Metalworking fluids (MWF) are used to lubricate and cool tools and the workpiece, as well as flush away metal chips during machining, cutting, grinding, and drilling of metals in many manufacturing processes, from small parts in the watch-making industry to large parts in the automotive or steel industries. MWF are classified into two main families [1]: (1) Straight MWF that is mineral oil containing no water; and (2) Aqueous MWF that regroup so-called soluble oils and semisynthetic fluids according to the amount of mineral oils emulsified in water, as well as synthetic fluids that contain no mineral oil. Depending on their type and use, MWF may contain lubricity, antimisting or antiwear additives, corrosion inhibitors and biocides, as well as perfumes or coloring agents.

Other substances potentially present in the aerosols from used MWF are the result of thermal degradation or contamination of the machined metal parts. Thus, MWF may contain: (1) polycyclic aromatic hydrocarbons (PAHs) due to lack of an initial refining stage or due to thermal degradation; (2) nitrosamines, present in aqueous MWF as by-products of the reaction between secondary amines and nitrite; or (3) microorganisms like bacteria or mycobacteria that may be growing in tanks containing aqueous MWF.

The physical process of metalworking generates a complex MWF aerosol consisting of droplets (the oil mist), which may contain solid particles (eg, metals), and a vapor phase (air or organic vapors). This vapor phase is the result of the evaporation of volatile or semivolatile constituents from the MWF in contact with the hot cutting zone. These aerosols can reach the workers’ breathing zone and may remain in suspension for several hours [1]. The size distribution of the MWF aerosols is highly variable (median aerodynamic diameters ranging from 1.8-17 µm) and may contain ultrafine particles (aerodynamic diameter <0.1µm) [2]. MWF aerosols may be inhaled or enter the body through skin contact. Exposure from soiled clothing and ingestion (hand to mouth contamination) are also possible. Consequently, assessing occupational exposure to MWF aerosols has many challenges.

Historically, exposure to poorly refined straight oil-mists has been related to cancer of the skin and the scrotum [3]. More recently, there is growing evidence of a relationship between exposure to straight MWF and bladder cancer [4,5]. As early as the 1990s, exposure to oil-mist was related to acute bronchial hyperresponsiveness, occupational asthma, hypersensitivity pneumonitis, ventilatory impairments and respiratory symptoms [6-9]. Recently, a causal model was applied [10] to explore the quantitative relationship between exposure to MWF aerosol and chronic obstructive pulmonary disease (COPD).

Inflammation is hypothesized to be an important process that explains some of the observed health outcomes. Indeed, in vivo chronic exposure to rather high concentrations of different semisynthetic MWF have resulted in inflammation in rat and mice lungs [11]. In addition, signs of oxidative stress have also been reported on the skin of vitamin E deficient mice exposed to MWF [12]. Based on this, Figure 1 (adapted from Ayres et al [13] who presented this mechanism for environmental particulate exposure) summarizes the presumed physiopathological mechanism. Given the multiplicity of health outcomes that have been related to oil-mist exposure, we assumed that the main, common mechanism of these effects was oxidative stress. Briefly, the exposure to the MWF aerosols generates free radicals in the lungs of both exogenous and endogenous origin and thereby causes oxidative stress. This oxidative stress induces inflammation, which ultimately increases the oxidative stress via a feedback mechanism. Thus, this chronic inflammation eventually leads to chronic adverse health effects.

Table 1 summarizes the different outcomes measured during the field study.

Exposure determinants are factors within the workplace that contribute to increasing or reducing exposure concentrations [14]. A questionnaire has been developed based on the literature [1,15] and will be used by the occupational hygienists during the field study.

EBC is an emerging technique that is simple, noninvasive and allows scientists to study processes in the lungs [18]. The metal concentrations in EBC (eg, Fe, Mn, Cr, Ni, Cu, Zn) will be determined with an inductively coupled plasma mass spectrometer (ICP-MS) using the analytical Totalquant technique with external calibration [19,20].

We will measure the following urinary exposure biomarkers: metals (27 metals), a nitrosamine (N-nitrosodiethanolamine [NDELA]) and two PAH metabolites (1-hydroxypyrene [1-OHP], a metabolite of pyrene, and 3-hydroxybenzo(a)pyrene [3-OHBaP], a metabolite of benzo(a)pyrene). Metals will be analyzed using ICP-MS [21], whereas the PAH metabolites will be analyzed using high-performance liquid chromatography with fluorescence detection (HPLC-Fluo) [22]. Finally, a more sensitive method for analyzing urinary NDELA will be developed based on high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS).

