Study design and setting
This prospective cohort study was conducted across successive 10 years. We conducted a baseline survey in 2003 and implemented follow-up surveys yearly from 2004 (first survey) to 2013 (tenth survey). Each participant received a periodic health check and completed 1) a toner-handling work status survey, 2) a questionnaire-based survey on self-reported respiratory symptoms and diseases, 3) chest radiography, 4) respiratory function tests, and 5) serum and urinary biomarker tests. We particularly examined the effects of toner-handling work on chest X-ray findings, respiratory function, inflammation, allergy, and oxidative stress.
Sample size calculation
The incidence of respiratory disease associated with toner exposure is not well known. Therefore, assuming that the prevalence of abnormal chest X-ray findings among the background characteristics was about 50 out of 100,000 toner-handling workers, and that the prevalence when the effect of toner exposure is significant is about 150 out of 100,000 toner-handling workers, we would need about 2,100 toner-handling-workers based on 90% power and 5% level of significance. When the background prevalence was set at < 10 out of 100,000 toner-handling workers, about 860 toner-handling workers were needed. We therefore estimated that it would be desirable to have about 1,000 toner-handling-workers in this study.
Participants
A total of 918 participants who were 19-50 years of age in 2003, worked in one toner and copier manufacturing enterprise, and handled toner particles at work, were potentially eligible for this study (toner-handling group). Their toner-handling work included toner development, toner manufacturing, toner or copy machine development, toner or copy machine recycling, and customer service. Additionally, we recruited gender-matched non-toner-handling workers aged 19-50 years who also worked in the same business sites as those in the toner-handling group. A total of 586 non-toner handlers were enrolled as controls (non-toner-handling group). We confirmed that the control group mainly engaged in desk work not often involving copy printing and had never engaged in toner-handling work. The toner-handling area and the area where the control group worked were physically separated. Participants were excluded from the analysis if they lacked a detailed work history or if they had already been diagnosed with chronic granulomatous pneumonia, pneumoconiosis, or lung cancer at the time of the baseline survey.
Chest X-ray examination
We performed a yearly chest X-ray examination on each participant following the standard examination method regulated by the Pneumoconiosis Law in Japan [17, 18]. The chest X-ray images were interpreted following the panel reading by two skilled readers, based on the international classification of pneumoconiosis (a 12-point scale from 0/- to 3/+) [19], and were electronically stored using a film digitizer. To avoid differential misclassification, the readers of the X-ray images were not given information about the toner-handling status of the participants.
Respiratory function tests: spirometry and flow-volume curve
We conducted yearly respiratory function tests for each participant, including the following parameters: vital capacity (VC), percentage of VC to predicted VC value (%VC), forced expiratory volume in 1 s (FEV1), percentage of FEV1 to predicted FEV1 value (%FEV1), percentage of forced expiratory volume in 1 s to forced vital capacity (FEV1/FVC), percentage of FEV1/FVC to predicted FEV1/FVC value (%FEV1/FVC), maximal expiratory flow at 25% FVC (V25), and percentage of V25 to predicted V25 value (%V25). The respiratory function tests were performed using Microspiro HI-701 and Microspiro HI-801 (CHEST Corporation, Tokyo, Japan), which are pneumotach-type spirometry measuring units that meet the standards regulated by the American Thoracic Society [20]. We measured each parameter three times on the same day to obtain adequate values. To ensure consistent and valid measurement, a skilled examiner at the same medical institution conducted the respiratory function tests throughout each 1-yearly study period. We calculated the predicted values for VC, FEV1, FEV1/FVC, and V25 for each participant using the formula based on sex, age, and height indicated by the Japanese Respiratory Society [21, 22].
Serum and urinary biomarker tests
Each participant underwent yearly biomarker tests for inflammation, allergy, and oxidative stress, such as those for C-reactive protein (CRP), immunoglobulin E (Ig E), interleukin (IL)-4, IL-6, IL-8, and interferon-gamma (IFN-γ) in serum, and 8-hydroxy-2′-deoxyguanosine (8-OHdG) in urine. To maintain accuracy and precision throughout the whole survey, we requested the OHG Institute Co., Ltd. (Kitakyushu, Japan), to perform the analysis of 8-OHdG, and SRL Inc. (Tokyo, Japan) to analyze other biomarkers.
We used latex immunoagglutination assays for analyzing CRP; fluorescent enzyme immunoassays for IgE; chemiluminescent enzyme immunoassays for IL-4 and IL-6; enzyme-linked immunosorbent assays for IL-8; enzyme immunoassays for IFN-γ; and high-performance liquid chromatography for 8-OHdG. Spot urinary 8-OHdG concentrations could be unstable due to the participants’ physical activity intensity, urine collection time, and other factors. Hence, creatinine-corrected 8-OHdG values were adopted in this study. The limits of detection (LODs) at SRL Inc. were 0.02 mg/dL for CRP, 5.00 IU/mL for IgE, 2.00 pg/mL for IL-4, 0.20 pg/mL for IL-6, 2.00 pg/mL for IL-8, and 0.10 IU/mL for IFN-γ. We allotted the values of LOD/2 to the undetectable values of each biomarker.
Toner particle
The toner-handling workers were exposed to two types of toner particles during the study period. Convention toner (C toner) and emulsion aggregation toner (EA toner) were manufactured (C toner is produced by pulverizing raw materials) in the toner- and copy- machine-manufacturing enterprise wherein this study was conducted. This factory produced less EA toner than C toner from 2004 to 2006. However, the proportion of production was reversed in 2007; the production of EA toner steadily increased [23].
