Occupational and Environmental Medicine 1995;52:508-514

Hepatic function in workers occupationallyexposed to carbon tetrachloride

John A Tomenson, Charles E Baron, John J O'Sullivan, John C Edwards,Michael D Stonard, Robin J Walker, Deborah M Feamley

ICI EpidemiologyUnit, PO Box 7,Brunner House,Winnington,Northwich, CheshireCW8 4DJJ A TomensonPersonnelDepartment,Occupational HealthSection, ClwydCounty Council, ShireHall, Mold, ClwydCH7 6NBC E BaronAshling OccupationalHealth, AshlingHouse, Works Lane,Lostock Gralam,Northwich, CheshireCW9 7FAJ J O'SullivanHealth and SafetyDepartment, BritishSteel, Swinden House,Moorgate, RotherhamS60 3ARJ C EdwardsZeneca CentralToxicologyLaboratory, AlderleyPark, Macclesfield,Cheshire SK10 4TJM D StonardWalton Hospital, RiceLane, LiverpoolL9 lAER J WalkerOccupational HealthDepartment, H J

Heinz and Company,Kitt Green, Wigan,LancashireD M FearnleyCorrespondence to:Dr J A Tomenson, ICIEpidemiology Unit, PO Box7, Brunner House,Winnington, Northwich,Cheshire CW8 4DJ.Accepted 28 April 1995

AbstractObjectives-To identify any differencesin hepatic function between workersexposed to carbon tetrachloride and con-

trols, and to identify the best variablewith which to examine any effects.Methods-In a cross sectional study of

hepatic function in workers occupation-ally exposed to carbon tetrachloride, 135exposed employees were compared with276 non-exposed controls. The exposedgroup was taken from three sites in thenorth west of England and the controlgroup included non-exposed workersfrom one of these sites and another sitelocated nearby. Demographic and alco-hol consumption data were collectedfrom both groups by questionnaire. Eachmember of the study group was allotted a

notional estimated exposure to carbontetrachloride, calculated from historicpersonal monitoring data and job cate-gory. A fasting sample ofblood was takenfrom all participants and analysed for a

variety of biochemical and haematologi-cal variables. The techniques of univari-ate and multivariate analysis of variancewere used to investigate the effect on bio-chemical and haematological indices of arange of factors.Results-Multivariate analysis of vari-ance of four core liver function variables,alanine transaminase, aspartatetransaminase, alkaline phosphatase, andy-glutamyl transferase, showed a signifi-cant difference between exposed andnon-exposed workers. The univariateanalyses identified increases in only alka-line phosphatase and ,'oglutamyl trans-ferase within the exposed group and thesedid not show a significant dose-responserelation. Univariate analysis of variancedid show effects of alcohol and age on

several variables. Significant differencesbetween exposed and control groups forthree haematological variables, haemo-globin, packed cell volume, and red bloodcount, were thought not to be due to theeffects of exposure. Clinical review ofexposed subjects with abnormal resultsdid not show clinically evident diseasethat could have been associated withexposure to carbon tetrachloride. Also, a

follow up study conducted three yearsafter the cross sectional study at the sitewith highest exposures to carbon tetra-chloride showed no evidence of any fur-

other changes in liver function variables.Conclusions-The most sensitive statisti-cal methods have shown significant dif-ferences in the liver fiction variablesmeasured between people exposed to car-bon tetrachloride and the control group.The interpretation of the data collectedwas that these differences may be due toexposure to carbon tetrachloride but thiswas not clearly shown. Furthermore, thechanges found have not given rise to anyclinical disease.

