased obvious psychiatric illnesses.

3.4. Results of laboratory tests A variety of laboratory tests was evaluated.

3.4.1. Serum li6er enzymes and serum-cholesterol There was no association of GOT (glutamic– oxaloacetic transaminase) or GPT (glutamic– pyruvic transaminase) with the duration of toluene exposure, nor when the group of long-term and higher toluene-exposed volunteers (printers and their helpers) was compared to employees with a much shorter and lower toluene exposure (Fig. 5A– D). These variables also exhibited no significant age-dependency (Fig. 5C). We have also for all the

variables of clinical chemistry analyzed the number of outliers, and the proportion of highly and long-term exposed persons among these values outside the reference range (Fig. 5D). There was no higher percentage of outliers for printers and their helpers. The activity of the k-GT (g-glutamyltransferase) showed a small trend to higher values with increased exposure time (Fig. 6A, P= 0.003, r 2 [adj.]: B1%) with a good number of outliers. However, this variable exhibited a clear-cut age-dependency (Fig. 6B). Analysis of the outliers (Fig. 6C) revealed no higher percentage of printers and their helpers among the volunteers employed 240 months or longer. In the multiple regression analysis neither age nor toluene exposure revealed statistically significant contributions (P= 0.098 and 0.535, respectively). There was no association between the serum uric acid level and the duration of employment or toluene exposure (Fig. 7A). Volunteers with the highest ten values (out of \ 1000) were all printers or their helpers, although there was no dose-dependency. A slight but significant upward trend was revealed with respect to the body mass index,

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Table 6 Number and percentage of male volunteers with very high and long toluene exposure showing abnormal neurological/psychiatric findings All volunteers (n)

Age (years) B30 (%)

30–39 (%)

40–49 (%)

550 (%)

Depression a All volunteers Printers and their helpersb

1062 717

2.3 1.6

1.4 1.0

1.6 1.0

2.1 2.2

Altered superficial sensation All volunteers Printers and their helpersb

1061 716

1.2 1.6

3.4 3.5

6.9 5.4

8.2 8.2

Sensiti6e misperception All volunteers Printers and their helpersb

1061 716

1.2 0.8

3.7 4.0

3.3 2.9

7.9 7.6

Altered coordination (standing and gait) All volunteers 1060 Printers and their helpersb 715

1.2 1.6

0.7 0.5

2.3 2.0

4.8 2.7

Reduced or (missing) monosynaptic strech reflexes All volunteers 1061 Printers and their helpersb 716

9.9 (0) 10.2 (0)

9.8 (0) 10.0 (0)

7.9 (1.7) 7.8 (2.0)

11.6 (2.1) 8.7 (2.2)

Reduced or (missing) polysynaptic reflexes All volunteers 1061 Printers and their helpersb 716

10.5 (1.8) 9.4 (1.6)

10.2 (1.7) 10.0 (1.0)

14.9 (2.0) 14.7 (2.9)

11.6 (1.4) 12.0 (1.1)

a b

Additionally, there was one volunteer with schizophrenia and four with convulsive diseases. The percentage is not increased in printers and their helpers.

indicating that the laboratory value is responding (Fig. 7B). A small upward trend was also observed with respect to total serum cholesterol, which could again be explained by the age-dependency. Among the outliers was no increased percentage of highly and long-term exposed printers and their helpers. There was also no effect of toluene exposure on fasting blood glucose. Cholesterol and blood sugar have not been mentioned before in the literature to be susceptible to toluene exposure.

3.4.2. Renal function (glomerular filtration rate) No association between exposure time and serum creatinine was found (Fig. 7C, r 2 [adj.]: B 1%, P=0.79), but a downward trend was noticed when the glomerular filtration rate was assessed according to the Cockroft formula,4 a trend which is fully 4 Glomerular filtration rate is: (140 −age)body weight (kg)/ 72creatinine in serum (mg/100 ml).

explained by the well-known simultaneous age-dependency, and no higher percentage of printers and their helpers was found among the outliers.

