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Toxicological information

Genetic toxicity: in vivo

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Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
no details given
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
acute toxicity: other routes
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
no details given
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Principle of test: acute toxicity of the test item graphene oxide in mice after intratracheal instillation
- Short description of test conditions: Mice were exposed to either GO by single intratracheal instillation (50 µl/mouse) under isoflurane sedation between 9 to 11 a.m.. The applied doses in main study were 18 µg, 54 µg and 162 µg/mouse. Mice were euthanized at 1, 3, 28 or 90 days post exposure.
- Parameters analysed / observed: body weight, clinical signs
GLP compliance:
not specified
Limit test:
no
Specific details on test material used for the study:
GO was prepared in 0.2 µm filtered, y-irradiated Nanopure Diamond UV water (Pyrogens: < 0,001 EU/mL, total organic carbon: < 3.0 ppb) added 0.1% Tween80® (TW80) to a final concentration of particles of 3.24 mg/mL. To achieve a homogenous dispersion, the final solution was then prepared by probe sonication on ice for 16 min with 10% amplitude (Branson Sonifier S-450D, Branson Ultrasonics Corp., Danbury, CT, USA) equipped with disruptor horn (model number 101-147-037). Following sonication, solution was further diluted to 1.08 mg/mL and sonicated for 2 minutes. Dilution was further diluted to 0.36 mg/mL and sonicated for 2 minutes. As vehicle control (VC), Nanopure water added 0.1% TW80 was prepared by procedure as described above. Suspensions were instilled in mice within 20 minutes after sonication.

The test item GO was characterised as shown in the table below.
Species:
mouse
Strain:
C57BL
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Taconic Europe (Ejby, Denmark)
- Age at study initiation: 7-weeks old
- Weight at study initiation: 19.7 ± 1 g
- Assigned to test groups randomly: grouped based on exposure and dose level (VC groups n = 8, exposed groups n = 7)
- Fasting period before study: not specified
- Housing: polypropylene cages with sawdust bedding and enrichment
- Diet (e.g. ad libitum): food (Altromin 1324) ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 1°C
- Humidity (%): 50 ± 10%
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12-h light and 12-h dark cycle
Route of administration:
other: intratracheal
Vehicle:
other: Nanopure water added 0.1% Tween80
Doses:
18 µg, 54 µg and 162 µg/mouse (corresponding to 0.91, 2.74 and 8.22 mg/kg bw, respectively)
No. of animals per sex per dose:
vehicle control groups n = 8, test item-exposed groups n = 7
Control animals:
yes
Details on study design:
- Duration of observation period following administration: 90 days
- Frequency of observations and weighing: Daily observations of clinical signs of stress and discomfort were performed. Bodyweight was monitored at 3-5 time points (n > 6) throughout the study period.
- Necropsy of survivors performed: yes, BAL samples
- Other examinations performed: clinical signs, body weight, histopathology of the lung
Statistics:
Statistical analysis was performed in Minitab v.17.1.0 (Minitab Inc., State College, PA, USA), except for neutrophils where SAS®, version 9.3 for the Windows platform was used. All data are presented as mean ± standard error of the mean (Mean±SEM).
All statistical analyses were performed on log-transformed data. The analyses of GO and rGO were performed for each day separately using two-way Analysis of Variance (ANOVA) with particle and dose level set as fixed category variables interacting with each other. In case of interaction, one-way ANOVA was performed including pairwise comparisons using Tukey's adjusted p-values (significance level: 0.05). Analysis of data for P90 was performed using unpaired t-test at all time points. For cells in BAL, based a very high variation caused by numerous zero-values in the dataset for number of eosinophils and lymphocytes, statistical analysis was only performed on number of neutrophils, macro¬phages and total cells. For neutrophils, there were some samples with no neutrophils among the 200 cells counted. The total number of neutrophils in each of these samples was considered to be left censored at the total number of cells in the sample divided by 200. For neutrophils, the two-way ANOVA for each day with interaction between particle and dose were performed using the Lifereg procedure in SAS®.
Mortality:
no mortality observed
Clinical signs:
Signs of discomfort including energy loss, back arching and piloerection were observed. The mice did not interact with each other.
Body weight:
In general, mice exposed to either the vehicle control or P90 increased their body weight at day 1 after instillation until day 90. In contrast, exposure to 54 or 162 µg/mouse of GO resulted in weight loss at day 1. The weight loss continued until day 3 with a total weight loss of ~15% and ~20% in bodyweight for the mice instilled with 54 and 162 µg/mouse, respectively.
Other findings:
Analysing the bronchoalveolar lavage revealed that at days 1 and 3, GO induced a potent and significant neutrophil influx at all doses compared to VC, and a dose-response relationship was observed at day 3. In addition, the number of macrophages was clearly increased at 1 and 3 days after GO exposure.

