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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
publication
Title:
28-Day inhalation toxicity of graphene nanoplatelets in Sprague-Dawley rats
Author:
Kim J.K. et al.
Year:
2016
Bibliographic source:
Nanotoxicology, 10:7, 891-901, DOI: 10.3109/17435390.2015.1133865

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
GLP compliance:
not specified
Remarks:
no information on GLP compliance available in this publication
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: nanomaterial
Details on test material:
- Surface area of particles: 750 m2/g
- Density: 0.2 g/mL
- Average lateral dimension: <2 µm
- Average thickness: 20-30 layers
Specific details on test material used for the study:
Aerosolized graphene nanoplatelets had various thicknesses, ranging from 0.350 to 0.380 nm. The TEM-EDS analysis indicated the presence of two elements (i.e. C and O), in the following atomic percentages:
C (96.28%) and O (3.72%).

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
no details given
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
OrientBio (Seongnam, Korea)
- Age at study initiation:
six weeks
- Fasting period before study:
- Housing:
polycarbonate cages (maximum of three rats per cage) installed in individually ventilated cage racks
- Diet (e.g. ad libitum):
rodent diet (Woojung BSC, Suwon, Korea) ad libitum
- Water (e.g. ad libitum):
filtered water ad libitum
- Acclimation period:
2 weeks before initiating the inhalation exposure; during the acclimation period, the animals were trained to adapt to the nose-only inhalation chamber for 6 h/day

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
22 ±0.65 °C
- Humidity (%):
56 ±0.08%
- Photoperiod (hrs dark / hrs light):
12-h light/dark cycle

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
clean air
Mass median aerodynamic diameter (MMAD):
123 nm
Geometric standard deviation (GSD):
3.63
Remarks on MMAD:
The MMAD, measured using a 13-stage DLPI cascade impactor, was 123 nm with a GSD of 3.63
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Nose-only exposure system (HCT, Incheon, Korea)
- System of generating particulates/aerosols: the graphene nanopowder was generated using an atomizer (AG-01, HCT, Icheon, Korea), with purified air as the carrier gas
- Air flow rate: The gas flow was maintained at 30 liters per minute (L/min) using a mass flow controller (MFC, FC-7810CD-4V, AERA, Tokyo, Japan), and the flow rate to each nose port was 1 L/min
- Method of particle size determination: measured with a scanning nanoparticle spectrometer (SNPS, HCT Co., Ltd., Icheon, Korea) and dust monitor (OPC, Model 1.1.09, Grimm Technologies Inc., Douglasville, GA, USA)

TEST ATMOSPHERE
- Brief description of analytical method used: The mass concentration of graphene was determined gravimetrically (as post-weight minus pre-weight) by sampling on a PC filter (polycarbonate filter, size: 27 mm and pore size 0.2 pm) and MSA Escort ELF sampling pump (MSA, Pittsburgh, PA, USA) from the top and bottom parts of the port, at a flow rate of 1.0 L/min.
- Samples taken from breathing zone: yes

VEHICLE (if applicable)
- Concentration of test material in vehicle: the suspension in water used to generate the aerosol contained fresh air for the control, 1.5 mg/mL for the low concentration, 5 mg/mL for the moderate concentration, and 16 mg/mL for the high concentration
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The number concentration of airborne graphene aerosols was measured with a condensation particle counter (CPC, model 3775, TSI Inc., Shoreview, MN, USA): see table 1 below (Concentration and size distribution of aerosolized graphene nanoparticles inside the nose-only exposure chambers)
Duration of treatment / exposure:
28 days (5 days/week for 4 weeks)
Frequency of treatment:
The animals were exposed to the graphene nanopowder for 6 h/day for 5 days/week, for four weeks; the control group received filtered fresh air.
Doses / concentrationsopen allclose all
Dose / conc.:
0.125 mg/m³ air (nominal)
Remarks:
target mass concentration - delivered mass concentration: 0.12 ± 0.00 mg/m³
Dose / conc.:
0.5 mg/m³ air (nominal)
Remarks:
target mass concentration - delivered mass concentration: 0.47 ± 0.03 mg/m³
Dose / conc.:
2 mg/m³ air (nominal)
Remarks:
target mass concentration - delivered mass concentration: 1.88 ± 0.18 mg/m³
No. of animals per sex per dose:
15
Control animals:
yes, concurrent vehicle
Details on study design:
The rats were divided into four groups: control (unexposed, n = 15), low concentration group (n = 15), moderate concentration group (n = 15), and high-concentration group (n = 15). For each group of 15 animals, 5 animals were killed 1-day post-exposure, 5 were killed 28-day post-exposure, and 5 were killed 90-day post-exposure. The animals were examined daily for any evidence of exposure-related toxic responses. The body weights were measured at the time of purchase, at the time of grouping, once during the inhalation period, and before necropsy. The food consumption (g/ rat/day) was measured once a week. At killing (1-, 28-, and 90-day post-exposure, n = 5 each), gross observations of the organs were recorded, plus the testes, kidneys, spleen, liver, lungs, and brain were all carefully removed and weighed. All animal testing protocols were approved by the Hanyang University's Institutional Animal Care and Use Committee.

