Graphene

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Jan 2020 to 23 Apr 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Combined In Vivo Mammalian Erythrocyte Micronucleus Test and Alkaline Comet Assay in Rat

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Feb 2020 to 28 Apr 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes

Remarks:

The study was conducted according to GLP, except for characterization and stability of the test item, which was performed according to established SOPs, controls, and approved test methodologies to ensure integrity and validity of the results generated

Type of assay:

mammalian erythrocyte micronucleus test

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD (SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Canada Inc, St-Constant, QC, Canada.
- Age at study initiation: 49-50 days old
- Weight at study initiation: 181 to 216 (males) and 163 to 184 g (females)
- Assigned to test groups randomly: yes, under following basis: the weight variation of animals was confirmed to be minimal as it did not exceed ± 20% of the mean weight
- Housing: Animals were group housed (up to 3 animals of the same dosing group together) in polycarbonate cages containing appropriate bedding equipped with an automatic watering valve. These housing conditions were maintained for the duration of the study. Control group animals were housed on a separate rack from the test item-dosed animals.
- Diet (e.g. ad libitum): ad libitum throughout the study, except during designated procedures
- Water (e.g. ad libitum): Municipal tap water after treatment by reverse osmosis and ultraviolet irradiation was freely available to each animal via an automatic watering system (except during designated procedures).
- Acclimation period: A period of 7 to 8 days was allowed between animal receipt and the start of treatment in order to acclimate the animals to the laboratory environment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19°C to 25°C
- Humidity (%): 30% to 70%
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

Route of administration:

inhalation

Details on exposure:

TYPE OF INHALATION EXPOSURE: nose only

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Flow-through chamber, chamber type 4 with 2 levels per chamber and 40 breathing ports
- Method of holding animals in test chamber: Restraint tubes
- System of generating particulates/aerosols: Extended Duration Powder Delivery System with Jet Mill and Cycloneb
- Temperature, humidity, pressure in air chamber: 19-25°C, 30-70% relative humidity, and at least 19% O2

TEST ATMOSPHERE
- Brief description of analytical method used: Real-time Qualitative Monitoring: microdust; Aerosol Concentration Analysis, (Substrate/Diameter): Glass fiber/25 mm; Particle Size Analysis (Substrate/Diameter): Stainless steel plates/34 mm

Duration of treatment / exposure:

3 days at 0, 24 and 45 hours (±15 minutes), once daily for up to 240 minutes

Frequency of treatment:

3 days at 0, 24 and 45 hours (±15 minutes), once daily for up to 240 minutes

Dose / conc.:

0 mg/L air

Remarks:

Control

Dose / conc.:

0.5 mg/L air

Dose / conc.:

1 mg/L air

Dose / conc.:

2 mg/L air

Remarks:

according to the acute inhalation study, 2 mg/L air was the maximum respirable practical dose.

No. of animals per sex per dose:

5 per sex per dose

Control animals:

yes

Positive control(s):

Positive control: Cyclophosphamide (CP)
- Doses / concentrations: 20 mg/kg at 10 mL/kg given 24 hours prior to sampling

Tissues and cell types examined:

Both femurs (groups 1 to 4) were dissected from each main study animal. Several bone marrow cell smears were prepared from each animal. The bone marrow from both femurs of each animal was pooled/eluted in 5 mL Hanks Balance Salts Solution. The resulting cell suspensions were processed for the micronucleus test.

Microscopic examination: A total of 4000 immature erythrocytes per animal were examined for the presence of micronuclei.
In addition, the proportion of immature erythrocytes was assessed by examination of a total of at least 500 erythrocytes per animal. The incidence of any micronucleated mature erythrocytes observed during this assessment was recorded as a check for potential micronucleus-like artifacts.

Details of tissue and slide preparation:

PROCESSING
Several bone marrow cell smears were prepared from each animal.

DETAILS OF SLIDE PREPARATION
The slides were fixed in methanol and stained with the fluorescent metachromatic dye, acridine orange for micronucleus test evaluation.
Positive control slides taken from 3 Sprague Dawley male rats previously dosed with positive control (cyclophosphamide, 20 mg/kg at 10 mL/kg given 24 hours prior to sampling), as part of a CR-MTL GLP-compliant study, were added to the study slides for evaluation as scoring controls. Data from these animals were entered in the study as a positive control group called Group 6 for use in statistical evaluation. Arbitrary animal numbers were given based on the standard animal identification scheme. No slides from the positive control Group 6 were archived.

