Standard Review for P09-0054/55/56/57 

25 February 2009 Page 1 of 71 

CONTAINS TSCA CBI

TI: S. Cragg SAT Date: 21 November 2008 

PM: Z. Kosim RAD RA: 25 February 2009 

STANDARD REVIEW RISK ASSESSMENT 

FOR P09-0054 THROUGH P09-0057 

BACKGROUND 

PMN P09-0054 through P09-0057, submitted by Nano-C, Inc on 5 November 2008, are 4 

“fullerenes” defined as a family of carbon compounds in the form of a hollow sphere, comprised 

of 5, 6, or sometime 7-membered rings. The fullerene was named by the chemists who 

discovered them after Richard Buckminster Fuller, architect/inventor of the Geodesic Dome 

which fullerenes resemble in 1985 (http://en.wikipedia.org/wiki/Fullerenes). Fullerenes are also 

known as “Buckyballs.” Molecular structures, chemical names, and CAS Numbers for all four 

PMNs are shown below. 

P09-0054 

Chemical Name: [5,6]Fullerene-C60-lh 

Case RN: 99685-96-8 

Number of Carbons: 120 

Molecular Weight: 721 

P09-0055 

Chemical Name: [5,6]Fullerene-C70-D5h(6) 

Case RN: 115383-22-7 

Number of Carbons: 140 

Molecular Weight: 841 
Standard Review for P09-0054/55/56/57 

25 February 2009 Page 2 of 71 

P09-0056 

Chemical Name: [5,6]Fullerene-C84-D2 

Case RN: 145809-19-4 

Number of Carbons: 168 

Molecular Weight: 841 

PO9-0057 

Chemical Name: [5,6]Fullerene-C84-D2d 

Case RN: 145809-20-7 

Molecular Weight: 841

The two structures for each PMN depict the side and top views of the same fullerene. The 

fullerenes are used as:

These fullerenes are solid with negligible solubility in water and negligible vapor pressure (SAT 

Report). Their boiling points are estimated to be greater than 400°C with unknown POTW 

removal, time for complete ultimate aerobic biodegradation, and ability to sorb to soils. Due to 

the novel nature of nanomaterials, it is not possible at this time to make assumptions about their 

behavior in the environment without the collection of physicochemical (p-chem) and fate data. 

The p-chem properties which seem most important for fate assessment are: surface reactivity, 

aggregation/agglomeration, physical form, particle size distribution, surface area solubility, 

dispersability, shape, adsorption tendency and surface charge porosity. 
Standard Review for P09-0054/55/56/57 

25 February 2009 Page 3 of 71 

Chemical properties affecting exposure and releases (see attached chemistry report): 

Physical Form: Brown powder 

Sublimation Point: 527-660oC at 0.4 Pa (submitter estimate) 

Density: 1.41 - 1.69 g/cc (literature values) 

Electrical Conductivity: 3.6 E-4 to 1 E-6 S/cm (literature values) 

Electrical Resistance: 900 – 2400 ohm (literature values) 

Electrical Resistivity: 1 E-7 to 0.35 ohm*cm (literature values) 

Glass Transition Temp: 804 - 1427o

C (literature values) 

Particle Size: 80–350 microns (for P09-54 others should be similar) 

 

Due to the novel nature of nanomaterials, it is not possible at this time to make assumptions 

about their behavior in the environment without the collection of physicochemical (p-chem) and 

fate data. The p-chem properties which seem most important for fate assessment are: surface 

reactivity, aggregation/agglomeration, physical form, particle size distribution, surface area 

solubility, dispersability, shape, adsorption tendency and surface charge porosity. 

These PMN’s are undergoing standard review because of their unique physical and chemical 

properties and lack of knowledge for this class of materials. 

However, because they are not actually nano-sized (verified by D. Darling), and are in fact of an 

average size in excess of 10 microns, they are not considered deep lung respirable and therefore 

not a significant hazard via inhalation. Moreover, materials this large in diameter would not be 

expected to penetrate the skin. Since ingestion is not considered likely in an occupational 

setting, the oral route of exposure also is eliminated. The population at large is not considered at 

risk from these materials also for these reasons and because exposures would be negligible. 