(Occup Environ Med 1995;52:508-514)

Keywords: occupational exposure; hepatic function;carbon tetrachloride

Carbon tetrachloride has been known formany years to be toxic to the liver. In animalexperiments it has been shown to producehepatic damage including necrosis and fattydegeneration in various species.' 2 Many casesof human over exposure leading to liver dam-age have been documented.3 6 These have ingeneral been cases of acute overexposure tomassive levels of carbon tetrachloride,whereas studies of long term exposure tolower levels contain little information aboutthe magnitude of exposure to carbon tetra-chloride and are in general, inadequate inanswering questions related to assessment ofhealth risk. 10

Experiments on laboratory animals haveshown minor effects to occur in liver tissue onrepeated daily exposure to vapour at concen-trations as low as 10 parts per million (ppm).' 2At the time of the study, the occupationalexposure limit in the United Kingdom was setat 10 ppm (8 hour time weighted average).The threshold limit value set by the AmericanConference of Industrial Hygienists was 5ppm" and the internal hygiene standard rec-ommended by the Occupational HealthDepartment within ICI Chemicals andPolymers (C and P) was also set at 5 ppm. Inthe years preceding the study, compliancewith this figure has not always been achievedat ICI C and P plants where occupationalexposure to carbon tetrachloride occurs.

Because the liver is one of the target organsfor carbon tetrachloride, monitoring of bio-logical effects has formed part of the medicalsurveillance of workers exposed to carbontetrachloride in the past. The monitoring ofliver function tests has involved the assay of a

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single enzyme, alanine transaminase with afull liver function screen on workers who haveshown an abnormality in this variable. Inrecent years, workers with potential exposureto carbon tetrachloride have had periodicmeasurements of several plasma enzymes.The data obtained from this biological surveil-lance have generally been used on an individ-ual basis to identify either people who haveunderlying liver pathology and who are thus atgreater risk from working in this environment,or to those who may be more sensitive to theeffects of carbon tetrachloride and who areshowing abnormalities in these tests due toexposure.The plasma enzyme assays carried out in

recent years have the advantage that they havebeen well tried and tested in clinical practice.An increase in measured enzyme activitiesmay be due to a variety of causes. For example,it has been postulated that cellular damage inthe liver or enzyme induction may result inincreased enzyme activity after exposure tocertain liver toxins.'2 13 The main disadvantageis that raised enzymes are not all organ spe-cific and this may cause occasional diagnosticproblems in clinical practice.The measurement of total bile acids in

serum may be a more sensitive indicator ofhepatic function and has the advantage ofbeing organ specific. Measurement of bileacids has not been standard clinical practice,however, and interpretation of high resultsmight present difficulties.The objective of the study was primarily to

identify any difference in hepatic functionbetween a work group with occupationalexposure to carbon tetrachloride and a controlgroup, which might indicate an adverse effectof exposure to carbon tetrachloride on liverfunction. In the event of such an effect beingdetected, it was also intended to investigatewhether it was related to clinically detectableliver disease. A secondary objective was toidentify which plasma measurements are themost sensitive indicators of any effect of car-bon tetrachloride on liver function that mayexist. As the testing of hepatic function neces-sitated the collection of a blood sample, it wasdecided to measure haematological variablesalthough there was no previous evidence ofchanges in haematological variables afterinhalation of carbon tetrachloride.

Subjects and methodsSTUDY DESIGNThis was a cross sectional study that com-pared biochemical and haematological vari-ables in two groups of workers in a chemicalplant. The study group consisted of process,maintenance, and other workers occupation-ally exposed to carbon tetrachloride and thecontrol group consisted of process, mainte-nance, and other workers with no occupa-tional exposure to carbon tetrachloride.A short questionnaire was given to all

potential study and control group members inadvance of the study and was used to select

eligible participants. It was also used to ensurethat study and control groups were wellmatched for demographic details (age, height,weight, and type of job) and alcohol con-sumption.

Participation in the study was voluntaryand the study group contained 135 workers,83% of those eligible for inclusion. The con-trol group was drawn from a target populationof 370 employees who worked at two sites. Atone site, a series of communication exerciseswere held and workers were encouraged tovolunteer and complete the short question-naire. At the second site, workers were sent aletter describing the study and a copy of theshort questionnaire. Three hundred andtwenty five employees completed the shortquestionnaire and 88% of those selected toparticipate on the basis of their responses tothe short questionnaire, agreed to take part.The overall participation in the control groupwas 276 (77%).