The daily lung burden per rat was estimated for 6 h of continuous exposure, a minute ventilation of 0.19L/min, and the following aerosol properties (see Results section): particle MMAD of 123 nm, GSD 3.63, lung deposition efficiency of 7.7% based on MPPD (2002), and graphene aerosol concentrations were 1.88, 0.47, and 0.12mg/m3 for high, moderate, and low concentrations, respectively. The following calculations were made:
Daily deposited dose (mg/day) = average graphene aerosol concentration (mg/m3) x minute volume (L/min = 0.06 m3/h) x exposure duration (h/day) x deposition efficiency (1)
Deposition at the high dose = 1.88 x (0.19 x 0.06) x 6 x 0.077 = 0.0099 mg/day
Deposition at the moderate dose= 0.47 x (0.19 x 0.06) x 6 x 0.077 = 0.0025 mg/day
Deposition at the low dose = 0.12 x (0.19 x 0.06) x 6 x 0.077 = 0.0006 mg/day

Cumulative dose (mg)/animal = daily deposited dose (mg/day) x number of doses (days)
For high exposure: 0.0099 mg/day x 20 days = 0.198 mg
For moderate exposure: 0.0025 mg/day x 20 days = 0.05 mg
For low exposure: 0.0006 mg/day x 20 days = 0.012 mg
Positive control:
no details given