Because of their extremely low water solubility, they are not likely to pose a hazard to aquatic 
Standard Review for P09-0054/55/56/57 

25 February 2009 Page 4 of 71 

species. Threats to terrestrial wildlife likely are considered negligible based on reasoning similar 

to that described above for occupational exposures and also due to further dilution if released 

off-site. 

These materials are not considered to pose an unreasonable risk to human health or the 

environment. 
Standard Review for P09-0054/55/56/57 

25 February 2009 Page 5 of 71 

Appendices 

Structure Activity Reports (SAT) (V. Nabholz) Page 6 

Genotoxicity Review (M. Cimino) Page 8 

Carcinogenity Review (Y. Woo) Page 14 

Immunotoxicity Review (R. Ward) Page 17 

Chemistry Report (D. Darling) Page 20 

Engineering Reports P09-0054 (F. Arnold) Page 22 

Engineering Reports P09-0055(F. Arnold) Page 31 

Engineering Reports P09-0055/56 (F. Arnold) Page 39 

Qualitative Environmental Exposure Assessment (C. Cinalli) Page 47 

Economics Report (W. Hoffman) Page 50 
Standard Review for P09-0054/55/56/57 

25 February 2009 Page 6 of 71 

 SAT Report 

PMN Number: P-09-0054/55/56/57

SAT Date: 11/21/2008 

Print Date: 3/3/2009 

Related cases: CBI: Yes 

Health related cases: 

 Ecotox related cases: 

Concern levels:

Type of Concern: Health Eco Comments

Level of Concern: 2 1 

 

 Persistence Bioaccum Toxicity Comments

 U U 1 

Exposure Based Review:

Health: 

Ecotox: No 

Routes of exposure: Health: Dermal, inhalation 

Ecotox: No releases to water 

Fate: U; 

P2Rec Comments:

Comment: SAT had no comment 

Keywords:

 Keywords: LUNG OVERLOAD; ONCO; IMMUNOTOX 

Summary of Assessment:

 Fate:

Fate Summary: P-09-0054-57 

FATE: 

Solid 

S < 0.001 mg/L at 25 EC (E) 

VP < 1.0E-6 torr at 25 EC (E) 

BP > 400 EC (E) 
Standard Review for P09-0054/55/56/57 

25 February 2009 Page 7 of 71 

H < 1.00E-8 (E) 

POTW removal (%) = U 

Time for complete ultimate aerobic biodeg = U 

Sorption to soils/sediments = U 

PBT Potential: PUBU 

*CEB FATE: Migration to ground water = U 

 

 Health:

 Health Summary: Expect poor absorption via all routes (pchem/analog). 

Concern for lung toxicity if inhaled based on lung overload for poorly soluble particulates. 

Also concern for oncogenicity and immunotoxicity based on carbon nanomaterials. 

 Ecotox:

Test 

Organis

m 

Test 

Type 

Test End 

Point 

Predicted Measured Comments 

fish 96-h LC50 * 

daphnid 48-h LC50 * 

green 

algal 

96-h EC50 * 

fish _ chronic 

value 

* 

daphnid _ chronic 

value 

* 

algal _ chronic 

value 

* 

Sewage 

Sludge 

3-h EC50 _ 

Sewage 

Sludge 

_ Chronic 

Value 

_ 

Ecotox Values Comments: Predictions are based on 

; MW 721; pH7; effective concentrations 

based on 100% active ingredients and nominal concentrations; hardness <180.0 mg/L as 

CaCO3; and TOC <2.0 mg/L; 

Factors Values Comments 

Assessment Factor 10 

Concentration of Concern (ppb) * 

SARs 

SAR Class 

Ecotox Factors Comments: 

 

 SAT Chair: L Keifer 564-8916 
Standard Review for P09-0054/55/56/57 

25 February 2009 Page 8 of 71 

December 29, 2008 

MEMORANDUM

SUBJECT: Mutagenicity Hazard Review of P09-54, P09-55, P09-56 and P09-57 

FROM: Michael C. Cimino, Ph.D. 