METHOD OF ANALYSIS
Microscopic Examination: The slides were encoded to minimize potential operator bias and then examined by fluorescence microscopy using a blue excitation filter and a yellow barrier filter. A total of 4000 immature erythrocytes per animal were examined for the presence of micronuclei. One smear was examined per animal, the remaining smears being held temporarily as reserves in case of technical problems with the first smear.

Evaluation criteria:

CRITERIA FOR BOTH COMET AND MICRONUCLEUS ASSAY

ACCEPTABILITY CRITERIA: concurrent positive and negative controls are within the distribution of the historical control data and positive control induce a statistically significant increase compared with the concurrent negative control; an appropriate number of doses and cells has been analysed (4000 PCE per animal for micronucleus test or 150 cells per tissue per animal for comet assay); The maximum dose tested is one that allows maximum exposure up to 2000 mg/kg/day for non-toxic compounds, or the limit of solubility, respirability or MTD based on available toxicity data, or the highest dose may also be defined as a dose that produces toxicity in the bone marrow.

CLEARLY POSITIVE
1. At least one of the test item groups exhibits a statistically significant increase in the frequency of MN-PCE or % tail DNA compared with the concurrent negative control.
2. The increase is dose-related when evaluated with an appropriate trend test.
3. Any of the results are outside the distribution of the historical negative control data (e.g. 95% control limits).
Conclusion: The test item is then considered able to induce DNA strand breakage/chromosomal damage or damage to the mitotic apparatus in the tissue studied in this test system.

CLEARLY NEGATIVE
1. None of the test item groups exhibits a statistically significant increase in the frequency of MN-PCE or % tail DNA compared with the concurrent negative control.
2. All results are inside the distribution of the historical negative control data (e.g. 95% control limits).
3. Direct or indirect evidence supportive of exposure of, or toxicity to, the target tissue(s) has been demonstrated.
Conclusion: The test item is then considered unable to induce DNA strand breakage/chromosomal damage or damage to the mitotic apparatus in the tissue studied in this test system.

Statistics:

All statistical tests were conducted at the 5% significance level. The pairwise comparisons were reported at the 0.1%, 1%, and/or 5% levels. Result was considered to be significant if p ≤ 0.05.

Numerical data collected on scheduled occasions were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation were reported whenever possible.
STATISTICS FOR BOTH COMET AND MICRONUCLEUS ASSAY

The numerical data corresponding to the MN-PCE frequency and % tail DNA was statistically evaluated using two separate datasets. The first one included the negative control group and the test item treated groups, whereas, the second one included only the negative control group and the positive control group.

For the first dataset, Levene’s test was used to assess the homogeneity of group variances. If the result of Levene’s test was not found to be significant (p > 0.05), then the groups were compared using an overall one-way ANOVA F-test, otherwise they were compared using Kruskal-Wallis test. If the overall ANOVA F-test or Kruskal-Wallis test was found to be significant (p ≤ 0.05),conducted using Dunnett’s or Dunn’s test, respectively.

For the second dataset, Levene’s test was used to compare the variances of negative and positive control groups. If the result of Levene’s test was not found to be significant (p > 0.05), then the groups were compared using t-test, otherwise, they were compared using Wilcoxon Rank-Sum
test.

Whenever the results of MN-PCE and % tail DNA, corresponding to test item treated groups, reveal significant mean increase when compared to the negative control group, then a doserelated increase trend in means across the negative control group and the test item treated groups would be conducted using Cochran-Armitage’s one-sided.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

There was no mortality, and no remarkable treatment-related clinical signs were noted during the study. Low body weight gain was observed at the highest respirable dose level (actual 1.92 mg/L) which is indicative of systemic toxicity.

Negative controls validity:

valid

Positive controls validity:

valid

Achieved chamber conditions:

Individual mass median aerodynamic diameter (MMAD) of generated aerosols ranged from 2.9 to 5.2 μm. Although this was higher than the targeted 4.0 μm aerosol, generated aerosols were considered acceptable given that the majority of particle size assessments described at least 30% of particles with a diameter < 3.5 μm. On the three consecutive days of inhalation dosing animals in Groups 1 to 4 were exposed to a stable and respirable aerosols of the test item at either 0, 0.547, 1.00 or 1.92 mg/L.

