Abstract
Titanium nitride (TiN) is a ceramic material with physical properties such as extreme hardness, high decomposition temperature, defect structure, and gold-yellow color. TiN is generally considered non-toxic and safe; however, hazards have not been identified, especially in workers after inhalation exposure. Here, we conducted a four-week inhalation toxicity study of TiN using a nose-only inhalation exposure system in Sprague–Dawley rats. Rats were exposed to TiN for 4 weeks (6 h a day, 5 days per week) at target concentrations of 45, 90, and 180 mg/m3. Clinical signs, mean body weight changes, hematology, blood biochemistry, necropsy, organ weight, bronchoalveolar lavage fluid analysis, and histopathological findings were observed. Analytical concentrations of the low, middle, and high-concentration groups were 45.55 ± 3.18 mg/m3, 90.69 ± 7.30 mg/m3, and 183.87 ± 15.21 mg/m3, respectively. The mass median aerodynamic diameter (MMAD) for the low, middle, and high-concentration groups were 1.44 ± 0.07 μm, 1.47 ± 0.18 μm, and 1.68 ± 0.16 μm, and the geometric standard deviation (GSD) was 2.24 ± 0.03, 2.31 ± 0.16, and 2.43 ± 0.11, respectively. No systemic adverse effects were observed after inhalation exposure to TiN; however, histopathological findings (increased phagocytic macrophages and alveolar/bronchiolar epithelial hyperplasia) and Bronchoalveolar Lavage Fluid (BALF) analysis (elevated lactate dehydrogenase and gamma-glutamyltransferase values) showed adverse effects on the lungs in the middle and high-concentration groups. Based on these results, the no observed adverse effect concentration (NOAEC) is suggested to be 45 mg/m3.
Keywords: Titanium nitride, Inhalation toxicity study, No-observed-adverse-concentration level, Lowest observed adverse effect concentration level, Occupational exposure
Introduction
Titanium nitride (TiN, CAS No. 25583-20-4) is a ceramic material with physical properties such as extreme hardness, high decomposition temperature, defect structure, and gold-yellow color [1]. It has been used as a coating on titanium and its alloys, stainless steel, carbide, and aluminum components, including machining tools, costume jewelry, automotive trim, and medical implants to improve surface properties, lifetime, and decoration [2–4]. Thin TiN films have been used in microelectronic and bioelectronic applications [2]. TiN is considered a non-toxic and good candidate for biomedical purposes because it has been extensively studied for biomedical applications and accepted by the U.S. Food and Drug Administration (FDA) for medical applications [5, 6]. In addition, the European Food Safety Authority (EFSA) concluded that TiN nanoparticles have no safety concerns for the consumer if the nanoparticulate substance is used up to 20 mg/kg in polyethylene terephthalate plastics intended for contact with all types of foodstuffs under conditions of any duration of time and at temperatures up to and including hot-fill [7]. Although generally considered safe for these reasons, hazards, especially for workers, have not been identified. Many researchers have mentioned that workers may be exposed to inhalation routes in the form of dust and mist during work, including the deposition process, cleaning, and application of cutting fluids within a machining process [8, 9]. Recently, a 2-month feeding study of nano-TiN showed genotoxic activities in mice [10], and adverse developmental effects on the heart, liver, and nervous system were observed after exposure to nano-TiN in a zebrafish model [11]. These studies suggest the possibility of adverse health effects due to TiN exposure and the need for safety evaluation. However, information on the toxicity of TiN and nano-TiN remains scarce. For this reason, we conducted a 4-week repeated inhalation toxicity study to obtain toxicity information on TiN through inhalation exposure for safety evaluation as a basic target for workers.
Materials and methods
Test substance
TiN powder was purchased from US Research Nanomaterials Inc. (Houston, TX, USA). The purities were 77.87% (Ti) and 21.88% (N), and the size was 2 μm (aerodynamic particle sizer). As the repeated inhalation toxicity study considered workers in the workplace, the test was conducted on a size suitable for the Organization for Economic Co-Operation and Development (OECD) guideline Test No. 412, not the nano size [12]. The test substance was not subjected to any special procedure (e.g., milling) for inhalation exposure.
Generation, analysis, and inhalation exposure systems
The test substance was generated using a dust generator (solid aerosol generator, Series SAG 410, TOPAS, GmbH, Dresden, Germany). TiN powder was placed into the dust generator, clean dry air was injected to generate the test substance, and the substance concentration was adjusted by mixing it again with clean dry air. To analyze the concentration in the inhalation exposure systems, a personal air sampling pump (Air Chek XR, SKC, Somerset, NJ, USA) and a 25-mm glass fiber filter (GF-C, HG00025C, Hyundai Micro, Seoul, Korea) were used. Substance concentrations were measured at least three times during the exposure day for each concentration in the vicinity of the animals’ breathing zone at a flow rate of 1 L/min. The mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) of the test substance were measured using a cascade impactor (Model-135 Mini MOUDI Impactor; MSP, MN, USA) at a flow rate of 1 L/min. Animals were exposed to nose-only inhalation exposure systems (HCT, Icheon, Korea) at 1 L/min flow/animal and acclimated to the restraining tubes before exposure to the test substance. The environment of the inhalation exposure systems, including temperature, relative humidity, and oxygen concentration, was monitored to meet OECD Guideline Test No. 412 [12].
