TABLE OF CONTENTS
1. OCCUPATIONAL EXPOSURE TO DIISOCYANATES IN POLYURETHANE FOAM FACTORY WORKERS........ 1
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OCCUPATIONAL EXPOSURE TO DIISOCYANATES
IN POLYURETHANE FOAM FACTORY WORKERS
Swierczynska-Machura, Dominika; Brzeznicki, Slawomir; Nowakowska-Swirta, Ewa; Walusiak-Skorupa,
Jolanta; Wittczak, Tomasz; Dudek, Wojciech; Bonczarowska, Marzena; Wesolowski, Wiktor; Czerczak,
Slawomir; Palczynski, Cezary . International Journal of Occupational Medicine and Environmental Health
; Heidelberg Vol. 28, Iss. 6, (2015): 985-998.
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Objectives: The aim of the study was to evaluate health effects of occupational exposure to diisocyanates (DIC)
among polyurethane foam products factory workers. Material and Methods: Thirty workers had a physical
examination, skin prick tests with common allergens, allergen-specific immunoglobulin E (IgE) antibodies to
diisocyanates and pulmonary function tests. Concentrations of selected isocyanates in the workplace air samples
as well as concentration of their metabolites in the urine samples collected from the workers of the plant were
determined. Results: The most frequent work-related symptoms reported by the examined subjects were rhinitis
and skin symptoms. Sensitization to at least 1 common allergen was noted in 26.7% of the subjects. Spirometry
changes of bronchial obstruction of a mild degree was observed in 5 workers. The specific IgE antibodies to
toluene diisocyanate (TDI) and 4,4'-methylenebis(phenyl isocyanate) (MDI) were not detected in any of the
patients' serum. Cellular profiles of the collected induced sputum (ISP) did not reveal any abnormalities. Air
concentrations of TDI isomers ranged 0.2-58.9 µg/m^sup 3^ and in 7 cases they exceeded the Combined Exposure
Index (CEI) value for those compounds. Concentrations of TDI metabolites in post-shift urine samples were
significantly higher than in the case of pre-shift urine samples and in 6 cases they exceeded the British Biological
Monitoring Guidance Value (BMGV - 1 µmol amine/mol creatinine). We didn't find a correlation between urinary
concentrations of TDI, concentrations in the air and concentrations of toluenediamine (TDA) in the post shift urine
samples. Lack of such a correlation may be an effect of the respiratory protective equipment use. Conclusions:
Determination of specific IgE in serum is not sensitive enough to serve as a biomarker. Estimation of
concentrations of diisocyanate metabolites in urine samples and the presence of work-related allergic symptoms
seem to be an adequate method for occupational exposure monitoring of DIC, which may help to determine
workers at risk as well as to recognize hazardous workplaces.
Biomarkers, Diisocyanates, Asthma, Polyurethanes, Biological monitoring, Occupational exposure, Occupational
Diisocyanates (DIC), such as: toluene diisocyanate (TDI), 4,4'-methylenebis(phenyl isocyanate) (MDI) and
hexamethylene diisocyanate (HDI), are commonly used in manufacture of many products, including flexible and
rigid polyurethane foams, polyurethane rubbers and elastomers, adhesives, paints, coatings, insecticides, and rock
consolidation media [1,2].
Diisocyanates at high concentrations can have direct toxic effects on mucous membranes or can act at low
concentrations as sensitizing agents after binding to different proteins. Concentration of isocyanate as low as 1
ppm has been confirmed to induce significant functional changes in humans and inflammation processes in the
lung tissues .
Diisocyanates are still an important cause of occupational asthma in most industrialized countries, with a
prevalence rate of 2.9-13% [4,5]. Inhalation of diisocyanate vapours is also associated with numerous pulmonary
disorders, such as eosinophilic airway inflammation, airway hyperresponsiveness and hypersensitivity
pneumonitis [4,6,7]. Clinical diagnosis and the differential identification of isocyanates as the cause of workrelated disorders are often difficult because of complexity of exposures. Exposure monitoring may recognize risk
factors for a disease development and help prevent the onset or aggravation of the disease [8,9]. Efficient methods
are needed to improve both primary preventive measures and surveillance of the exposed workers.
The route of exposure largely depends on workplace conditions, especially concentration and temperature during
the manufacturing process. It is believed that allergy to diisocyanates occurs primarily via the respiratory system.