The mean particle diameters of the C and EA toners manufactured by this enterprise were 6.5 µm and 5.8 µm, respectively. Black C toner is composed of 70–80% polyester resin, 10–20% ferrite powder (iron oxide and manganese oxide), <10% amorphous silica, <10% carbon black, and <1% titanium dioxide. Black EA toner is composed of 60–70% styrene-acrylate resin, 10–20% ferrite powder (iron oxide and manganese oxide), <10% polyethylene, <10% amorphous silica, <10% carbon black, and <1% titanium dioxide [24].
Toner exposure assessment
We have previously reported our findings following detailed assessments of toner exposure levels [19, 21–24]. In particular, Matsuda et al. described the details of the actual state of toner exposure in workers who handled toner in the same enterprise where this study was conducted.
In previous studies [23, 25–28], participants were randomly selected from among workers who engaged in five categories of work. Their toner exposures were measured using a personal dust sampler every year between 2003 and 2011. In fiscal years 2003 and 2004, we useda Roken-type Filter Holder for Personal Total and Respirable Dust Sampler (Model PS-43; Shibata Scientific Technology Ltd., Soka, Saitama, Japan) to measure the particles. These samplers were equipped with glass-fiber filters (PTFE binding and T60A20 type ϕ 25 mm; Tokyo Dylec Corp., Tokyo, Japan). An AirChek 2000 Sample Pump (SKC Inc., Pennsylvania, USA) or Gilian GilAir-5 Air Sampling Pumps (Sensidyne, St. Petersburg, Florida, USA) was used, with a flow rate of 1.5 L/min. These instruments collected particles with a size classification that was characteristically set at 5 µm (50% cutoff-point). In the fiscal years 2005 to 2010, we used a Model NWPS-254 Filter Holder for Personal Dust Sampler (Shibata Scientific Technology). This sampler was equipped with glass-fiber filters (PTFE binding and T60A20 type ϕ 25 mm; Tokyo Dylec.), and AirChek 2000 Sample Pumps or Gilian GilAir-5 Air Sampling Pumps were used, with a flow rate of 2.5 L/min. These instruments collected particles with a size classification that was characteristically set at 4 µm (50% cutoff- point).
The levels of personal exposure to toner particles were different for each type of toner-handling work; being significantly higher in machine-recycling work and toner-manufacturing work than in three other types. The mean 8-h time-weighted average (TWA-8h) (SD) of each worker according to the five types of toner-handling work at the baseline survey was 0.989 (0.786) mg/m3 for toner and copy machine recycling (hereafter referred to as “recycling”), 0.203 (0.441) mg/m3 for toner manufacturing, 0.034 (0.030) mg/m3 for toner development, 0.019 (0.063) mg/m3 for toner and copy machine development, and 0.020 (0.060) mg/m3 for customer service. In all types of toner-handling work, the TWA-8h value was much lower than the 3.0 mg/m3 maximum level allowed for unspecified particles, defined as the threshold limit value–time-weighted average (TLV-TWA), recommended by the American Conference of Governmental Industrial Hygienists (ACGIH) [29].
Subgrouping according to toner exposure assessment
We divided the toner-handling group into two groups based on the toner exposure assessment, namely the high-concentration toner exposure group, who engaged in recycling and toner manufacturing, and the low-concentration toner exposure group, who engaged in the other three types of toner-handling work. Thus three groups in total were created, including the non-toner-handling group. We then evaluated the health effects of toner particle exposure among the three groups.
Statistical analysis
To compare between two independent groups, qualitative variables were analyzed using the chi-square test or Fisher's exact test, and quantitative variables were analyzed using the simple t-test and Welch's t-test. The mean values of each parameter over the 10-year period were compared between the two groups by performing a two-way repeated measures analysis of variance with each parameter as the dependent variable and toner handling status as the independent variable. We used a linear mixed model (LMM) [30] to analyze the longitudinal change. Dependent variables consisted of the respiratory function test parameters and the biomarker values, and the following four models were analyzed. In model 1, we treated toner-handling work, the survey year, and the interaction between toner-handling work and survey year as fixed effects and treated only the individual differences at baseline as the random effects (random intercept model). In model 2, we added both individual differences at baseline and responses to toner exposure as random effects (random intercept and slope models). Akaike’s Information Criterion (AIC) was used to determine the model with high fitness. In model 3, we adapted a model with lower AIC values, and adjusted the model using age at baseline, body mass index, smoking, asthma, allergic rhinitis, pneumonia, sinusitis, exposure to dust other than toner at work, and organic solvent-handling work as confounding factors. Baseline surveys [25, 31] and interim reports [26-28] have suggested that these variables may influence the dependent variables. Additionally, in model 4, with regard to toner-handling work, analysis was performed using the three groups, that is, the high-concentration toner exposure group, low-concentration toner exposure group, and non-toner-handling group. We also adapted a higher-fit model of the random intercept model and the random intercept and slope model for model 4.
If any significant effects of toner exposure on each parameter were observed, we also performed LMM analysis adjusted for the same confounding factors as models 3 and 4, respectively for each exposure concentration level group. In all analyses, the threshold for significance was at P<0.05. IBM SPSS Statistics for Windows 23(IBM Corp., Armonk, N.Y., USA) was used.
Definition of confounding factors
Individuals who declared that they were currently smoking were considered as smokers. Those who had never smoked and those who had quit smoking before the study began were considered as non-smokers. The presence or absence of asthma, allergic rhinitis, pneumonia, and sinusitis, which were included as confounding factors in the statistical analyses, were self-reported by the participants. The medical history of pneumonia was investigated with the intention of community-acquired pneumonia and did not include chronic granulomatous pneumonia.