Test animals and animal husbandry
Specific pathogen-free (SPF) Sprague–Dawley (SD) rats were purchased from Japan SLC Inc. (Tokyo, Japan). Animals were obtained at 7-weeks-old, acclimatized for 7 days, and exposed at 8-weeks-old. Animals were housed in poly-sulfone solid bottom cages with stainless steel grid tops (up to three animals of the same sex), and the animal room was maintained at 22 ± 3 °C, 50 ± 20% relative humidity, and a 12-h light/dark cycle. The rats were fed a radiation-sterilized laboratory diet (18% protein Rodent Diet 2918 C; Envigo RMS Inc., IN, USA) and filtered water ad libitum. This study was approved by the Institutional Animal Care and Use Committee of the Chemical Research Bureau, Occupational Safety, and Health Research Institute prior to obtaining the rats, and all tests were conducted in accordance with established animal care protocols.
Test groups and exposure concentration
The test groups consisted of three test substance concentration groups and one concurrent control group. Fifteen animals were randomly assigned to each group (10 male and 5 female). The test groups were exposed to 0 mg/m3 (control, filtered dry air), 45 mg/m3 (low), 90 mg/m3 (middle), or 180 mg/m3 (high) TiN for 6 h a day, 5 days per week, for 4 weeks. The low-concentration group was selected for stable exposure and analysis for 6 h (chamber concentration samples should deviate from the mean chamber concentration by no more than ± 20%) and technical limitations such as meeting the MMAD range (≤ 2 μm with a geometric standard deviation of 1–3) of OECD guidelines [12, 13], and other concentration groups were selected at doubling intervals based on a previous report of a structural analog (TiO2) with reference to no clinical signs, gross observation (white foci in the lung) in a two-year inhalation study, and significant changes in BALF analysis results and alveolar macrophages following exposure to 250 mg/m3 in a four-week inhalation study [14, 15].
Clinical signs, mean body weights, and food consumptions
All animals were observed twice a day (before and after exposure) and once a day on non-exposure days for clinical signs including mortality, general appearance, and change of behavior. Changes in individual mean body weights were measured using an electronic balance (QUINTIX3102, Sartorius Co., Göttingen, Lower Saxony, Germany) twice per week for the first 2 weeks and once per week for 2 weeks. Food consumption was calculated individually by dividing the number of housed animals after measuring the food consumption per poly-sulfone cage weekly using the same electronic balance that was used for mean body weight measurement.
Hematology and blood biochemistry
At the end of the 4-week exposure period, all animals were fasted overnight and sacrificed via exsanguination from the abdominal aorta and vein after anesthesia with isoflurane (Il-sung Pharm, Seoul, Korea). Blood samples were collected from the abdominal aorta while maintaining anesthesia and prepared in test tubes containing ethylenediaminetetraacetic acid (EDTA) or serum-separating tubes for analysis. Hematological parameters were analyzed using a blood cell analyzer (ADIVA 2120i, Siemens Diagnostics, Tarrytown, NY, USA) or coagulation analyzer (ACL Elite Systems, Instrumentation Laboratory, Massachusetts, USA) with the following parameters: white blood cell (WBC) count, red blood cell (RBC) count, hemoglobin (HGB) concentration, hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet (PLT) count, reticulocyte (RET) count, differential WBC count (neutrophils, lymphocytes, monocytes, eosinophils, and basophils), activated partial thromboplastin time (APTT), and prothrombin time (PT). Blood biochemical parameters were measured using a blood chemistry analyzer (TBA-120FR, Toshiba Co., Tokyo, Japan) using the following parameters: sodium (Na), potassium (K), chloride (Cl), total protein (TP), albumin (ALB), creatinine (CREA), blood urea nitrogen (BUN), glucose (GLU), calcium (Ca), inorganic phosphorus (IP), total bilirubin (TBIL), total cholesterol (TCHO), triglyceride (TG), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and albumin/globulin (A/G) ratio.
Necropsy, organ weight, and histopathology
All animals were subjected to gross necropsy, which involved examination of gross pathological changes in the body surface, subcutis, and internal organs in the cranial, thoracic, and abdominal cavities.
The adrenals, brain, heart, kidneys, liver, lungs, spleen, testes, thymus, epididymides, ovaries, and uterus were removed and weighed using an electronic balance (QUINTIX313; Sartorius Co., Göttingen, Lower Saxony, Germany) after trimming. Bilateral organs were measured together because there were no abnormal findings; however, the lungs were separated into the left and right lobes, and only the left lobe was measured.
For histopathological examination, the left lung was preserved in 10% neutral buffered formalin solution, paraffin-embedded, sectioned using microtome, and stained with hematoxylin and eosin. Slides were microscopically examined at 100× or 200× magnification.