However, dermal exposure may also induce respiratory sensitization [10,11]. Depending on the requirements of the
technological process, diisocyanates may occur as liquids, vapors or aerosols. That is why measurement of
diisocyanate levels in the air is complicated and requires application of different sampling methods. Another
important thing related to the evaluation of occupational exposure to DIC via inhalation route is possibility of
respiratory protective equipment (RPE) use. Taking into account the mentioned above problems and possibility of
additional skin adsorption of DIC, biological monitoring seems to be a better way for occupational exposure
assessment or for identification of susceptible subjects among the exposed workers.
At present, biological monitoring of exposure to isocyanates is carried out based on the measurement of
concentrations of the corresponding amines (4,4'-methylenedianiline - MDA, 2,4-toluenediamine - 2,4-TDA, 2,6toluenediamine - 2,6-TDA, and 1,6-hexanediamine - HDA) [12-23] in urine or determination of the adducts of
diisocyanates with hemoglobin or albumin in blood [14,24-27].
Also the presence of specific immunoglobulin E (IgE) antibodies in serum is suggested as an indicator of exposure
to diisocyanates . For practical reasons (convenience and non-invasive sampling), determination of selected
diamines in urine samples seems to be more useful for risk assessment related to occupational exposure to
diisocyanates than measurements of DIC adducts in blood. Until now, biological monitoring hasn't been used in
Poland for the purpose of evaluation of occupational exposure to DIC. Estimation of occupational exposure to
selected isocyanates has been conducted solely on the basis of measurements of their concentration in the work
atmosphere and on the comparison of the obtained results with the values of maximum admissible concentrations
(MAC) valid in Poland.
The aim of the study was to assess, for the first time in Poland, occupational exposure to DIC using environmental
and biological monitoring, and to evaluate health effects associated with exposure among polyurethane foam
MATERIAL AND METHODS
The study population consisted of 30 workers (male) who have been working in a plant manufacturing TDI-based
flexible polyurethane (PUR) foam in continuous foam blocks. In the manufacturing process a technical mixture of
TDI isomers (2,4-TDI and 2,6-TDI), and (in some cases) MDI is used. 4,4'-Methylenebis(phenyl isocyanate), however,
was used during only 1 day for production of 4 blocks of PUR. The production process consists of the preparation
of components for manufacturing, programming the proportion of each component and the foaming process,
which takes place in a closed ventilated tunnel on a moving conveyor lined with craft paper. At the end of the
tunnel, expanded block is periodically cut into 60-m-long pieces. Foaming process requires periodic presence of
some workers in the tunnel where the expansion process occurs (tunnel workers performing folding paper task or
maintenance workers). Because of high concentrations of diisocyanates, during their presence in the tunnel, the
workers should wear respiratory protective equipment. Workers employed in production of polyurethane foam
blocks are not assigned to perform specific tasks during their working week. Depending on the needs, each of
them can perform various operations related to the production process.
Each subject had a medical history collected to gain information on the possible respiratory symptoms,
occupational exposure, history of atopy, the smoking status and exposure to domestic animal allergens.
A clinical examination was also performed in all the subjects.
Skin prick tests
Skin prick tests (SPT) were performed on the volar part of the forearm with common allergens, which included tree
and grass pollens, Dermatophagoides pteronyssinus, Dermatophagoides farinae, Lepidoglyphus destructor,
moulds and feathers (Allergopharma, Germany). The SPTs were performed according to the standardized
techniques . All SPTs included positive (10 mg/ml histamine hydrochloride, Stallergenes, France) and negative
controls (phenylated glycerol-saline, Stallergenes, France). The results were assessed after 15 min. Positive
reaction was defined as a wheal diameter of at least 3 mm in the absence of reaction to the diluent and in the
presence of a positive reaction to histamine.
The level of allergen-specific IgE antibodies (asIgE) in serum
The allergen-specific IgE antibodies to TDI (k75) and to MDI (k76) (Phadia, Uppsala, Sweden) were evaluated in
each patient's serum.
Pulmonary function tests
Resting spirometry using MasterScope PC spirometer equipment (Jaeger, USA) was performed in all the subjects
(reference values for Caucasian population according to the European Respiratory Society - ERS). Metacholine
challenge was performed in selected persons according to Cockroft .
Induced sputum analysis
Cellular profiles of induced sputum (ISP) were analyzed in 18 workers. The whole process of collecting ISP has
been described elsewhere .
Air measurements of diisocyanates
Air samples (individual samples: N = 20) for determination of diisocyanates (2,4-TDI, 2,6-TDI, MDI) were collected in
the breathing zone of the workers using Gilian GilAir-3 personal samplers. Measurement period was always close
to the nominal time or no less than 75% of the time of the 8-h work shift, according to the adopted uniform criteria
for monitoring work environment .
