Breath, urine, and blood measurements as biological exposure indices of short-term inhalation exposure to methanol

Due to their transient nature, short-term exposures can be difficult to detect and quantify using conventional monitoring techniques. Biological monitoring may be capable of registering such exposures and may also be used to estimate important toxicological parameters. This paper investigates relationships between methanol concentrations in the blood, urine, and breath of volunteers exposed to methanol vapor at 800 ppm for periods of 0.5, 1, 2, and 8 h. The results indicate factors that must be considered for interpretation of the results of biological monitoring. For methanol, concentrations are not proportional to the exposure duration due to metabolic and other elimination processes that occur concurrently with the exposure. First-order clearance models can be used with blood, breath, or urine concentrations to estimate exposures if the time that has elapsed since the exposure and the model parameters are known. The 0.5 to 2-h periods of exposure were used to estimate the half-life of methanol. Blood data gave a half-life of 1.44±0.33 h. Comparable but slightly more variable results were obtained using urine data corrected for voiding time (1.55±0.67 h) and breath data corrected for mucous membrane desorption (1.40±0.38 h). Methanol concentrations in blood lagged some 15–30 min behind the termination of exposure, and concentrations in urine were further delayed. Although breath sampling may be convenient, breath concentrations reflect end-expired or alveolar air only if subjects are in a methanol-free environment for 30 min or more after the exposure. At earlier times, breath concentrations included contributions from airway desorption or diffusion processes. As based on multicompartmental models, the desorption processes have half-lives ranging between 0.6 and 5 min. Preliminary estimates of the mucous membrane reservoir indicate contributions of under 10% for a 0.5-h exposure and smaller effects for longer periods of exposure. Received: 1 August 1996 / Accepted: 24 January 1998     

Monitoring of occupational exposure to dichloromethane by diffusive vapor sampling and urinalysis

Objective: The aim of the present study was to develop valid methods for monitoring of occupational exposure to dichloromethane (DCM). Methods: Carbon cloth as an adsorbent in diffusive sampling was tested for its capacity to adsorb DCM vapor and to retain adsorbed DCM after termination of the exposure. Urine samples collected from DCM-exposed workers were analyzed for DCM by the head-space technique. After extraction with carbon disulfide, DCM in the cloth was analyzed on a DB-WAX capillary column by flame-ionization detection gas chromatography (FID-GC) and DCM in urine was analyzed by electron-capture detection (ECD)-GC. Results: The diffusive sampling with carbon cloth as an adsorbent is applicable to 4-h monitoring of exposure to up to 100 ppm DCM vapor. DCM concentrations detected in end-of-shift urine samples correlated linearly with time-weighted average DCM concentrations measured in the breathing-zone␣air␣of the exposed workers; essentially the same exposure-excretion relationship was obtained by vapor monitoring for the afternoon 4-h period as compared with a whole day (8-h) of vapor monitoring. There was no sex difference in the exposure-excretion relation. Conclusions: Both personal diffusive sampling (at up to 100 ppm DCM and for up to 4 h) and biological exposure monitoring by urinalysis for DCM are applicable in occupational health as reliable measures of exposure to this chlorinated hydrocarbon solvent. Received: 22 October 1997 / Accepted: 26 February 1998     

Urinary excretion of unmetabolized acetone as an indicator of occupational exposure to acetone

Summary Acetone concentrations in urine samples from 28 workers exposed to acetone in a fiber-reinforced plastics factory were determined by directly injecting urine supernate into a gaschromatograph with FID detectors. Acetone concentrations in the urine from ten nonexposed subjects were also determined. The 8-h time-weighted exposure intensity of individual workers was monitored by means of diffusive sampling. Acetone concentration in urine and acetone concentration in the breathing zone showed a linear correlation to each other. The study results indicate that the correlation coefficient is high enough to enable use of the urinary level of acetone as an indicator of occupational exposure to acetone.     

