TI: RAD Disposition 

PM: Zofia Kosim August 11, 2009 

Risk Assessment for P09-0416 

This Report Contains CBI Information 

Background Information 

Physicochemical Properties

MW: 

Physical State: 

BP: 

VP: 

neat solid 

> 400 

< 0.000001 

neat solid 

> 400 

< 0.000001 

 
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Other General Information

 

 

 

 
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Health Hazard Assessment 

Nanoparticle absorption/deposition/removal 

For insoluble particles with size less than 10 micrometers, penetration into the deep lung (i.e., 

alveoli and terminal bronchioles) is assumed to occur. Removal of deep-lung respirable particles 

deposited within the alveoli and terminal bronchioles is via phagocytosis (engulfment) by 

macrophages, a type of white blood cell, followed by migration of the particle-containing 

macrophage upward to the ciliary escalator to exit the lung, followed either by swallowing or 

expectorating. Particle-containing macrophages are also capable of penetrating lung tissue and 

ultimately entering the blood or lymphatic system. In experimental animals exposed via 

inhalation, nano-particles have been found in the lymph nodes near the lung and other organs, 

presumably via transport in macrophages through the blood and/or lymphatic systems. 

For particles with size greater than 10 micrometers, deposition in the upper respiratory passages 

may occur and removal by the ciliary escalator is expected. These larger particles are not 

believed to reach the deep lung because they are too large. 

For either size particle, the two physiological defense mechanisms described above may be 

overwhelmed by significant exposures over a prolonged period of time resulting in pathology in 

a process informally referred to as “lung overload” and otherwise known as chronic obstructive 

pulmonary disease (COPD). In this process, particles may build-up in the alveoli and terminal 

bronchioles. When taken up by macrophages, particles also may build up in the interstitial 

spaces between lung cells after the macrophage has moved out of the alveoli/bronchioles. 

Overloaded macrophages may die, releasing enzymes that cause a chronic inflammatory 

response, itself leading to destruction of lung tissue. More lymphocytes may collect at 

particulate laden foci further exacerbating the inflammatory process. For any poorly soluble 

particle (PSP), there is potential concern for adverse lung effects via this process, sequentially 

leading to chronic lung inflammation followed by progressive fibrosis (even sometimes after 

exposures are discontinued), emphysema, and potentially to lung cancer and other systemic 

effects distant from the lung. The process of migration of nanomaterials to tissues outside the 

lung after inhalation exposure is not well understood currently. 

Dermal absorption of nanoparticles has not been well studied but it is believed that aggregated 

particles would not penetrate the skin significantly. The absorption, distribution, metabolism, 

and excretion of nanoparticles via the oral route of exposure, also is not well understood at this 

time. Neither dermal nor oral routes of exposure are believed to play a significant role in this 

risk assessment for worker or public exposures. 

 
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Carcinogenicity (D. Lai – See attached report) 

There are no data on the potential human health effects of itself (that comprise 

). These aggregates/agglomerates have health concerns for particles under the PSP (poorly 

soluble particles) category. A number of PSP such as 

have been shown to be carcinogenic to the lung. Particle surface 

characteristics are considered key factors in free radical and reactive oxygen specie (ROS) 

formation and in the development of fibrosis and cancer by quartz By 

analogy to these carcinogenic particles, there is a concern for the PMN for pulmonary fibrosis 

and cancer if inhaled (see attached toxicity and carcinogenicity hazard review). 

In a subchronic inhalation toxicity study, (2.22 mg/m3, 55 nm in 

diameter) and microparticles (2.35 mg/m3, 0.93 μm in diameter) were exposed to rats for 3 

hr/day for 10 consecutive days. No gross or microscopic lesions were observed in either group 

of treated rats, and only minimal changes in hematology and serum chemistry, and in 

bronchoalveolar lavage (BAL) fluid parameters were noted. In a second longer-term inhalation 

study of 90-days duration, Wistar-Han rats and B6C3F1 mice were exposed by nose-only 

inhalation to two sizes of (0.05 um or 1 um) 3 hours per day 5 days per week for 90 days. 

Exposure concentrations were 0, 0.5, 2.5 mg/m3 for 0.05 um and 0, 5, 15, and 30 mg/m3 for 

the 1 um There were no biologically significant exposure-related changes in these 

parameters in rats or mice exposed to either 0.05 um or 1 um 

As indicated above, no chronic exposure inhalation studies on are available. However, 

several long-term inhalation studies have been conducted with ultrafine particulates similar to 

these . Lifetime exposure studies for 

have been assessed for their potential to serve as surrogates for with 

which to derive a New Chemical Exposure Level (NCEL). Evaluated studies include those by 

 The study for diesel exhaust soot 

was selected as a basis for deriving a NCEL for this risk assessment and, as explained in the 

following sections, a NCEL was derived from it. 