Examinations

Observations and examinations performed and frequency:
The animals were examined daily for any evidence of exposure-related toxic responses.
The body weights were measured at the time of purchase, at the time of grouping, once during the inhalation period, and before necropsy.
The food consumption (g/rat/day) was measured once a week.
At killing (1-, 28-, and 90-day post-exposure, n = 5 each), gross observations of the organs were recorded, plus the testes, kidneys, spleen, liver, lungs, and brain were all carefully removed and weighed.
All animal testing protocols were approved by the Hanyang University's Institutional Animal Care and Use Committee.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Other examinations:
After collecting the blood, the rats were killed by enotobar anasthesia, and the adrenal glands, bladder, testes, epididymis, heart, thymus, thyroid gland, trachea, esophagus, prostate, lungs, nasal cavity, kidneys, spleen, liver, pancreas, and brain were carefully removed. These organs were then weighed and fixed in a 10% formalin solution containing neutral phosphate-buffered saline (PBS). The organs were embedded in paraffin and stained with hematoxylin and eosin (BBC biochemical, Washington, DC). All the animal organs were examined using light microscopy. At killing, the left lungs were removed and fixed in a 10% formalin solution (BBC biochemical, Washington, DC) containing neutral PBS under 25 cm of water pressure for the histopathological evaluation. Thereafter, the lungs were embedded in paraffin, stained with hematoxylin and eosin, and examined under a light microscope. In addition, the graphene imaging was examined using a high-resolution illuminator (Cytoviva, Auburn, AL, USA) attached to a dark field microscope.
Statistics:
The statistical analysis of outcome parameters was performed using SPSS version19 (SPSS Inc., Chicago, IL). The statistical evaluation was performed using an analysis of variance (ANOVA) following multiple comparison tests using Duncan's method. The level of statistical significance was set at p < 0.05 and p < 0.01.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Significant body weight losses were observed in the moderate-concentration group at 2 weeks and in the high-concentration group at 1, 5, 6, 11, and 13 weeks from the start of the exposure when compared with the control.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Significant decreases in food consumption were noted in the low-concentration group at 2, 6, 12, 16, and 17 weeks and in the high-concentration group at 1, 7, 9, 12, 13, 14, 16, and 17 weeks from the start of the exposure when compared with the control.
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
No concentration-related changes in the blood biochemical parameters were observed.
The hematology values at the 1-day post-exposure and 90-day post-exposure did not show any significant changes in the hematological parameters when compared with the control.
The thrombin and activated partial thrombo-plastin times were measured for the blood at 1-day post-exposure and at 90-day post-exposure to evaluate the effect of the graphene exposure on blood coagulation; yet, no significant effects were found.
The blood glucose and total bilirubin (TBIL) were significantly increased in the moderate-concentration group, while the blood potassium was significantly decreased in the moderate-concentration group at the 1-day post-exposure.
The LDH (lactate dehydrogenase) was significantly decreased in the moderate-concentration group, the TBIL and chloride significantly increased in the high-concentration group, and the potassium significantly decreased in the high-concentration group when compared with the control at the 28-day post-exposure.
The blood urea nitrogen (BUN) was significantly decreased in the moderate-concentration group, the sodium (Na) significantly increased in the low-concentration group, chloride (Cl) was significantly increased in the high-concentration group, and the ratio of albumin to glucose was also significantly increased in the moderate-concentration group when compared with the control at 90-day post-exposure.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Effects on BAL fluid
The BUN, UrDF, LDH, mALB, and mTP were all measured in the BAL fluid from the rat lungs. The mALB was significantly decreased in the moderate-concentration group and the LDH significantly increased in the low-concentration group at 28-day post-exposure when compared with the control. No concentration-related effects were observed for the inflammatory biomarkers in the BAL fluid (Table 3). Furthermore, no other oxidative stress markers, such as H2O2, GSH, and MDA, measured in the BAL fluid were significantly elevated after the 1-day post-exposure or 28- and 90-day post-exposure, indicating the graphene exposure had no effect on oxidative stress (Table 3). Notably, the MDA levels at 90-day post-exposure were significantly lower in the low- and moderate-concentration groups relative to the control.
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
no effects observed
Description (incidence and severity):
Various cytokines were measured, including the granulocyte-colony stimulating factor, interferon-gamma, IL-1P, IL-6, IL-18, monocyte chemotactic protein-1, macrophage-colony stimulating factor, macrophage inflammatory protein-1, TNF-a, and vascular endothelial growth factor (VEGF) in the lung tissue lysates and TNF-a, IL-18, VEGF (ng/mL/UrDF), and transforming growth factor-beta1 (ng/mL/UrDF) in the BAL fluid. The cytokines measured in the lung lysates at 1-day post-exposure and 28- and 90-day post-exposure did not show any significant changes.
In BAL fluid the total cell counts and macrophage counts were significantly decreased in all the exposed groups at the 1-day postexposure and 28-day post-exposure when compared with the control (Table 2). The lymphocyte counts were also significantly decreased in the all exposed groups at the 1-day postexposure when compared with the control (Table 2).
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
A significant organ weight loss was observed for the thymus in the high-concentration group when compared with the control after the 28-day graphene exposure.
Significant organ weight losses were observed for the liver in the moderate-concentration group, and for the left lung in the low-and moderate-concentration groups when compared with the control at 28-day post-exposure.
The brain weight was significantly increased in the high-concentration group, and the thymus weight significantly increased in the low-concentration group when compared with the control at 90-day post-exposure.
Gross pathological findings:
no effects observed
Description (incidence and severity):
The gross findings for the rat organs, including the testes, kidneys, spleen, liver, lungs, thymus, eyeballs, and brain, after the 28-day graphene exposure and during the 90-day recovery period
revealed no particular changes resulting from the graphene exposure when compared to the control.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No inflammatory-related histological evidence, such as the increase and migration of PMN cells or proliferation of pneumocytes, a change in the thickness of the alveolar wall, or the formation of granulomatous regions, were observed in the lungs at 1-day post-exposure, and 28- and 90-day post-exposure.
Black particles ingested by the alveolar macrophages were observed at 1-day post-exposure and at 28- and 90-day post-exposure. Translocation of graphene was observed in lung-associated lymph nodes at 1-day post-exposure and at 28- and 90-day post-exposure. Hyperspectral images taken at 1-day post-exposure and at 28-and 90-day post-exposure showed a concentration-dependent deposition of graphene in mostly lung macrophages and some deposition in lung epithelial cells. The ingested graphene in the lung macrophages persisted even after the 90-day post-exposure period.
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1.88 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical biochemistry
immunology
histopathology: neoplastic

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
NOAEL of no less than 1.88 mg/m3.
Executive summary:

The current study evaluated subacute inhalation toxicity of graphene according to the OECD testing guideline 412 (28-day study) (OECD, 2009). In this study, male SD rats were subjected to inhalation exposure to 0.12, 0.47 and 1.88 mg/m3 of graphene nanomaterials for 28 days (6 h/day, 5 days/week, for 4 weeks) and then allowed to recover for 28 or 90 days. No dose-dependent effects were recorded up to the highest test concentration of graphene for the body weights, organ weights, bronchoalveolar lavage fluid inflammatory markers, and blood biochemical parameters at 1-day post-exposure and 28-day post-exposure. The inhaled graphenes were mostly ingested by macrophages yet there was no increase in the inflammatory cell numbers, markers, and cytokines resulting from the graphene-ingested lung macrophages. No distinct lung pathology was observed at the 1-, 28- and 90-day post-exposure. The inhaled graphene was translocated to lung lymph nodes.

The results of this 28-day inhalation study suggests a low toxicity of graphene and a NOAEL of no less than 1.88 mg/m3.