Multi-Walled Carbon Nanotubes (MWCNT), synthetic graphite in tubular shape

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

not indicated, year of publication: 2019

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

not indicated, year of publication: 2019

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 412 (28-Day (Subacute) Inhalation Toxicity Study

Version / remarks:

27 Jun 2018

Deviations:

yes

Remarks:

only male rats tested

GLP compliance:

not specified

Remarks:

Published study performed by University staff in the Republic of Korea

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: JEIO CO., LTP, 69, Namdong-daero 49beon-gil, Namdong-gu, Incheon, Korea. Lot: JC162
- The test material is identical to Jenotube 10B
- Purity, including information on contaminants, isomers, etc.: 98.6%

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: according to ISO TR 19601 (see reference below) under use of an acoustic dry aerosol generator (HCTm, Icheon, Korea): Aggregates or agglomerates of the test material were dispersed with acoustic energy. The amount of generated aerosol was adjusted by altering the frequency of the acoustic waves, the air supply flow rate and the quantity of the test material. A buffer chamber and cyclone trapped larger test materials. 2.5 g of MWCNT were in the generator. The generator used 8.5 LPM, and the total volume of air in each chamber was 25 LPM controlled by a mass flow controller (MFC, FC-7810CD-4V, AERA, Tokyo, Japan).

FORM AS APPLIED IN THE TEST:
aerosol
MMAD: low dose: 381 nm (GSD 2.34), moderate dose: 495 nm (GSD 3.08), high dose 1015 nm (GSD 2.81)
The difference in the MMAD among the concentrations was because one generator was used to dilute the high concentration to the moderate concentration and then to the low concentration.

INFORMATION ON NANOMATERIALS
Please refer to information in tables 1 and 2.

REFERENCE
ISO/TR 19601. Nanotechnologies International Organization for Standardization, Aerosol Generation for Air Exposure Studies of Nano-Objects and Their Aggregates and Agglomerates (NOAA). Geneva, Switzerland: ISO. 2017

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Specific Pathogen Free

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: OrientBio (Kyeonggi-do, Korea)
- Age at study initiation: 6 weeks
- Weight at study initiation: 179.75 +/- 0.34g
- Housing: polycarbonate cage (max. 3 rats /cage)
- Diet (e.g. ad libitum): no details available
- Water (e.g. ad libitum): no details available
- Acclimation period: 2 weeks, in a nose-only tube at 2 h/day during 4 days before initiation of the MWCNT exposure.
- Health status: specific-pathogen-free

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2 °C (housing); 22 ± 3 °C (inhalation chamber)
- Humidity (%): 55 ± 7% (housing); 30-70% (inhalation chamber)
- Air changes (per hr): not indicated
- Photoperiod (hrs dark / hrs light): 12/12
- Fasting period: Food and water were withheld during the 6-h exposure period

IN-LIFE DATES: not indicated; period of 56 days (28 d exposure, 28 d recovery period)

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

clean air

Remarks:

fresh filtered air

Mass median aerodynamic diameter (MMAD):

> 381 - < 1 015 nm

Geometric standard deviation (GSD):

3.08

Remark on MMAD/GSD:

For each dose, there exists one MMAD with GSD, respectively. Please see table 2.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: dry aerosol generator with exposure system
- Method of holding animals in test chamber: not indicated
- Source and rate of air: fresh filtered air, 8.5 LPM (generator), 25 LPM (total volume of air in each chamber), controlled by a mass flow controller (MFC, FC-7810CD-4V, AERA, Tokyo, Japan)
- Method of conditioning air: according to ISO 19601
- System of generating particulates/aerosols: acoustic dry aerosol generator (HCTm, Icheon, Korea)
- Temperature, humidity, pressure in air chamber: 22 ± 3 °C; 30-70%; no pressure indicated
- Air flow rate: 1 L/min
- Air change rate: not indicated
- Method of particle size determination: At least 400 fibers were counted using a field emission scanning electron microscope FESEM, Hitachi, Tokyo, Japan) to determine the mean particle length and diameter. The analysis used a cumulative method based on the count median diameter (CMD) and geometric standard deviation (GSD). The rigidity of the MWCNTs was measured using the method described in ISO/TS 11888. The bending ratio (Db) and static bending persistence length (SBPL) were measured from end to end (R) and along the axis (L) of the MWCNTs using FESEM.
- Treatment of exhaust air: not indicated

TEST ATMOSPHERE
- Brief description of analytical method used: The mass concentration of MWCNTs in chamber was gravimetrically determined with aPVC filter (polyvinyl chloride, size: 37mm and pore size 5.0 µm). During 30 min of the 6-h exposure period, two samples were collected from each concentration chamber. The MWCNT aerosol number concentrations were measured daily during the exposure period with a dust monitor (OPC, Model 1.1.09, Grimm Technologies Inc., Douglasville, GA, USA). The mass median aerodynamic diameter (MMAD) was measured using a foil fiber for each stage (diameter, 47 mm; pore size, 5 mm, SKC, Inc., Eighty Four, PA) with a Nano MOUDI impactor (MOUDI 125 NR, MSP Co., MN) composed of 13 stages (0.01, 0.018, 0.032, 0.056, 0.10, 0.18, 0.32, 0.56, 1.0, 1.8, 3.2, 5.6, and 10 mm). The geometric standard deviation (GSD) for the MMAD was derived from the cumulative mass distribution of the MOUDI. The elemental carbon (EC) concentrations in the generated MWCNT aerosols were measured in each chamber using an OC/EC analyzer (Sunset Laboratory Inc, SW) with a quartz filter (PallfelxVR tissuequartz filter, Pall Life Sciences, Port Washington, NY, USA) according to the NIOSH 5040. The MWCNT aerosols were collected on a TEM grid copper grid (Formvar/Carbon 200 mesh, TEDpella, CA) or holey grid (Quantifoil 656-200-Cu, Tedpella, Inc., Redding, CA) and were further examined under a field emission-transmission electron microscope (JEM2100F, JEOL, Tokyo, Japan) equipped with an energy dispersive X-ray analyzer (TM200, Oxford Instruments PLC, Oxfordshire, UK) at an acceleration voltage of 200 kV (according to NIOSH 7402).
- Samples taken from breathing zone: yes


REFERENCES
ISO/TR 19601. Nanotechnologies International Organization for Standardization, Aerosol Generation for Air Exposure Studies of Nano-Objects and Their Aggregates and Agglomerates (NOAA). Geneva, Switzerland: ISO. 2017
ISO/TS 11888. Nanotechnologies-Characterization of Multiwall Carbon Nanotubes- Mesoscopic Shape Factors. Geneva, Switzerland: ISO. 2017
NIOSH Manual of Analytical Methods 7402, Asbestos by TEM. Cincinnati, OH: NIOSH 1994
NIOSH Manual of Analytical Methods 5040, Diesel Particulate Matter (as Elemental Carbon). Cincinnati, OH: NIOSH 2003

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

6 h

Remarks on duration:

6h/day, 5 days/week, 4 weeks

Concentrations:

The analytical doses were 0, 0.257, 1.439 and 4.253 mg/m3

No. of animals per sex per dose:

20

Control animals:

yes

Details on study design:

- Dose selection rationale: not indicated
- Rationale for animal assignment: randomized by body weight
- After 28 days of exposure a 28-days post-exposure observation period (recovery period) was followed. At four time points (after one day of exposure (E-1), one day after 28 days of exposure (Post-exposure observation period, PEO-1), 7 days post exposure (PEO-7), 28 days post exposure (PEO-28)) during the study, 5 animals of each dose group were sacrificed, respectively.

Statistics:

SPSS version 19 (SPSS Inc, Chicago, IL) was used to perform the statistical analysis of the outcome parameters. Analysis of variance (ANOVA) following multiple comparison tests using the Dunnett T3 method was used for statistical evaluation. A p-value less than 0.05 was considered to be statistically significant.

Key result

Sex:

male

Dose descriptor:

LC0

Effect level:

4.253 mg/m³ air (analytical)

Based on:

test mat.

Mortality:

no mortality observed

Body weight:

no effects observed

Gross pathology:

no effects observed

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, this study conducted after OECD TG 412 assessing subacute inhalation toxicity of JENO TUBE 10B MWCNT in rats did not reveal any mortality nor signs of acute inhalation toxicity. Therefore, the LC 0 is concluded to be at the high dose level at 4.253 mg/m3.

