Graphene

Endpoint summary

Description of key information

Oral: Key, acute fixed dose method comparable to OECD TG 420, mouse 5000 mg/kg bw, single dose (gavage): LD50 > 2000 mg/kg bw (Mingjing, 2015)

Inhalation: Key, acute toxicity class method according to OECD TG 436, GLP, rat, single dose (inhalation): LC50 > 1.99 mg/L (the max. achieved aerosol concentration) (Zabaiou, 2020)

Dermal: no study required (Waiver)

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 Jun 2015 to 17 Jul 2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 420 (Acute Oral Toxicity - Fixed Dose Method)

GLP compliance:

not specified

Remarks:

no information given

Test type:

fixed dose procedure

Limit test:

yes

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Laboratory Animal Center of Jiangsu University (Certificate: SCXK (SU) 2013-0011)
- Weight at study initiation: 18.3 - 22.0 g
- Fasting period before study: animals fasted overnight
- Housing: housed in solid-floor polypropylene cages with sawdust bedding
- Diet (e.g. ad libitum): free access to food (Rat and Mouse Common Formula Diet, supplied by Suzhou Shuangshi Laboratory Animal Feed Science Co.,Ltd. (Certificate: SUSISHENG (2009) 05032).
- Water (e.g. ad libitum): free access to drinking water
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2 °C
- Humidity (%): 40 - 70 %

Route of administration:

oral: gavage

Vehicle:

not specified

Details on oral exposure:

MAXIMUM DOSE VOLUME APPLIED: 20 mL/kg bw

Doses:

5000 mg/kg bw

No. of animals per sex per dose:

5000 mg/kg bw: 10 female/ 10 male mice

Control animals:

not specified

Details on study design:

- Duration of observation period following administration: 14 days (5000 mg/kg bw)
- Frequency of observations: daily
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight, histopathology- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: within the first 4 hours, and thereafter at least once daily
- Other examinations performed: clinical signs

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Mortality:

5000 mg/kg bw: female: 0/10, male: 0/10

Clinical signs:

other: no sign of systemic toxicity detected

Interpretation of results:

other: EU GHS criteria not met

Conclusions:

For the test item graphene the oral LD50 of both male and female mice is higher than 2000 mg/kg bw.

Executive summary:

Study design

Acute oral toxicity was analysed according to the Chemical toxicity test instruction (2005), Ministry of Health of the People's Republic of China, which is in large part equivalent to method described in OECD TG 420.

Ten male and female ICR mice received doses of 5000 mg/kg bw graphene by gavage.

Results and discussion

No acute toxicity was observed at 5000 mg/kg b.w., the LD50 was found to be >2000 mg/kg b.w.

Conclusion

A classification according to Regulation (EC) 1272/2008 is not warranted.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

Both available studies are of high quality: Mingjing, 2015 is comparable with OECD TG study and Sanders 2018 is compliant with OECD TG method and GLP.

Acute toxicity: via inhalation route

Link to relevant study records

Reference

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

Reason / purpose for cross-reference:

reference to other study

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

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)

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.

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.

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

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.

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.

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).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LC50

Value:

1 990 mg/m³ air

Quality of whole database:

The available study is of high quality: it is a fully reliable OECD TG study in compliance with GLP.

Acute toxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Acute toxicity: oral

In a key study, acute oral toxicity analysed according to the Chemical toxicity test instruction (2005), Ministry of Health of the People's Republic of China, which is in large part equivalent to method described in OECD TG 420 (Mingjing, 2015). Ten male and female ICR mice received doses of 5000 mg/kg bw graphene by gavage.

No acute toxicity was observed at 5000 mg/kg b.w., the LD50 was found to be >2000 mg/kg bw.

 

A supporting study was performed according to OECD TG 420 and in compliance with GLP (Sanders, 2018). In the study five fasted female animals were given four oral doses (at hourly intervals) of the test item, as a suspension in arachis oil BP, at a dose level of 75 mg/kg body weight to give a total maximum attainable dose level of 300 mg/kg. Clinical signs and body weight development were monitored during the study. All animals were subjected to gross necropsy.

No deaths were observed and there were no signs of systemic toxicity. Black feces were noted during the observation period. All animals showed expected gains in body weight and no abnormalities were noted at necropsy.

The acute oral median lethal dose (LD50) of the test item in the female Wistar strain rat was found to be greater than 300 mg/kg body weight (maximum attainable concentration). The result can only be used to a limited extend for evaluation, because of the low maximum attainable dose level of 300 mg/kg.

 

 

Acute toxicity: inhalation

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).

Justification for classification or non-classification

Based on the available data, the registered substance does not require classification and labelling for lethal effects following a single oral or inhalation exposure according to Regulation (EC) No 1272/2008.