Mutagenicity (M. Cimino) 

No mutagenicity data were provided with Based upon their 

nanotech properties, the PMNs may induce gene mutation in bacterial cells and in mammalian 

cells in culture and in vivo, chromosomal effects in mammalian cells in culture and in vivo, and 

DNA effects in mammalian cells in culture and in vivo. They may not induce gene mutation and 

DNA effects in lower eukaryotes in vivo or cell transformation in mammalian cells in culture. 

Due to uncertainty about their ability to penetrate cells, and to caveats based upon the 

nonstandard methodologies employed to obtain the majority of the positive results so far 

demonstrated in nanotech materials, testing of these materials in genotoxicity assays remains 

problematic, and negative responses would not conclusively rule out the potential mutagenicity 

hazard of materials in humans. No testing of the PMNs is recommended at this time 

(see Attached Mutagenicity hazard review). 
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Immunotoxicity (R. Ward) 

Based on published scientific data, some nanoparticles appear to have immunotoxicity hazard 

potential. Pulmonary exposure to these PMNs may lead to toxicity to alveolar macrophage, 

impaired clearance of inhaled bacteria and possible systemic immunosuppression (see attached 

Immunotoxicity hazard review). 

Developmental Toxicity (C. Austin; W. Richards – SRC) 

This report summarizes the potential adverse developmental effects of 

 The three PMN substances are derivatives. No data were 

available on the developmental toxicity of the PMN substances themselves. There are limited 

data (one study, described below) for , an analogue of the PMN substance; 

however, this study had several deficiencies (see section IV of this report). Due to these 

deficiencies and the use of a route of administration (i.p.) that is not an expected route of human 

exposure, this study does not appear acceptable for assessing the potential developmental 

toxicity of . This study was previously reviewed in 2004 as part of a 

standard review for (see attached Developmental Toxicity hazard review). 

 

 

Health Risk Assessment

Nil-to-minor effects were seen with underivatized in the 10- and 90-day repeatedexposure studies. While NTP studies are generally well conducted, no report has been 

forthcoming to date making evaluation of the 90-day study problematic. Although a 

close structural analog to the PMN substance, it is not possible to draw conclusions about these 

derivitized PMN based on longer-term endpoints such as tumorigenicity. Longer-term 

studies on which are more distantly related to from a 

structural standpoint are also available to generate 

concern levels relevant to . 
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Some fraction of the PMN material is in the respirable range. Therefore, the concern for any 

poorly soluble particles (PSP) supports a health concern for these PMNs. In addition, there is a 

concern for cardiovascular effects. Dermal absorption of particles over 1 micrometer is not 

considered significant thus the dermal route of exposure is not assessed in this risk assessment. 

Likewise, oral exposures are not considered relevant for workers or the public. 

Table 1 indicates an inhalation exposure value (0.11 mg/day/individual) during processing for 

the PMN. This value of 0.11, divided by 10 m3

 inhaled by a worker each work day, yields an 

inhalation exposure concentration of 0.011 mg/m3. A NCEL of 0.025 mg/m3 may be derived 

from the on as explained in the footnote below.1 

This NCEL may be appropriate as it is derived from a chronic carcinogenesis study of 

. As indicated above, a discussion of this study is 

attached. 

The worst case worker inhalation exposure of 0.011 mg/m3

 for P09-416 during manufacturing is 

slightly less than the NCEL of 0.025 mg/m3 for diesel exhaust soot. 

 

Testing Recommendations

The standard in the New Chemicals Program is not to identify a significant risk to workers nor 

ask for testing when the NCEL is higher than the inhalation exposure estimate. However, this 

PMN case presents other factors that need to be considered. While the NTP study on 

underivitized examined the toxicity of a material that is more structurally similar to the 

PMN material, the NTP study found that there were no statistically significant adverse effects 

following a 90-day inhalation study. On the other hand, the DS study by found 

adverse effects in a longer-term study, but DS is more distantly related structurally to the PMN 

than the underivitized tested by NTP. In this particular PMN case, in order to resolve 

the inconsistencies in the few studies available on materials related to this PMN, testing is 

recommended. This finding is further supported by: 1) a low NCEL indicating significant 

toxicity for these particles; 2) the proximity of estimated exposure to the NCEL; and, 3) the 

possibility that exposures to respirable particulate are likely higher than estimated due to 

deficiencies in particle-sizing methods that under-estimate the smaller, respirable fraction of the 

particulate population. If tested, inhalation is the preferred method of exposure of the respiratory 

tract for hazard identification and to obtain dose-response data for use in quantitative risk 

assessment. A 90-day inhalation study by nose-only administration in male or female rats with 

up to 3 months observation post exposure, is recommended for evaluating the toxicity of 

 substances (CFR 870.3465). There are several additional considerations for using the 