Executive summary:

This study investigated the impact of sub-acute inhalation exposure of JENO TUBE 10B MWCNT on 80 male Sprague-Dawley rats after OECD TG 412. Rats were exposed nose-only for 6 h/day, 5 days/week for 4 weeks, followed by a post-exposure observation period (PEO) of 28 days. There were 20 animals per dosing group. The analytical doses were 0, 0.257, 1.439 and 4.253 mg/m3. At 4 time points during the study, 5 animals of each dose group were sacrificed, respectively and underwent necropsy procedure: organ weight, hematology, serum biochemistry, bronchoalveolar lavage, histopathology and lung burden was performed or examined. During the course of this study, there were neither signs of acute inhalation toxicity nor mortality observed. Therefore, the LC 0 is set to the high dose at 4.253 mg/m3.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Remarks:

sub-acute repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

not indicated, year of publication: 2019

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 412 (28-Day (Subacute) Inhalation Toxicity Study

Version / remarks:

27 Jun 2018

Deviations:

yes

Remarks:

only male rats tested

GLP compliance:

not specified

Remarks:

Published study performed by University staff in the Republic of Korea

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: JEIO CO., LTP, 69, Namdong-daero 49beon-gil, Namdong-gu, Incheon, Korea. Lot: JC162
- The test material is identical to Jenotube 10B
- Purity, including information on contaminants, isomers, etc.: 98.6%

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: according to ISO TR 19601 (see reference below) under use of an acoustic dry aerosol generator (HCTm, Icheon, Korea): Aggregates or agglomerates of the test material were dispersed with acoustic energy. The amount of generated aerosol was adjusted by altering the frequency of the acoustic waves, the air supply flow rate and the quantity of the test material. A buffer chamber and cyclone trapped larger test materials. 2.5 g of MWCNT were in the generator. The generator used 8.5 LPM, and the total volume of air in each chamber was 25 LPM controlled by a mass flow controller (MFC, FC-7810CD-4V, AERA, Tokyo, Japan).

FORM AS APPLIED IN THE TEST:
aerosol
MMAD: low dose: 381 nm (GSD 2.34), moderate dose: 495 nm (GSD 3.08), high dose 1015 nm (GSD 2.81)
The difference in the MMAD among the concentrations was because one generator was used to dilute the high concentration to the moderate concentration and then to the low concentration.

INFORMATION ON NANOMATERIALS
Please refer to information in tables 1 and 2.

REFERENCE
ISO/TR 19601. Nanotechnologies International Organization for Standardization, Aerosol Generation for Air Exposure Studies of Nano-Objects and Their Aggregates and Agglomerates (NOAA). Geneva, Switzerland: ISO. 2017

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Specific Pathogen Free

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: OrientBio (Kyeonggi-do, Korea)
- Age at study initiation: 6 weeks
- Weight at study initiation: 179.75 +/- 0.34g
- Housing: polycarbonate cage (max. 3 rats /cage)
- Diet (e.g. ad libitum): no details available
- Water (e.g. ad libitum): no details available
- Acclimation period: 2 weeks, in a nose-only tube at 2 h/day during 4 days before initiation of the MWCNT exposure.
- Health status: specific-pathogen-free

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2 °C (housing); 22 ± 3 °C (inhalation chamber)
- Humidity (%): 55 ± 7% (housing); 30-70% (inhalation chamber)
- Air changes (per hr): not indicated
- Photoperiod (hrs dark / hrs light): 12/12
- Fasting period: Food and water were withheld during the 6-h exposure period

IN-LIFE DATES: not indicated; period of 56 days (28 d exposure, 28 d recovery period)

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

clean air

Remarks:

fresh filtered air

Mass median aerodynamic diameter (MMAD):

>= 381 - <= 1 015 nm

Geometric standard deviation (GSD):

3.08

Remarks on MMAD:

For each dose, there exists on MMAD with GSD, respectively. Please see table 2.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: dry aerosol generator with exposure system
- Source and rate of air: fresh filtered air, 8.5 LPM (generator), 25 LPM (total volume of air in each chamber), controlled by a mass flow controller (MFC, FC-7810CD-4V, AERA, Tokyo, Japan)
- Method of conditioning air: according to ISO 19601
- System of generating particulates/aerosols: acoustic dry aerosol generator (HCTm, Icheon, Korea)
- Temperature in air chamber: 22 ± 3 °C; 30-70%
- Air flow rate: 1 L/min
- Method of particle size determination: At least 400 fibers were counted using a field emission scanning electron microscope FESEM, Hitachi, Tokyo, Japan) to determine the mean particle length and diameter. The analysis used a cumulative method based on the count median diameter (CMD) and geometric standard deviation (GSD). The rigidity of the MWCNTs was measured using the method described in ISO/TS 11888. The bending ratio (Db) and static bending persistence length (SBPL) were measured from end to end (R) and along the axis (L) of the MWCNTs using FESEM.


TEST ATMOSPHERE
- Brief description of analytical method used: The mass concentration of MWCNTs in chamber was gravimetrically determined with aPVC filter (polyvinyl chloride, size: 37mm and pore size 5.0 µm). During 30 min of the 6-h exposure period, two samples were collected from each concentration chamber. The MWCNT aerosol number concentrations were measured daily during the exposure period with a dust monitor (OPC, Model 1.1.09, Grimm Technologies Inc., Douglasville, GA, USA). The mass median aerodynamic diameter (MMAD) was measured using a foil fiber for each stage (diameter, 47 mm; pore size, 5 mm, SKC, Inc., Eighty Four, PA) with a Nano MOUDI impactor (MOUDI 125 NR, MSP Co., MN) composed of 13 stages (0.01, 0.018, 0.032, 0.056, 0.10, 0.18, 0.32, 0.56, 1.0, 1.8, 3.2, 5.6, and 10 mm). The geometric standard deviation (GSD) for the MMAD was derived from the cumulative mass distribution of the MOUDI. The elemental carbon (EC) concentrations in the generated MWCNT aerosols were measured in each chamber using an OC/EC analyzer (Sunset Laboratory Inc, SW) with a quartz filter (PallfelxVR tissuequartz filter, Pall Life Sciences, Port Washington, NY, USA) according to the NIOSH 5040. The MWCNT aerosols were collected on a TEM grid copper grid (Formvar/Carbon 200 mesh, TEDpella, CA) or holey grid (Quantifoil 656-200-Cu, Tedpella, Inc., Redding, CA) and were further examined under a field emission-transmission electron microscope (JEM2100F, JEOL, Tokyo, Japan) equipped with an energy dispersive X-ray analyzer (TM200, Oxford Instruments PLC, Oxfordshire, UK) at an acceleration voltage of 200 kV (according to NIOSH 7402).
- Samples taken from breathing zone: yes


REFERENCES
ISO/TR 19601. Nanotechnologies International Organization for Standardization, Aerosol Generation for Air Exposure Studies of Nano-Objects and Their Aggregates and Agglomerates (NOAA). Geneva, Switzerland: ISO. 2017
ISO/TS 11888. Nanotechnologies-Characterization of Multiwall Carbon Nanotubes- Mesoscopic Shape Factors. Geneva, Switzerland: ISO. 2017
NIOSH Manual of Analytical Methods 7402, Asbestos by TEM. Cincinnati, OH: NIOSH 1994
NIOSH Manual of Analytical Methods 5040, Diesel Particulate Matter (as Elemental Carbon). Cincinnati, OH: NIOSH 2003

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For the method, please see box Details on inhalation exposure.
- MWCNT aerosol mass concentrations in the nose-only chambers (low, moderate, high dose): 0.257±0.114mg/m3, 1.439±0.117mg/m3, 4.253±0.326mg/m3, respectively
- EC concentrations (low, moderate, high dose): 0.139±0.009mg/m3, 1.144±0.124mg/m3, and 4.233±0.903mg/m3, respectively
- Agglomerate concentrations (low, moderate, high dose): 3302 ±1.672 number/L, 16658 ±4.233 number/L, 70931 ±20.654 number/L, respectively

Duration of treatment / exposure:

6h/day, 5 days/week, 4 weeks

Frequency of treatment:

daily, 5 days/week

Dose / conc.:

0 mg/m³ air

Remarks:

control (fresh filtered air)

Dose / conc.:

0.257 mg/m³ air (analytical)

Remarks:

±0.114 mg/m3
low dose

Dose / conc.:

1.439 mg/m³ air (analytical)

Remarks:

± 0.117 mg/3
moderate dose

Dose / conc.:

4.253 mg/m³ air (analytical)

Remarks:

± 0.326 mg/m3
high dose

No. of animals per sex per dose:

20 rats/dose, 80 rats in total

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: not indicated
- Rationale for animal assignment: randomized by body weight
- After 28 days of exposure a 28-days post-exposure observation period (recovery period) was followed. At four time points (after one day of exposure (E-1), one day after 28 days of exposure (Post-exposure observation period, PEO-1), 7 days post exposure (PEO-7), 28 days post exposure (PEO-28)) during the study, 5 animals of each dose group were sacrificed, respectively.