Formic acid excretion in comparison with methanol excretion in urine of workers occupationally exposed to methanol

Summary A semiautomated head-space gas chromatographic (GC) method was developed for measuring formic acid in urine. The method consists of heating 1 ml urine sample in a 20-ml air-tight vial in the presence of 1 ml sulfuric acid and 2 ml ethanol at 60°C for 30 min for ethyl esterification and air-liquid equilibrium, followed by automatic injection of 1 ml head-space air into a flame ionization detector GC. The detection limit was 1 mg/l for formic acid. The method was applied to measure formic acid in the shift-end urine samples from 88 workers exposed to methanol at 66.6 ppm (as geometric mean) and in urine samples from 149 nonexposed controls. Methanol concentrations were also determined. Regression analysis showed that urinary formic acid concentrations, as observed or corrected for either creatinine concentration or specific gravity of urine (1.016), correlated significantly with time-weighted average intensities of exposure to methanol vapor. Men excreted significantly more formic acid than women. Comparison with methanol excretion suggested, however, that urinary formic acid is less sensitive than urinary methanol as an indicator of methanol vapor exposure, primarily because the background level for formic acid (26 mg/l as arithmetic mean, or 23 mg/l as geometric mean) is more than ten times higher than the level for methanol (1.9 mg/l as arithmetic mean, or 1.7mg/l as geometric mean). After theoretical methanol exposure at infinite concentration, the urinary formic acid/methanol ratio should be about 0.4.     

Urinary methylchloroform rather than urinary metabolites as an indicator of occupational exposure to methylchloroform

In order to compare methylchloroform (MC, or 1,1,1-trichloroethane) per se and its metabolites in urine as indicators of occupational exposure to this solvent, 50 male solvent workers were studied in the second half of a working week to evaluate the exposure-excretion relationship. The time-weighted average intensity of solvent exposure of individuals during an 8-h shift was monitored by personal diffusive sampling. Urine samples were collected near the end of the shift and were analyzed for MC and its metabolites [i.e., trichloroacetic acid (TCA), trichloroethanol (TCE) and total trichloro-compounds (TTC; the sum of TCA and TCE)] by head-space gas chromatography. MC per se, TCA, TCE, and TTC in urine correlated significantly (P < 0.01) with MC in ambient air, and among the four the correlation coefficient was highest for MC. The same results were obtained by multiple regression analysis in which ambient air MC was taken as the dependent variable and either the three indicators urinary MC, TCA, and TCE or the two indicators urinary MC and TTC were taken as independent variables. Taking the specificity and selectivity of the analyte as well as the simple and hazardous chemical-free procedure of analysis into consideration, it is concluded that MC is the analyte of choice as an indicator of occupational exposure to MC, when urine is selected as a specimen available by noninvasive sampling.     

Urinary methanol and formic acid as indicators of occupational exposure to methyl formate

Objective: To evaluate the validity of methanol (MeOH) and formic acid (FA) in urine as biological indicators of methyl formate (MF) exposure in experimental and field situations. Methods: The subjects were 28 foundrymen and two groups of volunteers (20 control and 20 exposed). Exposure assessment of the workers was performed by personal air and biological monitoring. Methyl formate vapour collected on charcoal tube was analysed by gas chromatography. The concentration of MF in the exposure chamber (volunteer-study) was monitored by two independent methods [flame ionisation detection (FID) and Fourier transformation infra-red detection (FTIR)]. Urinary metabolites (MeOH and FA) were analysed separately by head-space gas chromatography. Results: The volunteers exposed to 100 ppm MF vapour at rest for 8 h excreted 3.62 ± 1.13 mg MeOH/l (mean ± SD) at the end of the exposure. This was statistically different (P < 0.001) from pre-exposure MeOH excretion (2.15 ± 0.80 mg/l), or from that of controls (1.69 ± 0.48 mg/l). The urinary FA excretion was 32.2 ± 11.3 mg/g creatinine after the exposure, which was statistically different (P < 0.001) from pre-exposure excretion (18.0 ± 9.3 mg/g creatinine) or that of controls (13.8 ± 7.9 mg/g creatinine). In foundrymen, the urinary FA excretion after the 8 h workshift exposure to a time weighted average (TWA) concentration of 2 to 156 ppm MF showed a dose-dependent increase best modelled by a polynomial function. The highest urinary FA concentration was 129 mg/g creatinine. The pre-shift urinary FA of the foundrymen (18.3 ± 5.6 mg/g creatinine) did not differ from that of controls (13.8 ± 7.9 mg/g creatinine). The urinary MeOH excretion of the foundrymen after the shift, varied from <1 to 15.4 mg/l, while the correlation with the preceding MF exposure was poor. The foundrymen excreted more (P=0.01) FA (2.12 ± 3.56 mg/g creatinine) after the workshift than experimentally, once-exposed volunteers (0.32 ± 0.11 mg/g creatinine) at a similar inhaled MF level of 1 ppm). Conclusions: In spite of its high background level in non-exposed subjects, urinary FA seems to be a useful biomarker of methyl formate exposure. The question remains as to what is the reason for the differences in chronic and acute exposure respectively. Received: 27 September 1999 / Accepted: 25 March 2000     

