We observed that both dermal and inhalation exposure were significant predictors of urine HDA levels when used as single exposure variables in the multiple linear regression analysis ( and ). However, when BZC-APF and dermal exposure were considered in the same model, BZC-APF was always a significant predictor while dermal exposure was insignificant. This may reflect the fact that 64% of the dermal samples collected were below the LOD compared to 9% of the BZC samples, thereby allowing a more robust estimation of the model fit between BZC and urine HDA levels. On the contrary, in a subset of workers (n = 12) who did not wear gloves or coveralls and whose dermal exposure levels were above the LOD, neither BZC-APF nor dermal exposure was significant when placed into the same model. However, a significant relationship between dermal exposure and a borderline significant relationship between BZC-APF and urine HDA levels were observed in the cumulative exposure model when modeled alone with creatinine concentration.
When interpreting these results, caution is warranted due to the limitations of the methods used to measure dermal and inhalation exposure. First, the method employed to calculate the exposure period may have slightly biased the inhalation exposure estimates. The total task period monitored reflected the duration of time the air-sampling pump was operating and not the actual time worker was in contact with HDI-containing paints. Second, estimation of the inhalation exposure from BZC by adjusting for respirator APF is challenging and does not account for improper fit and/or maintenance. Adjustment with APF provides reliable protection estimates only when a respiratory protection program is employed that includes proper training, fit testing, maintenance, and use requirements (as described in OSHA, 2006
). Unfortunately, we did not have means to test the respiratory protection during this study, and it is unknown how rigorously the workers followed these requirements. However, our data indicated that adjustment of inhalation exposure level with the APF provided a better model fit than BZC without adjustment. The APF adjustment used assumes that all similar type respirators provide the same level of protection. For example, it is assumed that all half-face cartridge-type respirators reduce the BZC by a factor of 10. However, there may be considerable variation in the protection level of the individual respirators based on the mask fit, the cartridge change schedule, and even manufacturers. Therefore, the correlation between urine HDA level and inhalation exposure to HDI, adjusted by APF, should be confirmed in an investigation in which the workers’ respiratory protection programs are evaluated. Third, the poor correlation between urine HDA level and dermal HDI exposure may be affected by rapid absorption of HDI through the stratum corneum and/or conjugation of HDI to macromolecules in the skin, and thus contributing to the large number of nondetectable samples collected from the skin even though the tape-strips were collected immediately after each paint task. Further investigation on the dermal absorption of HDI and binding to macromolecules in human skin are warranted to increase our understanding of the contribution of this exposure route to urine HDA levels and potential ill effects due to dermal exposure.
All models in which creatinine concentration was accounted for as an independent variable along with inhalation and dermal exposure showed it to be a highly significant variable (P
< 0.0001; ). This indicates that the exposure models need to include creatinine concentration to account for the water content of urine. Furthermore, the parameter estimates and P
values of all variables were similar when un-normalized models were compared to the normalized models ( and ). If the creatinine concentration had a parameter estimate of 1 in the model, the model with creatinine as an independent variable would have been equivalent to the model with creatinine-normalized HDA levels. However, the creatinine parameter estimates ranged from 1.25 to 1.30 in the un-normalized models implying that normalization of urine HDA level with creatinine concentration may attenuate the external exposure biomarker relationship. The models with creatinine as an independent variable also had much higher marginal R2
s than models with traditional normalization. Since the marginal R2
is computed with only fixed effect components (i.e. no random effect components are included), much of the variance in the urine HDA levels may have been caused by variance in the creatinine concentrations. In the models where creatinine is an independent variable, any variance due to the creatinine is modeled as a fixed effect for urine HDA. However, in the traditional normalization models, any creatinine variance is included in variance for the overall model, and thus not modeled with the fixed effects. Therefore, as proposed by Barr et al. (2005)
and as indicated by this study, creatinine concentration should be used as an independent variable in linear mixed models, rather than the traditionally used method of normalization of urine HDA level with creatinine concentration.
