Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
no details given
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions

Data source

Reference
Reference Type:
publication
Title:
Pulmonary Responses of Sprague-Dawley Rats in Single Inhalation Exposure to Graphene Oxide Nanomaterials
Author:
Han S.G. et al.
Year:
2015
Bibliographic source:
BioMed Research International Volume 2015, Article ID 376756

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Principles of method if other than guideline:
not applicable
GLP compliance:
not specified
Remarks:
no information on GLP compliance available in this publication
Test type:
acute toxic class method
Limit test:
no

Test material

Constituent 1
Reference substance name:
Reaction product of Graphite, acid-treated and potassium permanganate
EC Number:
947-768-1
IUPAC Name:
Reaction product of Graphite, acid-treated and potassium permanganate
Test material form:
solid: particulate/powder
Details on test material:
The graphene oxide nanomaterials were kindly provided by Dr. Heon Sang Lee of Dong-A University (Busan, Korea).
Specific details on test material used for the study:
Characteristics of the test item are given below.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: OrientBio (Seongnam, Korea)
- Age at study initiation: 6 weeks old
- Housing: polycarbonate cages (maximum of 3 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

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.8 ± 0.17 °C
- Humidity (%): 49.16 ± 2.37 %
- 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):
> 50.6 - < 72.9 nm
Geometric standard deviation (GSD):
> 1.82 - < 2.02
Remark on MMAD/GSD:
The diameter (nm) and surface area (nm2/cm3) of the graphene oxide were 50.6 ± 1.82 nm and 6.45 E10 ± 7.04 E9 nm2/cm3,respectively, for the low-concentration chamber and 72.9 ± 2.02 nm and 2.72 E11 ± 3.70 E10 nm2/cm3,respectively, for the high-concentration chamber. The size distribution and number of graphene oxide particles in each chamber were also measured during the exposure period using SNPS. The particle size ranged from 10 to 120 nm, with the highest peak at 35 nm and 50 nm for the low-and high-concentration chambers, respectively.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: nose-only exposure system (NITC system, HCT Co., Ltd., Incheon, Korea)
- Source and rate of air: the gas flow was maintained at 16 liters per minute (L/min) using a mass low controller (MFC, AERA, FC-7810CD-4V, Japan), and the low rate to each nose port was 1 L/min.
- System of generating particulates/aerosols: graphene oxide was generated using an atomizer, and purified air was used as the carrier gas
- Method of particle size determination: the average flake size of the graphene oxide in a solution was measured using dynamic light scattering (DLS). The size distribution of the graphene oxide was measured using a scanning nanoparticle spectrometer (SNPS, HCT Co., Ltd., Korea) connected to a condensation particle counter (CPC, model 3022A, TSI Inc., Shoreview, MN, USA) and dust monitor (Model 1.1.09, Grimm Technologies Inc., Douglasville, GA, USA).
- Temperature, humidity, pressure in air chamber: the temperature and humidity were 23.10 ± 0.05 °C and 32.90 ± 0.21 % for the low-concentration inhalation chamber and 25.25 ± 0.07 °C and 34.40 ± 0.34%, respectively, for the high-concentration inhalation chamber.

TEST ATMOSPHERE
- Brief description of analytical method used: The concentrations of graphene oxide in the chambers were measured using polycarbon filters connected to a MAS Escort ELF sampling pump (MSA, Pittsburgh, PA, USA) at a flow rate of 1.0 L/min. The weight difference of the polycarbon filter before and after sampling was calculated.

VEHICLE
- Composition of vehicle (if applicable):
- Concentration of test material in vehicle (if applicable): the target concentrations of the generated graphene oxide were 0.3 mg/m and 3 mg/m for the low- and high-concentration chamber, respectively. The graphene oxide exposure concentrations (mg/m3)measured based on the weight of the polycarbon filter before and after sampling were 0.46 ± 0.06 and 3.76 ± 0.24 for the low- and high-concentration groups, respectively.