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Not specified

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: At time of necropsy: E-1, PEO-1, PEO-7 and PEO-28

HAEMATOLOGY: Yes, including blood coagulation
- Time schedule for examinations: At time of necropsy: E-1, PEO-1, PEO-7 and PEO-28
- Anaesthetic used for blood collection: Yes (pentobarbital sodium, i.p. 200 mg/kg bw)
- Animals fasted: No
- How many animals: all
- Parameters checked in table [No.3] were examined.

CLINICAL CHEMISTRY: Yes, serum biochemistry
- Time schedule for examinations: At time of necropsy: E-1, PEO-1, PEO-7 and PEO-28
- Animals fasted: No
- How many animals: all
- Parameters checked in table [No.4] were examined.


BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: At time of necropsy: E-1, PEO-1, PEO-7 and PEO-28
- Dose groups that were examined: all
- Number of animals: all


LUNG BURDEN: Yes
- Time schedule for analysis: At time of necropsy: E-1, PEO-1, PEO-7 and PEO-28
- Dose groups that were examined: all
- Number of animals: all

Sacrifice and pathology:

SACRIFICE:
Animals were anesthetized with pentobarbital sodium (i.p., 200 mg/kg bw), blood was drawn from the abdominal aorta and anmials were terminated by cervical dislocation afterwards.

GROSS PATHOLOGY: Yes, see table 5 for PEO-1, -7, -28. On E-1, only organ weight of left and right lungs was examined

HISTOPATHOLOGY: Yes
The left lungs were fixed in a 10% formalin solution (BBC Biochemical, Washington, DC) containing neutral phosphate-buffered saline under 25 cm of water pressure. They were trimmed, processed, embedded in paraffin, and stained with hematoxylin and eosin according to routine histological techniques. The histopathological changes were evaluated using light microscopy (ZEISS International, Oberkochen, Germany).

Other examinations:

Lung clearance rate:
The lung clearance rate was analyzed using the LB data at PEO-1, PEO-7, and PEO-28. First-order elimination kinetics were assumed, as shown in Formula 1 (see below), which was used to calculate the fractional clearance rate (at E-1) of lung compartment removal (d). The retention halftime (T1/2) was obtained by first-order plotting the post-exposure data. All of the calculations were based on computer software.
Formula 1: yt = y0 x (exp(-d x t))

Statistics:

SPSS version 19 (SPSS Inc, Chicago, IL) was used to perform the statistical analysis of the outcome parameters. Analysis of variance (ANOVA) following multiple comparison tests using the Dunnett T3 method was used for statistical evaluation. A p-value less than 0.05 was considered to be statistically significant.

Clinical signs:

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

None of the significant changes in haematological parameters were considered test-article-specific. Please refer to tables 8 and 9.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

None of the significant changes in serum biochemistry parameters were considered test-article-specific. Please refer to table 10.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Please refer to table 7.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Low Dose: agglomerated MWCNT observed in terminal bronchi, alveolar ducts and alveoli without any infiltration of inflammatory cells in the lung tissue or pneumocyte damage, thus not considered adverse.
Moderate and High Dose: granulomatous lesions sequestered by MWCNT-engulfed alveolar macrophages and thickened alveolar walls at PEO-1, -7, -28.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

BALF
The number of polymorphonuclear leukocytes (PMNs) was significantly higher than the control at PEO-1 and -7 for animals treated with moderate and high dose and at PEO-28 only for animals treated with the high dose.
Lactate Dehydrogenase (LDH) level was significantly elevated at PEO-1 in the moderate and high dose group when compared to the control.
In the high dose group, microalbumin level was significantly higher than the control at PEO-1, -7 and -28. Levels of microprotein were elevated at PEO-28 in both, moderate and high dose animals, when compared to the control.
For detailed information, please refer to table 6.

LUNG BURDEN & -CLEARANCE
The retained MWCNT lung burdens for the high dose group at E-1 and PEO-1, -7, and -28 were 6.61 ± 0.93, 73.35 ± 2.99, 49.25 ± 4.75, and 42.60 ± 3.81 µg/whole lung, respectively. The lung clearance rate of the deposited MWCNTs was 35 days for the high dose group, estimated using first-order kinetics.

Key result

Dose descriptor:

NOAEC

Effect level:

0.257 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

other: BALF

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1.439 mg/m³ air (analytical)

System:

respiratory system: lower respiratory tract

Organ:

alveolar duct

alveoli

bronchi

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

presumably yes

All of the following statistical information was obtained by testing with ANOVA following multiple comparison tests using the Dunnet T3 method.

 

Table 6: Change in inflammatory markers for cell distribution and chemistry in BALF (n = 5 for each group).

Post-exposure day	Group	Control	Low	Moderate	High
PEO-1	PMNs (x10^3)	1.96±0.24	5.48 ±1.55	7.08 ±0.96*	7.81± 1.07**
	LDH (IU/L)	19.40±1.78	22.20 ±1.07	27.00 ±2.30*	30.00± 2.30**
	mALB (µg/mL)	10.73±1.20	10.02±0.56	11.54±1.24	13.31 ±0.40*
	mTP (µg/mL)	7.80±0.35	7.98 ±0.15	8.51 ±0.50	9.51 ±0.13
PEO-7	PMNs (x10^3)	3.06±0.97	10.34±2.24	14.52 ± 2.35**	14.14± 1.74**
	LDH (IU/L)	29.20±2.52	25.00 ±2.27	26.20 ±2.31	34.60±4.49
	mALB (µg/mL)	10.51 ± 0.46	8.95±1.12	11.45 ± 0.66	13.81 ± 0.69*
	mTP (µg/mL)	9.62±0.30	9.16 ±0.46	9.81 ±0.31	10.83±0.44
PEO-28	PMN (x10^3)	4.67±0.92	6.36 ±0.19	8.04±2.12	14.12 ± 1.62***
	LDH (IU/L)	17.40±1.75	18.20 ±1.32	22.60 ±1.63	25.00± 2.70*
	mALB (µg/mL)	7.77±0.67	10.21 ±1.53	9.61 ±0.48	12.10± 3.17*
	mTP (µg/mL)	4.59±0.55	6.85 ±0.50	7.83 ±0.25*	8.87 ± 1.16**

(mean ± SE); PEO: post-exposure observation; PMN: polymorphonuclear leukocytes; LDH: lactate dehydrogenase; mALB: microalbu-

min; mP: microprotein.

*p<0.05 compared with control group.

**p < 0.01 compared with control group.

***p < 0.01 compared with control and low group

 

 

Table 7: Relative organ weights (mean ± SE) of male rats exposed to MWCNTs (n=5 for each group and Timepoint)