90-day subchronic protocol at CFR 870.3465: 

a. Evaluation includes markers of damage, oxidant stress, cell proliferation, the 

degree/intensity and duration of pulmonary inflammation, fibrosis, and cytotoxic effects 

 
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in the bronchoalveolar lavage fluid (BALF) and histopathology of pulmonary and expulmonary organs/tissues (cardiovascular, CNS, liver, kidney, etc., ). It is also 

recommended that the potential for cardiovascular toxicity be determined through 

monitoring of the most sensitive blood and/or plasma endpoints indicative of 

cardiovascular effects. 

b. Data on pulmonary deposition, clearance half-life (biopersistence) and translocation of 

the test material are desirable to aid in interpreting the test results. 

c. Size distributions should be determined prior to, and during, the inhalation tests. 

d. Differences in the physicochemical properties of the as-administered material – relative 

to the as-produced material -- should be provided Techniques used to produce the asadministered material should also be described. 

e. The proposed protocol for testing should be provided to EPA for review and comment 

prior to the initiation of testing. 

f. The results of the subchronic inhalation study may support the need to conduct a 2-year 

inhalation study bioassay in rats with the PMN substance. 

File:TSCA_PMN-90-day_InhalationTesting_12032008Sayre.doc 

Ecological Risk Assessment for P09-0416 

Hazard Assessment: 

Although available information is sparse to date regarding toxicity in the environment, 

some data has been developed. tested underivatized for acute 

aquatic toxicity in Daphnia magna, Hyalella azteca, benthic harpacticoid copepods (the only 

marine species tested), Fathead minnow, and medaka. Rather than use ultrasonication or solvent 

vehicles (tetrahydrofuran) to aid in solubilizing the stirred pure 

underivatized with sterile water for 2 months in the presence of sunlight, indicating 

that concentrations of 35 ppm were reached resulting in 10-200 nm aggregates with 

 on the outside of the aggregates and underivatized on the inside. The 

solutions containing the aggregates were further processed by centrifugation to remove larger 

aggregates and the resulting supernatant was used for testing with aquatic species. In salt water, 

aggregates came out of solution at 35 ppm resulting in a maximum of 22.5 ppm. 

In acute toxicity studies, could not determine EC/LC50 concentrations due to the 

poor solubility of in the test media. No effects were seen with Hyalella (35 ppm) or 

marine copepods (22.5 ppm). Daphnids were found to ingest without other effects 

from acute exposure. In a 21-day exposure of daphnia to at 5 ppm produced 40% 

mortality and concomitant reductions in reproduction and molting. In the acute Fathead minnow 

study, no mortality, effects on mRNA, P450 isozymes, or PMP70 (a peroxisome protein) were 

found nor on the up or down-regulation of these biomarkers at 0.5 ppm concentration. 

Likewise with Medaka, no effects were detected clinically or with biomarkers. 

The fate and effects of are reviewed in another relevant paper by 

This review discusses several studies employing to help solubilize 
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 It is suspected that may contribute to the toxicity found since tests without it, 

 show less or no toxicity. This overview of the literature should not be considered 

comprehensive. More data is being generated in this new and developing field of 

technology and will be incorporated into the environmental assessment as studies become 

available. 

If released into the aquatic environment, this PMN is unlikely to result in adverse effects to 

aquatic species. Materials with such low water solubility are categorized has having, “no effects 

at saturation” due to their poor initial solubility in water. 

 

Exposure Assessment: Although marginally more water soluble than it’s non-derivatized 

counterpart, this derivatized is also expected to have very low water solubility (personal 

communication: D. Darling). As a result, its measured concentration in the aqueous 

environment would be expected to be low. The average surface water concentration of these 

materials cannot be predicted with the present state of knowledge (EPA/EETD). 

Risk Characterization: Consequently, the risk of such materials to aquatic organisms in the 

environment cannot be determined quantitatively at this time by comparing predicted surface 

water concentrations with a concentration of concern. Qualitatively, risk to aquatic organisms is 

considered to be low because of the extremely low solubility of these materials in water, as 

exemplified by the . No ecotoxicity testing is recommended at 

this time. 

