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 Na no-C, Inc on 5 November 2008, are 4

“fullerenes” defined as a family of carbon compo unds 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 Full er, 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, ch emical 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: 841Standard 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 si de and top views of the same fullerene. The

fullerenes are used as:

These fullerenes are solid with negligible solubil ity in water and negligib le vapor pressure (SAT

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

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

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

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

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

aggregation/agglomeration, physical form, par ticle size distribution, surface area solubility,

dispersability, shape, adsorption tend ency 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: 527-660 o 

### C 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 - 1427 o 

### C (literature values)

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

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

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

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

reactivity, aggregation/agglomer ation, physical form, particle size distribution, surface area

solubility, dispersability, shape, adsorpti on tendency and surface charge porosity.

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

properties and lack of knowledge fo r this class of materials.

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

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

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

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

setting, the oral route of exposure also is eliminat ed. The population at larg e is not considered at

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

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

25 February 2009 Page 4 of 71 

### species. Threats to terrestrial wildlife likely ar e 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. W oo) 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 Assess ment (C. Cinalli) Page 47

Economics Report (W. Hoffman) Page 50Standard 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: Migratio n 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 immunot oxicity 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 *

Sewa g e

Sludge 3-h EC50 _

Sewa g e

Sludge _ Chronic

Value _

Ecotox Values Comments: Predictions are based on

; MW 721; pH7; effective concentrations

based on 100% active ingredients and nomina l 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 564-8916Standard 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 w ith 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 problema tic, and negative responses would not conclusively rule out the

potential mutagenicity hazard of nanotubule materi als in humans. No furthe r 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; s ynonym 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 

### ar e not gene mutagens in

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

WP2 uvrA , and not chromosome mutagens in mamma lian CHL cells, in both cases without and

with metabolic activation.

was not a gene mutagen in Salmonella

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

mammalian V79 cells, in both cases wit hout 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 acti vation, 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 ge ne 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 al so been discussed previously. [5,6]Fullerene-C60-ih

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

mutagen in Salmonella strains TA102, TA104 and YG3003 in th e 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 cel l transformation in BALBc/3T3 cells in vitro . These

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

Other nanotech products for whic h mutagenicity data exist are nanoparticles of Fe 3 

### O 4



### and Fe 3 

### O 4 

### 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)

Gene mutations Chromosome mutations DNA Other Chemical

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

Fe 3 O 4 neg neg pos

Fe 3 O 4 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

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

25 February 2009 Page 12 of 71 Gene mutations Chromosome mutations DNA Other Chemical

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 positive25 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 ma mmalian 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 problema tic, and negative responses would not conclusively rule out the

potential mutagenicity hazard of nanotubule materi als 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 CONF IDENTIAL 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 C 60 

### 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 a bove 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 asbest os-like characteristics a nd toxicity. Multi-wall

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

introduced into the abdominal cav ity of mice (Poland et al., 2 008) 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 com pounds 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 or ganism, fullerene can be either a powerful antioxidant or an

oxidative damaging agent (e.g., Sp oohn et al. 2008). Pristine C 60 

### 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 agains t chemically

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

et al., 2005;

(3) Low acute or sub-acute toxicity: Available data show that pristine C 60 

### has little or no acute

or sub-acute toxicity in a larg e variety of living organisms, fr om bacteria and fungi to human

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

al., 2005; Sayes et al., 2007: Baker et al., 2008). A short-term inhalation study of aerosolized C 60 

### nanoparticles showed that the lung burden of C 60 

### 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 carcin ogens, there are stillStandard 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 report s of antioxidant activities

mentioned above, in cell culture, C 60 

### has been shown to synergize th e effects of oxidative stressinducing agents and elicit cytot oxic action through cell membrane lipid peroxidation (Sayes et

al., 2005; In vivo, C 60 

### induced oxidative stress a nd lipid peroxidation in

the brain of juvenile largemouth bass (Oberdor ster, 2004). Using zebrafi sh embryos, Usenko, C.