Bronchoalveolar lavage fluid (BALF) analysis
At necropsy, the right lung was injected 4 °C with phosphate buffered saline (PBS) using a disposable syringe and an intubation tube (Polyethylene Tubing intramedicTM PE-90, ClayAdams, NJ, USA), and lavaged fluids (3 times, 4 mL/time) were collected. Collected lavaged fluids were centrifuged at 4 °C, 400×g, for 10 min (Labmaster ABC CB200R, HANLAB, Anyang, Korea). The supernatants were used for analysis of lactate dehydrogenase (LDH), gamma-glutamyltransferase (GGT), and total protein using a blood chemistry analyzer (TB-120FR, Toshiba Co., Tokyo, Japan). The pellets were smeared and fixed evenly on the slide using a cell centrifuge at 1000 rpm for 10 min (Cellspin, Hanil, Incheon, Korea), and stained with a Differential-Quick stain solution (Polyscience, Inc., Warrington, PA, USA). Over 300 cells were counted for analysis of differentials for macrophages, neutrophils, lymphocytes, and eosinophils. Total cell counts were measured using a blood cell analyzer (ADIVA 2120i, Siemens Diagnostics, Tarrytown, NY, USA).
Statistical analysis
Data are expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used to analyze the means of the groups. Dunnett’s test or Dunn’s rank sum test was used as a post hoc test. SPSS 22.0 K statistical software IBM Corp. IBM SPSS Statistics for Windows (Armonk, NY, USA) was used for all statistical analyses. Statistical significance was set at p < 0.05.
Results
Test substance concentration in inhalation exposure systems
The analytical mean concentrations of substance in the inhalation exposure systems during the exposure period for the low, middle, and high-concentration groups were 45.55 ± 3.18 mg/m3, 90.69 ± 7.30 mg/m3, and 183.87 ± 15.21 mg/m3, respectively (Fig. 1). The MMAD for the low, middle, and high-concentration groups were 1.44 ± 0.07 μm, 1.47 ± 0.18 μm, and 1.68 ± 0.16 μm, respectively, and the GSD was 2.24 ± 0.03, 2.31 ± 0.16, and 2.43 ± 0.11, respectively. The delivered doses were calculated using the formula presented by Alexander et al. [15]. Respiratory minute volume (RMV) was set to 0.2 L/min, duration of exposure was set to 360 min (6 h a day), and body weight was set to 0.3 kg and 0.2 kg for male and female, respectively. Proportion by weight of particles that are inhalable or the inhalable fraction (IF) was set to 1 because the MMAD was less than 2 μm and assumed to be 100% inhalable.
Fig. 1.
Concentration of titanium nitride in the inhalation exposure systems exposed to 45 mg/m3, 90 mg/m3, and 180 mg/m3 during the exposure period
The results of the delivered doses for male rats were 10.8 mg/kg/day (low concentration), 21.6 mg/kg/day (middle concentration), 43.2 mg/kg/day (high concentration), and for female rats were 16.2 mg/kg/day (low concentration), 32.4 mg/kg/day (middle concentration), 64.8 mg/kg/day (high concentration).
Clinical signs, mean body weight, and food consumption changes
No deaths or unusual clinical signs due to the test substance were observed during the study period (data not shown). No statistically significant differences in the mean body weight were observed in each sex groups, except for a statistically significant decrease (p < 0.01) in the male high-concentration group on the first day of exposure (Figs. 2, 3). In the food consumption results, no statistically significant differences were observed in the male and female groups, except for a statistically significant increase (p < 0.05) in the male high-concentration group in the first week (data not shown).
Fig. 2.
Changes in mean body weight of male rats after exposure to titanium nitride. Error bars indicate standard deviation. (n = 10). **Significantly different when compared to the control group, p < 0.01
Fig. 3.
Changes in mean body weight of female rats after exposure to titanium nitride. Error bars indicate standard deviation (n = 5)
Hematology and blood biochemistry
The HGB count was significantly diminished in the male low (p < 0.05) and high (p < 0.01)-concentration groups, and the MCHC count was significantly diminished in the male high (p < 0.01)-concentration group compared to the male control group, while PT was significantly increased in the male high (p < 0.01)-concentration group (Table 1). WBC and absolute neutrophil (NEUA) levels were significantly elevated in the female high-concentration group (p < 0.01), while MCV was significantly diminished in the female middle-concentration group (p < 0.05). APTT and PT were significantly increased in the female low- (p < 0.01, p < 0.05, respectively), middle- (p < 0.05), and high-concentration groups (p < 0.01) (Table 2).
Table 1.