 Biologist 

 Science Support Branch 

 Risk Assessment Division (7403M) 

TO: Steven Cragg, PhD, DABT 

 Technical Integrator 

 Existing Chemicals Assessment Branch 

 

THRU: Donald Rodier 

 Branch Chief 

 Science Support Branch 

 Risk Assessment Division (7403M) 

 

THIS MEMORANDUM CONTAINS 

TSCA CONFIDENTIAL BUSINESS INFORMATION

I. CONCLUSION

 No mutagenicity data were provided with PMNs 09-54, -55, -56 and -57. Based upon 

their nanotech properties, they may be weak inducers of aneuploidy, and induce DNA effects in 

mammalian cells in culture. They may induce gene mutations in mammalian cells in culture but 

not in prokaryotes, lower eukaryotes, or mammals in vivo. They may not induce DNA effects in 

lower eukaryotes in vivo, nor cell transformation in mammalian cells in culture. 

 

 Due to uncertainty about their ability to penetrate cells, testing of nanomaterials in 

genotoxicity assays is problematic, and negative responses would not conclusively rule out the 

potential mutagenicity hazard of nanotubule materials in humans. No further testing of the PMNs 

is recommended at this time. 
Standard Review for P09-0054/55/56/57 

25 February 2009 Page 9 of 71 

II. STRUCTURES OF P09-54 through -57 

Structures deleted as they are duplicates of those shown in the risk assessment (pages 1-11). 

NANOTECH ANALOGUES 

 

 Fullerene-C60 

III. BASIS FOR THE CONCLUSIONS

 No mutagenicity data were provided with the Premanufacturing Notices on: 

PMN 09-54: [5,6]Fullerene-C60-Ih; synonym nano-c(BF); CAS# 99685-96-8, 

PMN 09-55: [5,6]Fullerene-C70-D5h(6); synonym nano-c(SF); CAS# 115383-22-7, 

PMN 09-56: [5,6]Fullerene-C84-D2; synonym nano-c(EF); CAS# 145809-19-4, and 

PMN 09-57: [5,6]Fullerene-C84-D2d; synonym nano-c(EF); CAS#145809-20-7. 

Structures of the four PMNS are in Section II. 

 The only basis for concern for these PMNs is that they are nanotech materials. Nanotech 

analogues of the PMN have been reviewed in detail previously 

): 
Standard Review for P09-0054/55/56/57 

25 February 2009 Page 10 of 71 

are not gene mutagens in 

Salmonella strains TA98, TA100, TA102, TA1535 and TA1537 and in Escherichia coli strain 

WP2uvrA, and not chromosome mutagens in mammalian CHL cells, in both cases without and 

with metabolic activation. 

was not a gene mutagen in Salmonella 

strains TA98, TA100, TA102, TA1535 and TA1537, and was not a chromosome mutagen in 

mammalian V79 cells, in both cases without and with metabolic activation. 

 Another analogue is SWCNT (single-walled carbon nanotubes) was not a gene mutagen 

in Salmonella in strains YG1024 and YG1029 without activation, or an inducer of chromosome 

aberrations and/or aneuploidy in an in vitro micronucleus assay in V79 cells. It did induce DNA 

effects, with a dose response, in an in vitro comet assay in V79 cells. 

 Carbon nanotubes (CNTs) reportedly induce gene mutations in mouse embryonic cells. 

Also, carbon nanotubes reportedly did not induce urinary mutagenicity in Salmonella when 

administered orally to rats, nor micronuclei or DNA effects in the form of sister chromatid 

exchange (SCE) in vitro. Both of these reports are in the form of abstracts, and details were not 

available to review. 