TEST ATMOSPHERE (if not tabulated)
- Particle size distribution: the particle size ranged from 10 to 120 nm, with the highest peak at 35 nm and 50 nm for the low-and high-concentration chambers, respectively.
The XRD pattern for the natural graphite oxide revealed a sharp reflection at 2Θ = 26.4°, corresponding to the interlayer spacing (d = 0.34 nm). The peak in the XRD pattern for the dried graphene oxide particles showed broadening, as well as a shift to a lower angle (2Θ = 9.5°), indicating that the interlayers were 0.93 nm apart due to intercalation by the hydroxyl, carbonyl, and epoxide groups and moisture. The DLS measurement showed the average lake size of the graphene oxide in a solution. Plus, the equivalent hydrodynamic diameter of the graphene oxide was estimated using Stokes-Einstein equation to be 150-250 nm.
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
6 h
Concentrations:
control (fresh air), low concentration (0.46 ± 0.06 mg/m3), and high concentration (3.76 ± 0.24 mg/m3)
No. of animals per sex per dose:
12
Control animals:
yes
Details on study design:
During the acclimation period, the animals were trained to adapt to the nose-only inhalation chamber. The rats were divided into 3 groups: control (unexposed, n=12), low-concentration group (n=12), and high-concentration group (n=12). The low- and high-concentration groups were exposed to the graphene oxide for a single period of 6 hours, while the control group received filtered fresh air. 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, after the 6h inhalation exposure, and before necropsy. The food consumption (g/rat/day) was measured once a week. After the single 6 h inhalation exposure to graphene oxide, the rats were allowed to recover for 1 day, 7 days, or 14 days (n=4 per treatment group for each time period) to investigate the toxic responses. At sacrifice, gross observations of the organs were recorded, and the testes, kidneys, spleen, liver, lungs, and brain were all carefully removed and weighed. All the animal protocols were approved by Hanyang University Institutional Animal Care and Use Committee.
Statistics:
The statistical analysis was performed using SPSS (Version 19) and the statistical evaluation 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

Effect levels
Key result
Sex:
male
Dose descriptor:
LC50
Effect level:
> 3.76 mg/m³ air (analytical)
Based on:
test mat.
Exp. duration:
6 h
Mortality:
no mortality observed
Clinical signs:
other: no details given
Body weight:
No significant body weight changes were observed for the low- and high-concentration groups during the exposure and recovery periods.
Gross pathology:
No significant gross effects were observed during the exposure and recovery periods. The examination of the rat organs, including the testes, kidneys, spleen, liver, lungs, and brain, also revealed no significant clinical signs or organ weight changes during the observation period.
Other findings:
No significant differences was observed in food intake between the control and graphene oxide-treated groups.
The levels of microalbumin and LDH were measured in the BAL fluid as indicators of bronchoalveolar mucosal permeability and lung cell damage, respectively. The results indicated that the single 6-hour nose-only inhalation exposure of the rats to graphene oxide did not significantly change the levels of microalbumin and LDH in the BAL fluid (Table 3). Plus, the results from counting the BAL cells (i.e., total cells, macrophages, PMNs, and lymphocytes) showed no significant alterations following the graphene oxide exposure at all the time points tested (Table 4). In order to determine the level of inflammation and damage in the lung, a total of nine toxicity parameters were measured in the cell-free BAL fluid. The results showed that limited numbers of parameters were increased with graphene oxide inhalation in the cell-free BAL fluid. At day 1 after inhalation, MMP-9 was significantly increased in the high-concentration group compared to control and the low-concentration group. At day 7, IL-18 and TGFbeta1 were significantly higher than those in control.
The histo-pathological examination of the rat lungs did not reveal any pathological changes in the low- and high-concentration groups after the graphene oxide exposure. However, alveolar macrophages with ingested graphene oxide were visualized in the high-concentration group during the recovery period, that is, 1 day, 7 days, and 14 days after exposure.

Applicant's summary and conclusion

Conclusions:
Inhalation of graphene oxide in male Sprague-Dawley rats exerted minimal toxic responses. LC50 > 3.76 mg/m3
Executive summary:

In the present study (similar to OECD Guideline 403) a nose-only inhalation technique with male Sprague-Dawley rats was used to examine the pulmonary effects of graphene oxide. Inhalation is considered the major route of human exposure to graphene nanomaterials, particularly in occupational settings.

A total of three groups were compared: control (fresh air), low concentration (0.46 ± 0.06 mg/m3), and high concentration (3.76 ± 0.24 mg/m3). The exposure to graphene oxide did not induce significant changes in the body weights, organ weights, and food consumption during the 14 days of recovery time. The microalbumin and lactate dehydrogenase levels in the bronchoalveolar lavage (BAL) fluid were not significantly changed due to the exposure. Similarly, total cell count, macrophages, polymorphonuclear leukocytes, and lymphocytes were not significantly altered in the BAL fluid.

BAL fluid evaluation of nine inflammatory parameters (i.e., TNF-a, IL-1beta, IL-18, G-CSF, M-CSF, VEGF, TGF-beta1, MMP-9, and TIMP-1) demonstrated that only MMP-9 (day 1) and IL-18 and TGF-beta1 (day7) were significantly elevated due to exposure of graphene oxide at high concentrations. The dose-dependent particle-loaded macrophages in the lungs of the exposed rats were observed and the graphene oxide-loaded macrophages were only found in the high-concentration group at the selected recovery time points (1 day, 7 days, and 14 days). The gross findings for the rat organs, such as the testes, kidneys, spleen, liver, lungs, and brain, during the recovery time revealed no particular changes due to the graphene oxide exposure when compared to the control.

These results demonstrate that the single inhalation exposure to graphene oxide induce minimal toxic responses in rat lungs at the concentrations and time points used in the present study. Thus, the LC50 is > 3.76 mg/m3.

Categories Display