Results of In-life Procedures, Observations, and Measurements

- Mortality: No mortalities occurred during this study.

- Clinical Observations: There were no test item-related clinical signs observed during the dosing and observation period. Black fur staining appeared during the dosing period, and were no longer present at the end of dosing or remained present with slight severity 1 hour post the end of dosing. This observation can be attributed to the nose-only administration of the test item to the animals using a Flow-Through rodent system, and considered non-adverse because of their transient nature.

All other clinical observations were considered unrelated to the test item based on their low and/or sporadic incidence.

- Body Weights and Body Weight Gains: The body weight increased during the pre-treatment period for all the animals. During the treatment period, there was low body weight gain at 1.92 mg/L. The body weight gain values for Days -1 to 3 for the negative control group and test item dose level groups 0.547, 1.0, and 1.92 mg/L were for males: 37.0, 31.8, 35.4 and 23.2 g and for females: 19.8, 19.4, 17.6 and 16.8 g, respectively.

This low body weight gain is indicative of systemic toxicity.

Proportion of Polychromatic Erythrocytes

Animals treated with the test item did not show any notable decreases in %PCE/TE, indicating that graphene did not cause bone marrow toxicity following exposure by nose-only inhalation for 240 minutes for 3 days to male and female rats. Group mean values for %PCE/TE (51.7 to 61.4% for males and 49.3 to 54.7% for females) were within the historical negative control range of 27.2 to 72.9% and 28.7 to 62.9% for males and females, respectively.

Micronucleated Polychromatic Erythrocytes

Male and female rats treated with the test item did not show any statistically significant increases in the frequency of MN-PCE. The group mean values for negative and test item dose levels 0.547, 1.0, and 1.92 mg/L were 5.5, 3.4, 2.4 and 4.8 for males; and 4.0, 4.4, 5.2 and 4.2 for the females, respectively. These group mean values were below the 95% historical negative control upper limit (8.9 and 8.7 for males and females, respectively). Even though, a statistical significance was obtained in group 3 males, it is not considered biologically meaningful as the micronuclei frequency was even lower in this group than in the negative control group. Therefore, graphene did not induce chromosomal damage or damage to the mitotic apparatus as assessed by micronuclei formation in bone marrow polychromatic erythrocytes of male and female rats, when exposed by nose-only inhalation for 240 minutes for 3 days to male and female rats up to 1.92 mg/L which is considered the maximum respirable dose level. This dose level induced low body weight gain which is indicative of systemic toxicity.

Conclusions:

In conclusion, the graphene test material did not induce the formation of micronuclei in polychromatic erythrocytes in the micronucleus test, when administered by nose-only inhalation for 240 minutes for 3 days to male and female rats up to 1.92 mg/L, the maximum respirable practical dose. Also this dose level induced low body weight gain which is indicative of systemic toxicity.

Executive summary:

Study design

The objective of this GLP-study was to determine the potential genotoxicity of graphene when given by nose-only inhalation for 240 minutes for 3 days in rats using the bone marrow micronucleus test and the comet assay with the lung using OECD 474 and 489 guidelines.

In the study, rats were dosed using target concentrations of 0.5, 1, 2 mg/L of graphene (5 animals/sex/group). The dose levels were selected based on the results of acute inhalation toxicity study (Zabaiou, 2020), where it was determined that the test item at 2 mg/L is the maximum respirable practical dose. 20 mg/mL (10 mL/kg) Ethyl methanesulfonate (EMS) or cyclophosphamide was used as positive controls (3 male animals). Bone marrow and lung were collected 3 hours after the last treatment.

 