Group	Control (g)	Low (g)	Moderate (g)	High (g)
Timepoint E-1
BODY WEIGHT	215.06 ± 2.94	215.84 ± 2.69	217.34 ± 2.56	214.10 ± 2.94
LUNG (RIGHT)	0.31 ± 0.00	0.29 ± 0.00	0.30 ± 0.01	0.32 ± 0.02
LUNG (LEFT)	0.16 ± 0.00	0.15 ± 0.01	0.16 ± 0.0	0.19 ± 0.03
Timepoint PEO-1
BODY WEIGHT	371.72 ± 9.17	390.90 ± 11.53	393.60 ± 4.50	405.10 ± 20.71
BRAIN	0.56 ± 0.01	0.54 ± 0.01	0.53 ± 0.01	0.53 ± 0.03
EYE(LEFT)	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00
EYE(RIGHT)	0.10 ± 0.00	0.10 ± 0.00	0.09 ± 0.00	0.10 ± 0.00
SPLEEN	0.18 ± 0.01	0.18 ± 0.01	0.18 ± 0.01	0.19 ± 0.01
KIDNEY(LEFT)	0.33 ± 0.00	0.35 ± 0.01	0.34 ± 0.01	0.36 ± 0.01
KIDNEY(RIGHT)	0.33 ± 0.00	0.34 ± 0.01	0.33 ± 0.01	0.36 ± 0.01
THYMUS	0.14 ± 0.02	0.13 ± 0.01	0.11 ± 0.01	0.12 ± 0.01
TESTIS(LEFT)	0.45 ± 0.02	0.43 ± 0.01	0.41 ± 0.01	0.43 ± 0.03
TESTIS(RIGHT)	0.45 ± 0.02	0.43 ± 0.02	0.38 ± 0.03	0.43 ± 0.03
HEART	0.34 ± 0.01	0.33 ± 0.01	0.34 ± 0.01	0.34 ± 0.01
LIVER	2.71 ± 0.05	2.85 ± 0.08	2.82 ± 0.05	2.89 ± 0.12
LUNG(LEFT)	0.12 ± 0.00	0.12 ± 0.01	0.12 ± 0.00	0.13 ± 0.01
LUNG(RCL)	0.09 ± 0.00	0.09 ± 0.00	0.09 ± 0.00	0.10 ± 0.01
BRONCHUS	0.03 ± 0.00	0.02 ± 0.00	0.03 ± 0.00	0.02 ± 0.00
Timepoint PEO-7
BODY WEIGHT	421.36 ± 8.65	400.48 ± 19.28	409.02 ± 6.50	407.82 ± 7.99
BRAIN	0.51 ± 0.01	0.53 ± 0.02	0.51 ± 0.01	0.51 ± 0.01
EYE(LEFT)	0.10 ± 0.01	0.09 ± 0.01	0.10 ± 0.01	0.09 ± 0.01
EYE(RIGHT)	0.10 ± 0.00	0.08 ± 0.01	0.10 ± 0.00	0.09 ± 0.01
SPLEEN	0.22 ± 0.03	0.18 ± 0.01	0.20 ± 0.01	0.17 ± 0.01
KIDNEY(LEFT)	0.35 ± 0.01	0.33 ± 0.01	0.35 ± 0.02	0.34 ± 0.01
KIDNEY(RIGHT)	0.36 ± 0.01	0.35 ± 0.01	0.35 ± 0.02	0.34 ± 0.01
THYMUS	0.10 ± 0.00	0.14 ± 0.02	0.11 ± 0.01	0.11 ± 0.01
TESTIS(LEFT)	0.40 ± 0.02	0.43 ± 0.02	0.41 ± 0.00	0.41 ± 0.02
TESTIS(RIGHT)	0.40 ± 0.02	0.43 ± 0.03	0.41 ± 0.00	0.42 ± 0.02
HEART	0.33 ± 0.01	0.32 ± 0.01	0.31 ± 0.01	0.33 ± 0.01
LIVER	2.72 ± 0.10	2.70 ± 0.07	2.72 ± 0.07	2.74 ± 0.06
LUNG(LEFT)	0.12 ± 0.00	0.13 ± 0.01	0.11 ± 0.00	0.13 ± 0.00
LUNG(RCL)	0.09 ± 0.00	0.09 ± 0.00	0.09 ± 0.00	0.09 ± 0.00
BRONCHUS	0.03 ± 0.00	0.03 ± 0.00	0.03 ± 0.00	0.03 ± 0.00
Timepoint PEO-28
BODY WEIGHT	488.40 ± 10.26	474.18 ± 13.27	521.40 ± 19.20	524.68 ± 19.15
BRAIN	0.45 ± 0.01	0.44 ± 0.01	0.42 ± 0.01	0.43 ± 0.01
EYE(LEFT)	0.09 ± 0.01	0.09 ± 0.00	0.09 ± 0.01	0.09 ± 0.01
EYE(RIGHT)	0.09 ± 0.00	0.09 ± 0.00	0.08 ± 0.00	0.09 ± 0.01
SPLEEN	0.19 ± 0.01	0.18 ± 0.00	0.19 ± 0.01	0.20 ± 0.01
KIDNEY(LEFT)	0.27 ± 0.05	0.32 ± 0.01	0.31 ± 0.01	0.31 ± 0.01
KIDNEY(RIGHT)	0.34 ± 0.02	0.32 ± 0.01	0.31 ± 0.02	0.31 ± 0.01
THYMUS	0.15 ± 0.06	0.08 ± 0.00	0.09 ± 0.00	0.09 ± 0.02
TESTIS(LEFT)	0.34 ± 0.02	0.38 ± 0.02	0.34 ± 0.01	0.34 ± 0.01
TESTIS(RIGHT)	0.33 ± 0.02	0.38 ± 0.02	0.33 ± 0.02	0.34 ± 0.01
HEART	0.29 ± 0.01	0.31 ± 0.01	0.30 ± 0.01	0.30 ± 0.01
LIVER	2.90 ± 0.10	2.79 ± 0.16	2.79 ± 0.05	2.84 ± 0.11
LUNG(LEFT)	0.11 ± 0.00	0.11 ± 0.00	0.10 ± 0.00	0.11 ± 0.00
LUNG(RCL)	0.08 ± 0.00	0.08 ± 0.00	0.08 ± 0.00	0.08 ± 0.00
BRONCHUS	0.03 ± 0.00	0.03 ± 0.00	0.03± 0.00	0.03 ± 0.00

RCL: Right caudal lobe; PEO, post-exposure observation

 

Table 8: Hematology of male (mean ± SE) rats exposed to MWCNTs (n=5 for each group and timepoint if not indicated otherwise)