 Other nanotech materials have also been discussed previously. [5,6]Fullerene-C60-ih 

(Buckerminsterfullerene: CAS# 99685-96-8; structure in Section II) reportedly is a gene 

mutagen in Salmonella strains TA102, TA104 and YG3003 in the presence of activation when 

irradiated with visible light, but is reported as negative in Salmonella strain BA13. It reportedly 

did not induce gene mutation or recombination in the Drosophila melanogaster somatic mutation 

and recombination (SMART) test, nor cell transformation in BALBc/3T3 cells in vitro. These 

data are cited as reported in abstracts and summaries, and were not reviewed in detail. 

 Other nanotech products for which mutagenicity data exist are nanoparticles of Fe3O4

and Fe3O4 coated with glutamic acid and carbon black (CAS# 1333-86-4), and and 

its analogues titanium (IV) oxide (CAS# 13463-67-7), titanium tetrachloride (CAS# 7550-

45-0; structure in Section II), also previously reviewed. 

 The data on nanoparticles are summarized in the Table on the following page (all data 

except for and SWCNT are as reported in abstracts): 
Standard Review for P09-0054/55/56/57 

25 February 2009 Page 11 of 71 

TABLE I: MUTAGENICITY DATA ON NANOTECH PRODUCTS 

(Most results as reported in abstracts) 

Chemical Gene mutations Chromosome mutations DNA Other 

SAL E 

co

li 

lower 

eukaryote

s 

mammali

an in vitro 

CA 

vitro 

MN 

vitro 

CA 

vivo 

MN 

vivo 

 Cell 

transfor

mation 

Sperm

head 

abnor

malitie

s 

 neg neg 

 neg neg 

 neg neg 

SWCNT neg weak pos 

come

t vit 

 

CNT pos ES vit 

neg rat 

BFU viv 

 neg neg 

SCE 

vit 

 

[5,6]FullereneC60-ih pos 

neg 

 neg 

SMART 

 neg 

SMA

RT 

neg 

BALBc 

nanoparticles of 

Fe3O4

 neg neg pos 

Fe3O4 coated with 

glutamic acid 

 neg neg pos 

carbon black pos pos L51 pos 

SCE 

vitro 

 

 neg ne

g 

 

Titanium (IV) 

oxide 

neg neg SRL neg L51 neg neg pos 

BM 

IP 

neg 

PB IP 

neg 

SCE 

vitro 

neg 

SCE 

vivo 

neg 

SA7/SH

E 

Titanium 

tetrachloride 

 neg CHO neg 

bacte

rial 

 
Standard Review for P09-0054/55/56/57 

25 February 2009 Page 12 of 71 

Chemical Gene mutations Chromosome mutations DNA Other 

SAL E 

co

li 

lower 

eukaryote

s 

mammali

an in vitro 

CA 

vitro 

MN 

vitro 

CA 

vivo 

MN 

vivo 

 Cell 

transfor

mation 

Sperm

head 

abnor

malitie

s 

Titanium 

trichloride 

pos neg 

SCE 

vitro 

 

Abbreviations: 

BFU = body fluid urine

BM = bone marrow 

CA = chromosome aberrations 

CHO = Chinese hamster ovary cells 

ES = mouse embryonic stem cells

IP = intraperitoneal injection 

L51 = mouse lymphoma cells 

MN = micronucleus 

neg = negative 

PB = peripheral blood 

pos = positive 

SAL = Salmonella 

SCE = sister chromatid exchange 

SMART = somatic mutation and recombination test in Drosophila

SRL = sex-linked recessive lethal in Drosophila

vit = in vitro 

vv = in vivo 

weak = weakly positive 
25 February 2009 Page 13 of 71 

As a nanotech material, PMNs 09-54 through -57 may be weak inducers of aneuploidy, and 

inducers of DNA effects, both in mammalian cells in culture. Based upon reports on similar 

materials, they may induce gene mutations in mammalian cells in culture but not in prokaryotes, 

lower eukaryotes, or mammals in vivo; they may not induce DNA effects in lower eukaryotes in 

vivo, nor cell transformation in mammalian cells in culture. 