Results

The generated aerosols of graphene were considered to be within the respirable range for the rats and the target concentrations were achieved during the three consecutive days of dosing by inhalation. There was no mortality, and no remarkable treatment-related clinical signs were noted during the study. Low body weight gain was observed at the highest respirable dose level (actual 1.92 mg/L) which is indicative of systemic toxicity. A total of 4000 immature polychromatic erythrocytes (PCEs) were evaluated for presence of micronuclei indicative of chromosome damage. In addition, the proportion of immature erythrocytes in the total of at least 500 erythrocytes per animal was assessed as a measure of potential bone marrow toxicity. The proportion of polychromatic erythrocytes and the frequencies of micronucleated polychromatic erythrocytes (MN-PCE) in the negative control group were within the laboratory historical negative control range. The positive control, cyclophosphamide, induced a clear and unequivocal increase in micronuclei. The results from both the negative and positive controls confirmed the validity of the assay. Animals treated with the test item did not show statistically significant increases in the incidence of MN-PCE, indicating that graphene did not induce chromosome damage in this in vivo assay. In addition, there were no substantial decreases in the proportion of immature erythrocytes (PCE) indicating that the test item was non-toxic to the bone marrow at the levels tested. For the Comet assay, the lung was collected 3 hours after final dose administration from each animal. A total of 150 cells per animal (149 cells for one animal of group 2) were analyzed for % tail DNA. The positive control, EMS, induced a clear and unequivocal increase in the incidence of % tail DNA. The mean % tail DNA in the negative control group was within the laboratory historical negative control range. The results from both the negative and positive control for the Comet assay confirmed the validity of the assay. Animals treated with graphene did not show any notable increases in the % tail DNA in the lung tissue. Additionally, individual and group mean values for animals treated with graphene were close to the laboratory historical control range. Thus, there was no evidence of an increase in DNA damage attributed to exposure to the test item in this assay.

 

Conclusion

In conclusion, the graphene test material did not induce the formation of micronuclei in polychromatic erythrocytes in the micronucleus test, when administered by nose-only inhalation for 240 minutes for 3 days to male and female rats up to 1.92 mg/L, the maximum respirable practical dose. Also this dose level induced low body weight gain which is indicative of systemic toxicity.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)

Version / remarks:

adopted 7 September 2009

Deviations:

no

GLP compliance:

yes

Test type:

acute toxic class method

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

carbon

EC Number:

801-282-5

Cas Number:

1034343-98-0

Molecular formula:

Cx

IUPAC Name:

carbon

Test material form:

solid: nanoform

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Canada Inc., St. Constant, QC, Canada.
- Age at study initiation: 10 to 11 weeks old and
- Weight at study initiation: animals weighed between 390 to 483 g (males) and 282 to 308 g (females)
- Housing: Group-housed (3 animals of the same sex and same dosing group together) in Polycarbonate cages containing appropriate bedding (Color-coded cage card indicating study, group, animal number(s), and sex)
- Diet: ad libitum (Lab Diet Certified CR Rodent Diet 5CR4)
- Water: ad libitum (Municipal tap water, treated by reverse osmosis and ultraviolet irradiation)
- Acclimation period: acclimation period of 18 or 22 days was allowed between animal receipt and the start of dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25°C
- Humidity (%): 30% to 70%
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark (except during designated procedures)

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 3.5 - <= 5.3 µm

Geometric standard deviation (GSD):

>= 2.1 - <= 2.5

Remark on MMAD/GSD:

The mass median aerodynamic diameter (MMAD) of generated aerosols ranged from 3.5 to 5.3 μm for Group 1 and 3.9 to 4.2 μm for Group 2. Although Group 1 and 2 was higher than the targeted 4.0 μm aerosol, they were considered respirable given that 31% and 43% was of a diameter < 3.5 μm.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Flow-Through Chamber
- Source and rate of air (airflow): Input Flow rate (L/min): 40, Exhaust Flow rate (L/min): 50
- System of generating particulates/aerosols: Extended Duration Powder Delivery System with Jet Mill and Cyclone
- Method of particle size determination: Particle Size Analysis (substrate and diameter): Stainless steel plates/34 mm, Cascade Impactor (Particle Size Analysis): Marple-style
- Temperature, humidity, pressure in air chamber: 19-25°C, 30-70% Relative Humidity, and at least 19% O2 (During acclimation on air, chamber humidity environmental condition was out of range. It was recorded to be 21.7% and 22.6% but the minimum allowed is 30%. There was no impact on resultant aerosol, or therefore on the integrity of the study)

TEST ATMOSPHERE
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): The mass median aerodynamic diameter (MMAD) of generated aerosols ranged from 3.5 to 5.3 μm for Group 1 and 3.9 to 4.2 μm for Group 2. Although Group 1 and 2 was higher than the targeted 4.0 μm aerosol, they were considered respirable given that 31% and 43% was of a diameter < 3.5 μm.