Group	Control (g)	Low (g)	Moderate (g)	High (g)
Timepoint PEO-1				n=4
WBC (×10^3/uL)	3.64 ± 0.66	4.76 ± 0.11	3.86 ± 0.36	5.87 ± 0.83
Abs-NEUT (×10^3/uL)	0.74 ± 0.10	0.98 ± 0.09	0.67 ± 0.09	0.67 ± 0.01
Abs-LYM (×10^3/uL)	2.81 ± 0.56	3.63 ± 0.01	3.07 ± 0.27	5.05 ± 0.83
Abs-MONO (×10^3/uL)	0.04 ± 0.01	0.06 ± 0.00	0.05 ± 0.01	0.07 ± 0.00
Abs-EOS (×10^3/uL)	0.04 ± 0.01	0.07 ± 0.01^*	0.05 ± 0.01	0.06 ± 0.01
Abs-BASO (×10^3/uL)	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00
NEUT (%)	21.80 ± 2.70	20.40 ± 1.64	17.14 ± 1.37	12.30 ± 1.74^*
LYM (%)	75.64 ± 2.94	76.58 ± 1.77	79.86 ± 1.34	84.98 ± 1.99^*
MONO (%)	1.26 ± 0.17	1.14 ± 0.03	1.22 ± 0.10	1.23 ± 0.20
EOS (%)	1.04 ± 0.33	1.52 ± 0.09	1.36 ± 0.08	1.00 ± 0.09
BASO (%)	0.00 ± 0.00	0.02 ± 0.00	0.04 ± 0.02	0.08 ± 0.02
RBC (×10^6/uL)	7.81 ± 0.25	7.78 ± 0.22	7.95 ± 0.12	7.81 ± 0.12
Hb (g/dL)	14.96 ± 0.37	14.80 ± 0.19	15.14 ± 0.16	14.88 ± 0.17
Hct (%)	45.82 ± 1.25	44.84 ± 0.60	45.94 ± 0.56	45.48 ± 0.50
MCV (fL)	58.70 ± 0.89	57.64 ± 0.85	57.80 ± 0.33	58.25 ± 0.99
MCH (pg)	19.20 ± 0.45	19.02 ± 0.28	19.02 ± 0.15	19.03 ± 0.16
MCHC (g/dL)	32.70 ± 0.39	33.00 ± 0.00	32.86 ± 0.16	32.68 ± 0.33
RDW (%)	11.08 ± 0.15	11.26 ± 0.03	11.20 ± 0.29	11.55 ± 0.31
PLT (×10^6/uL)	955.60 ± 121.52	1127.00 ± 24.35	1012.20 ± 19.64	981.00 ± 37.50
Retic (%)	2.52 ± 0.14	2.39 ± 0.12	2.58 ± 0.16	2.87 ± 0.14
Timepoint PEO-7
WBC (×10^3/uL)	3.99 ± 0.34	4.27 ± 0.97	6.52 ± 0.36	4.82 ± 0.71
Abs-NEUT (×10^3/uL)	0.83 ± 0.05	0.68 ± 0.16	0.59 ± 0.09	0.75 ± 0.09
Abs-LYM (×10^3/uL)	3.05 ± 0.33	3.48 ± 0.80	5.55 ± 0.27	3.94 ± 0.64
Abs-MONO (×10^3/uL)	0.04 ± 0.00	0.05 ± 0.02	0.32 ± 0.01	0.05 ± 0.01
Abs-EOS (×10^3/uL)	0.04 ± 0.01	0.05 ± 0.01	0.04 ± 0.01	0.04 ± 0.01
Abs-BASO (×10^3/uL)	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00
NEUT (%)	21.38 ± 2.00	16.02 ± 1.68	9.05 ± 1.37^**	16.30 ± 2.14
LYM (%)	75.90 ± 1.99	81.26 ± 1.62	84.93 ± 1.34^*	81.12 ± 2.21
MONO (%)	1.08 ± 0.10	1.10 ± 0.11	5.05 ± 4.12	1.16 ± 0.13
EOS (%)	1.10 ± 0.08	1.06 ± 0.24	0.58 ± 0.08	0.92 ± 0.17
BASO (%)	0.02 ± 0.02	0.04 ± 0.02	0.00 ± 0.02	0.06 ± 0.02
RBC (×10^6/uL)	7.85 ± 0.15	7.65 ± 0.13	7.39 ± 0.12	7.88 ± 0.18
Hb (g/dL)	14.52 ± 0.10	14.58 ± 0.22	14.13 ± 0.16	14.88 ± 0.29
Hct (%)	44.46 ± 0.32	44.44 ± 1.12	42.48 ± 0.56	44.38 ± 0.78
MCV (fL)	56.70 ± 0.74	58.08 ± 0.77	57.55 ± 0.33	56.32 ± 0.68
MCH (pg)	18.48 ± 0.30	19.10 ± 0.21	19.13 ± 0.15	18.88 ± 0.22
MCHC (g/dL)	32.60 ± 0.13	32.88 ± 0.55	33.23 ± 0.16^*	33.50 ± 0.10^**
RDW (%)	11.40 ± 0.09	11.46 ± 0.18	11.53 ± 0.29	11.36 ± 0.35
PLT (×10^6/uL)	1029.40 ± 60.54	1057.40 ± 55.84	1159.75 ± 19.64	1161.60 ± 34.62
Retic (%)	2.79 ± 0.09	2.65 ± 0.14	3.01 ± 0.16	3.06 ± 0.37
Timepoint PEO-28
WBC (×10^3/uL)	4.66 ± 0.60	5.58 ± 0.41	7.05 ± 0.75	6.39 ± 0.86
Abs-NEUT (×10^3/uL)	0.88 ± 0.08	0.85 ± 0.05	1.22 ± 0.19	0.86 ± 0.10
Abs-LYM (×10^3/uL)	3.63 ± 0.55	4.61 ± 0.40	5.62 ± 0.64	5.37 ± 0.80
Abs-MONO (×10^3/uL)	0.07 ± 0.01	0.05 ± 0.01	0.11 ± 0.03	0.07 ± 0.01
Abs-EOS (×10^3/uL)	0.06 ± 0.01	0.04 ± 0.01	0.07 ± 0.01	0.06 ± 0.01
Abs-BASO (×10^3/uL)	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00
NEUT (%)	19.44 ± 2.00	15.50 ± 1.37	17.62 ± 2.66	14.12 ± 1.60
LYM (%)	77.30 ± 2.23	82.28 ± 1.48	79.46 ± 2.71	83.40 ± 1.77
MONO (%)	1.56 ± 0.25	0.94 ± 0.11	1.56 ± 0.31	1.16 ± 0.11
EOS (%)	1.30 ± 0.33	0.74 ± 0.11	0.94 ± 0.17	0.92 ± 0.12
BASO (%)	0.04 ± 0.02	0.06 ± 0.02	0.04 ± 0.02	0.02 ± 0.02
RBC (×10^6/uL)	7.97 ± 0.23	7.98 ± 0.10	8.05 ± 0.15	8.12 ± 0.23
Hb (g/dL)	14.64 ± 0.37	14.54 ± 0.24	14.86 ± 0.16	14.72 ± 0.28
Hct (%)	44.18 ± 1.36	44.20 ± 0.55	44.86 ± 0.56	44.10 ± 1.00
MCV (fL)	55.40 ± 0.57	55.42 ± 0.57	55.72 ± 0.78	54.36 ± 0.96
MCH (pg)	18.40 ± 0.32	18.22 ± 0.24	18.48 ± 0.19	18.18 ± 0.35
MCHC (g/dL)	33.22 ± 0.39	32.90 ± 0.22	33.16 ± 0.17	33.40 ± 0.23
RDW (%)	12.46 ± 0.28	13.18 ± 0.26	12.84 ± 0.38	12.44 ±0.20
PLT (×10^6/uL)	996.80 ± 47.05	1108.20 ± 88.87	1112.00 ± 40.36	1085.00 ± 37.77
Retic (%)	2.84 ± 0.23	3.22 ± 0.16	2.87 ± 0.21	2.94 ± 0.12

^*p<0.05 compared with control group; ^**p<0 compared with control group; PEO, post-exposure observation; WBC, white blood cells; Abs, Absolute; NEUT, Neutrophils; LYM, Lymphocytes; MONO, Monocytes; EOS, Eosinophils; BASO, Basophils; RBC, Red blood cells; Hb, Hemoglobin; Hct, Hematocrit; MCV, Mean corpuscular volume; MCH, Mean corpuscular hemoglobin; MCHC, Mean corpuscular hemoglobin concentration; RDW, Red cell distribution width; Retic, Reticulocyte; PLT, Platelets

 

Table 9: Blood coagulation times (mean ± SE) of male rats exposed to MWCNTs at PEO-1, -7, and -28 (n=5 for each group)

Post-exposure day		Control	Low	Moderate	High
PEO-1	PT (sec)	9.84 ± 0.36	9.18 ± 0.24	9.37 ± 0.25	9.41 ± 0.15
	aPTT (sec)	19.22 ± 2.53	18.70 ± 1.48	18.72 ± 1.23	21.48 ± 2.84
PEO-7	PT (sec)	9.51 ± 0.11	9.80 ± 0.13	9.34 ± 0.17	9.75 ± 0.40
	aPTT (sec)	14.22 ± 0.66	14.60 ± 1.02	13.09 ± 1.18	13.42 ± 0.70
PEO-28	PT (sec)	9.21 ± 0.18	9.39 ± 0.20	9.24 ± 0.38	9.30 ± 0.13
	aPTT (sec)	9.21 ± 0.18	9.39 ± 0.20	9.24 ± 0.38	9.30 ± 0.13

PT, Prothrombin time; aPTT, activated partial thromboplastin time; PEO, post-exposure observation

 

Table 10: Serum biochemistry (mean ± SE) of male rats exposed to MWCNTs (n=5 for each group and timepoint).