Determinations of selected isocyanates were performed using high performance liquid chromatography (HPLC)
with spectrofluorometric detection (FLD) and/or spectrophotometric detection (PAD) according to the fully
validated, accredited method (the Polish Centre for Accreditation, Certificate No. 215) used in the Department of
Chemical Hazards of the Nofer Institute of Occupational Medicine (NIOM) in Lodz, Poland.
Briefly, a known volume of air (-2001,11/min) was passed through glass fiber filters coated with l-(2Pyridyl)piperazine (1-2-PP). Then the filters were extracted with mixture (2 ml) of acetonitrile and dimethyl
sulfoxide (9:1 v:v) on a rotary shaker (1 h). Extracts were transferred to the autosampler vial and analysed using
the Waters Alliance 2695 HPLC system equipped with Waters 2475 FLD and Waters 2996 PAD detectors.
Calibration standards were prepared on 1,2-PP coated filters spiked with subsequent dilutions of 2,4-TDI, 2,6-TDI
and MDI derivatives mixture. After evaporation of the solvent, the filters were treated the same way as the sample
filters. Analytical conditions are presented in Table 1.
Determination of DIC metabolites in urine samples
Urine samples for determination of diisocyanates metabolites (2,4-TDA, 2,6-TDA and MDA) were collected before
and immediately after the shift into polypropylene cups pretreated with 1 g of citric acid. The samples were stored
at -20°C until an instrumental analysis. Determinations of concentrations of 2,4-TDI, 2,6-TDI and MDI metabolites
were performed using the capillary gas chromatography with mass spectrometry (GC/ MS) techniques, according
to the method described by Williams for HDI metabolite determination  and adapted by Creely  and Budnik
 for determination of TDI and MDI metabolites in urine. This method is also proposed by The Health and Safety
Laboratory (Agency of the Health and Safety Executive) for determination of DIC metabolites in urine .
Briefly, 2 ml of urine sample was hydrolysed in sealed tubes with 0.2 ml of concentrated sulfuric acid (90 min,
100°C). The cooled samples were then alkalyzed with 2 ml of 10 M sodium hydroxide and extracted with 4 ml of
diethylether. Three milliliters of the resulted extract were transferred to clean tubes and diethylether was
evaporated (N2) to dryness. To the residue, heptafluorobutyric anhydride (HFBA) (50 pi in 0.5 ml toluene) was
added as a derivatizating agent and the reaction of derivatization was carried out in closed tubes for 1 h at 55°C.
After evaporation of the excess of HFBA, residue was dissolved in 0.1 ml of toluene and transferred to the
autosampler vial. Instrumental analyses were performed using a HP 6890N gas chromatograph equipped with HP
5973 mass detector. Calibration standards were prepared by spiking urine of the unexposcd person with
subsequent dilution of 2,4-TDA, 2,6-TDA (5 data points at range 0.125-2.5 ug/1) and MDA (5 data points at range
0.2-4 ug/1) mixture. So prepared solutions were treated the same way as the urine samples. All the analytical
conditions are presented in Table 2.
Determination of creatinine
One milliliter of each urine sample was transferred to 2 ml Eppendorf tubes, frozen and submitted for analysis to
the SYNEVO Polska Medical Diagnostics Laboratory.
Validation of DIC determination method was performed according to the rules described in the European Standard
EN 482:2012 . Validation steps covered determinations of recovery of DIC from sampling media, linearity,
precision, quantitation and detection limits and sample storage. Extended uncertainty of the method calculated for
the pre-analytical and analytical stages was 27%.
The method for determination of DIC metabolites in urine has been partially validated (linearity, precision,
detection limit), because we have assumed, that the former use of this method in the other already published
works reporting relevant validation data, in combination with our use of similar analytical equipment and reagents
causes that the method may be reliably applied in our assays. Limit of detection (LOD) for each metabolite was
calculated according to the equation:
(ProQuest: ... denotes formulae omitted.)(1)
SDr - standard deviation of the response of the lowest standard from calibration range diluted 10 times,
b - the slope of the calibration curve.
Extended uncertainty covering uncertainty associated with precisions of determinations, linearity, weighing
accuracy, standards purity, sample storage and glassware characteristic calculated for this method was 21%.
During analysis of DIC and their metabolites, after analysis of 20 study samples, 1 reagent sample and one control
sample were determined.