Hematology data of male rats exposed to titanium nitride for 4 weeks
| Titanium nitride (mg/m3) | ||||
|---|---|---|---|---|
| Control | 45 | 90 | 180 | |
| Male (No. of Animals) | 5 | 5 | 5 | 5 |
| WBC (×103/µL) | 5.01 ± 0.79 | 4.75 ± 0.87 | 5.12 ± 0.66 | 5.63 ± 1.01 |
| RBC (×106/µL) | 7.92 ± 0.13 | 7.80 ± 0.31 | 7.81 ± 0.33 | 7.95 ± 0.41 |
| HGB (g/dL) | 15.54 ± 0.18 | 14.98 ± 0.47* | 15.02 ± 0.38 | 14.68 ± 0.48** |
| HCT (%) | 41.48 ± 0.88 | 40.74 ± 1.25 | 40.64 ± 1.31 | 41.04 ± 1.37 |
| MCV (fL) | 52.34 ± 1.07 | 52.22 ± 0.54 | 52.08 ± 0.77 | 51.60 ± 1.48 |
| MCH (pg) | 19.60 ± 0.44 | 19.22 ± 0.44 | 19.22 ± 0.36 | 18.46 ± 1.04 |
| MCHC (g/dL) | 37.48 ± 0.61 | 36.80 ± 0.76 | 36.94 ± 0.36 | 35.78 ± 0.99** |
| PLT (×103/µL) | 888.60 ± 93.58 | 926.20 ± 83.36 | 942.25 ± 70.31a | 972.40 ± 98.49 |
| NEU% (%) | 19.64 ± 2.37 | 24.18 ± 7.59 | 23.58 ± 1.09 | 23.92 ± 5.11 |
| LYM% (%) | 76.40 ± 2.26 | 74.60 ± 5.51a | 72.70 ± 0.75 | 74.43 ± 1.17a |
| MON% (%) | 2.04 ± 0.43 | 2.16 ± 0.43 | 1.56 ± 0.44 | 1.74 ± 0.22 |
| EOS% (%) | 1.32 ± 0.29 | 1.32 ± 0.38 | 1.48 ± 0.31 | 1.46 ± 0.45 |
| BAS% (%) | 0.16 ± 0.05 | 0.16 ± 0.09 | 0.18 ± 0.08 | 0.24 ± 0.05 |
| RET% (%) | 3.03 ± 0.35 | 3.13 ± 0.65 | 3.12 ± 0.45 | 3.18 ± 0.28 |
| NEUA (×103/µL) | 0.98 ± 0.18 | 1.14 ± 0.27 | 1.20 ± 0.12 | 1.32 ± 0.13 |
| LYMA (×103/µL) | 3.83 ± 0.63 | 3.43 ± 0.86 | 3.73 ± 0.50 | 4.10 ± 0.95 |
| MONA (×103/µL) | 0.10 ± 0.02 | 0.10 ± 0.02 | 0.08 ± 0.03 | 0.10 ± 0.03 |
| EOSA (×103/µL) | 0.07 ± 0.02 | 0.06 ± 0.02 | 0.07 ± 0.02 | 0.08 ± 0.03 |
| BASA (×103/µL) | 0.01 ± 0.01 | 0.00 ± 0.01 | 0.01 ± 0.01 | 0.01 ± 0.01 |
| RETA (×109/µL) | 240.30 ± 31.09 | 243.80 ± 45.83 | 243.38 ± 33.64 | 252.94 ± 21.08 |
| APTT (sec) | 17.00 ± 1.15 | 16.74 ± 1.09 | 16.63 ± 0.94a | 15.38 ± 1.30 |
| PT (sec) | 9.90 ± 0.12 | 10.08 ± 0.20 | 10.40 ± 0.54 | 10.56 ± 0.44* |
All values are expressed as mean ± standard deviation
WBC white blood cell count, RBC red blood cell count, HGB hemoglobin, HCT hematocrit, MCV mean corpuscular volume, MCH mean corpuscular hemoglobin, MCHC mean corpuscular hemoglobin concentration, PLT platelet, NEU% neutrophil relative, LYM% lymphocyte relative, MON% monocyte relative, EOS% eosinophil relative, BAS% basophil relative, RET% reticulocyte relative, NEUA absolute neutrophil count, LYMA lymphocyte absolute, MONA monocyte absolute, EOSA eosinophil absolute, BASA basophil absolute, RETA reticulocyte absolute, APTT activated partial thromboplastin time, PT prothrombin time
Significant differences compared to the control group (*p < 0.05; **p < 0.01)
aAnimal number is 4
Table 2.
Hematology data of female rats exposed to titanium nitride for 4 weeks
| Titanium nitride (mg/m3) | ||||
|---|---|---|---|---|
| Control | 45 | 90 | 180 | |
| Female (No. of Animals) | 5 | 5 | 5 | 5 |
| WBC (×103/µL) | 3.63 ± 1.10 | 4.45 ± 0.54 | 4.43 ± 0.68 | 5.14 ± 0.54** |
| RBC (×106/µL) | 7.46 ± 0.26 | 7.33 ± 0.36 | 7.34 ± 0.11 | 7.50 ± 0.23 |
| HGB (g/dL) | 14.32 ± 0.40 | 14.60 ± 0.32a | 14.54 ± 0.30 | 14.26 ± 0.27 |
| HCT (%) | 39.28 ± 1.07 | 39.23 ± 1.24a | 39.18 ± 0.75 | 38.52 ± 1.13 |
| MCV (fL) | 52.66 ± 0.58 | 52.36 ± 0.88 | 53.40 ± 0.50 | 51.36 ± 0.48** |
| MCH (pg) | 19.22 ± 0.36 | 19.52 ± 0.46 | 19.82 ± 0.31* | 19.04 ± 0.34 |
| MCHC (g/dL) | 36.48 ± 0.39 | 37.24 ± 0.50 | 37.12 ± 0.51 | 37.04 ± 0.55 |
| PLT (×103/µL) | 867.40 ± 104.20 | 991.40 ± 82.12 | 1015.60 ± 110.42 | 956.80 ± 93.57 |
| NEU% (%) | 19.40 ± 3.62 | 16.74 ± 4.08 | 19.92 ± 4.76 | 24.04 ± 3.04 |
| LYM% (%) | 75.26 ± 4.77 | 78.90 ± 3.91 | 75.68 ± 4.92 | 71.44 ± 3.62 |
| MON% (%) | 2.10 ± 0.60 | 2.14 ± 0.52 | 2.18 ± 0.08 | 2.02 ± 0.50 |
| EOS% (%) | 1.78 ± 0.77a) | 1.26 ± 0.40 | 1.56 ± 0.58 | 1.58 ± 0.30 |