Significance of both dermal and BZC-APF exposure increased with increasing half-life estimate for HDA level, and the significances in the 174 min (2.9 h) half-life model (D) were similar to the cumulative exposure model (model A; and ). A similar half-life was also observed by Liu et al. (2004)
who estimated a half-life of 2.8 h for subjects exposed to HDI vapor using a method that precluded dermal exposure. Our observation is also similar to the 2.5 h half-life derived from a study in which both dermal and inhalation exposure occurred (Tinnerberg et al., 1995
As illustrated in , some workers exhibited decreasing urine HDA levels over the course of a day. The reason for this is unclear but may reflect biphasic elimination kinetics. A slow clearance phase would also explain the detectable HDA levels in the 64 (53%) first urine samples of the day when exposure was not known to have occurred yet. It is possible that some of the first urine samples of the day with detectable HDA level may have reflected HDI exposure received the day before. However, calculation using the GM HDA level of the first urine samples (0.08 μg l−1) along with the estimated half-life of 2.9 h and assuming that the first urine sample was taken at 8 am, point to a urine HDA level of 2.89 μg l−1 at 5 pm the day before. Since the GM HDA level of the last urine samples of the day was 0.14 μg l−1 and 97% of all last urine samples were below 2.89 μg l−1, this would appear to be an unlikely scenario. The results of these calculations are similar for creatinine-normalized HDA levels. In addition, secondary exposure to HDI (i.e. not due to painting) may also contribute to the morning HDA levels. The secondary exposure may be caused by touching contaminated surfaces or entering the mixing area without dermal or inhalation protection. The detectable HDA levels in all the urine samples of two of the five workers who were monitored on days when they were not painting may be explained by either biphasic elimination of HDA or secondary exposure to HDI (i.e. not due to painting).
Further evidence for the biphasic elimination kinetics is provided by the seven workers who had detectable HDA levels in their first urine sample before exposure occurred on a Monday. While it is conceivable that some of the painters worked on Sundays or on weeknights, 36 different subjects had at least one urine sample with detectable HDA before exposure was known to have occurred and, thus, it seems unlikely that this many painters would have had a second employment in another painting facility. As evidenced by and and the estimated half-life of 2.9 h for HDA, collecting one urine sample at the end of the workday may not provide a reliable exposure estimate for that day. Furthermore, a biphasic elimination pattern is indicated as seen with TDA (Brorson et al., 1991
; Tinnerberg et al., 1995
; Lind et al., 1996
; Lind et al., 1997
). Therefore, further studies are needed to investigate the potential biphasic elimination kinetics of HDA in spray painters and to determine the half-life of the slow elimination phase.
There are no regulatory guidelines regarding safe HDA levels in urine in the USA. In the UK, however, a biological monitoring guidance level of 1 μmol HDA per mole creatinine exists (HSE, 2006
). Among our subjects, the average HDA level was 0.29 μmol HDA per mole creatinine and the median value was 0.10 μmol HDA per mole creatinine. This indicates that by UK standards, the subjects are adequately protected. It should be noted that this guidance value is based on an recommendation for good occupational hygiene practices achievable by most of UK industry and not based on protection from health effects caused by HDI exposure (HSE, 2006
). This guidance document also states that urine samples should be collected immediately after the task or shift. In several diisocyanate studies, only one urine sample per worker was collected at the end of the workday (Maitre et al., 1996
; Rosenberg et al., 2002
; Sabbioni et al., 2007
). Our results indicate that this methodology may miss the timeframe when the highest diamine level after exposure occurs. As indicated by our results and stated in the HSE guidelines, the optimal sampling strategy would be collection and analysis of all urine voids during the day. This, however, may prove to be impractical, and thus collecting a urine sample within 2 h after exposure ceased may be sufficient for monitoring HDA as a biomarker for HDI exposure in an occupational exposure setting.
The quantitative relationship between dermal and inhalation exposure to HDI and urine HDA levels has not been characterized previously. Here, we report a significant association between inhalation and dermal exposure to HDI and urine HDA level in occupationally exposed workers. The results indicate biphasic elimination kinetics with a fast phase of 2.9 h. Further study is necessary to determine the half-life of the slow phase. The results of our investigation indicate that biological monitoring of the workers should be tailored to reliably capture the intermittent exposure pattern typical in this industry as well as the use of personal protective equipment. Furthermore, when urine HDA levels are used as biomarkers of exposure in this occupational group, urine creatinine concentration should be included as an independent variable in subsequent statistical exposure assessment analysis.