3.4.3. FSH, LH and testosterone le6els in blood serum A very small apparent trend for an increased FSH serum concentration, again confounded by a slight age-dependency, might be present. However, the trend is barely detectable within the data cloud (Fig. 8A). When the outliers are considered, a very small tendency for a higher percentage of lower than reference values may be detectable for the printers and their helpers versus non-printers (Fig. 8B). However, a difference is no longer detectable when workers employed 5 240 months are considered (Table 7). Thus, no clear-cut toluene-associated effect on serum FSH can be verified with reasonable certainty. A similar situation exists with respect to serum LH (Fig. 8C and D). Again, a small tendency of a few printers exposed to more

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197

Fig. 5. Association between the activity of liver enzymes (GOT and GPT) in serum and duration of toluene exposure (months) or age (years) of male volunteers of the printing industry (n= 1056). (A) Liver enzyme (GOT) in serum vs. exposure: no effect of exposure can be recognized. (B) Liver enzyme (GPT) in serum vs. exposure: no effect of exposure can be recognized. (C) Liver enzyme (GPT) in serum vs. age: no age-dependency can be recognized. (D) Values (GPT) above the reference range of the different laboratories vs. exposure: dark spots — values for printers and their helpers. No effect of extended exposure can be recognized.

than 20 years for low values seems to be present (Fig. 8D), but no clear-cut effect of a high and long-term toluene exposure can be detected with reasonable certainty (Table 7). Serum testosterone levels seem to show a slight trend for reduced values for volunteers with longterm employment (Fig. 9A, r 2 [adj.]: 4%, P = 0.0001), but all values of the data cloud stay within the limits of the workers with short-term exposure. Furthermore, an at least as pronounced trend exists with respect to age (Fig. 9B), and multi-variant analysis reveals a significant age-de-

pendency, but no longer a significant effect of the toluene exposure.5 The outliers for the long-term toluene-exposed printers are not remarkable when compared with those of shorter-term exposure (Fig. 9C), or when the highly exposed printers were compared with workers with negligible toluene exposure (Table 7).

5

Multi-variance analysis: testosterone = 6.8 − 0.04age+ 0.0004duration of exposure. P (age) = 0.0001, P (exposure) = 0.63.

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Fig. 6. Association between the activity of a liver enzyme (g-GT) in serum and duration of toluene exposure (months) or age (years) of male volunteers of the printing industry (n = 1056 and 1224). (A) Liver enzyme (g-GT) in serum vs. duration of exposure: a very slight trend for an increase might be present. The outliers 1 and 2 had hepatomegaly and an extended alcohol consumption. (B) Liver enzyme (g-GT) in serum vs. age: a similar slight trend as in Fig. 6A can be recognized. The outliers 1, 2 and 3 had hepatomegaly and an extended alcohol consumption. (C) Values (g-GT) above the reference range of the different laboratories vs. duration of exposure: dark spots: values for printers and their helpers. No effect of extended exposure can be recognized.

There was no effect on serum TSH values which could be associated with toluene exposure.

3.4.4. Systolic blood pressure There was a slight trend toward an increase in the association of systolic blood pressure with the duration of possible exposure (Fig. 10A), with a r 2 [adj.]: of 7% and P B0.0001 in the analysis of

variance. However, a clear-cut influence of age was also obvious (Fig. 10B). Multi-variance analysis confirmed the contribution of age, but the influence of toluene exposure also remained statistically significant (P=0,036).6 Of the 284 printers 6 Multi-variance analysis: systolic RR = 115 + 0.29age+ 0.013exposure. P (age) = 0.0001, P (exposure) = 0.036.

C. Gericke et al. / Toxicology 168 (2001) 185–209

exposed to toluene for more than 20 years (Fig. 10C and Table 8), 14 volunteers (5%) showed values outside the reference range (\160 mmHg), and this was not significantly different from the less exposed volunteers (4/115 = 4%). However, all volunteers with an exposure exceeding 20 years and a systolic blood pressure above 175 mmHg (n = 10) were printers or their helpers. A similar, but less pronounced situation was found with respect to the diastolic blood pressure (Table 8). Although many factors may contribute to hypertension, and the majority of these confounding factors could not be controlled in our trial, a

199

small effect of very long toluene exposure on systolic blood pressure of a few exposed persons cannot be excluded.