| BAS% (%) | 0.16 ± 0.11 | 0.16 ± 0.11 | 0.14 ± 0.05 | 0.18 ± 0.04 |
| RET% (%) | 2.50 ± 0.11 | 2.79 ± 0.60 | 3.01 ± 0.63 | 2.69 ± 0.60 |
| NEUA (×103/µL) | 0.72 ± 0.23 | 0.76 ± 0.31 | 0.90 ± 0.25 | 1.26 ± 0.21** |
| LYMA (×103/µL) | 2.74 ± 0.90 | 3.50 ± 0.25 | 3.35 ± 0.53 | 3.67 ± 0.40 |
| MONA (×103/µL) | 0.08 ± 0.03 | 0.09 ± 0.03 | 0.10 ± 0.01 | 0.11 ± 0.03 |
| EOSA (×103/µL) | 0.06 ± 0.02a | 0.05 ± 0.02 | 0.07 ± 0.04 | 0.08 ± 0.02 |
| BASA (×103/µL) | 0.01 ± 0.01 | 0.01 ± 0.01 | 0.01 ± 0.00 | 0.01 ± 0.00 |
| RETA (×109/µL) | 186.74 ± 11.46 | 203.02 ± 36.88 | 220.56 ± 44.85 | 200.62 ± 39.20 |
| APTT (sec) | 15.30 ± 0.32a | 17.74 ± 0.95** | 16.58 ± 0.30* | 16.74 ± 0.90** |
| PT (sec) | 9.35 ± 0.26a | 9.98 ± 0.51* | 10.02 ± 0.40* | 10.66 ± 0.29** |
All values are expressed as mean ± standard deviation
WBC white blood cell count, RBC red blood cell count, HGB hemoglobin, HCT hematocrit, MCV mean corpuscular volume, MCH mean corpuscular hemoglobin, MCHC mean corpuscular hemoglobin concentration, PLT platelet, NEU% neutrophil relative, LYM% lymphocyte relative, MON% monocyte relative, EOS% eosinophil relative, BAS% basophil relative, RET% reticulocyte relative, NEUA absolute neutrophil count, LYMA lymphocyte absolute, MONA monocyte absolute, EOSA eosinophil absolute, BASA basophil absolute, RETA reticulocyte absolute, APTT activated partial thromboplastin time, PT prothrombin time
Significant differences compared to the control group (*p < 0.05; **p < 0.01)
aAnimal number is 4
No significant changes were observed in the blood biochemical parameters in male and female rats, except for significantly diminished (P < 0.01) TG values in the female high-concentration group (Tables 3 and 4).
Table 3.
Blood biochemical data of male rats exposed to titanium nitride for 4 weeks
| Titanium nitride (mg/m3) | ||||
|---|---|---|---|---|
| Control | 45 | 90 | 180 | |
| Male (No. of Animals) | 5 | 5 | 5 | 5 |
| Na (mmol/L) | 142.44 ± 1.10 | 143.14 ± 0.83 | 144.80 ± 0.87b | 144.04 ± 1.76 |
| K (mmol/L) | 4.80 ± 0.25 | 4.82 ± 0.16 | 4.58 ± 0.22 | 4.98 ± 0.27 |
| Cl (mmol/L) | 103.20 ± 1.41 | 104.44 ± 0.85 | 105.38 ± 2.38a | 103.66 ± 2.36 |
| TP (g/dL) | 6.34 ± 0.17 | 6.28 ± 0.19 | 6.42 ± 0.33 | 6.20 ± 0.19 |
| ALB (g/dL) | 4.12 ± 0.08 | 4.12 ± 0.13 | 4.18 ± 0.23 | 4.06 ± 0.15 |
| CREA (mg/dL) | 0.49 ± 0.02 | 0.49 ± 0.02 | 0.46 ± 0.04 | 0.47 ± 0.02 |
| BUN (mg/dL) | 17.92 ± 0.54 | 17.98 ± 0.82 | 17.12 ± 1.48 | 17.95 ± 0.49a |
| GLU (mg/dL) | 186.30 ± 13.06 | 188.26 ± 16.00 | 189.18 ± 21.59 | 191.88 ± 17.50 |
| Ca (mg/dL) | 10.64 ± 0.27 | 10.64 ± 0.18 | 10.82 ± 0.55 | 10.70 ± 0.20 |
| IP (mg/dL) | 6.78 ± 0.47 | 6.68 ± 0.48 | 7.04 ± 0.43 | 7.62 ± 1.04 |
| TBIL (mg/dL) | 0.20 ± 0.01 | 0.21 ± 0.03 | 0.21 ± 0.02 | 0.22 ± 0.02 |
| TCHO (mg/dL) | 83.18 ± 7.56 | 75.06 ± 6.72 | 75.64 ± 8.96 | 88.40 ± 12.88 |
| TG (mg/dL) | 104.60 ± 26.66 | 85.64 ± 27.88 | 96.76 ± 11.21 | 88.90 ± 12.32 |
| AST (IU/L) | 93.70 ± 19.96 | 85.32 ± 12.98 | 88.88 ± 19.64 | 87.28 ± 14.58 |
| ALT (IU/L) | 50.44 ± 9.75 | 48.96 ± 6.46 | 55.46 ± 8.65 | 48.66 ± 5.01 |
| ALP (IU/L) | 1035.30 ± 194.84 | 1017.34 ± 186.43 | 927.58 ± 164.67 | 997.38 ± 136.69 |
| A/G ratio | 1.86 ± 0.05 | 1.90 ± 0.10 | 1.90 ± 0.07 | 1.92 ± 0.11 |
All values are expressed as mean ± standard deviation
Na sodium, K potassium, Cl chloride, TP total protein, ALB albumin, CREA creatinine, BUN blood urea nitrogen, GLU glucose, Ca calcium, IP, norganic phosphorus, TBIL total bilirubin, TCHO total cholesterol, TG triglyceride, AST aspartate aminotransferase, ALT alanine aminotransferase, ALP alkaline phosphatase, A/G ratio albumin/globulin ratio
aAnimal number is 4
bAnimal number is 3
Table 4.