The statistical analysis will follow the presumed causal model (Figure 2) and will be performed using the Stata (StataCorp LP, College station, Texas) statistical software. The first analysis will model the exposure measurements as a function of the determinants using linear models, after suitable transformations (logarithmic) if necessary. The exposure biomarkers in EBC and urine will be modelled as a function of the exposure measurements as well as possibly the exposure determinants. These analyses will focus on the within-shift evolution of these markers and will be based on mixed models. Measurements below the limits of detection will be included using specific models (random effect Tobit or Bayesian models) [34]. The biomarkers of effect and the FeNO will be analyzed as a function of the exposure measurements and determinants using similar statistical models. Again, the focus will be on the within-shift evolution but also on the evolution over the three days. The effects from the circadian cycle will be controlled by the simultaneous modelling of the nonexposed subjects. The prevalence of symptoms and the frequency of micronuclei will be analyzed using logistic regression as a function of the exposed or nonexposed status and the chronic occupational exposure that will be estimated from the job history and the exposure determinants. Note that in the analysis of the frequency of micronuclei, the number of collected cells per subject will be included as an offset and that this analysis will account for a possible overdispersion using a negative binomial regression. Possible confounders such as smoking, age, sex and diet will be accounted for in the different statistical analyses.

The study size was determined based on the longitudinal evolution of FeNO and the comparison of the frequency of micronuclei in two exposure groups. According to Bohadana et al [35], a 10% increase of FeNO between 2 measurements corresponds to an 80% power at a 5% significance level with a sample of 30 subjects.

With respect to micronuclei, assuming a 0.74% [36] baseline prevalence among controls, we have an 80% power at a 5% significance level with two samples of 30 subjects. This allows us to detect a 2.2 rate ratio between an exposed and a nonexposed group assuming a 1.5 between-subject geometric SD.

The rationale behind choosing the number of controls in a 1:2 ratio to exposed workers is to get equal sized exposure groups between the aqueous exposed works, the straight oil exposed workers, and the controls.

The present study is carried out by a consortium of three organizations: (1) Team 1, the research team EA 4483 from the IMPact de l’Environnement Chimique sur la Santé humaine (IMPECS; Impact of the chemical environment on human health) of the University of Lille in France; (2) Team 2, Unisanté, the department of occupational and environmental health from the University of Lausanne in Switzerland; and finally (3) Team 3, the Institut National de Recherche et de Sécurité (INRS; French National Institute for Research and Safety) in France.

The study is coordinated by the INRS. A Consortium Agreement specifying the legal, financial and scientific framework of the cooperation regarding the present study was signed by the three organizations on March 22, 2017.

Two parallel groups of scientists in Switzerland and France with common operational procedures carry out the on-site data collection. To minimize laboratory bias, most of the samples are dispatched to and analyzed by one laboratory only. All organic biomarkers in urine and EBC, as well as the micronuclei frequency in buccal cells, will be analyzed by team 3. The metals in EBC will be analyzed [14] by team 1. Team 2 will characterize MWF aerosol for its oxidative potential, organic carbon, nitrate, nitrite and aldehyde content as well as the formate concentration in EBC. Both Team 2 and 3 will determine the particulate exposure.

All electronic documents related to the study are deposited in a structured, encrypted extranet located on a server of team 3. The access to this extranet is strictly restricted to the study team members by an individual password. Measurement data from each lab will be deposited by the laboratories generating them. All deposited data will be anonymous, and the rules for generating the identification codes are defined by an operational procedure. The data on the extranet will be saved daily and backups will be kept in a separate building from the server. The deposited data will be organized and prepared for data analysis in the three months after collection by the same data manager, who will provide regular feedback on the data collection.

This study was approved by the ethical committee of the canton Vaud, Switzerland (Commission cantonale d’éthique de la recherche sur l’être humain CER-VD) on June 13, 2017, project number 2017-00630, and by France (Comité de Protection des Personnes Sud-Est) on May 17, 2017, project number 17-02-EE-VD/OXIGENOCOM.

Several scientific, peer-reviewed publications are planned. The first ones will correspond to the preparatory phases (eg, a scientific article on the determination of biomarkers in EBC, a paper on automatic versus human-based counting of micronuclei in buccal cells). The other planned papers will respectively correspond to the objectives described in the beginning of this paper.

The ultimate objective is to develop relevant exposure reduction strategies. The results will be published in a nonscientific format and will be accessible by environmental health and safety professionals.

Depending on the corresponding legal authorizations, parts or all of the data will be deposited on a public data repository after the final publications.

This study is unique, as it comprises human biological samples, questionnaires, and MWF exposure measurement. The aim is to develop preventative strategies based on exposure determinants related to health outcomes. To achieve this goal, this integrative multidisciplinary approach quantifies the relationships between exposure determinants, exposure measurements, biomarkers of exposure, biomarkers of effect, and early effect outcomes.