Group	Control	Low	Moderate	High
Timepoint PEO-1
GLU (mg/dL)	127.00 ± 4.87	138.40 ± 10.10	114.80 ± 4.26	127.40 ± 2.58
CHO (mg/dL)	70.80 ± 7.82	69.60 ± 6.17	69.60 ± 3.28	53.20 ± 4.93
TP (g/dL)	5.72 ± 0.18	5.08 ± 0.13	5.38 ± 0.10	5.30 ± 0.26
BUN (mg/dL)	16.58 ± 1.33	14.28 ± 0.57	17.86 ± 1.60	15.48 ± 0.99
TBIL (mg/dL)	0.05 ± 0.01	0.02 ± 0.01	0.03 ± 0.01	0.01 ± 0.01^*
CRE (mg/dL)	0.45 ± 0.02	0.43 ± 0.02	0.46 ± 0.02	0.42 ± 0.02
ALB (g/dL)	2.36 ± 0.09	2.10 ± 0.07	2.22 ± 0.09	2.16 ± 0.11
ALT (IU/L)	35.80 ± 3.28	30.60 ± 2.68	34.20± 1.59^**	28.60 ± 3.44
AST (mg/dL)	159.00 ± 10.60	122.60 ± 17.45	178.40 ± 9.42	130.40 ± 14.40
ALP (IU/L)	582.60 ± 15.13	432.00 ± 61.60	409.00± 20.14^*	406.60± 47.91
K (mmol/L)	4.76 ± 0.22	4.38 ± 0.16	4.82 ± 0.09	4.64 ± 0.26
Na (mmol/L)	159.00 ± 6.38	140.00± 1.79^*	144.40 ± 3.44	142.60 ± 3.66^*
Ca (mg/dL)	9.12 ± 0.39	8.06 ± 0.21	8.28 ± 0.27	8.32 ± 0.26
IP (mg/dL)	8.88 ± 0.34	7.82 ± 0.18^*	8.36 ± 0.34	7.84 ± 0.18^*
Cl (mmol/L)	105.60 ± 5.10	90.60 ± 1.36^*	92.20 ± 2.35^*	91.60 ± 3.06^*
Timepoint PEO-7
GLU (mg/dL)	100.00 ± 3.65	125.80 ± 6.26^*	122.00 ± 5.39	140.20 ± 8.58^*
CHO (mg/dL)	71.80 ± 3.38	60.20 ± 3.44	59.00 ± 3.44	68.20 ± 8.36
TP (g/dL)	5.84 ± 0.10	5.82 ± 0.07	5.66 ± 0.10	5.58 ± 0.07
BUN (mg/dL)	17.96 ± 1.29	17.16 ± 0.64	15.48 ± 0.69^**	18.98 ± 0.22
TBIL (mg/dL)	0.05 ± 0.00	0.05 ± 0.00	0.05 ± 0.01	0.05 ± 0.01
CRE (mg/dL)	0.49 ± 0.01	0.51 ± 0.02	0.47 ± 0.02	0.50 ± 0.01
ALB (g/dL)	2.28 ± 0.06	2.30 ± 0.03	2.22 ± 0.04	2.26 ± 0.02
ALT (IU/L)	31.00 ± 1.76	31.00 ± 1.48	30.60 ± 2.50	31.00 ± 0.63
AST (mg/dL)	173.40 ± 15.69	151.20 ± 6.67	146.80 ± 20.58	135.60 ± 7.13
ALP (IU/L)	504.60 ± 67.23	440.40 ± 57.76	423.00 ± 37.62	487.20 ± 53.12
K (mmol/L)	4.64 ± 0.09	4.52 ± 0.04	4.70 ± 0.13	4.58 ± 0.10
Na (mmol/L)	147.60 ± 0.51	148.80 ± 0.73	147.80 ± 0.58	147.60 ± 0.98
Ca (mg/dL)	8.68 ± 0.07	8.68 ± 0.11	9.08 ± 0.23	8.64 ± 0.02
IP (mg/dL)	8.66 ± 0.22	8.84 ± 0.14	8.40 ± 0.18	8.48 ± 0.26
Cl (mmol/L)	94.00 ± 0.55	93.80 ± 0.37	95.00 ± 0.63	94.00 ± 0.84
Timepoint PEO-28
GLU (mg/dL)	146.00 ± 3.75	154.60 ± 12.57	160.60 ± 14.47	167.60 ± 6.31
CHO (mg/dL)	62.20 ± 2.71	70.00 ± 3.52	72.00 ± 4.38	68.60 ± 2.62
TP (g/dL)	6.12 ± 0.15	6.02 ± 0.20	6.00 ± 0.10	6.08 ± 0.07
BUN (mg/dL)	16.90 ± 0.67	19.34 ± 0.90	17.50 ± 0.67^**	21.92 ± 1.04
TBIL (mg/dL)	0.04 ± 0.01	0.06 ± 0.01	0.06 ± 0.02	0.05 ± 0.01
CRE (mg/dL)	0.46 ± 0.02	0.49 ± 0.02	0.48 ± 0.02	0.56 ± 0.05
ALB (g/dL)	2.38 ± 0.05	2.32 ± 0.06	2.30 ± 0.03	2.32 ± 0.04
ALT (IU/L)	37.40 ± 2.38	37.80 ± 2.63	33.80 ± 4.59	33.80 ± 1.71
AST (mg/dL)	154.60 ± 4.74	171.00 ± 20.68	136.00 ± 21.38	174.00 ± 13.15
ALP (IU/L)	382.40 ± 50.90	326.00 ± 13.40	298.60 ± 18.40	333.80 ± 35.30
K (mmol/L)	4.88 ± 0.10	5.04 ± 0.23	4.88 ± 0.12	4.84 ± 0.08
Na (mmol/L)	149.60 ± 0.51	148.60 ± 0.40	148.20 ± 0.37	147.80 ±0.49^***
Ca (mg/dL)	9.36 ± 0.11	9.58 ± 0.08	9.46 ± 0.15	9.42 ± 0.07
IP (mg/dL)	7.62 ± 0.17	8.26 ± 0.29	7.46 ± 0.14	7.64 ± 0.20
Cl (mmol/L)	96.00 ± 0.00	95.20 ± 0.58	96.20 ± 0.20	94.20 ± 0.86

^*p<0.05 compared with control group; ^** p<0.01 compared with high group; ^*** p<0.05 compared with control group; PEO, post-exposure observation; GLU, Glucose; CHO, Cholesterol; TP, Total protein; BUN, Blood urea nitrogen; TBIL, Total bilirubin; CRE, Creatinine; TP, Total protein; ALB, Albumin; ALT, Alanine aminotransferase; AST, Aspartate aminotransferase; ALP, Alkaline phosphatase; K, Potassium; Na, sodium; Ca, Calcium; IP, Inorganic phosphorus; Cl, Chloride

Conclusions:

In conclusion, this study conducted after OECD TG 412 assessing subacute inhalation toxicity of JENO TUBE 10B MWCNT in rats revealed a dose-dependent increase of LDH concentration and of polymorphonuclear leukocyte count at bronchoalvelaor lavage fluid examination in animals dosed with 1.439 and 4.253 mg/m3 (moderate and high dose groups). In addtition, granulomatous lesions sequestered by MWCNT-engulfed alveolar macrophages and thickened alveolar walls were observed during the post exposure observation period in these dose groups, as well. Hence, the NOAEC was set to the low dose level at 0.257 mg/m3.

Executive summary:

The study of Kim et al. investigated the impact of sub-acute inhalation exposure of JENO TUBE 10B MWCNT on 80 male Sprague-Dawley rats after OECD TG 412. Rats were exposed nose-only for 6 h/day, 5 days/week for 4 weeks, followed by a post-exposure observation period (PEO) of 28 days. There were 20 animals per dosing group. The analytical doses were 0, 0.257, 1.439 and 4.253 mg/m³. At 4 time points during the study, 5 animals of each dose group were sacrificed, respectively and underwent necropsy procedure: organ weight, haematology, serum biochemistry, bronchoalveolar lavage, histopathology and lung burden was performed or examined.

There were significant changes in haematological and biochemical parameters noted which were not considered to be treatment related.

Besides, histopathology revealed agglomerated MWCNT in terminal bronchi, alveolar ducts and alveoli without any infiltration of inflammatory cells in the lung tissue or pneumocyte damage, in the low dose group. This effect was not considered adverse. However, in the moderate and high dose group, granulomatous lesions sequestered by MWCNT-engulfed alveolar macrophages and thickened alveolar walls at PEO-1, -7, -28 were observed and considered adverse.

In addition to that, BALF analysis showed a significant increase in polymorphonuclear leukocyte count in animals treated with moderate and high dose was at PEO-1 and -7 and at PEO-28 only in animals treated with the high dose. Lactate Dehydrogenase (LDH) level was significantly elevated at PEO-1 in the moderate and high dose group when compared to the control, as well. In the high dose group, microalbumin level was significantly higher than the control at PEO-1, -7 and -28. Levels of microprotein were elevated at PEO-28 in both, moderate and high dose animals, when compared to the control.

The retained MWCNT lung burdens for the high dose group at E-1 and PEO-1, -7, and -28 were 6.61 ± 0.93, 73.35 ± 2.99, 49.25 ± 4.75, and 42.60 ± 3.81 µg/whole lung, respectively. The lung clearance rate of the deposited MWCNTs was 35 days for the high dose group, estimated using first-order kinetics.