EXPOSED GROUPSubjects in the exposed study group were vol-unteers drawn from three plants in the northwest of England belonging to ICI C and P. Toqualify for inclusion in the study groupemployees were required to work on one ofthe processes with potential exposure to car-bon tetrachloride either full time (in the caseof process personnel and dedicated mainte-nance personnel) or on a regular basis (in thecase of other maintenance personnel).

CONTROL GROUPA control population was chosen fromemployees of ICI C and P who worked onplants where there was no risk of exposure tocarbon tetrachloride or other hepatotoxicchemicals. Control subjects came from one ofthe sites that provided workers for theexposed group and a further site locatednearby where carbon tetrachloride is nothandled. Workers were excluded from thecontrol group if they had worked in or on anyof a redefined list of workplaces within ICIwhere there was a potential for exposure tocarbon tetrachloride or other known hepa-totoxins, during the previous five years.The study was scheduled to start in

November 1986 but after a two week periodof sample collection (about 60 subjects) therewas a problem with availability of controls dueto plant breakdown. In view of this it wasdecided to restart the study in February 1987and the rest of the samples were taken during aperiod of about eight weeks.

QUESTIONNAIREAn extensive questionnaire was given to eachmember of the study and control groups. Thequestionnaire was given by one occupationalhealth nursing officer trained for this purpose.The data obtained on the questionnaireincluded age, height, weight, job description,plant, hobbies, medical history, and a detailedalcohol history. Also, the length of service in ajob exposed to carbon tetrachloride wasobtained for the exposed workers.

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BIOCHEMICAL AND HAEMATOLOGICALMEASUREMENTSAll members of the study and control groupshad a blood sample taken on the day on whichthey completed the questionnaire. This bloodsample was taken after the subject had noteaten or drank beverages containing milk,sugar, or alcohol for the previous 12 hours.The sample comprised 15 ml (10 ml in alithium heparinised tube, 4 ml in a plain glasstube for serum, and 1 ml in an EDTA tubefor a full blood count). Samples were trans-ported to the ICI central toxicology laboratoryfor analysis on the morning of collection. Tominimise any effect of laboratory variation,blood samples were taken from a roughly con-stant ratio of study and control group subjectson each day.The variables measured on plasma, serum,

or EDTA samples consisted of alaninetransaminase, aspartate transaminase, alkalinephosphatase, y-glutamyl transferase, gluta-mate dehydrogenase, 5' nucleotidase, totalbile acids, cholesterol, and triglycerides.The haematological variables consisted of

haemoglobin, packed cell volume, meancorpuscular volume, red blood count, meancorpuscular haemoglobin concentration,platelets, white blood count, lymphocyte,monocyte, eosinophil, neutrophil, andbasophil counts.

Bile acids were measured with a commer-cial radioimmunoassay and other biochemicalvariables were measured with either a VitatronPA800 analyser or a Kone CD analyser.Haematological variables were measured withan ELT 800 analyser.

EXPOSUREEach member of the study group was allotted anotional estimated exposure to carbon tetra-chloride that was calculated from historicalpersonal monitoring data for each job cate-gory. According to this estimate, study groupmembers were allotted to an exposure cate-gory (high, medium, or low).Most work groups had historical personal

monitoring data and the mean of these resultswas calculated and the group categorised. The

Table 1 Alcohol consumption of exposed and control populations

Alcohol conslliptiol

Low:Never drink1 2 Units no more than 1-2 times/week3 7 Units no more than 2-3 times/month> 8 Units no more than once/month

Medium:1 2 Units at least 3-4 times/week5 7 Units 3 -4 times/week> 8 Units 1 2 times/week

High:3 4 Units at least 3--4 times/week5-7 Units 3 4 times/week> 8 Units 1 2 times/week

Very high:5 7 Units every day> 8 Units at least 3-4 times/week

Control (0O

26 5

26 9

27 3

19 6

low category contained those with meanresults of 1 ppm or less, the medium categoryincluded groups with mean results from morethan 1 ppm to 3 ppm and the high category,those with mean results of 4 ppm or more.For groups or workers where no monitoringhad taken place, categorisation was allotted byjudgement of likely exposure from compari-son with other groups. This judgement wasmade by a professional industrial hygienist inassociation with each plant manager. Theappendix shows a summary of the distributionof results on which the categorisation wasbased.Members of the study group were also cate-

gorised according to length of time in the job(< 1 year, 1-5 years, and > 5 years).