 

 Due to uncertainty about their ability to penetrate cells, testing of nanomaterials in 

genotoxicity assays is problematic, and negative responses would not conclusively rule out the 

potential mutagenicity hazard of nanotubule materials in humans. No testing of the PMNs is 

recommended at this time. 

 
Standard Review for P09-0054/55/56/57 

25 February 2009 Page 14 of 71 

From: Yin-tak Woo 

To: Steve Cragg 

Thru: Don Rodier 

Re: P09-54 to P09-57: Cancer Concern 

THIS MEMORANDIUM CONTAINS CONFIDENTIAL BUSINESS INFORMATION 

The PMN chemicals P09-54 to P09-57 are a series of fullerene (buckyball) nanomaterials of 

increasing sizes. Their chemical names and CAS numbers are summarized below: 

PMN No. Chemical Name CAS RN Common Name 

P09-54 [5,6]Fullerene-C60-Ih 99685-96-8 C60 

P09-55 [5,6]Fullerene-C70-D5h(6) 115383-22-7 

P09-56 [5,6]Fullerene-C84-D2 145809-19-4 

P09-57 [5,6]Fullerene-C84D2d 145809-20-7 

There is no cancer bioassay data available for assessing the carcinogenic potential of fullerenetype nanomaterials. A cancer concern for the above PMN chemicals was raised at the SAT, in 

part, by comparison to carbon nanotubes. Depending on the dimensions and durability, some 

carbon nanotubes have been shown to have asbestos-like characteristics and toxicity. Multi-wall 

carbon nanotubes (MWCNT) have been shown to induce asbestos-like pathogenicity when 

introduced into the abdominal cavity of mice (Poland et al., 2008) as well as to induce 

mesotheliomas when injected intraperitoneally to p53 heterozygous mice that have been reported 

to be sensitive to asbestos (Takagi et al., 2008) 

Several lines of evidence, however, suggest that the analogy between fullerene type 

nanomaterials and carbon nanotubes is limited and that the carcinogenic potential of fullerene 

type nanomaterials should be lower than that of carbon nanotubes. 

(1) Dimension not asbestos-like: In contrast to the fibrous or rod-shaped carbon nanotubes, 

fullerene (buckyballs) type compounds are spherical. Therefore, fullerene type compounds are 

not expected to be asbestos-like. 

(2) Evidence of antioxidant activities: Depending on the specific test conditions, the type of 

fullerene compounds and the test organism, fullerene can be either a powerful antioxidant or an 

oxidative damaging agent (e.g., Spoohn et al. 2008). Pristine C60 and derivatives have been 

shown to have antioxidant activities in a variety of mammalian cells under in vitro conditions 

 as well as in rodents protecting against chemically 

(e.g., alcohol, carbon tetrachloride, doxorubicin) or radiation induced oxidative damage (Gharbi 

et al., 2005; 

(3) Low acute or sub-acute toxicity: Available data show that pristine C60 has little or no acute 

or sub-acute toxicity in a large variety of living organisms, from bacteria and fungi to human 

leukocytes, and also in drosophila, mice rats and guinea pigs (Kolosnjaj et al., 2007; Gharbi et 

al., 2005; Sayes et al., 2007: Baker et al., 2008). A short-term inhalation study of aerosolized C60

nanoparticles showed that the lung burden of C60 nanoparticles did not appear to be very high; 

the half-life was 26 days. 

Although the PMN chemicals are not expected to be asbestos-like carcinogens, there are still 
Standard Review for P09-0054/55/56/57 

25 February 2009 Page 15 of 71 

uncertainties about other potential mechanisms of carcinogenesis and the concern for potential 

carcinogenicity after long term exposure cannot be excluded. In this respect, the following 

findings may provide some support 

(1) Evidence of causing oxidative stress: In contrast to reports of antioxidant activities 

mentioned above, in cell culture, C60 has been shown to synergize the effects of oxidative stressinducing agents and elicit cytotoxic action through cell membrane lipid peroxidation (Sayes et 

al., 2005; In vivo, C60 induced oxidative stress and lipid peroxidation in 

the brain of juvenile largemouth bass (Oberdorster, 2004). Using zebrafish embryos, Usenko, C. 

et al. (2008) showed evidence of global expression of genes indicative of oxidative stress 

response after C60 exposure. 