4. Discussion There is no doubt that exposure to very high concentrations of toluene may cause significant adverse health effects, predominantly in the central nervous system (e.g. King et al., 1981; Streicher et al., 1981; Hormes et al., 1986; Byrne et al., 1991). Information on chronic effects of long-

Fig. 7. Association between the activity of uric acid in serum and of renal function vs. duration of toluene exposure (months) or age (years) of male volunteers of the printing industry (n = 1056). (A) Uric acid in serum vs. duration of exposure: no effect of exposure can be recognized. (B) Uric acid in serum vs. body mass index: there might be a very slight association between uric acid in serum and the body mass index. (C) Creatinine in serum vs. duration of exposure: no effect of exposure can be recognized.

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Fig. 8. Association between the FSH and LH in serum and the duration of toluene exposure (months) of male volunteers of the printing industry (n =1057). (A) FSH in serum vs. duration of exposure: there may be a very slight trend for an increased FSH level with respect to the duration of exposure. (B) Values (FSH) above the reference range of the different laboratories vs. duration of exposure: there may be a very slight apparent increase in the number of printers with high FSH levels. (C) LH in serum vs. duration of exposure: no effect of exposure can be recognized. (D) Values (LH) above the reference range of the different laboratories vs. duration of exposure: dark spots: values for printers and their helpers. There may be a very slight apparent increase in the number of printers with low LH levels.

term exposure under occupational conditions is less reliable. The study design selected in our trial represents a compromise, as is the case of virtually all studies of this type. Ideally, all workers of the rotogravure factories would have to be studied, and not a (large-numbered) selection of them. However, this was not feasible with this study design. Nevertheless, this is the largest

evaluation performed on occupational toluene exposure. As to be expected, the results of this large field study show a number of peculiarities typical for many clinical trials on occupational exposure to chemicals, including: 1. Limitations in the predictive value of the results must be recognized, which are due to shortcomings in the study design.

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2. Results are clearly limited to the exposure scenario of the volunteers studied. 3. The influence of a confounding factor, in our evaluation predominantly age, is obvious, often exhibiting a larger influence than the exposure, in our study to toluene, might possibly have. This confounder will be largely responsible for some small but statistically significant trends observed, e.g. effects on clinical end points versus duration of exposure (e.g. Figs. 3B,C, 4C,D and 9B). 4. There is considerable inter-individual variability, leading to data clouds with little or no association with the period of exposure (e.g. Figs. 3D, 4A,B). 5. Although the majority of data points stays within the previously defined reference range, those participants with values outside this range are of special interest, and they have

6.

7.

8.

9.

201

been separately analyzed by us (e.g. Figs. 5D, 6C, 8B,D, 9C, 10C). An increased variability of the data may possibly be due to the fact that in a multi-center study data are generated by different physicians at various locations. Only volunteers still active in the working process were evaluated, and a ‘healthy workers effect’ cannot be excluded. The possibility exists that adverse effects may have occurred at previous periods of high exposure, and that such deviations from a reference range have been reversible by now at today’s lower exposure scenario. There are some indications in the literature for such an assumption. Because the situation of occupational exposure is rather complex, unknown confounding factors may exist which may be

Table 7 Number and percentage of male volunteers with very high and long toluene exposure showing abnormal serum hormone values All volunteers (n)

All 6olunteers FSH Increased Reduced LH Increased Reduced Testosterone Increased Reduced