STATISTICAL METHODSThe techniques of univariate and multivariateanalysis of variance were used to investigatethe effect on biochemical and haematologicalindices of a range of factors, in particularthat attributable to exposure to carbontetrachloride. For certain biochemical andhaematological measurements a logarithmictransformation gave closer agreement with theassumptions of the analysis of variancemodel that is, normally distributed errorsand a linear model. All analyses of thesevariables were performed on the logarithmi-cally transformed measurements. The SASpackage was used to fit the univariate andmultivariate analysis of variance models.'4

Linear models were fitted to either the rawor logarithmically transformed data. Theterms in the models included exposure cate-gory, age, sampling time (first or secondphase) and a measure of alcohol intake basedon the quantity and frequency of consump-tion. The possibility of a synergistic reactionbetween exposure to carbon tetrachloride andalcohol consumption was examined byincluding an interaction term between the twofactors in the linear model.

For each exposure category, further com-parisons were made between workers at thefour sites. Also, a multivariate analysis of vari-ance was performed for the four variablesalkaline phosphatase, aspartate transaminase,alanine transaminase, and )4glutamyl trans-ferase, with the same linear models as the uni-variate analysis.The means presented in tables are least

Exposed (/o) square means.'5 These means correct forimbalance in the numbers of workers in the

23 0 different categories when broken down by thefactors present in the analysis of variancemodel.

21-5 As well as comparing mean levels of thebiochemical and haematological variables inthe exposed and non-exposed groups, thespread of results was also compared. For each

28 9 variable a normal range was constructed forthe non-exposed workers, which ranged fromthe 2-5% quantile up to the 9755% quantile.

26 7 The percentage of exposed and non-exposedworkers lying above and below the range werecalculated for each variable.

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Table 2 Exposure effects-means (SEM)jt ofselected biochemical and haematological indices for different exposurecategories

None Low Medium High

Mean (SEM) Mean (SEM) Mean (SEM) Mean (SEM)

Alanine transaminaset (mU/ml) 20-54 (1-03) 20 35 (1-08) 20-82 (1-05) 19-39 (1-06)Aspartate transaminaset (mU/ml) 16-48 (1-02) 15-25 (1-05) 15-88 (1-04) 15 62 (1-04)Alkaline phosphataset (mU/ml) 125-79 (1-02) 122-20 (1-05) 137-10* (1-04) 135-10 (1-04)y-Glutamyl transferaset (mU/ml) 26-89 (1-05) 26-89 (1-11) 33-17* (1-08) 31-50 (1-08)Totalbile acidst (umolIl) 1-06 (1-06) 1-00 (1-14) 1-25 (1-10) 1-28 (1 10)5'-Nucleotidase (mU/ml) 5-89 (1-03) 6-54 (1-08) 6-25 (1-06) 5-75 (1-06)Glutamate dehydrogenaset (mU/ml) 3-00 (1-05) 3-26 (1 10) 3-57* (1-07) 2-98 (1-07)Haemoglobin (g/dl) 15-97 (0 08) 15-60 (0-19) 15-39*** (0-14) 15-71 (0-14)Packed cell volume 48-54 (0-23) 47.32* (0-54) 47-32** (0-39) 48-05 (0-41)Red blood count (x 1012/1) 5-61 (0-03) 5-50 (0-08) 5-47* (0-06) 5-50 (0-06)

*P < 0-05; **P 0-01; ***P < 0-001; v control group.t Geometric means (SEM) are presented for variables analysed after a logarithmic transformation.16

ResultsThe study and control groups were wellmatched for age, height, weight, work pat-terns and alcohol consumption. The ages ofsubjects in the exposed and control groupswere roughly normally distributed with meansbetween 40 and 41 and ranges of 18-63 and17-62, respectively. Virtually all control(97%) and exposed (98%) subjects were cur-rent drinkers. Table 1 shows the drinkinghabits and the alcohol consumption as mea-sured by the variable measuring the quantityand frequency of alcohol consumption.