Methyltetrahydrophthalic acid in urine as an indicator of occupational exposure to methyltetrahydrophthalic anhydride

Objective: To investigate whether methyltetrahydrophthalic acid (MTHP acid) in urine can be used as a biomarker for exposure to methyltetrahydrophthalic anhydride (MTHPA). Methods: Workers occupationally exposed to MTHPA were studied in combination with one of the authors, who was experimentally exposed to MTHPA. Air levels of MTHPA were determined by personal sampling in the breathing zone. The MTHPA in air was sampled by silica gel and analyzed by gas chromatography (GC) with electron-capture detection. Urinary levels of MTHP acid, a metabolite of MTHPA, were determined in 15 subjects in total. Urine was collected from 14 workers immediately before the start of the work shift and then after 4 and 8 h, and from one of the authors at intervals during 24 h. MTHP acid in urine was analyzed by GC with mass spectrometric detection. Results: The time-weighted average (TWA) air levels ranged from 1.0 g to 200 g MTHPA/m³ during 8 h work shifts. The urinary levels of MTHP acid increased during exposure and decayed after the end of exposure, with an estimated half-time of about 3 h. A close correlation was found between the TWA air levels of MTHPA and creatinine-adjusted MTHP acid levels in urine collected at the end of the shift (r=0.955; P<0.0001). The current occupational exposure limit of 50 g MTHPA/m³ (Japan Society for Occupational Health) corresponded to about 1300 g MTHP acid/g creatinine, which was equivalent to about 900 nmol/mmol creatinine in the International System of Units (SI). Conclusions: These results indicate that the determination of MTHP acid in urine is suitable for use in the biological monitoring of MTHPA exposure.     

尿邻甲酚作为接触甲苯生物监测指标的探讨

目的探讨尿邻甲酚作为接触甲苯生物监测指标的可能性。方法建立柱前衍生高效液相色谱法测定人体尿中邻甲酚,且使用该方法测定非职业及职业接触甲苯人群尿中邻甲酚水平,并进行接触评定。结果甲苯接触者尿邻甲酚水平为(2.61±1.94)mg/L,明显高于对照组[(0.32±0.23)mg/L],差异有显著性(P<0.001),且接触甲苯工人班后尿邻甲酚水平比班前明显升高,最高可达29倍。接触甲苯者尿邻甲酚水平与个体接触甲苯浓度明显相关(r=0.6295,P<0.01)。结论尿邻甲酚可以作为接触甲苯的生物监测指标。     

Monitoring of occupational exposure to tetrachloroethene by analysis for unmetabolized tetrachloroethene in blood and urine in comparison with urinalysis for trichloroacetic acid

Objective: The present study was initiated to examine a quantitative relationship between tetrachloroethene (TETRA) in blood and urine with TETRA in air, and to compare TETRA in blood or urine with trichloroacetic acid (TCA) in urine as exposure markers. Methods: In total, 44 workers (exposed to TETRA during automated, continuous cloth-degreasing operations), and ten non-exposed subjects volunteered to participate in the study. The exposure to vapor was monitored by diffusive sampling. The amounts of TETRA and TCA in end-of-shift blood and urine samples were measured by either head-space gas chromatography (HS-GC) or automated methylation followed by HS-GC. The correlation was examined by regression analysis. Results: The maximum time-weighted average (TWA) concentration for TETRA-exposure was 46 ppm. Regression analysis for correlation of TETRA in blood, TETRA in urine and TCA in urine, with TETRA in air, showed that the coefficient was largest for the correlation between TETRA in air and TETRA in blood. The TETRA in blood, in urine and in air correlated mutually, whereas TCA in urine correlated more closely with TETRA in blood than with TETRA in urine. The TCA values determined by colorimetry and by the GC method were very similar. The biological marker levels at a hypothetical exposure of 25 ppm TETRA were substantially higher in the present study than were the levels reported in the literature. Possible reasons are discussed. Conclusions: Blood TETRA is the best marker of occupational exposure to TETRA, being superior to the traditional marker, urinary TCA. Received: 11 October 1999 / Accepted: 3 December 1999     