Taken together, the effects observed in animals treated at moderate or high dose level led to the NOEAC being set at 0.257 mg/m³, corresponding to the low dose.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Objective of study:

toxicokinetics

other: repeated dose (28-d) inhalation toxicity

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

27 Jun 2018

Deviations:

yes

Remarks:

only male rats tested

GLP compliance:

not specified

Remarks:

Published study performed by University staff in the Republic of Korea

Test material

Test material information

Constituent 1

Reference substance name:

carbon

EC Number:

936-414-1

Molecular formula:

C

IUPAC Name:

carbon

Test material form:

solid: nanoform, no surface treatment

Details on test material:

Lot / Batch : JC162
Date : 2016-03-14
BET surface area (m²/g) : NA
Purity (wt%) : 98.6
Bulk density (g/ml) : 0.011
Moisture content (wt%) : NA

Jenotube 10B - SIP
Shape category : elongated
Shape of particles : tube
Pure shape : yes
Crystal structure : crystalline
Pure structure : yes
Crystal system : armchair, zig-zag and chiral
Surface treatment/functionalisation : no
State of aggregation : agglomerated
Bundle length : 10.5 μm – 117.7 μm
Bundle diameter : 1.1 μm – 15.4 μm
Tube diameter : 8.3 nm – 17.9 nm
BET surface area (m²/g) : 181 – 249
Number of walls : 7 – 18

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: JEIO CO., LTP, 69, Namdong-daero 49beon-gil, Namdong-gu, Incheon, Korea. Lot: JC162
- The test material is identical to Jenotube 10B
- Purity, including information on contaminants, isomers, etc.: 98.6%

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: according to ISO TR 19601 (see reference below) under use of an acoustic dry aerosol generator (HCTm, Icheon, Korea): Aggregates or agglomerates of the test material were dispersed with acoustic energy. The amount of generated aerosol was adjusted by altering the frequency of the acoustic waves, the air supply flow rate and the quantity of the test material. A buffer chamber and cyclone trapped larger test materials. 2.5 g of MWCNT were in the generator. The generator used 8.5 LPM, and the total volume of air in each chamber was 25 LPM controlled by a mass flow controller (MFC, FC-7810CD-4V, AERA, Tokyo, Japan).

FORM AS APPLIED IN THE TEST:
Aerosol
MMAD: low dose: 381 nm (GSD 2.34), moderate dose: 495 nm (GSD 3.08), high dose 1015 nm (GSD 2.81)
The difference in the MMAD among the concentrations was because one generator was used to dilute the high concentration to the moderate concentration and then to the low concentration.

INFORMATION ON NANOMATERIALS
Please refer to information in tables 1 and 2.

REFERENCE
ISO/TR 19601. Nanotechnologies International Organization for Standardization, Aerosol Generation for Air Exposure Studies of Nano-Objects and Their Aggregates and Agglomerates (NOAA). Geneva, Switzerland: ISO. 2017

Radiolabelling:

no

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

specific-pathogen-free animals purchased from OrientBio (Kyeonggi-do, Korea)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: OrientBio (Kyeonggi-do, Korea)
- Age at study initiation: 6 weeks
- Weight at study initiation: 179.75 +/- 0.34g
- Housing: polycarbonate cage (max. 3 rats /cage)
- Diet (e.g. ad libitum): no details available
- Water (e.g. ad libitum): no details available
- Acclimation period: 2 weeks, in a nose-only tube at 2 h/day during 4 days before initiation of the MWCNT exposure.
- Health status: specific-pathogen-free

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2 °C (housing); 22 ± 3 °C (inhalation chamber)
- Humidity (%): 55 ± 7% (housing); 30-70% (inhalation chamber)
- Air changes (per hr): not indicated
- Photoperiod (hrs dark / hrs light): 12/12
- Fasting period: Food and water were withheld during the 6-h exposure period

IN-LIFE DATES: not indicated; period of 56 days (28 d exposure, 28 d recovery period)

Administration / exposure

Route of administration:

inhalation: aerosol

Vehicle:

other: fresh filtered air

Details on exposure:

TYPE OF INHALATION EXPOSURE: nose only

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: dry aerosol generator with exposure system
- Method of holding animals in test chamber: not indicated
- Source and rate of air: fresh filtered air, 8.5 LPM (generator), 25 LPM (total volume of air in each chamber), controlled by a mass flow controller (MFC, FC-7810CD-4V, AERA, Tokyo, Japan)
- Method of conditioning air: according to ISO 19601
- System of generating particulates/aerosols: acoustic dry aerosol generator (HCTm, Icheon, Korea)
- Temperature, humidity, pressure in air chamber: 22 ± 3 °C; 30-70%; no pressure indicated
- Air flow rate: 1 L/min
- Air change rate: not indicated
- Method of particle size determination: At least 400 fibers were counted using a field emission scanning electron microscope FESEM, Hitachi, Tokyo, Japan) to determine the mean particle length and diameter. The analysis used a cumulative method based on the count median diameter (CMD) and geometric standard deviation (GSD). The rigidity of the MWCNTs was measured using the method described in ISO/TS 11888. The bending ratio (Db) and static bending persistence length (SBPL) were measured from end to end (R) and along the axis (L) of the MWCNTs using FESEM.
- Treatment of exhaust air: not indicated

TEST ATMOSPHERE
- Brief description of analytical method used: The mass concentration of MWCNTs in chamber was gravimetrically determined with aPVC filter (polyvinyl chloride, size: 37mm and pore size 5.0 µm). During 30 min of the 6-h exposure period, two samples were collected from each concentration chamber. The MWCNT aerosol number concentrations were measured daily during the exposure period with a dust monitor (OPC, Model 1.1.09, Grimm Technologies Inc., Douglasville, GA, USA). The mass median aerodynamic diameter (MMAD) was measured using a foil fiber for each stage (diameter, 47 mm; pore size, 5 mm, SKC, Inc., Eighty Four, PA) with a Nano MOUDI impactor (MOUDI 125 NR, MSP Co., MN) composed of 13 stages (0.01, 0.018, 0.032, 0.056, 0.10, 0.18, 0.32, 0.56, 1.0, 1.8, 3.2, 5.6, and 10 mm). The geometric standard deviation (GSD) for the MMAD was derived from the cumulative mass distribution of the MOUDI. The elemental carbon (EC) concentrations in the generated MWCNT aerosols were measured in each chamber using an OC/EC analyzer (Sunset Laboratory Inc, SW) with a quartz filter (PallfelxVR tissuequartz filter, Pall Life Sciences, Port Washington, NY, USA) according to the NIOSH 5040. The MWCNT aerosols were collected on a TEM grid copper grid (Formvar/Carbon 200 mesh, TEDpella, CA) or holey grid (Quantifoil 656-200-Cu, Tedpella, Inc., Redding, CA) and were further examined under a field emission-transmission electron microscope (JEM2100F, JEOL, Tokyo, Japan) equipped with an energy dispersive X-ray analyzer (TM200, Oxford Instruments PLC, Oxfordshire, UK) at an acceleration voltage of 200 kV (according to NIOSH 7402).
- Samples taken from breathing zone: yes

REFERENCES
ISO/TR 19601. Nanotechnologies International Organization for Standardization, Aerosol Generation for Air Exposure Studies of Nano-Objects and Their Aggregates and Agglomerates (NOAA). Geneva, Switzerland: ISO. 2017
ISO/TS 11888. Nanotechnologies-Characterization of Multiwall Carbon Nanotubes- Mesoscopic Shape Factors. Geneva, Switzerland: ISO. 2017
NIOSH Manual of Analytical Methods 7402, Asbestos by TEM. Cincinnati, OH: NIOSH 1994
NIOSH Manual of Analytical Methods 5040, Diesel Particulate Matter (as Elemental Carbon). Cincinnati, OH: NIOSH 2003

Duration and frequency of treatment / exposure:

6h/day, 5 days/week, 4 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/m³ air

Remarks:

control (fresh filtered air)

Doses / concentrations 2

Dose / conc.:

0.257 mg/m³ air (analytical)

Remarks:

±0.114 mg/m3
low dose

Doses / concentrations 3

Dose / conc.:

1.439 mg/m³ air (analytical)

Remarks:

± 0.117 mg/3
moderate dose

Doses / concentrations 4

Dose / conc.:

4.253 mg/m³ air (analytical)

Remarks:

± 0.326 mg/m3
high dose

Doses / concentrations 5

Dose / conc.:

0.313 mg/m³ air (nominal)

Remarks:

low dose

Doses / concentrations 6

Dose / conc.:

1.25 mg/m³ air (nominal)

Remarks:

moderate dose

Doses / concentrations 7

Dose / conc.:

5 mg/m³ air (nominal)

Remarks:

high dose

No. of animals per sex per dose / concentration:

20 rats/dose, 80 rats in total

Control animals:

yes, concurrent vehicle

Positive control reference chemical:

no

Details on study design:

- Dose selection rationale: not indicated
- Rationale for animal assignment: randomized by body weight

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: blood, eyes, testes, epididymis, heart, thymus, trachea, lungs, bronchus, kidneys, spleen, liver, and brain including the olfactory bulb, bronchoalveolar lavage fluid (BALF)
- Time and frequency of sampling: samples were taken after sacrifice of 5 animals per group at 4 time points during the study (20 rats in total per group): after 1 day of exposure (E-1), 1-day postexposure (PEO-1), 7-days post-exposure (PEO-7), and 28-days post-exposure (PEO-28)
- 5 animals per dosing group were anesthesized i.p. with 200 mg/kg pentobarbital sodium, blood was drawn from the abdominal aorta. Afterwards, animals were sacrificed by cervical dislocation and the organs enumerated above were collected, weighed, and fixed in 10% neutral buffered formalin, picric acid (testes) or Davidson’s solution (eyes).