Preliminary analyses were first performedincorporating an interaction term betweenexposure to carbon tetrachloride and alcoholconsumption. No evidence of such an effectwas found in these analyses and the term wasdropped from the analyses presented below.Univariate analyses of variance for all 21 ofthe biochemical and haematological variablesshowed significant differences betweenexposed and non-exposed workers for onlythree variables, haemoglobin (P < 0 001),packed cell volume and red blood count (P <0-0 1). None of these variables showed any sig-nificant differences between exposed workersin the three exposure categories.

Significant relations with alcohol consump-tion were found for total bile acids (P <0-001), yrglutamyl transferase (P < 0-01), redblood count, mean corpuscular volume, andtriglycerides (P < 0 05). There were also sig-nificant differences for several variablesbetween samples taken in November 1986 andthose taken in spring 1987. These differenceswere particularly obvious for the haematologi-cal variables. Several variables showed signifi-cant relations with age. The strongest relations

with age were seen for y4glutamyl transferase,cholesterol, and triglycerides (P < 0 001) butmean corpuscular volume (P < 0.01) andaspartate transaminase, 5' nucleotidase, redblood count, and neutrophil count (P < 0-05)were also related to age.

Table 2 shows mean results by exposurecategory for the targeted variables alaninetransaminase, aspartate transaminase, alkalinephosphatase, yglutamyl transferase, total bileacids, 5'-nucleotidase, and glutamate dehy-drogenase and those variables for which therewas a significant difference between exposedand non-exposed workers, haemoglobin,packed cell volume, and red blood count.The multivariate analysis of variance of the

variables alkaline phosphatase, aspartatetransaminase, alanine transaminase, and r'glutamyl transferase showed a significant dif-ference (P < 0 05) between exposed andnon-exposed workers. In the exposed groupthere were no significant differences betweendifferent exposure categories. The univariateanalyses of variance of these four variables didnot show significant differences betweenexposed and non-exposed workers. Pairwisecomparisons showed a significant increase (P< 0.05) in the concentrations of alkaline phos-phatase and yglutamyl transferase in themedium exposure group and a comparable,but non-significant increase in the high expo-sure group (table 2). There were no similarpatterns in the concentrations of aspartatetransaminase and alanine transaminase.

Table 3 shows mean results by alcohol cate-gory for alanine transaminase, aspartate trans-aminase, alkaline phosphatase, yglutamyltransferase and total bile acids and the othervariables that showed a significant alcohol

Table 3 Alcohol effects-means (SEM) t of selected biochemical and haematological indices for different alcoholconsumptions

Low Medium High Very high

Mean (SEM) Mean (SEM) Mean (SEM) Mean (SEM)

Alanine transaminaset (mU/ml) 20-40 (1-05) 21-10 (1-04) 20-75 (1-04) 19-22 (1-04)Aspartate transaminaset (mU/ml) 15-76 (1-03) 16-19 (1-03) 16-12 (1-03) 16-04 (1-03)Alkaline phosphataset (mU/ml) 128-23 (1-03) 126-38 (1-03) 126-84 (1-03) 132-69 (1-03)y-Glutamyl transferaset (mU/ml) 24-60 (1-07) 27-97 (1-06) 30 05** (1-06) 32.32*** (1-06)Total bile acidst (umol/l) 1-02 (1-08) 0-94 (1-07) 1-19 (1-07) 1-35** (1-08)Triglyceridest (mg/dl) 125-08 (1-07) 122-87 (1-06) 139-16 (1-06) 149.77* (1-06)Mean corpuscular volume 86-11 (1-01) 85-73 (1-01) 87-22 (1-01) 87-59* (1-01)Red blood count (x 102/1) 5-60 (0-05) 5-61 (0-04) 5.48* (0-04) 5-52 (0-05)*P < 0-05; **P < 0-01; ***P < 0-001; v low group.t Geometric means (SEMs) are presented for variables analysed after a logarithmic transformation.6

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Table 4 Normal ranges derivedfrom results of control group with percentages above and below in exposed andnon-exposed groups