Histopathological analysis found that at day 3, GO was observed as light brownish granular pigments. Granulocytes and macrophages with or without GO were mostly seen in the alveolar walls followed by alveolar sacs and alveolar ducts, and to some extent in the terminal bronchioles including adjacent interstitium. Observations indicated severe acute inflammatory response, involving air passages and respiratory segments distal to the ciliated airways. GO was more prominent and visible in the 162 µg/mouse group compared to 54 µg/ mouse group, and was observed mainly as free deposits or in aggregates in macrophages within the alveoli and the alveolar ducts. In addition, inflammation at sites with GO deposits was prominent and patchy and located to the respiratory parenchyma with a larger total area affected with increased dose (~ 20-30% and 30-40% of the total cut lung surface for groups 54 and162 µg/mouse, respectively). The alveolar walls were enlarged and neutrophils were found together with congested capillaries and alveolar granular exudate as part of the acute inflammation. Hyperplastic Type II cells were also observed. These pathological findings were notably found in group 162 µg/mouse as part of the acute inflammation. In general, areas devoid of dusts were mainly without pathological findings. Perivascular lymphocytic accumulations were also clearly observed.
At day 90, GO appeared as smaller and more brown pigments. Fewer macrophages and complete engulfment of GO in macrophages were also observed. Remnants from the acute inflammatory response were minimal and observed mainly as small spots with chronic inflammation in alveolar walls affecting approximately 2-4% of the total examined lung section surface area. Chronic inflammatory cells loaded with GO in the alveolar walls were observed (~ 2-4% of the cut lung surface). Perivascular lymphocytic accumulations were also observed. Birefringent collagen was not observed and there were no findings indicating lung fibrosis.

Measuring Saa3 mRNA levels as marker for an acute phase response in the lung found revealed that GO was a strong inducer of Saa3 expression. At day 1, GO induced statistically significantly increased Saa3 expression with 113, 45, and 9-fold increase compared to the vehicle control for 18, 54 and 162 µg/mouse, respectively. At day 3, the increase in mRNA expression levels of Saa3 was also statistically significant compared to the vehicle control, with 22, 33 and 49-fold increases in Saa3 for 18, 54 and 162 µg/mouse, respectively. At day 28 and day 90, no statistical significant increase in Saa3 was observed for GO compared to the vehicle control.
Furthermore, at day 3, a statistically significantly increased level of SAA3 protein in blood was observed for groups exposed to 54 and 162 µg GO/mouse compared to the vehicle control.
In addition, Saa1 mRNA levels were measured as markers for an acute phase response in the liver. GO induced statistically significantly increased Saa1 mRNA expression compared to the vehicle control. The highest induction of Saa1 mRNA was observed at day 1 post exposure, and maximal response was observed after treatment with 18 µg/mouse. At day 3, a statistically significant increased Saa1 expression was observed for the highest dose of GO (162 µg/mouse) compared to the vehicle control. At day 28 and day 90, no statistically significantly increased Saa1 expression was observed.
Conclusions:
Intratracheal instillation of GO in mice induced acute pulmonary inflammation and a strong pulmonary and hepatic acute phase response.
Executive summary:

The present study investigated the toxicity of 2-3 layered >1 µm sized graphene oxide (GO) in mice with respect to pulmonary inflammation and acute phase response (biomarker for risk of cardiovascular disease) following a single intratracheal exposure. Toxicity was evaluated at day 1, 3, 28 and 90 days (18, 54 and 162 µg/ mouse), except for GO exposed mice at day 28 and 90 where only the lowest dose was evaluated due to the discomfort and weight loss of mice following exposure to higher doses of GO.

Histopathological analysis and the measurement of markers for an acute phase response revealed a potent acute inflammatory response caused by GO, peaking at day 3 post exposure to GO.

Histopathological analyses of lung sections showed a severe acute inflammation at day 3, and large focal deposits of GO in the lung parenchyma. The alveolar walls were enlarged and neutrophils were found together with congested capillaries and alveolar granular exudate as part of the acute inflammation, whereas areas devoid of dusts were mainly without pathological findings. At day 90, GO agglomerates were still present in the lungs, though only minimal inflammation and no sign of fibrosis were observed. This effect was accompanied by a strong and transient increase in pulmonary and hepatic mRNA levels of acute phase response genes (Saa3 and Saa1, respectively), and in addition also increased levels of SAA3 protein were found in blood at day 3. The observed high acute toxicity of GO peaking at day 3 was further reflected in the decrease in bodyweight and signs of discomfort for mice exposed to 54 and 162 µg/mouse.

In conclusion, intratracheal instillation of GO induced inflammation and an acute phase response in mice.