Pa

With values outside the reference range

All volunteers

Printers and helpers

All volunteers

n

n

n

%

n

%

n

%

1057

755 79 17

8 2

51 14

7 2

28 3

9 1

0.42

12 71

1 7

11 48

2 6

1 23

0.3 8

0.47

19 78

2 8

13 50

2 7

6 28

2 11

0.07

44 1

11

28 1

10

16 0

14

6 36

2 9

5 25

2 9

1 11

1 10

3 42

1 11

3 29

1 11

0 13

11

1052

995

Exposure ]240 months FSH 402 Increased Reduced LH 402 Increased Reduced Testosterone 390 Increased Reduced

Printers and helpers

Non-printers

751

731

286

286

276

a There are no statistically significant ( 2- or Fisher’s-exact tests) differences (printers vs. non-printers). The general outcome is not changed when the 45–46 printers and their helpers of the longitudinal part are included in the evaluation.

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Fig. 9. Association between the testosterone levels in serum and the duration of toluene exposure (months) and of age (years) of male volunteers of the printing industry (n = 995). Multiple regression analysis: testosterone = 6.8 − 0.04 age + 0.0004exposure; r 2[adj.]: 4%; P (age) = 0.0001; P (exposure)= 0.626. (A) Testosterone level in serum vs. duration of exposure: there may be a very slight ostensible trend for a decrease when testosterone levels are plotted against the duration of exposure. r 2[adj.]: 4%; P =0.0001. (B) Testosterone level in serum vs. age: there is a trend for a decrease when testosterone levels are plotted against age. (C) Values (testosterone) above the reference range of the different laboratories vs. duration of exposure: dark spots — values for printers and their helpers. The number of low values is not higher in printers, exposed for a long period.

responsible for deviations observed, but cannot be taken into account. 10. The general inability of any medical study must be recognized, and especially of those concerned with exposure to environmental substances, to verify apparent ‘border-line effects’. Conflicting results of different studies are bound to occur.

In fact, the epidemiological methodology was initially designed for studying infectious diseases, and it may be excellently suited for analyzing special effects of well-defined doses of medicinal drugs. It is barely capable of providing satisfactory answers with respect to risk assessment on chronic effects of variable long-term exposures to agents with comparatively short elimination half-

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lives. Furthermore, important and crucial confounding factors are difficult to recognize, and it may be impossible to control them or estimate their contribution to differences observed. Often poly-exposure occurs, which forbids conclusions on the contribution of single components. Longterm exposure is mostly ill-defined, and only pronounced health effects can be recognized with reasonable certainty. Therefore, it is not surpris-

203

ing that with respect to relatively small and less pronounced effects of ‘environmental agents’ contradictory results are obtained by various investigators. Many of these aspects are also relevant to our evaluations. We have not evaluated synergistic effects, possibly occurring in combinations of toluene with other factors. Synergism between toluene and noise has been demonstrated before at overdoses

Fig. 10. Association between the systolic blood pressure and the duration of toluene exposure (months) and of age (years) of male volunteers of the printing industry (n= 1052). (A) Systolic blood pressure vs. duration of exposure: there may be a very slight trend for a increase in systolic blood pressure with respect to the duration of exposure. (B) Systolic blood pressure vs. age: there may be an age-dependent trend for a increase in systolic blood pressure at least as pronounced as an apparent effect of exposure (Fig. 10A). (C) Values (systolic blood pressure) above the reference range vs. duration of exposure: dark spots — values for printers and their helpers. The number of high values seems to be somewhat higher in printers, exposed for a long period.

C. Gericke et al. / Toxicology 168 (2001) 185–209

204

Table 8 Number and percentage of male volunteers with very high and long toluene exposure showing abnormal values in blood pressure

All 6olunteers Systolic values Increased Diastolic values Increased

Pa

With values outside the reference range

All volunteers (n) All volunteers

Printers and helpers

All volunteers

n

n

n

%

n

%

n

%

1052

750 26

3

18

2

8

3

1052

750 69

7

47

6

22

7

18

5

14

5

4

4

33

8

26

9

7

6

Exposure 5240 months Systolic values 399 Increased Diastolic values 399 Increased

Printers and helpers

Non-printers

284 284 0.33

a There are no statistically significant ( 2- or Fisher’s-exact tests) differences (printers vs. non-printers). The general outcome is not changed when the 45–46 printers and their helpers of the longitudinal part are included in the evaluation.

in animal studies (Johnson et al., 1988; Lataye and Campo, 1997). There is also evidence of the relevance of such a synergistic action in humans, as demonstrated in two publications by the same group of investigators (Morata et al., 1993, 1997).