Non-exposed ExposedRange

Below Above Below AboveVariable (with manufacturers range) Lower Upper (Os) (%) n (%) (%) n

Alanine transaminase (mU/mI) (< 29) 9-6 40 0 2-3 2-7 262 1 6 7-8 129Aspartate transaminase (mU/ml) (< 25) 11 0 30 4 3-8 2-3 262 6-2 1 6 129Alkaline phosphatase (mU/ml) (< 170) 80-0 206-0 2-7 3-0 263 0-8 4-7 129y-Glutamyl transferase (mU/ml) (< 50) 10-6 68-0 2-3 3 0 263 0-8 10.9 129Total bile acids (iinol/l) (< 6) 0 3 3-3 6-2 2-5 242 1-6 4 1 1225'-Nucleotidase (mU/ml) (2-10) 3 0 13-0 6-8 3 0 263 5-4 3-1 129Glutamate dehydrogenase (mU/ml) (< 4) 1-6 8-0 2-3 3 0 263 3-9 7-0 129Haemoglobin (g/dl) (140-18-0) 13-9 17-6 3-4 2-3 264 3-9 3-1 130Packed cell volume (39-52) 40-2 54-3 2-7 2 7 264 1-5 0.0 130Red blood count ( x 1012/1) (4-5-6 3) 4-5 6-3 2-3 2-3 264 3-9 0-8 129

effect, triglycerides and mean corpuscular vol-ume. The significance of comparisons of themedium, high, and very high consumptiongroups with the low consumption group arealso given.

For each exposure category (high, medium,low, or unexposed) a comparison was madebetween workers at the four sites. Theseanalyses showed no significant differencesbetween biochemical and haematological vari-ables in workers at the four sites.

Table 4 shows the results of the normalrange analysis for the targeted variables andthose showing a significant difference in con-centrations between exposed and non-exposed workers. The ranges are presentedtogether with the percentage less than orequal to the lower limit or greater than orequal to the upper limit. The reference rangessupplied by the manufacturers of the tests arealso included for comparison. The proportionof exposed workers above the upper limit wassignificantly raised for two variables, alaninetransaminase (P < 005) and yglutamyltransferase (P < 0001). There was little dif-ference between the proportions of exposedand non-exposed workers below the lowerlimits of the haematological variables, haemo-globin, packed cell volume, and red bloodcount.

DiscussionThe finding of a significant differencebetween exposed and control groups in themultivariate analysis of alanine transaminase,aspartate transaminase, alkaline phosphatase,and yglutamyl transferase is consistent withthe excess of abnormal results in the exposedgroup for two of these variables, alaninetransaminase and yglutamyl transferase(table 4). The univariate analysis of variancedid not show significant differences betweenthe exposed and non-exposed workers inthese four variables, but there was a sugges-tion of a dose response for alkaline phos-phatase and yglutamyl transferase.The lack of a significant dose-response

effect may be due to imprecision in the expo-sure ranking of each worker. Unfortunately,few measurements were available for thelower exposure groups as the monitoringstrategy mainly targets the higher exposuregroups. In general, however, workers in the

high group will have had greater exposurethan those in the medium group who in turnhave had greater exposure than those in thelow group. Alternatively the finding of a lackof dose-response effect but a differencebetween the group exposed to carbon tetra-chloride and the control group could havebeen due to something other than carbontetrachloride.The comparison of alcohol consumption in

the two groups (table 1) shows that theexposed group admitted to drinking five toseven units of alcohol every day or eight ormore units at least three or four times a week(very high category) more frequently than thecontrol group. The results shown in table 3 ofmean values of several liver function variablesaccording to alcohol consumption are broadlyin line with those that would have beenexpected from a clinical viewpoint. This find-ing gives some confidence to the accuracy ofthe alcohol data.From a theoretical understanding of the

metabolism of carbon tetrachloride and alco-hol one might expect that alcohol and carbontetrachloride would have a synergistic effecton liver function.'7-19 This was tested byincluding an interaction term between alcoholand exposure in the linear model. No syner-gistic effect could be detected.To determine the extent to which the effect

of exposure on the multivariate analysis ofliver function could have been related to clini-cally detectable liver disease, all participantsfrom both study and control groups with oneor more results in excess of 3 SDs outside thecontrol group mean were reviewed in a clini-cal gastroenterology department by a special-ist. The result of this review was that one caseof liver disease (as defined clinically by thegastroenterologist) was identified but thiscould not be explained by exposure to carbontetrachloride at work. Another study subjectwas found to have non-Hodgkin's lymphomaand one control had haemochromatosis.