Blood biochemical data of female rats exposed to titanium nitride for 4 weeks
| Titanium nitride (mg/m3) | ||||
|---|---|---|---|---|
| Control | 45 | 90 | 180 | |
| Female (No. of Animals) | 5 | 5 | 5 | 5 |
| Na (mmol/L) | 143.82 ± 3.10 | 143.90 ± 5.05a | 142.50 ± 1.31 | 146.15 ± 0.64c |
| K (mmol/L) | 4.62 ± 0.44 | 4.44 ± 0.69 | 4.42 ± 0.33 | 5.00 ± 1.09 |
| Cl (mmol/L) | 103.38 ± 2.61 | 104.70 ± 3.41a | 102.60 ± 0.98 | 106.25 ± 0.49c |
| TP (g/dL) | 6.90 ± 0.20 | 6.60 ± 1.03 | 6.82 ± 0.19 | 7.88 ± 1.36 |
| ALB (g/dL) | 4.52 ± 0.13 | 4.38 ± 0.58 | 4.54 ± 0.11 | 5.12 ± 0.78 |
| CREA (mg/dL) | 0.47 ± 0.01 | 0.51 ± 0.02a | 0.50 ± 0.03 | 0.51 ± 0.03b |
| BUN (mg/dL) | 18.34 ± 2.50 | 16.78 ± 4.27 | 17.32 ± 2.02 | 19.60 ± 2.75 |
| GLU (mg/dL) | 188.10 ± 13.19 | 199.28 ± 32.85 | 210.14 ± 36.31 | 232.04 ± 42.60 |
| Ca (mg/dL) | 11.40 ± 0.34 | 10.66 ± 1.63 | 11.44 ± 0.36 | 13.32 ± 2.41 |
| IP (mg/dL) | 6.94 ± 0.44 | 6.20 ± 1.12 | 7.04 ± 0.96 | 8.00 ± 1.10 |
| TBIL (mg/dL) | 0.19 ± 0.01 | 0.18 ± 0.02 | 0.18 ± 0.01 | 0.20 ± 0.02 |
| TCHO (mg/dL) | 108.74 ± 4.02 | 106.23 ± 15.57a | 111.00 ± 13.37 | 121.08 ± 16.23 |
| TG (mg/dL) | 86.46 ± 15.58 | 61.66 ± 29.91 | 68.66 ± 22.81 | 43.58 ± 8.99** |
| AST (IU/L) | 82.94 ± 16.74 | 88.88 ± 25.37 | 70.96 ± 8.54 | 116.82 ± 38.02 |
| ALT (IU/L) | 49.06 ± 8.68 | 52.50 ± 8.93 | 47.08 ± 1.89 | 69.52 ± 14.98 |
| ALP (IU/L) | 613.80 ± 140.63 | 549.30 ± 124.08 | 669.78 ± 191.05 | 857.56 ± 201.58 |
| A/G ratio | 1.92 ± 0.13 | 1.90 ± 0.08a | 2.00 ± 0.00 | 1.88 ± 0.13 |
All values are expressed as mean ± standard deviation
Significant difference from the control group (**p < 0.01)
Na sodium, K potassium, Cl chloride, TP total protein, ALB albumin, CREA creatinine, BUN blood urea nitrogen, GLU glucose, Ca calcium, IP inorganic phosphorus, TBIL total bilirubin, TCHO total cholesterol, TG triglyceride, AST aspartate aminotransferase, ALT alanine aminotransferase, ALP alkaline phosphatase, A/G ratio albumin/globulin ratio
aAnimal number is 4
bAnimal number is 3
cAnimal number is 2
Organ weight and histopathological examination
No significant differences were observed in the absolute and relative organ weights in each sex group (data not shown). Histopathological examination of the lungs revealed increased numbers of phagocytic macrophages and alveolar/bronchiolar epithelial hyperplasia in male and female rats (Figs. 4, 5; Table 5).