Data source

Reference
Reference Type:
publication
Title:
Differences in inflammation and acute phase response but similar genotoxicity in mice following pulmonary exposure to graphene oxide and reduced graphene oxide
Author:
Bengtson S. et al.
Year:
2017
Bibliographic source:
PLOS ONE 12(6): e0178355. https://doi.org/10.1371/journal.pone.0178355

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Deviations:
yes
Remarks:
only one exposure, no early sampling at 2-6 h after treatment but sampling at 1, 3, 28 or 90 days post exposure
GLP compliance:
not specified
Type of assay:
mammalian comet assay

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
Details on test material:
Graphene oxide (GO) was manufactured and supplied by Graphenea (San Sebastian, Spain). GO was synthesized by chemical exfoliation of synthetic graphite using a modified Hummer's method and later chemically reduced with ascorbate. GO was delivered in a water suspension.
Specific details on test material used for the study:
GO was prepared in 0.2 µm filtered, y-irradiated Nanopure Diamond UV water (Pyrogens: < 0,001 EU/mL, total organic carbon: < 3.0 ppb) added 0.1% Tween80® (TW80) to a final concentration of particles of 3.24 mg/mL. To achieve a homogenous dispersion, the final solution was then prepared by probe sonication on ice for 16 min with 10% amplitude (Branson Sonifier S-450D, Branson Ultrasonics Corp., Danbury, CT, USA) equipped with disruptor horn (model number 101-147-037). Following sonication, solution was further diluted to 1.08 mg/mL and sonicated for 2 minutes. Dilution was further diluted to 0.36 mg/mL and sonicated for 2 minutes. As vehicle control (VC), Nanopure water added 0.1% TW80 was prepared by procedure as described above. Suspensions were instilled in mice within 20 minutes after sonication.

Test animals

Species:
mouse
Strain:
C57BL
Details on species / strain selection:
C57BL/6J mice
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Taconic Europe (Ejby, Denmark)
- Age at study initiation: 7-weeks old
- Weight at study initiation: 19.7 ± 1 g
- Assigned to test groups randomly: grouped based on exposure and dose level (VC groups n = 8, exposed groups n = 7)
- Fasting period before study: not specified
- Housing: polypropylene cages with sawdust bedding and enrichment
- Diet (e.g. ad libitum): food (Altromin 1324) ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 1°C
- Humidity (%): 50 ± 10%
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12-h light and 12-h dark cycle

Administration / exposure

Route of administration:
intratracheal
Vehicle:
0.2 pm filtered, y-irradiated Nanopure Diamond UV water (Pyrogens: < 0,001 EU/ml, total organic carbon: < 3.0 ppb) added 0.1% Tween80
Details on exposure:
single intratracheal instillation (50 pl/mouse) under isoflurane sedation
Duration of treatment / exposure:
not specified
Frequency of treatment:
single treatment
Post exposure period:
1, 3, 28 or 90 days
Doses / concentrationsopen allclose all
Dose / conc.:
18 other: µg/mouse
Remarks:
corresponds to 0.91 mg/kg bw
Dose / conc.:
54 other: µg/mouse
Remarks:
corresponds to 2.74 mg/kg bw
Dose / conc.:
162 other: µg/mouse
Remarks:
corresponds to 8.22 mg/kg bw
No. of animals per sex per dose:
only female mice, vehicle control groups n = 8, exposed groups n = 7
Control animals:
yes, concurrent vehicle
Positive control(s):
Carbon Black Printex90 (P90), provided by Degussa (Frankfurt, Germany) was included as reference material (162 µg/mouse) based on findings from previous studies showing inflammatory and genotoxic response in mice following single instillation (Bourdon et al., 2012).
- Justification for choice of positive control(s): The authors routinely include P90 as a reference material (Kyjovska et al., 2015a, Kyovska et al., 2015b, Poulsen, 2015, Wallin et al., 2016, Saber et al., 2012a, Saber et al., 2012b, Saber et al., 2016, Saber et al., 2012c) and P90 has been described and characterized in detail (Saber, 2012a, Jacobsen et al., 2008).
- Route of administration: intratracheal
- Doses / concentrations: 162 µg/mouse, corresponds to 8.22 mg/kg bw

Examinations

Tissues and cell types examined:
Bronchoalveolar lavage, lung, liver
Details of tissue and slide preparation:
All mice were anesthetized by i.p. injection of 0.1 mL ZRF solution (Zoletil 250 mg, Rompun 20 mg/mL, Fentanyl 50mg/mL in sterile isotone saline). Blood was withdrawn from the heart and stabilized using 36 µl K2EDTA and followed by collection of BAL where lungs were flushed twice with 0.8 ml 0.9% sterile saline. Total BAL recovery was about 1.4 mL. BAL samples were immediately stored on ice until further preparation. BAL cells were prepared on glass slides and stained with May-Grünewald-Giemsa staining. Details about preparation method have been described previously (Kyovska et al. 2015a). Images of BAL cells were acquired at 100x on an Olympus BX 43 microscope with a Qimaging Retiga4000R camera. Uneven illumination in brightfield images was corrected using ImageJ (Schneider et al., 2012) and the Calculator Plus plugin via the formula: Corrected image = (Image / background) * 255. The background image was a maximum projection of 3 background brightfield images without BAL cells.
Statistics:
Statistical analysis was performed in Minitab v.17.1.0 (Minitab Inc., State College, PA, USA), except for neutrophils where SAS®, version 9.3 for the Windows platform was used. All data are presented as mean ± standard error of the mean (Mean±SEM).
Statistical analysis of DNA damage (%DNA) was performed on data normalized to the mean %DNA of PBS-exposed A549 cells on slides included in each electrophoresis.