CLASS METHOD (if applicable)
- Rationale for the selection of the starting concentration: Initially one group of 3 males and 3 females will be treated with the mid-high aerosol concentration (1.0 mg/L) for 240 minutes. Depending on the results obtained after 72 hours, if well tolerated the next group should be treated (5.0 mg/L) for 240 minutes. However, the maximum target aerosol concentration that could be achieved while maintaining a particle size (mass median aerodynamic diameter) that ranged from between 1 to 4 μm was 2 mg/L instead of 5.0 mg/L.

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

240 min

Concentrations:

1 mg/L, 2 mg/L (nominal) / 0.878 mg/L, 1.99 mg/L (analytical)

No. of animals per sex per dose:

3/sex/dose

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Mortality/Moribundity was checked twice daily (morning and afternoon) starting upon arrival through termination. Detailed clinical observations were made twice during the prestudy period, and weekly thereafter. Cage side observations were made immediately post each dose after the end of each animal (within 5 minutes), and at 30 and 60 minutes post each dose after the end of each animal, and daily thereafter. Body weights were observed twice during the prestudy period, on Days 1, 2, 4, 8, and 14
- Necropsy of survivors performed: yes
- Clinical signs including body weight
- Other examinations performed: food consumption, histopathology (Lung, Lymph node tracheobronchial)

Statistics:

All results presented in the tables of the report were calculated using non-rounded values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.
Any data collected during the prestudy period was tabulated, summarized. Numerical data collected on scheduled occasions was summarized and statistically analyzed as indicated below according to sex and occasion.

Results and discussion

Preliminary study:

The maximum target aerosol concentration that could be achieved while maintaining a particle size (mass median aerodynamic diameter) that ranged from between 1 to 4 μm was 2 mg/L. Additional aerosol optimization of 0.5 and 2 mg/L was performed post dosing and in the context of Study No. 9800693 (pre-study). The mass median aerodynamic diameter (MMAD) of generated aerosols ranged from 3.5 to 5.3 μm for Group 1 and 3.9 to 4.2 μm for Group 2. Although Group 1 and 2 were higher than the targeted 4.0 μm aerosol, they were considered respirable given that 31% and 43% was of a diameter < 3.5 μm.

Effect levels

Effect levels 1

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 1.99 mg/L air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Remarks on result:

not determinable due to absence of adverse toxic effects

Effect levels 2

Sex:

male/female

Dose descriptor:

other: NOAEL

Effect level:

1.99 mg/L air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Mortality:

There were no deaths during the course of this study.

Clinical signs:

other: please refer to 'any other information on results'

Body weight:

There were no test item-related changes in body weight parameters observed during the dosing and observation period. A transient weight loss was observed in females at ≥ 0.878mg/L on Day 2. The animals had regained their weight as on Day 3.
Any other differences noted were considered related to individual animal variability.

Gross pathology:

Test item-related gross pathology findings were noted in the lung (mottled discoloration) of males at 1.99 mg/L and females at ≥ 0.878 mg/L and tracheobronchial lymph node (dark focus) in 1 male at 0.878 mg/L. The mottled discoloration of the lung and dark focus in the tracheobronchial lymph node were correlated microscopically with the presence of pigments. Enlargement of the tracheobronchial lymph node was also noted in 1 male at 1.99 mg/L and 1 female at 0.878 mg/L. There were, however, no microscopic correlates and it was therefore considered unlikely related to the administration of the test item. Other gross findings observed were considered incidental or of the nature commonly observed in this strain and age of rats and, therefore, were considered unrelated to administration of the test item.

Other findings:

- Histopathology: Pigmented macrophages were noted within the alveoli in all animals at ≥ 0.878 mg/L. The macrophages were often forming small aggregates and occasionally increased in numbers (mild in severity). The observed pigment was black, granular or crystalloid in appearance. Granulomas were also noted at ≥ 0.878 mg/L in males and at 1.99 mg/L in females. The granulomas observed were minimal in severity in all animals except in 1 male at 1.99 mg/L (Animal No. 2001) (mild). The granulomas often contained foreign material which were considered exacerbation due to the load of the test item or the experimental procedure (inhalation). Pigmented macrophages were correlated with the observed gross findings (discoloration, mottled).
Similar pigment was also noted in the tracheobronchial lymph node of both males and females at ≥ 0.878 mg/L. The exact location of the pigment deposition remains uncertain, but likely located in the cytoplasm of macrophages. The pigment was correlated with the gross finding noted in 1 male at 1.99 mg/L (focus, dark). Other microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rats and, therefore, were considered unrelated to administration of the test item.
- Other observations:
Food Consumption: There were no test item-related effects on food consumption observed during the dosing and observation period. Any differences noted were considered related to individual animal variability.