METABOLITE CHARACTERISATION STUDIES
not performed

ANALYTICAL METHOD
- Hematology:
The blood sample analysis was conducted using a biochemistry analyzer (Hitachi 7108, Hitachi, Tokyo, Japan) and blood cell counter (Hemavet 0950, CDC Tech., Dayton, OH). The coagulation time was evaluated using an ACL700 instrument (Instrumentation Laboratory, Bedford, MA). No further details indicated.

- BALF analysis:
After occluding the right caudal lobe (RCL) and left lung, the BALF was collected from the right lung 4 times using cold saline (0.9% NaCl, first lavage: 3 ml, subsequent lavages: 2 ml of saline). The BALF was collected in a conical tube, centrifuged for 7min at 500 g and the cell-free BALF was used to analyze the cytotoxicity markers: lactate dehydrogenase (LDH), microalbumin (mALB), and micro-total protein (mTP), with a Hitachi 7108 serum biochemistry analyzer (Hitachi, Tokyo, Japan). Thereafter, the BALF pellets were re-suspended in 1ml of saline to determine the total cell count using a hemocytometer and the differential cell count using light microscopy (x400) based on Wright-Giemsa staining.

- Histopathology:
The left lungs were fixed in a 10% formalin solution (BBC Biochemical, Washington, DC) containing neutral phosphate-buffered saline under 25 cm of water pressure. They were trimmed, processed, embedded in paraffin, and stained with hematoxylin and eosin according to routine histological techniques. The histopathological changes were evaluated using light microscopy (ZEISS International, Oberkochen, Germany).

- Lung Burden (LB) Measurement:
The RCL was weighed and fixed with 10% neutral buffer formalin and dried in a dry oven at 110 °C for 2 h. The dried lungs were weighed and digested with 5ml of a solvable solution (2.5% NaOH, 2.5~10% N,N-dimethyldodecylamine N-oxide, 2.5~10% polyethylene glycol trimethylnonyl ether) in a 15ml conical tube, and heated at 60 °C for one day using a hot plate oven (Perkin-Elmer). The digested lung samples were centrifuged at 2700 g for 15 min, the supernatant was discarded, and the pellet containing MWCNTs washed with distilled water by vortexing (1500 g and 15 s). Afterwards, the pellet was centrifuged at 2700 g for 15 min, washed with distilled water, and placed on a quartz filter (Pallfelx (R) tissuequartz filter, Pall life sciences) for analysis with an organic carbon/elemental carbon (OC/EC) analyzer (Sunset laboratory Inc, SW) based on NIOSH 5040 (NIOSH 2003). A spiked standard curve was obtained from the RCL tissues under use of a known mass of MWCNTs of 0, 13.5, 53, and 206.5 mg/g. Two replicates were measured at each concentration. The polynomial linearity of the standard curve was y = 0.505x + 1.93 and the R2 value was 0.997. The recovery (%) for 0, 13.5, 53, and 206.5 mg/g was 0, 45.55, 59.13, and 51.22%, respectively. The quantitative analyses of the LB were then corrected using the spiked standard curve (recovery). The limit of detection (LOD) and limit of quantitation (LOQ) were obtained using LOD = 3SD/b and LOQ = 10SD/b, respectively, where SD is the standard deviation of the lowest concentration and b is the slope of the calibration curve. The LOD and LOQ measured using dry lung tissue from the control group was 1.96 mg/g and 6.52 mg/g, respectively. After analyzing the RCL LB, the total LB was obtained based on the control dry whole-lung weight: average 0.235 g at age 7weeks, 0.272 g at age 11 weeks, 0.309 g at age 12 weeks, and 0.368 g at age 15 weeks. The dry whole-lung weight was measured for five animals. The actual LB was then corrected after subtracting the background obtained from the control group.

- Lung clearance rate:
The lung clearance rate was analyzed using the LB data at PEO-1, PEO-7, and PEO-28. First-order elimination kinetics were assumed, as shown in Formula 1 (see below), which was used to calculate the fractional clearance rate (at E-1) of lung compartment removal (d). The retention halftime (T1/2) was obtained by first-order plotting the post-exposure data. All of the calculations were based on computer software.
Formula 1: yt = y0 x (exp(-d x t))

REFERENCE
NIOSH Manual of Analytical Methods 5040, Diesel Particulate Matter (as Elemental Carbon). Cincinnati, OH: NIOSH 2003

Statistics:

SPSS version 19 (SPSS Inc, Chicago, IL) was used to perform the statistical analysis of the outcome parameters. Analysis of variance (ANOVA) following multiple comparison tests using the Dunnett T3 method was used for statistical evaluation. A p-value less than 0.05 was considered to be statistically significant.

Results and discussion

Preliminary studies:

Not indicated

Main ADME results

Type:

clearance

Results:

Estimated using first-order kinetics: T1/2 = 35 days for the high dose group. Fractional clearance rate of lung compartment removal for the high dose group: 0.0195/day

Toxicokinetic / pharmacokinetic studies

Details on absorption:

No investigation on absorption was performed.

Details on distribution in tissues:

Only the lung was investigated. In this tissue the substance was distributed to and found as agglomerated MWCNTs in the terminal bronchi, alveolar ducts and alveoli.
The assessed retained MWCNT lung burden for the high dose group can be accessed in table 3. Alvoelar macrophages with engulfed MWCNTs were observed in the moderate and high dose group together with thickened alveolar walls and granulomatous lesions.
No treatment related changes were observed in the blood or serum parameters.

Transfer into organs

Transfer type:

other: respiration

Observation:

other: agglomerated MWCNTs in terminal bronchi, alveolar ducts, alveoli

Details on excretion:

The lung clearance rate of the deposited MWCNTs, estimated using first-order kinetics and based on data from LB measurement (see table 3) was 35 days for the high dose group. In addition, the fractional clearance rate of lung compartment removal for the same group was 0.0195/day.

Toxicokinetic parameters

Toxicokinetic parameters 1

Key result

Toxicokinetic parameters:

half-life 1st: 35 days for high dose group

Toxicokinetic parameters 2

Toxicokinetic parameters:

other: fractional clearance rate of lung compartment removal for high dose group: 0.0195/day

Metabolite characterisation studies

Metabolites identified:

not measured

Enzymatic activity

Enzymatic activity measured:

not measured

Bioaccessibility (or Bioavailability)

Bioaccessibility (or Bioavailability) testing results:

not measured

Any other information on results incl. tables

Only results regarding toxicokinetic endpoint are reported here. Further results of this study regarding repeated dose toxicity are reported in the respective section 'Repeated dose toxicity'. Results for low and moderate dose group are not reported in the study for the toxicokinetic endpoint.

 

Table 3: Lung Burden in the high dose group; MWCNT-burden measured as elemental carbon-burden.

Time point	E-1	PEO-1	PEO-7	PEO-28
Elemental carbon [µg/whole lung]	6.61 ± 0.93	73.35 ± 2.99	49.25 ± 4.75	42.60 ± 3.81

Applicant's summary and conclusion

Conclusions:

In conclusion, it could be shown that the test substance was deposited in the rat lung tissues and showed a clearance half-life of 35 days after 28-days of inhalative exposure to 4.253 ± 0.326 mg/m3 test substance.

Executive summary:

In a 28-days repeated dose inhalation toxicity study conducted in 80 male, SPF Sprague Dawley rats, lung burden and lung clearance of MWCNTs using elemental carbon analysis were evaluated. The retained MWCNT lung burdens for the high dose group (4.253 ± 0.326 mg/m3) at E-1, PEO-1, PEO-7, and PEO-28 were 6.61 ± 0.93, 73.35 ± 2.99, 49.25 ± 4.75, and 42.60 ± 3.81 µg/whole lung, respectively. The lung clearance rate of the deposited MWCNTs, estimated using first-order kinetics, was 35 days for the high dose group. In addition, the fractional clearance rate of lung compartment removal for the same group was 0.0195/day.