Results and discussion

Test results
Sex:
female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
other: Significant increases in DNA strand breaks in BAL at day 3 and in liver at day 90.

Any other information on results incl. tables

Table 4. DNA strand breaks. Level of DNA damage (Mean ± SEM) in BAL, lung and liver assessed with the comet assay (% DNA) 1, 3, 28 and 90 days post exposure to VC, GO, rGO or P90 (n = 7-8).

    Day 1 Day 3 Day 28 Day 90
  Dose BAL
VC 0 0.44 ± 0.06 0.60 ± 0.04 1.19 ± 0.17 1.00 ± 0.11
GO 18 0.61 ± 0.03 0.98 ± 0.04* 1.87 ± 0.15* 1.18 ± 0.13
54 0.54 ± 0.03 0.68 ± 0.03  -   - 
162 0.55 ± 0.07 0.51 ±0.06  -   - 
P90 162 0.39 ± 0.02 0.75 ± 0.02** 0.87 ± 0.06 0.81 ± 0.05
    Lung
VC 0 0.54 ± 0.02 0.97 ± 0.08 1.47 ± 0.12 2.05 ± 0.16
GO 18 0.46 ± 0.04 0.99 ± 0.07 1.67 ± 0.18 1.64 ± 0.13
54 0.42 ± 0.03 1.09 ± 0.06  -   - 
162 0.43 ± 0.04 1.21 ± 0.14  -   - 
P90 162 0.78 ± 0.12 1.04 ± 0.07 1.58 ± 0.08 2.15 ± 0.19
    Liver
VC 0 1.75 ± 0.12 1.56 ± 0.21 1.04 ± 0.10 0.66 ± 0.03
GO 18 1.71 ± 0.30 1.62 ± 0.13 0.87 ± 0.09 0.72 ± 0.03
54 2.06 ± 0.20 1.92 ± 0.26  -   - 
162 1.56 ± 0.22 1.65 ± 0.15  -   - 
P90 162 1.43 ± 0.19 1.62 ± 0.19 0.80 ± 0.10 0.91 ± 0.04**

Data were normalized to the mean level of %DNA of H2O2-exposed A549 cells included on each slide during each electrophoresis. All samples from BAL, lung and liver were divided onto different electrophoresis and further divided according to time point post exposure to minimize day-to-day variation.

*, **, ***: statistically significantly different from corresponding VC at level p < 0.05, p < 0.01, p < 0.001, respectively.

Applicant's summary and conclusion

Conclusions:
The test item graphene oxide did not show a consistent pattern of a genotoxic potential in this in vivo Comet assay in mice.
Executive summary:

The genotoxic potential of the test item graphene oxide was examined in an in vivo Comet assay in mice. The study conduct was generally comparable to OECD TG 489, however sampling was at 1, 3, 28 and 90 days after a single intratracheal exposure, and Carbon Black Printex90 was used as positive control.

Mice were exposed to 18, 54 and 162 µg graphene oxide/animal. DNA strand breaks were examined in bronchoalveolar lavage cells, lung and liver cells. At 1 day post exposure, no significant increase was observed for any concentration of graphene oxide in BAL, lung or liver. At day 3 post exposure, no statistically significant increase was observed in lung and liver, while the result obtained for 18 µg/mouse reached statistical significance. However, this effect was not concentration-dependent and was accompagnied by an acute inflammatory response in lung cells at day 3. DNA strand breaks induced by 18 µg graphene oxide/mice in BAL cells were also statistically significant (p < 0.05) at day 28 post-exposure, however due to the acute toxicity observed at day 3, only animals exposed to the lowest concentration could be further observed after day 3. Therefore, a concentration-dependence cannot be determined from these data at the later time points, and in addition, cytotoxicity was not determined. Therefore, the biological significance and relevance of this effect cannot be assessed from these data. No statistically significant increase in DNA strand breaks was observed in BAL cells after 90 days, and in lung and liver at 28 days and 90 days post exposure to graphene oxide.

Therefore, these data do not provide a clear evidence of a genotoxic potential of the test item graphene oxide in mice after intratracheal instillation.

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