(2) Evidence of photo activation: Although pristine C60 is relative nontoxic under dark 

conditions, there is evidence that in the presence of light or uv, C60 can be highly toxic through 

generation of singlet oxygen (Kolosanjaj et al., 2007). C60 and derivatives have been shown to be 

phototoxic to human keratinocytes and lens epithelial cells 

(3) Potential DNA effect: Computer simulation studies showed that C60 buckyballs could enter 

into cells and latch onto spiral DNA molecule, making it change shape 

(http://www.bloggernews.net/115920) 

Given the fact that there is a lack of information on long-term toxicity and biodurability of the 

PMN chemicals, to be prudent, a 2-year cancer bioassay of at least one of the PMN chemicals is 

recommended if there are of inhalable size and exposure is deemed substantial. As a trigger for 

the long term assay, a 90-day inhalation study is recommended for at least two of the PMN 

(preferably, C60 and one of the C80). In addition to assessing carcinogenic potential, these studies 

can also be used to assess the potential systemic toxicity (e.g., cardiotoxicity, neurotoxicity, 

immunotoxicity) as well as biodurability and body burden. 

References 

Baker,G.L. et al. (2008). Inhalation toxicity and lung toxicokinetics of C60 fullerene 

nanoparticles and microparticles. Tox. Sci. 101: 122-131 

Gharbi, N. et al. (2005). [60]fullerene is a powerful antioxidant in vivo with no acute or subacute 

toxicity. Nano Lett. 5: 2578-2585.. 

Kolosnjaj, J. et al. (2007). Toxicity studies of fullerenes and derivatives. Adv. Exp. Med. Biol. 

620: 168-180. 
Standard Review for P09-0054/55/56/57 

25 February 2009 Page 16 of 71 

Oberdorster, E. (2004). Manufactured nanoomaterials (fullerene, C60) induce oxidative strss in 

the brain of juvenile largemouth bass. Environ. Health Persp. 112: 1058-1062. 

Poland, CA et al. (2008). Carbon nanotubes introduced into the abdominal cavity of mice show 

asbestos-like pathogenicity in a pilot study. Nat. Nanotechnol. 3: 423-428. 

Sayes, C.M. et al. (2004). The differential cytotoxicity of water-soluble fullerenes. Nano Lett. 4: 

1881-1887. 

Sayes, C.M. et al. (2007) Comparative pulmonary toxicity assessments of C60 water 

suspensions in rats: few differences in fullerene toxicity in vivo in contrast to in vitro profiles. 

Nana Lett. 7:2399-2406. 

Spohn, P. et al. (2008). C(60) fullerene: a powerful antioxidant or a damaging agent? The 

importance of an in-depth material characterization priorto toxicity assays. Environ. Pollut. 

[Sep 26, 2008 Epub ahead of print] 

Tkahagi, A. et al. (2008). Inudction of mesotheliomas in p53+/- mouse by intraperitoenal 

application of multi-wall carbon nanotube. J. Toxicol. Sci. 33: 105-116. 

Usenko, CY et al. (2008) Fullerene C60 exposure elicits an oxidative stress in embryonic 

zebrafish. Toxicol. Appl. Pharmacol. 229: 44-55. 
Standard Review for P09-0054/55/56/57 

25 February 2009 Page 17 of 71 

02/05/09 

MEMORANDUM

SUBJECT: P09-54-57: Immunotoxicity Standard Review 

 

FROM: Ronald E. Ward Ph.D., Microbiologist/Immunologist 

 RAD/SSB (7403M) 

THRU: Donald Rodier, Branch Chief 

 RAD/SSB (7403M) 

 