Cholinesterase activity as a biological indicator of exposure to pesticides

Summary Cholinesterase activity measurement should be considered in relationship to its proposed use, whether for diagnostic information, a monitor of health status or because of its logistical and economic practicality. To prevent confusion one must consider that measurement of cholinesterase activity may confirm the existence of poisoning by an inhibitor, may measure an individual worker's overall exposure or his characteristic individual response. Where groups of workers are involved a group response may be derived to identify the magnitude of the response at some preset value of significance from which a biological threshold limit value may be established. The population at risk, the intensity and frequency of exposure dictate the frequency of sampling. Pesticide exposed workers are grouped according to confined and unconfined environments. Confined environments are those associated with manufacturing, formulating and warehousing of pesticidal products. Unconfined environments are those associated with application and postapplication exposures. All of the aforementioned factors must be considered before employing cholinesterase activity measurement as an index of exposure to pesticides.     

Biological monitoring of occupational exposure to isopropyl alcohol vapor by urinalysis for acetone

Summary The relationship of the intensity of occupational vapor exposure to isopropyl alcohol (IPA) with urinary excretion of acetone and unmetabolized IPA was studied in 99 printers of both sexes, who were exposed to up to 66 ppm IPA (as time-weighted average), together with toluene, xylenes, methyl ethyl ketone and/or ethyl acetate. Acetone and IPA concentrations in urine were studied also in 34 non-exposed subjects. Acetone was detectable in the urine of most of the non-exposed, and the urinary acetone concentration increased in proportion to the IPA exposure intensity (r = 0.84 for observed, non-corrected values), whereas the correction for creatinine concentration or specific gravity of urine did not give a larger correlation coefficient. IPA itself was not found in the urine of the non-exposed, and was detectable in urine of only those who were exposed to IPA above a certain level, e.g. 5 ppm. The present study results suggest that urinary acetone is a valuable index for biological monitoring of occupational exposure to IPA as low as 70 ppm.A part of this work was presented at 62nd Annual Meeting of Japan Association of Industrial Health, held in Hirosaki, Japan, on 27th–30th, April, 1989     

Monitoring of workers exposed to a mixture of toluene,styrene and methanol vapours by means of diffusive air sampling,blood analysis and urinalysis

Summary Exposure of 34 male workers to combined toluene, styrene and methanol was monitored by personal diffusive sampling of solvent vapours in breathing zone air, analysis of shift-end blood for the 3 solvents and analysis of shift-end urine for hippuric, mandelic and phenylglyoxylic acids and methanol. The exposure of most of the workers was below current occupational exposure limits. Regression analysis showed that a linear correlation exists for each of the 3 solvents between any pairs of the concentrations in air, blood and urine. Namely, toluene, styrene and methanol concentrations in blood obtained at the end of a shift are linearly related to the time-weighted average intensity of exposure to corresponding solvents, and also hippuric, mandelic and phenylglyoxylic acids as well as methanol in shift-end urine. The concentrations of hippuric, mandelic and phenylglyoxylic acids as well as methanol in urine correlated with the respiratory exposure intensity. Comparison of the present results with the exposure — excretion relationship after occupational exposure to the individual solvent showed that no modification in metabolism is induced by the combined exposure when exposure is low, as in the present case.     

The method of choice for the determination of 2,5-hexanedione as an indicator of occupational exposure to n-hexane

Summary To identify the method of choice for analysis of urine for 2,5-hexanedione (2,5-HD) as an indicator of occupational exposure to n-hexane, the end-of-shift urine samples of 36 n-hexane exposed male workers and 30 non-exposed male workers were analyzed for 2,5-HD under three conditions of hydrolysis, i.e. enzymic hydrolysis at pH 4.8, acid hydrolysis at pH 0.5, and without hydrolysis. The 2,5-HD concentrations thus determined were examined for correlation with 8-h, time-weighted average exposure concentrations of n-hexane measured by diffusive sampling. The regression analysis showed that the 2,5-HD concentrations without any hydrolysis correlated best with the intensity of exposure to n-hexane. No 2,5-HD was detected in the urine of the non-exposed subjects under the analytical conditions with no hydrolysis. Thus, the analysis without hydrolysis was considered to be the method of choice from the viewpoint of simplicity in analytical procedures, sensitive separation of the exposed from the non-exposed, and quantitative increase in the amount of 2,5-HD after n-hexane exposure.A part of this work was presented at the 63rd Annual Meeting of Japan Association of Industrial Health, held in Kumamoto, Japan, on 3rd–6th April, 1990     