Fig. 4.
Histopathological changes in the lungs of the male rats at control (CON, 0 mg/m3) and low (T1, 45 mg/m3), middle (T2, 90 mg/m3), and high (T3, 180 mg/m3) concentration titanium nitride. Increased phagocytic macrophage (arrow); Alveolar/bronchiolar epithelial hyperplasia (asterisk). Magnification, 200×
Fig. 5.
Histopathological changes in the lungs of the female rats at control (CON, 0 mg/m3) and low (T1, 45 mg/m3), middle (T2, 90 mg/m3), and high (T3, 180 mg/m3) concentration titanium nitride. Increased phagocytic macrophage (arrow); Alveolar/bronchiolar epithelial hyperplasia (asterisk). Magnification, 200×
Table 5.
Histopathological findings of male and female rats exposed to titanium nitride
| Histopathological findings | Titanium nitride (mg/m3) | ||||
|---|---|---|---|---|---|
| Severity | control | 45 | 90 | 180 | |
| Male | |||||
| Lung including bronchi | |||||
| Increased phagocytic macrophage, alveolus | + | 0/5 | 5/5 | 2/5 | 0/5 |
| ++ | 0/5 | 0/5 | 3/5 | 0/5 | |
| +++ | 0/5 | 0/5 | 0/5 | 5/5 | |
| Alveolar/bronchiolar epithelial hyperplasia | + | 0/5 | 0/5 | 0/5 | 4/5 |
| ++ | 0/5 | 0/5 | 0/5 | 1/5 | |
| Female | |||||
| Increased phagocytic macrophage, alveolus | + | 0/5 | 4/5 | 0/5 | 0/5 |
| Alveolar/bronchiolar epithelial hyperplasia | ++ | 0/5 | 1/5 | 4/5 | 0/5 |
| +++ | 0/5 | 0/5 | 1/5 | 5/5 | |
| ± | 0/5 | 0/5 | 0/5 | 4/5 | |
Data was presented as incidence/examined animals
± minimal, + mild, ++ moderate, +++ marked
Bronchoalveolar lavage fluid (BALF) analysis
In male rats, the macrophage count was significantly diminished (p < 0.01), while the neutrophil count and LDH and GGT values were significantly elevated (p < 0.01) in the high-concentration group. In female rats, macrophage count was significantly diminished (p < 0.01) at low concentration, LDH level was significantly elevated (p < 0.01) in the high-concentration group, and GGT level was significantly elevated (p < 0.01) in the middle and high-concentration groups (Table 6).
Table 6.
Analysis of bronchoalveolar lavage fluid of male and female rats exposed to titanium nitride
| Titanium nitride (mg/m3) | ||||
|---|---|---|---|---|
| Control | 45 | 90 | 180 | |
| Male (Animal No.) | 5 | 5 | 5 | 5 |
| Total cells | 448.60 ± 65.06 | 489.00 ± 49.93 | 487.40 ± 75.72 | 434.60 ± 41.60 |
| Macrophage | 425.00 ± 60.91 | 444.60 ± 46.00 | 428.60 ± 59.56 | 214.60 ± 62.99** |
| Neutrophil | 11.20 ± 5.67 | 18.20 ± 11.01 | 44.00 ± 19.74 | 212.00 ± 50.20** |
| Lymphocyte | 12.20 ± 2.17 | 26.20 ± 9.96 | 14.80 ± 6.30 | 8.00 ± 3.46 |
| Eosinophil | 0.20 ± 0.45 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 |
| TP (g/dL) | 0.019 ± 0.009 | 0.009 ± 0.007 | 0.010 ± 0.008 | 0.006 ± 0.013 |
| LDH (IU/L) | 100.560 ± 35.251 | 92.000 ± 6.727 | 90.380 ± 7.729 | 248.240 ± 37.229** |
| GGT (IU/L) | 4.980 ± 0.799 | 6.000 ± 0.644 | 6.380 ± 1.040 | 8.050 ± 1.475** |
| Female (Animal No.) | 5 | 5 | 5 | 5 |
| Total cells | 541.20 ± 97.13 | 412.40 ± 21.17 | 521.80 ± 151.94 | 557.00 ± 157.55 |
| Macrophage | 468.20 ± 40.39 | 389.40 ± 23.08** | 309.00 ± 99.85 | 372.60 ± 96.94 |
| Neutrophil | 49.20 ± 58.03 | 11.00 ± 4.80 | 194.80 ± 176.37 | 159.20 ± 143.69 |
| Lymphocyte | 23.80 ± 9.20 | 12.00 ± 4.90 | 18.00 ± 14.54 | 25.20 ± 8.90 |
| Eosinophil | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 |
| TP (g/dL) | 0.018 ± 0.012 | 0.007 ± 0.002a | 0.026 ± 0.013 | 0.018 ± 0.011 |
| LDH (IU/L) | 87.380 ± 14.346 | 77.260 ± 5.996 | 102.060 ± 11.791 | 204.420 ± 27.329** |
| GGT (IU/L) | 4.420 ± 0.795 | 5.400 ± 1.025 | 7.460 ± 1.197** | 8.900 ± 1.654** |
All values are expressed as mean ± standard deviation
TP total protein, ALB albumin, ALP alkaline phosphatase, LDH lactate dehydrogenase, GGT gamma-glutamyltransferase
Significantly different from the control (*p < 0.05; **p < 0.01)
aAnimal number is 4
Discussion
Titanium nitride (TiN) is generally considered safe and is used in a wide range of fields such as alloys, metal and machine tool coatings, jewelry, medical devices, automobiles, aerospace and military, and 3D printers [1–4]. However, many people handling them, especially workers, are potentially exposed. Although there are many, inhalation is considered the main route of exposure. For this reason, the toxicity information of TiN for repeated inhalation studies is limited, and more information, including toxicity data, is needed to evaluate its human health effects. Therefore, we conducted a 4-week inhalation toxicity study to obtain toxicity information on TiN through inhalation exposure for safety evaluation as a basic target for workers. No deaths or unusual clinical signs related to the test substances were observed during the study period. In the mean body weight changes, temporary weight loss was observed in the male high-concentration group. The exact cause of temporary weight loss is unknown, but it is presumed to be due to individual differences based on grouping and holder adaptation