Before the study there was an expectationthat total bile acids might represent a moresensitive test of liver function than the morecommonly used variables of alanine transami-nase, aspartate transaminase, alkaline phos-phatase, and rglutamyl transferase. In theevent, the two variables that seem to have beenmost affected were alkaline phosphatase andrglutamyl transferase. These two variables

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contributed most to the differences noted inthe multivariate analysis although whenanalysed on a univariate basis, the differencebetween exposed and control groups did notreach significance. It could be argued that theappearance of enzymes such as alkaline phos-phatase, alanine transaminase, aspartatetransaminase, or yglutamyl transferase inplasma at higher activities reflects an increasein cell turnover or cellular leakage of theseenzymes. In contrast, total bile acids are spe-cific endogenous materials that are synthesisedand secreted by the liver and can be used as afunctional index of the liver. An explanation ofthe results of this study could thus be thatthere has been some enzyme leakage from cellswithout measurable deficit in function.The haematology results presented a differ-

ent picture to those of the liver function tests.Differences between exposed and controlgroups noted in the univariate analysis forhaemoglobin, packed cell volume, and redblood count were not accompanied by an

equivalent increase in the number of abnor-mally low results in the exposed group butrather by a decrease in the number of abnor-mality high results for two of the variables(table 4). There is no theoretical basis for areduction of haemoglobin, packed cell vol-ume, or red blood count as a result of expo-sure to carbon tetrachloride. Indeed, Loyke ina study of the effects of subcutaneous carbontetrachloride on normotensive Sprague-Dawley rats showed an increase in red bloodcell count in treated animals and no signifi-cant change in packed cell volume or haemo-globin.20 Finally there is no indication of anyexposure-effect relation for these variables(table 2).

It seems unlikely that the differencesbetween exposed and control groups in thethree haematological variables were caused byexposure to carbon tetrachloride but there isno obvious explanation. There were signifi-cant differences in many haematological vari-ables between samples taken in November1986 and those taken in February or March1987 and there is a possibility that some sea-

sonal or laboratory effect could have intro-duced a systematic bias.2' The later resultswere analysed separately and were consistentwith the overall result.

Biochemical and haematological variabilityof workers from different sites could havecaused exposure like effects. The hygieneresults (appendix) show, for instance, thatthere were no workers in the highest exposurecategory at one site. There was no evidence tosuggest that the biochemical and haematolog-ical variables differed between workers in thesame category at different sites, although thenumbers of workers in the different exposurecategories were sometimes too small to per-form sensitive comparisons between the foursites.

Despite reservations about the accuracy ofthe assessment of exposure to carbon tetra-chloride, there was no evidence of effects ofclear clinical significance on the liver functionof workers exposed to carbon tetrachloride atthe levels indicated. Nevertheless, there is thepossibility that exposure to carbon tetrachlo-ride was responsible for some of the effectsseen in liver function enzymes. Before thestudy started, considerable efforts were beingmade to identify and implement improve-ments in plant and procedures to reduceexposure. These were continued at the threeplants involved.As a postscript to the study, a follow up

study was conducted at one of the sites threeyears after the cross sectional study. A largeproportion of the workers at this site had beencategorised in the high exposure group in thecross sectional study but considerable reduc-tions in exposures at this site had since beenachieved. Changes in haematological and bio-chemical variables were compared between 26exposed workers and 43 unexposed workerswho had participated in the cross sectionalstudy. There was clear evidence of differencesin laboratory procedure between the laborato-ries who had performed the testing of bloodsamples in the cross sectional and follow upstudies. The changes in variables over thethree year period were remarkably consistent

APPENDIX: Distribution ofpersonal monitoring results for exposure to carbon tetrachloride in three plants