Enzymatic assay of formic acid and gas chromatography of methanol for urinary biological monitoring of exposure to methanol

Summary An enzymatic assay method for the determination of urinary formic acid is described. Formic acid in urine was cleaved to carbon dioxide and water by formic acid dehydrogenase, whereby NAD⁺ was converted to NADH, which reacted with INT (p-iodonitrotetrazolium violet) in the presence of NAD-diaphorase. The color thus produced was determined at 500 nm. In addition, a simple gas chromatographic method of urinary formic acid is described, in which head space gas of formic acid methylester was applied into the wide bore column. The urinary formic acid concentrations by the enzymatic method agreed well with that by the gas chromatographic method. A simple gas chromatographic method for urinary methanol assay is also described. Acetonitrile was added to an equal volume of urine containing methanol. After centrifugation, the supernatant was injected into gas chromatography (GC). The peaks of urinary methanol and ethanol were separated by GC. Formic acid and methanol in urine of unexposed healthy subjects and workers exposed to methanol were analyzed by the colorimetric and gas chromatographic methods. Geometric mean concentrations of urinary formic acid and methanol in the healthy subjects were 7.82 mg/g creatinine and 1.34 mg/l, respectively. The concentration ratio of formic acid to methanol in the urine of the workers exposed to methanol was calculated to be 3.67 ± 2.10, which agreed with the ratio under a controlled exposure experiment. A slower excretion of formic acid than that of methanol in the urine of a volunteer was also observed.     

n-Hexane urine elimination and weighted exposure concentration

Summary The concentration of n-hexane in urine was determined in 30 subjects occupationally exposed to n-hexane (median value 59.6 mg/m³) in a shoe factory. The measurement of the substance was performed by means of a Hewlett-Packard 5880 gas chromatograph supplied with a Hewlett-Packard 5970 Mass Selective Detector. The analyses were performed by the head space method (constant volume method, after determination of the urine partition coefficient by the multiple phase equilibration method). The authors found a significant correlation between the n-hexane urine concentrations (g/1, Cu) and the n-hexane environmental concentrations (mg/m³, Ci) (r = 0.84; Cu = 0.0669 x Ci + 0.8396).This work was supported by the research funds given to Fondazione Clinica del Lavoro, Pavia by the Health Council     

Urinary cyclophosphamide assay as a method for biological monitoring of occupational exposure to cyclophosphamide

Summary Urine of twenty hospital workers was monitored for the excretion of the cytostatic drug cyclophosphamide using GC-MSD. The drug was found to be present above the detection limit of 0.5 g/24 h urine in five cases. A clear relationship between cyclophosphamide handling and the detectability of excretion existed. This method developed can be of use for biological monitoring studies directed toward the finding of exposure hazards.     

Urinary hexane diamine as an indicator of occupational exposure to hexamethylene diisocyanate

The occupational exposure of 19 men to hexamethylene diisocyanate (HDI) vapour was monitored during one 8-h shift. It ranged from 0.30 to 97.7 μg/m³. This was compared with the urinary output of hexane diamine (HDA) liberated by acid hydrolysis from its conjugates in post-shift samples. The excretion varied from 1.36 to 27.7 μg/g creatinine, and there was a linear association of HDI air concentration with urinary HDA excretion. The validity of the urinary analysis was confirmed by simultaneous blind analysis in another laboratory. The results had an excellent linear concordance. Thus, it seems that while the gas chromatographic-mass spectrometric detection method requires sophisticated apparatus, the results are very useful to occupational health practices. A biological exposure index limit of 19 μg HDA/g creatinine in a post-shift urine specimen is proposed as an occupational limit level of HDI monomer (time-weighted average=75 μg/m³). Most importantly, biological monitoring of HDA is sensitive enough to be used at and below the current allowable exposure limit levels.     