training. In male rats, a decrease in HGB and MCHC values was not considered to be an effect of the test substance, as there was no difference in diet, no observed bleeding, and the values of change were minor. In female rats, an increase in WBC and NEUA values usually indicates infection and tissue damage, and pathological causes include bacterial infection, inflammation or necrosis, and metabolic disorders [17, 18]. Based on the correlation with the results of bronchoalveolar lavage fluid analysis, it could be determined as a change by the test substance. The changes in MCV and MCH values were not related to other parameters, and thus they were not considered to be affected by the test substance. Changes in PT and APTT in male and female rats could show hepatic failure or blood clotting factor deficiencies [17, 18]; however, because of the correlation with other items and short time, it was not considered as a change by test substance. The decrease in TG value in blood biochemical analysis in female rats may be owing to general metabolic status [17, 18], so it was not considered as a change by the test substance. Effects on the liver could not be confirmed without histopathological examination. These results suggest that there are no systemic adverse effects after inhalation exposure to TiN. Conversely, histopathological findings and BALF analysis showed effects of the test substance on the lungs after inhalation exposure to TiN. Histopathological findings confirmed that the frequency and severity increased in a dose-dependent manner, and BALF analysis showed an increase in LDH, which is used as an indicator of tissue damage, and GGT, which is related to oxidative stress [19]. The changes in macrophages and neutrophils in BALF analysis were similar to those in our previous studies on particulate matter (data not shown). Furthermore, Wang et al. [11] concluded that nano-TiN may cause toxicity in zebrafish development through the elevation of oxidative stress, which is considered to be associated with increased GGT in BALF analysis in our study. However, because BALF is an extracellular fluid that surrounds the cells in the airway-alveoli and the outside of the cells and does not reflect the characteristics or tissue inflammation inside the lung interstitial or alveolar wall [19], it is an indicator that can be used as a limited reference.
Meanwhile, lung burden measurement (only male rats) was performed to confirm the lung deposition of titanium using inductively coupled plasma optical emission spectrometer (ICP-OES, PerkinElmer 4300 DV, PerkinElmer, Waltham, MA, USA), and the results for the control, low, middle, and high-concentration groups were 0.005 ± 0.0049 mg/g, 17.600 ± 2.4931 mg/g, 33.445 ± 3.0083 mg/g, and 36.929 ± 12.4103 mg/g, respectively. A statistically significant increase was observed in all exposure groups, but the clearance of titanium could not be confirmed because subsequent measurements were not performed. Based on the results, macrophage infiltration in the lung did not lead to histological lesions in the lungs, and the BALF analysis results were considered to be due to change within the physiological range in the low-concentration group, and in view of the lack of information on recovery after termination of exposure, the no observed adverse effect concentration (NOAEC) is suggested to be 45 mg/m3. Further studies on the effect of TiN, including recovery, clearance, long-term effects, and target organ toxicity studies, are required.
Acknowledgements
This study was supported by the Korea Occupational Safety and Health Agency, Ministry of Labor, Republic of Korea, and Grant-in-Aid for Chemical Hazard Assessment.
Author contributions
Y-SK wrote the original draft of this manuscript. Y-SK, C-HP, and E-SC contributed to study planning and editing of the original draft. Y-SK, C-HP, H-GC, and E-SC performed the experiments. Y-SK and E-SC contributed to the discussion on the data, and supervision. All the authors have read and approved the final version of the manuscript.
Funding
This study was supported by the Korea Occupational Safety and Health Agency, Ministry of Labor, Republic of Korea, and Grant-in-Aid for Chemical Hazard Assessment.
Declarations
Conflict of interest
The authors have no relevant financial or non-financial interests to disclose.
Ethics approval
This study was approved by the Institutional Animal Care and Use Committee of the Chemical Research Bureau, Occupational Safety, and Health Research Institute, prior to obtaining the rats, and all tests were conducted in accordance with established animal care protocols.
Consent to participate
This manuscript does not require IRB approval because there are no human participants.
Consent to publish
This manuscript does not require IRB approval because there are no human participants.
Footnotes
Publisher’s Note
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