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

response after C 60 

### exposure.

(2) Evidence of photo activation : Although pristine C 60 

### is relative nontoxic under dark

conditions, there is evidence that in the presence of light or uv, C 60 

### can be highly toxic through

generation of singlet oxygen (Kolosanjaj et al., 2007). C 60 

### and derivatives have been shown to be

phototoxic to human keratinocytes and lens ep ithelial cells

(3) Potential DNA effect: Computer simulation studies showed that C 60 

### 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 informa tion on long-term toxicity and biodurability of the

PMN chemicals, to be prudent, a 2-year cancer bi oassay 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 i nhalation study is recommended for at least two of the PMN

(preferably, C 60 

### and one of the C 80 

### ). In addition to assessing car cinogenic potential, these studies

can also be used to assess the potential system ic toxicity (e.g., cardi otoxicity, neurotoxicity,

immunotoxicity) as well as biodurability and body burden.

References

Baker,G.L. et al. (2008). I nhalation toxicity and lung toxi cokinetics of C60 fullerene

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

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

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

Kolosnjaj, J. et al. (2007). Toxi city 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). Ma nufactured nanoomaterials (fullerene , C60) induce oxidative strss in

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

Poland, CA et al. (2008). Carbon na notubes introduced into the a bdominal cavity of mice show

asbestos-like pathogenic ity in a pilot study. Na t. Nanotechnol. 3: 423-428.

Sayes, C.M. et al. (2004). The differential cytotoxi city of water-soluble fu llerenes. Nano Lett. 4:

1881-1887.

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

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

Nana Lett. 7:2399-2406.

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

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

[Sep 26, 2008 Epub ahead of print]

Tkahagi, A. et al. (2008). Inudction of meso theliomas in p53+/- m ouse by intraperitoenal

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

Usenko, CY et al. (2008) Fulle rene 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., Microbiol ogist/Immunologist

RAD/SSB (7403M)

THRU: Donald Rodier, Branch Chief

RAD/SSB (7403M)

TO: Steve Cragg Ph.D., T echnical 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 P 09- 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 b acteria, inflammation, and possible systemic

immunosuppression.

Basis for Conclusion:

(1,2). These PMNs are poorly soluble particul ates (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 a ssociated with these PMNs, the conclusions

reached here are based on analog published scientific data.

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

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

into the lung to such an exte nt that the capacity of the al veolar 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 particle s, 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 re search. In addition, seve ral 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, in flammation, and cell

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

25 February 2009 Page 18 of 71 

### al.(6) lists 7 published rodent pul monary 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 followi ng CNT inhalation. Alve olar macrophages are

important in both innate and adapti ve immunity in the lung. These ce lls play a central role in the

defense against respiratory bacteria , virus, and fungal infections.

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

of SWCNT in mice not only elicited inflamma tion 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 μ g/mouse via pharyngeal aspirations ) and 3 days later exposed to Listeria

monocytogenes (10 3 

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

amplified lung inflammation and collagen form ation. Despite this inflammatory response,

SWCNT pre-exposure significantly decr eased the pulmonary clearance of Listeria

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

bacterial clearance in SWCNT pre-exposed mice wa s associated with decr eased phagocytosis of

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

results support the idea that SWCNT exposure may lead to in creased susceptib ility to lung

infections in exposed populations.

Systemic immunotoxicity to inhaled multiw alled 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/m 3 respirable aggregates of

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

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

this period, pulmonary injury was analyzed us ing standard measures including histopathology,

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

ability of this substance to cause sy stemic immunosuppression was analyzed.

The histopathology results of th is study showed that particul ate material was contained in

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

lavage fluid analysis also dem onstrated particle-laden macropha ges, 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 conc anavalin 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 inju ry 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 oxidor eductase 1 (an indicator of

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

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

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

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

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

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

beneficial in vivo effects; whereas, at high doses, inha lation 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 th e 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 Cu rrent Applications to Respiratory

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

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