TO: Steve Cragg Ph.D., Technical Integrator 

 NCSAB (7403M) 

 

TSCA CONFIDENTIAL BUSINESS INFORMATION 

 

Conclusion: P09-54 ([5,6]Fullerene-C60-Ih), P09-55 ([5,6]Fullerene-C70-D5h(6)), P09-56 

([5,6]Fullerene-C84-D2), and P09- 57 ([5,6]Fullerene-C84-D2d) are potential immunotoxicity 

hazards. Pulmonary exposure to these PMNs may lead to reduced alveolar macrophage 

phagocytosis, impaired clearance of inhaled bacteria, inflammation, and possible systemic 

immunosuppression. 

Basis for Conclusion:

(1,2). These PMNs are poorly soluble particulates (2). P09-54 has a molecular weight of 721, 

and PMN-55-57 have a molecular weight of 841. These PMNs will be poorly absorbed into the 

body via all routes (2). 

 Since there were no immunotoxicity data associated with these PMNs, the conclusions 

reached here are based on analog published scientific data. 

 One group of potential effects caused by exposure to the new chemical substance is due 

to “lung overload” (2, 3). Lung particle overload occurs when insoluble particles are inhaled 

into the lung to such an extent that the capacity of the alveolar macrophages to clear the 

deposited particles efficiently is overwhelmed (3). This overload ultimately leads to prolonged 

particle clearance, inflammation, cell proliferation, and chronic lung disease. The resultant 

inflammation may damage cells of the lung and may lead to the exacerbation of asthma (4). 

 However, these PMNs consists of nano-sized (fullerene-derived) particles. There is a 

concern that nano-sized particles, having an increased surface area per unit mass, may be more 

toxic than larger particles. Due to the concerns over the risk of nanomaterials, there has been a 

recent increase in nanomaterial-related safety research. In addition, several related reviews have 

been published. 

 According to two fairly recent reviews (5,6), most research supports the idea that both 

multi-walled and single-walled carbon nanotubes have the potential to cause pulmonary toxicity. 

Once taken up by humans, carbon nanotubes may cause oxidative stress, inflammation, and cell 

damage leading to effects like granulomas, fibrosis and wall thickening. The review by Stern et 
Standard Review for P09-0054/55/56/57 

25 February 2009 Page 18 of 71 

al.(6) lists 7 published rodent pulmonary CNT toxicity studies. All but one showed evidence of 

lung toxicity. 

 Studies in the literature also support the idea that both local pulmonary immunity and 

systemic immunity can be impacted following CNT inhalation. Alveolar macrophages are 

important in both innate and adaptive immunity in the lung. These cells play a central role in the 

defense against respiratory bacteria, virus, and fungal infections. 

 In a recent publication, Shvedova and coworkers (7) showed that intratracheal instillation 

of SWCNT in mice not only elicited inflammation and lung damage, but also caused a delayed 

clearance of Listeria monocytogenes. In their experiments, C57Bl/6 mice were given SWCNT 

(0, 10 and 40 μg/mouse via pharyngeal aspirations) and 3 days later exposed to Listeria 

monocytogenes (103 bacteria/mouse). Exposure to both SWCNT and Listeria monocytogenes 

amplified lung inflammation and collagen formation. Despite this inflammatory response, 

SWCNT pre-exposure significantly decreased the pulmonary clearance of Listeria 

monocytogenes measured 3 - 7 days after microbial infection relative to controls. Decreased 

bacterial clearance in SWCNT pre-exposed mice was associated with decreased phagocytosis of 

bacteria by macrophages and a decrease in nitric oxide production by these phagocytes. These 

results support the idea that SWCNT exposure may lead to increased susceptibility to lung 

infections in exposed populations. 