4.1. Rejection or confirmation of the hypotheses formulated Results of previously published studies on this topic have been quite contradictory (Antti-Poika et al., 1985; Juntunen et al., 1985; Ha¨ nninen et al., 1987). Many examinations suffered from a too small number of volunteers, a too small reference range taken into account, and the inability of considering important confounding factors, including age. Besides performing group analyses of printers and their helpers versus ‘non-printers’, scatter plots showing the individual data were provided by us for all the relevant end points. According to a suggestion by Professor Nathan Mantel (on the occasion of evaluating the Seveso data, Mocarelli et al., 1986), additionally the outliers from a defined reference range were analyzed with respect to the proportion of highly exposed volunteers

within this group, and for possible special confounding factors. We strongly feel that considering such criteria for analysis is essential for this type of studies. Most often, simple statistics, e.g. just providing median-values and ranges and even less mean-values and S.D., do not provide a sufficient toxicological and medical information for risk assessment. They may even be misleading when evaluating small deviations from a reference range. When considering the hypotheses put forward before the initiation of the study, the following results were obtained.

4.1.1. Hypothesis 1a Psycho-physiological evaluations are generally considered to be especially sensitive markers of toluene-induced acute effects. This may not be surprising, when considering the well-known resemblance of toluene with acute effects of alcohol. A possible association of long-term exposure with deficits in the corresponding performances has been much less convincing. Without considering confounding factors, a number of psycho-physiological tests showed a statistically significant decrease in overall perfor-

C. Gericke et al. / Toxicology 168 (2001) 185–209

mance (P=0.003–0.0001) in a linear regression analysis of the scores versus duration of toluene exposure. Examples are digit span forward and backward tests (Fig. 2A and B), the digit symbol test (Fig. 3A), and the visual reproduction test (Fig. 3D). However, the coefficients of determination were extremely low and the vast majority of the values of higher and longer exposed volunteers stayed within the range of the persons with negligible and short-term toluene exposure. As has been described before, age was found to be a strong confounding factor in several of the tests. When this confounder was taken into account in a multiple regression analysis, the contribution of toluene exposure became statistically insignificant in the case of all the performance tests. Interestingly, in the multiple regression analyses the contribution of the duration of toluene exposure in several cases (e.g. Fig. 3A and D) became opposite (with statistical significance) to the overall trend, this trend apparently being exclusively influenced by the confounding factor age. Because no one would take such statistically significant opposite trends as being indicative of a beneficial effect of the toluene exposure, the examples illustrate the dubious nature and the lack of medical relevance of borderline effects or results occurring by chance (in our case regression lines with very low coefficients of determination within data clouds), despite a statistical significance. Further analyses of the data are indispensable for such conditions. The examples also illustrate the necessity of taking crucial confounding factors into account. A similar situation was described by Ørbæk and Nise (1989). Results of previous studies reported in the literature are quite controversial. Most studies suffer from relatively small numbers of (often probably highly selected) volunteers, problems with references (a too small group, and in several studies volunteers exposed to other solvents), and the documentation of data often was inadequate. The important confounding factor age was not adequately considered in several of the previous publications, probably because the number of volunteers was too small. In the largest study, performed on 262 selected workers of a Danish printing factory (Mørck et al., 1988), an overall