Any other information on results incl. tables

Clinical signs:

There were no test item-related clinical signs observed during the dosing and observation period.

Animals given 0.878 or 1.99 mg/L were noted with black fur staining (muzzle, lower jaw, cranium, periorbital areas, forelimbs, dorsal cervical and thoracic areas, ventral cervical and thoracic areas, and/or abdominal regions). These clinical signs appeared during the dosing period, and were no longer present at the end of dosing or remained present with slight severity 1 hour post the end of dosing. These clinical signs described can be attributed to the nose-only administration of the test item to the animals using a Flow-Through rodent system, and considered nonadverse because of their transient nature.

All other clinical observations were considered unrelated to the treatment based on their low and/or sporadic incidence.

Applicant's summary and conclusion

Interpretation of results:

other: EU GHS criteria not met

Conclusions:

In conclusion, administration of the test item by nose-only inhalation in male and female rats at achieved aerosol concentration of 0.878 and 1.99 mg/L when given as a single dose was well tolerated. Given the lack of any adverse finding at any dose, the no-observed-adverse-effect-level (NOAEL) was considered to be 1.99 mg/L (the maximum achieved aerosol concentration feasible).

Executive summary:

Study design

The objectives of this study were to determine the acute toxicity of dry powder of graphene (nanoform), when given to the rat via nose-only inhalation for 240 minutes in single escalating doses (total of 2 doses) with at least 72-hour observation period between each dose, and to evaluate the potential reversibility of any findings following a 14-day observation period. The design of this study was based on the study objectives, the overall product development strategy for the test item, and OECD TG 436: Acute Inhalation Toxicity - Acute Toxic Class Method, in compliance with GLP.

Initially, 1 group of 3 males and 3 females received an aerosol concentration of 0.878 mg/L (target concentration of 1 mg/L) for 240 minutes. After 72 hours observation period, animals had tolerated the dose well, the next group received an aerosol concentration of 1.99 mg/L (target concentration of 2 mg/L) for 240 minutes. Each animal was observed for mortality and signs of a reaction for 14 days following dosing. A minimum of 72 hours was allowed between each group. The following parameters and end points were evaluated in this study: mortality, clinical observations, postdose observations, body weights, food consumption, macroscopic examinations, and microscopic evaluations of the lung and tracheobronchial lymph node. On the two discrete single dosing occasions, rats were exposed to a stable and respirable aerosol of the test item at either 0.878 or 1.99 mg/L.

Results

All rats survived to scheduled euthanasia. There were no treatment-related effects on the assessment of clinical observations, postdose observations, body weights, or food consumption. Administration of the test item resulted in macroscopic changes in the lung (mottled discoloration) in males at 1.99 mg/L and females at ≥ 0.878 mg/L and tracheobronchial lymph node (dark focus) in 1 male at 1.99 mg/L.

Administration of the test item resulted in microscopic changes in the lung and tracheobronchial lymph node. Presence of pigmented macrophages in the lungs, and pigments in the tracheobronchial lymph nodes were observed in both sexes at ≥ 0.878 mg/L.

Conclusion

In conclusion, administration of the test item by nose-only inhalation in male and female rats at achieved aerosol concentration of 0.878 and 1.99 mg/L when given as a single dose was well tolerated. The inhalation of the test item was associated with macroscopic findings in the lung (mottled discoloration) in males at 1.99 mg/L and females at ≥0.878 mg/L and tracheobronchial lymph node (dark focus) in 1 male at 1.99 mg/L. Microscopic findings in the lungs and tracheobronchial lymph nodes included the presence of pigmented macrophages in the lungs at ≥ 0.878mg/L, and granulomas at ≥ 0.878 mg/L, were considered exacerbation due to the experimental procedure (inhalation), and therefore, considered non-adverse. The mottled discoloration of the lung and dark focus in the tracheobronchial lymph node were correlated microscopically with the presence of pigments.

Given the lack of any adverse finding at any dose, the no-observed-adverse-effect-level (NOAEL) was considered to be 1.99 mg/L (the maximum achieved aerosol concentration feasible).