The study protocol was approved by the local Biomedical Ethics Committee.
The data were presented as mean ±standard deviations (M±SD). Statistical analyses were performed using the
non-parametric Mann-Whitney Rank Sum test. Correlations were assessed by the Pearson's rank method. A level
of p <0.05 was considered as significant.
Thirty workers aged 23 to 58 were evaluated. The characteristics of the study group is shown in Table 3.
Employment duration on the current work post was 9.63 ±8.7 years. The prevalence of allergic symptoms is
presented in Table 4. The most frequent symptoms reported by the examined subjects were: rhinitis (N = 7, 23.3%)
and skin symptoms (N = 5,16.7%).
Skin prick tests were performed in all the plant workers. Sensitization to at least 1 common allergen was observed
in 26.7% of the subjects (Table 5). Grass pollens (N = 5) and Dermatophagoides pteronyssinus (N = 4) were the
most frequent allergens that caused positive results.
Resting spirometry was carried out in all the 30 patients. Baseline values were normal in 25 persons (83.3%).
Spirometry changes of bronchial obstruction of a mild degree were observed in 5 workers. One of them has been
earlier diagnosed as asthmatic. Metacholine challenge tests were performed in those 5 subjects. This test
revealed non-specific bronchial hyperactivity (BHR) (PC20 = 4 mg/ml) in 1 patient, who has never been treated for
asthma. The specific IgE antibodies to TDI and MDI were not detected in any of the patients' serum.
Induced sputum samples from 18 workers were collected. Cellular profiles of ISP are shown in Figure 1. In each
sample, the dust of unknown origin in macrophages has been observed.
Twenty workers agreed to participate in the environmental and biological monitoring study. 4,4'Methylenebis(phenyl isocyanate) was detected only in the samples collected in the manufacturing department, in
which the MDI was used for the foam production. However, concentrations of the compound were below the lower
limit of the working range (0.0006 mg/m3) of the analytical method. The results of 2,4-TDI and 2,6-TDI
determinations in the workplace air during production of polyurethane foam blocks are presented in Table 6. The
highest concentrations of TDI (sum of 2,4-TDI and 2,6-TDI isomers) were found in the work stations of the
maintenance workers (9.9-41.5 pg/m3) and of paper folders (0.3-58.7 pg/m3). Lower concentrations of TDI were
found in the samples collected from the work stations of foaming head operators (0.6-11.3 pg/m3) and cutting
machine operators (0.2-6.5 pg/m3).
The results of TDI metabolites in the urine samples collected from the workers employed in the production of
polyurethane foam blocks are shown in Figure 2 and in Table 6. Data presented in Figure 2 were expressed as pg/1
because concentration of creatinine in 8 samples of the pre-shift urine exceeded the accepted  range of 0.3-3
A statistically significant increase in the concentrations of these metabolites in the samples collected at the end
of the work shift, compared with the samples collected before the work shift was observed (Figure 3). Among the
post-shift urine samples, only in 2 cases concentration of creatinine exceeded range of 0.3-3 pg/1, therefore,
urinary concentrations of TDI metabolites were adjusted to creatinine. The highest concentrations of TDI
metabolites were found in the urine samples from the maintenance workers (geometric mean (GM) = 2.6 pmol/mol
creatinine) and cutting machine operators (GM = 0.9 pmol/mol creatinine).
In 39% (7 of 18) of the post-shift urine samples, the TDI metabolite concentrations exceeded the Biological
Monitoring Guidance Value (BMGV) of 1 pmol TDA/mol creatinine . Taking into account all the obtained results,
no correlation was found between the concentrations of diiisocyanate (ITDI isomers) in the air and the
concentration of metabolites (2TDA isomers) in the post shift urine samples (r = 0.051). However, a positive
correlation (r = 0.84) was found for geometric means of TDI in the air and TDA in urine calculated for each of the
Diisocyanates are one of the leading occupational causes of respiratory disorders, predominantly asthma.
Because permanent impairment of lung function has been noted in the long-term follow up studies of
diisocyanate-induced asthma (DA), the development of biomarkers to identify susceptible subjects among the
exposed workers is essential. Only 5 to 10% of the exposed workers develop DA because a genetic predisposition
plays a substantial role in the development of the disease. An important risk factor for the DA is the presence of
certain polymorphisms associated with the xenobiotics metabolism, especially polymorphisms of genes encoding
glutathione-S-transferase. It plays an important role in the efficiency of the elimination of diisocyanates form the
body and therefore, might predict susceptibility for the induc ...
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