Serum fluoride as an indicator of occupational hydrofluoric acid exposure

Summary To define the relationship between ionic fluoride concentration in the serum of workers and the amount of hydrofluoric acid (HF) in the work environment, pre-and postshift serum and urine samples of 142 HF workers and 270 unexposed workers were examined. The maximum and minimum concentrations of HF in the air in each workshop varied from the mean by less than 30%. The pre-exposure levels of serum and urinary fluoride in HF workers were higher (P < 0.001) than the control values. This suggests that fluoride excretion from the body continues for at least 12 h. The postshift serum and urinary fluoride concentrations of these workers were significantly higher (P < 0.001) than the preshift concentrations. A good correlation (r = 0.64) was obtained between postshift serum fluoride and postshift urine fluoride. There was a linear relationship between mean serum fluoride concentration and HF concentration in the workshop. A mean fluoride concentration of 82.3 g/l with a lower fiducial limit (95%, P = 0.05) of 57.9 g/l was estimated to correspond to an atmospheric HF concentration of 3 ppm. This is the maximum allowable environmental concentration recommended by the Japanese Association of Industrial Health, and it is also the threshold limit value suggested by the American Conference of Governmental Industrial Hygienists. The results demonstrate that exposure to HF can be monitored by determining the serum fluoride concentration.     

High-pressure liquid chromatographic determination of toluenein urine as a marker of occupational exposure to toluene

Objective: To establish a convenient method by high-pressure liquid chromatography (HPLC) to measure toluene in urine as a marker of occupational exposure to toluene. Methods: As soon after sampling as possible, 1 ml of urine was mixed with an equal volume of acetonitrile in a 2.2-ml HPLC glass bottle, and the bottle was tightly sealed and stored at 4 °C. Immediately before HPLC determination, 100 μl methanol was added to the mixture to prevent confounding effects of glycosuria, and the bottle was spun to remove any suspended matter. An aliquot of the supernate was introduced into the HPLC system and analyzed on a PRODIGY column, with an acetonitrile – perchloric acid – phosphoric acid – water mixture serving as the mobile phase. The effluent was monitored at 191 nm. Results: The method can measure toluene in urine every 20 min; the detection limit was 2 μg/l, the coefficient of variation was less than 5%, and the recovery rate was 100%. No significant reduction in toluene concentration was observed for 1 week after storage at 4 °C. When the method was applied to end-of-shift urine samples from 13 male workers exposed to toluene at 18–140 ppm and also to urine samples from 10 nonexposed male controls, toluene in urine was linearly related to toluene exposure concentration, with a regression line passing close to the origin. The correlation coefficient was as high as 0.97 (n = 23). No toluene was detected in control urine samples. Calculations suggest that urinary toluene accounts for as little as less than 0.01% of the toluene absorbed via inhalation and that the absorbed toluene is converted almost quantitatively to hippuric acid and, by less than 0.1%, to o-cresol. Received: 25 August 1997 / Accepted: 13 February 1998     

Occupational dimethylformamide exposure

Summary The relationship between the 8-h time-weighted average (TWA) intensity of exposure toN, N-dimethylformamide (DMF) vapor (with little possibility of skin contact with liquid DMF) and the subsequent excretion ofN-monomethylformamide (MMF) precursor in shift-end urine samples was examined in 116 workers exposed to DMF and 92 workers exposed to DMF in combination with toluene. Urinary MMF level was examined also in 42 non-exposed subjects. The TWA vapor concentration in breathing zone air of each worker was successfully measured by means of a recently developed diffusive sampler in which water was used as an absorbent. The examination of gas chromatographic (GC) conditions for MMF determination showed that the formation of MMF was not saturated when the injection port temperature was set at 200°C, reached a plateau at 250°C, and showed no additional increase at 300°C. There was a linear relationship between DMF in air and MMF in urine with a regression equation ofy =1.65x + 1.69 (r = 0.723,P<0.01), wherey is MMF (unit; mg/l, uncorrected for urine density) in urine andx is DMF (ppm) in air, when only those exposed to DMF were selected, and the injection port temperature was set at 250°C. From this equation, it was possible to estimate that about 10% of the DMF absorbed will be excreted into urine as the MMF precursor. The slope of the regression line was significantly smaller among those exposed to DMF and toluene in combination as compared with those with DMF exposure only.