 Systemic immunotoxicity to inhaled multiwalled carbon nanotubes was the subject of a 

recent published study (8). In this study, C57BL/6 adult (10-12 weeks old) male mice were 

exposed via whole body inhalation to control air or 0.3, 1, or 5 mg/m3 respirable aggregates of 

MWCNTs (diameter=10-20 nm and length=5-15 μm) for 7 or 14 days (6 hr/day). (In a later 

publication, this material was shown to be a mixture of MWCNTs and nanofibers.) At the end of 

this period, pulmonary injury was analyzed using standard measures including histopathology, 

WBC count, and some measurements of oxidative stress and cytokine induction. In addition, the 

ability of this substance to cause systemic immunosuppression was analyzed. 

 The histopathology results of this study showed that particulate material was contained in 

alveolar macrophages, but no inflammation or tissue damage was observed. Bronchial alveolar 

lavage fluid analysis also demonstrated particle-laden macrophages, with no increase in WBC 

count. However, MWCNT/nanofiber exposure of 0.3 mg/m3 and higher concentrations caused 

systemic immunosuppression after exposure for 14, but not 7, days. The immunosuppression 

was characterized as a reduced T-cell dependent antibody response to sheep erythrocytes, as well 

as a reduced T cell proliferation induced by concanavalin A, a T cell mitogen. Animals exposed 

to 1 mg/m3 also had decreased NK cell function. These results showed that, even though the 

nanoparticle material caused no pulmonary injury or inflammation, MWCNT/nanofiber mixture 

exposure resulted in systemic immunosuppression. 

 To help elucidate a mechanism, the authors of this report found that the mRNA levels for 

the cytokine interleukin-10 (IL-10) and the enzyme NAD(P)H oxidoreductase 1 (an indicator of 

oxidative stress) were increased in the spleen, but not the lung, upon 14 day exposure. These 

authors suggest that oxidative stress and IL-10 may play roles in inducing immunosuppression. 

 There seems to be no direct immunotoxicity testing of fullerenes in the published 

literature (9). In addition, there are only a few experimental repeat-dose studies of fullerenes 

(reviewed in 10). Interestingly, existing data show that fullerenes have a low acute toxicity 

(reviewed in 10). Nielson and colleagues (10) postulate that, at low doses, fullerenes may have 

beneficial in vivo effects; whereas, at high doses, inhalation exposures may cause inflammation 

and production of ROS, 
Standard Review for P09-0054/55/56/57 

25 February 2009 Page 19 of 71 

 Collectively, these results support the idea that P09-54-57 would be potential 

immunotoxicity hazards. Pulmonary exposure to these PMNs may lead to reduced alveolar 

macrophage phagocytosis, impaired clearance of inhaled bacteria, inflammation, and possible 

systemic immunosuppression. 

 References

1.P09-54-57 

2.P09-54-57- 

3. 5.Oberdorster, G. Lung Particle Overload: Implications for Occupational Exposures to 

Particles. Regulatory Tox. And Pharm. 27, 123-135. (1995). 

4. Luster, M. et al. Overview of Immunotoxicology and Current Applications to Respiratory 

Diseases. Immunopharmacology 48 (2000), 311-313. 

5. Helland, A. et al. Reviewing the Environmental and Human Health Knowledge Base of 

Carbon Nanotubes. EHP 115 (8), 1125-1131 (2007). 

6.Stern, S, and McNeil, S. Nanotechnology Safety Concerns Revisited. Tox. Sci. 101, 4-21 

(2008). 

7. Shvedova, A. et al. Sequential Exposure to Carbon Nanotubes and Bacteria Enhances 

Pulmonary Inflammation and Infectivity. Am. J. Respir. Cell Mol. Biol., Volume 38, Number 5, 

May 2008, 579-590. 

8. Mitchell, L. A. et al. Pulmonary and Systemic Immune Response to Inhaled Multiwalled 

Carbon Nanotubes. Toxicological Sciences 100 (1), 203-214 (2007). 

9) Ward. R. E. 2009, Pubmed search for Fullerene and Immunotoxicity. 

10) Nielsen, G. D. et al. In vivo Biology and Toxicology of Fullerenes and Their Derivatives 

2008 Basic and Clinical Pharmacology and Toxicology 103, 197-298.
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