205

reduced ability of the volunteers to memorize and to concentrate was reported. Exposure at the time of the study and for the past 2 decades was assumed to be 50– 100 ppm, but it was much higher during the periods before. When neuropsychological functions were tested in 29 workers with acute blood toluene levels of 1250 mg/l (S.D.: 370) who had been exposed to an average of only about 5 years (Boey et al., 1997), the volunteers were reported to perform poorer than ‘controls’ with a mean blood toluene level of 160 mg/l (S.D.: 60) in short-term memory tests, etc. The data of the abstract are difficult to interpret because of documentation deficits. In a Swedish study (Ørbæk and Nise, 1989), long-term toluene-exposed printers performed worse in psychological tests than the references, but these differences disappeared when important confounding factors were considered (alcohol, age, expressiveness). In a number of other publications, no convincing effects were obtained in psycho-physiological tests (Kempe et al., 1980; Cherry et al., 1985; AnttiPoika et al., 1985; Juntunen et al., 1985 [probably the same data]), or claims of effects by the authors were not justified or not convincing (Ha¨ nninen et al., 1987; Foo et al., 1990).

4.1.2. Hypothesis 1b: psycho-motoric e6aluations Poorer performance had been reported in some preliminary reports (Boey et al., 1997). Our investigations gave no statistical significance in the regression analysis against the duration of exposure (Fig. 5A and B), and no clue from group analyses of printers versus ‘non-printers’. 4.1.3. Hypothesis 2 In previous studies a number subjective complaints was found to be increased in rotogravure printers when compared with references. One of the previous investigators even expressed the, highly debatable, opinion that subjective disorders might be more sensitive for detecting possible deficits after toluene exposure than objective measures (Echeverria et al., 1989). As end points, dizziness and fainting as well as fecal irregularities (Mørck et al., 1988), sleepiness, poor short-term memory, difficulties to concentrate, labile mood, depressive mood, headache, sleep disturbance,

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paresthesia were mentioned (Ørbæk and Nise, 1989). An ‘organic brain syndrome’ (mostly neurasthenic complaints, anxiety, depression) was stated to be much more frequent in longer than 12 years toluene-exposed rotogravure workers than in a control group (Larsen and Leira, 1988). Exposure at the time of the examination was moderate, but it had previously been extensive (1000 ppm). The effects mentioned were not regularly reported in other publications and with such a high frequency, possibly because the exposures have been smaller. While most publications were concerned with male workers, subjective symptoms were also assessed by questionnaires in toluene-exposed women (Lee et al., 1988), and a dose-dependent increase in the frequency of types of discomfort mentioned by the volunteers was reported in the exposure range of 50 to \ 150 ppm. Our data do not second the assumption of an increased frequency of subjective disorders in the toluene-exposed volunteers studied by us. We realized that the evaluation of such complaints is difficult and biased by many factors.

4.1.4. Hypothesis 3 Data on serum transaminases in toluene-exposed workers are contradictory. Alterations were found in some studies (Svensson et al., 1992a,b), but not in others (Waldron et al., 1982). The extent of exposure may have been different, and there is a clue that an adverse effect induced by toluene may be reversible (Fornazzari et al., 1983). In our evaluation, there was no indication for a higher incidence of abnormal liver enzymes in serum, when confounding factors were taken into account. 4.1.5. Hypothesis 4 Renal function has been reported to be irreversibly altered following massive toluene exposure (Russ et al., 1981), and also reversible impairment of kidney function was reported in persons highly toluene-exposed by glue sniffing (O’Brian et al., 1971; Patel and Benjamin, 1986). On the other hand, there have been a number of publications on painters and printers with toluene

exposure reporting no effect on renal functions, including no effect on excretion of b-2-microglobulin (Askergren, 1982; Franchini et al., 1983). In our trial, kidney function, evaluated as serum creatinine levels and calculated as glomerular filtration rate, was not found to be different in long-term and highly exposed volunteers when compared with participants with negligible toluene exposure. Our evaluation was confined to assessing kidney function with the conventional method, the clinical relevance of which can easily be assessed. Micro-albumin or micro-globulin excretion were not included in our studies, because confounding factors are difficult to control.