Number of subjects (number of results)

Category Results mean Plant 1 Plant 2 Plant 3 Total

Low: Measured range:< I ppm 0 0 0 01 ppm 0 0 17 (52) 17Estimated 6 6 11 23Total 40

Medium: Measured range:11 1-9ppm 4 (76) 0 0 42-2-9ppm 8 (190) 2 (26) 0 103-3-9ppm 4(1) 1(5) 0 5Estimated 8 1 26 35Total 54

High: Measured range:4-59 ppm 12 (588) 2 (11) 0 146-7-9 ppm 12 (407) 2 (16) 0 148-99 ppm 4 (135) 12 (56) 0 1610-11 9 ppm 0 15 (173) 0 15Estimated 2 0 0 2Total 61

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Tomenson, Baron, O'Sullivan, Edwards, Stonard, Walker, Fearnley

in the exposed and unexposed groups. Thefollow up study has provided no evidence ofany further changes in biochemical andhaematological indices since the cross sec-tional study.

Finally, a comparison of the ranges calcu-lated from the control group with the refer-ence ranges supplied from the manufacturersof the biochemical tests show that there weresome substantial differences between the twosets of ranges (table 4). In general the controlgroup (and the study group) had higher aver-age results for all the liver function variablesthan would have been expected from the ref-erence ranges supplied with the tests. The rea-son for this difference is not completely clear,although it could be due to the use of hospitalor laboratory populations to formulate refer-ence ranges. If this is the case, these popula-tions are clearly not comparable with a groupof working men in the north west of Englandand it does emphasise the importance of hav-ing a proper control group in this type ofstudy.

We express our particular gratitude to Sisters DebbieFearnley, Doreen Evans, Win Ashcroft, and Margaret Rileyfor their role in local organisation, data collection, and vene-section. Thanks also to Sue Braithwaite for her work in datacoordination and to Angela Benson for her contribution to thepreparation of the report.

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8 Saric M, Prpic-Majic D, Beritic T. Activity of serumtransaminase in workers exposed to carbon tetrachlo-ride. Arh HigRada Toksikol 1962;13:19-27.

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_Ind Hyg Toxicol 1936;18:277-98.

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11 American Conference of Government IndustrialHygienists. Documentation of the threshold limit values andbiological exposure indices, 5th ed Cincinatti: ACGIH,1986.

12 Misslbeck NG, Campbell TC, Roe DA. Increase inhepatic gamma-glutamyl transferase activity followingchronic ethanol intake in combination with a high fatdiet. Biochem Pharmacol 1986;35:399-404.

13 Teschke R, Ramen J, Nenefiend M, et al. Alcoholic liverdisease associated with increased gamma-glutamyl trans-ferase activities in serum and liver. Adv Exp Med Biol1980;132:647-54.

14 SAS Institute. The users guide version 5th ed. NorthCarolina: SAS Institute, 1985.

15 Searle SR, Speed SH, Milliken GA. Population marginalmeans in the linear model: an alternative to least squaremeans. The American Statistician 1980;34:216-21.

16 Kirkwood TBL. Geometric means and measures of disper-sion. Biometrics 1979;35:908-9.

17 Folland DS, Schaffer W, Gunn HE, et al. Carbon tetra-chloride toxicity potentiated by isopropyl alcohol. JAMA1976;236: 1853-6.

18 Strubelt 0, Obermeier F, Siegers CP, et al. Increased car-bon tetrachloride hepatotoxicity after low-level ethanolconsumption. Toxicology 1978;lO:261-70.

19 Wei E, Wong LCK, Hine CH. Potentiation of carbontetrachloride hepatotoxicity by ethanol and cold. ToxicolAppl Pharmacol 197 1;18:329-34.

20 Loyke HF. Haematological and blood pressure studies incell treated rats. Y Environ Pathol Toxicol Oncol 1986;7:1-8.

21 Lee CJ, Lawler GS, Panemangalore M. Nutritional status ofmiddle-aged and elderly females in Kentucky in two sea-sons: part 2. Haematological parameters. YAm Coll Nutr1987;6:217-22.

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