4.1.6. Hypothesis 5 Decreased concentrations of certain male sex hormones in serum of workers of rotogravure printing companies have been reported in two publications by the same investigators. This concerned follicle stimulating hormone (FSH), luteinizing hormone (LH) and testosterone levels. In 20 male volunteers employed an average of 25 years (B1–37) and showing body burdens of 1.7 mmol toluene/l and rather low-level ambient air exposure, all three hormones were considered by the authors to be decreased (Svensson et al., 1992a,b). However, most values remained within the reference range, and the toxicological relevance is questionable. There was some clue for a reversibility of the effect, and no correlation with cumulative exposure (Mørck et al., 1988). In our studies, no clear-cut alteration in the levels of these hormones was found in long-term and apparently highly toluene-exposed workers. Minor effects, more often in the opposite direction, might have occurred, but statistical analyses do not second convincing evidence for reduced values. 4.1.7. Hypothesis 6 With respect to possible changes in systolic blood pressure, there is one report suggesting a significant decrease in a number of printers with such an outcome (Mørck et al., 1988). No effect was observed by these authors on diastolic blood pressure.

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In our studies, a slight trend for an increased systolic blood pressure might be present in the regression analysis, with a very small coefficient of determination (r 2 [adj.]: 7%, P B0.0001). Age certainly is a confounding factor, but in the analysis of variance duration of exposure remains a contributor (P = 0.04). When all long-term exposed printers and their helpers are evaluated against the ‘non-printers’, no statistically significant difference is found. However, a few of the many long-term (more than 20 years) toluene-exposed printers and their helpers are those with the highest systolic blood pressure (Fig. 10C) measured in the study. Since the many confounding factors associated with hypertension were not taken into account by us, the question of a causal relationship must remain unanswered.

5. Conclusions The study design of this type of epidemiological evaluation of occupational exposures must remain sub-optimal, and conclusions are limited because of a number of reasons. We must limit our conclusions to the end points evaluated, the participants selected, and the assessment of exposure chosen, because we are faced with the usual shortcomings when attempting to assess possible adverse health effects of long-term environmental exposure. With respect to exposure assessment, according to previous MAK-values, exposures up to 200 ppm are quite likely during past decades (i.e. before 1985). When accepting the duration of exposure as a biomarker of exposure, we did not find convincing evidence for a higher incidence of defined chronic diseases or serum surrogate markers in heavily toluene-exposed workers when compared with workers from the same factories with presumed very small toluene exposure, or with an external reference group with no special exposure to this solvent. In several of our evaluations of individual data, typical ‘data clouds’ are obtained. It is of little help to provide regression lines for such data. Such lines may even be misleading, despite some ‘statistical significance’ demonstrated, especially

207

when all the data points stay within the reference range, which is most often the case. Most important was the medical relevance, and not necessarily the statistical significance. With all the limitations mentioned in mind, overall no clear-cut adverse effects could be verified in our very large trial on many volunteers after very long-term exposure to toluene (more than 20 years). This included in past decades exposure to much higher levels than the present concentrations at the work-place. Our evaluation refers to the exposure scenario of the volunteers studied in the Western part of Germany, and the results of our study are limited to male workers. In the future, it does not seem advisable to conduct similar but much smaller-scale trials on this topic and on rotogravure workers, because the present exposure is expected to be lower when compared with our volunteers, and it might become even lower in the following years. According to our experience, crucial confounding factors can hardly be controlled in a study with rather few, highly selected, volunteers. A healthy workers effect cannot be excluded for some of the chronic effects evaluated in our study, since some very sick persons may have left the factories. Furthermore, previous effects which occurred in the past at periods of much higher exposure, might have been reversible and therefore no longer detectable. For some of the end points there are clues for reversibility in the literature. The results of our studies by no means contradict the possibility of chronic adverse health effects at very high toluene exposure levels, as amply described in the literature.

Acknowledgements We would like to thank all volunteers participating in this trial. The study was initiated by the German Professional Association of the Printing and Paper Processing Industry (responsible office), the German Association of Printing Ink Industry, the German Association of Magazine Editors, and the Industrial Trade Union Mass Media. It was financed by these organizations

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together with the participating factories, and by considerable funding of the Free University Berlin.

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