 

D. Darling has requested water solubility data from the submitter but it has not yet been provided.
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Appendices 

Structure Activity Report (SAT) (L. Keifer) Page 11 

Human health review of (D. Lai) Page 14 

Immunotoxicity (R. Ward) Page 21 

Developmental Effects (C. Austin/W. Richards - SRC) Page 24 

Genotoxicity (M. Cimino) Page 27 

Chemistry Report (D. Darling) Page 35 

Engineering Report (F. Arnold) Page 38 

Exposure Report (S. Flessner) Page 42 

Economics Report (W. Hoffman) Page 49 
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cases: I:

 

 

 

 

 

 

 

 

 
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February 26, 2009 

MEMORANDUM

SUBJECT: Health Hazard Review of 

FROM: David Lai (ECAB/RAD)

TO: Sylvon VonderPool (NCSAB/RAD) 

THRU: Jennifer Seed (ECAB/RAD) 

Attached is my review of the health hazard potential of the subject PMN. Please 

contact me should you have any questions regarding this review. 

This memo contains Confidential Business Information. 

Attachment 

cc: 

Bob Morcock 
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Health Hazard Review of 

I. CONCLUSION 

There are no data on the potential human health effects of the . Using energyfiltered transmission electron microscopy and electron tomography, uptake of 

 to the cytoplasm and nucleus of human macrophage cells has been reported. In a 

number of in vitro and in vivo studies utilizing the organic solvent to 

prepare water-soluble suspensions of cytotoxicity of to human cells or toxicity in the 

brain of juvenile largemouth bass due to lipid peroxidation and generation of reactive oxygen 

species (ROS) were reported. However, the use of suspensions prepared by solvent 

exchange methods in toxicity studies has been suspected to be due to the toxic effects of 

the toxicity data of remains a topic of debate within the scientific community. In a 

subchronic inhalation toxicity study, (2.22 mg/rn3

, 55 nm in diameter) 

and microparticles (2.35 mg/m3, 0.93 μm in diameter) were exposed to rats for 3 hr/day for 10 

consecutive days. No gross or microscopic lesions were observed in either group of treated rats, 

and only minimal changes in hematology and serum chemistry, and in bronchoalveolar lavage 

(BAL) fluid parameters were noted. No chronic exposure inhalation studies on are 

available. 

A number of mineral particles such as have been 

shown to be carcinogenic. Particle surface characteristics are considered to be key factors in free 

radicals and reactive oxygen species (ROS) formation and in the development of fibrosis and 

cancer by quartz By analogy to these carcinogenic mineral particles, there is a 

concern of the PMN for pulmonary fibrosis and cancer if inhaled. 

II. BASIS OF CONCLUSION 

 

They are virtually insoluble in water and tend to form 

aggregates . In aqueous systems they can form colloidal solutions which contains 

 of different sizes (20 nm and larger), shapes (facetted to spherical), 

surface charges and crystallinity, depending on the preparation methods 

Because of their nanoscale and unique physicochemical properties, there are concerns of 

 for human health effects. Many of the special properties of nanoparticles are due to 

the nano-size and an extremely large surface-to-volume ratio relative to bulk materials. As a 
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particle decreases in size, the surface area increases and a greater proportion of atoms/molecules 

are found at the surface compared to those inside. Thus nanoparticles have a much larger surface 

area per unit mass and a higher potential for biological interaction compared with larger 

particles. The increase in the surface-to-volume ratio results in the increase of the particle surface 

energy which may become reactive. Therefore, as materials reach the nano-scale, they often 

display different chemical and electronic properties/reactivity and can have toxicological 

properties that differ from their bulk materials. Data from limited pulmonary studies in rats have 

demonstrated that exposures to some nanoparticles produced enhanced 

toxicity responses when compared with larger-sized particles of similar chemical composition. 

For example, even a typically inert bulk compound, such as can elicit a biological response 

when it is introduced as a nanomaterial With the particle size reduced from 17 

μm (surface area: 3.99 x 102 cm/g) to 23.5 nm (surface area: 2.95 x 105 cm

2

/g), the toxicity 

(LD50) of particles sharply increased from >5000 mg/kg to 413 mg/kg in mice 

 In a subchronic inhalation study in rats, particles (20 nm) have been 

shown to elicit a persistently higher inflammatory reaction in the lungs compared to the largersizes (250 nm) 

Nanoparticles of various chemical compositions have been shown to generate reactive 

oxygen species (ROS) in both in vivo and in vitro studies . 

 demonstrated that nanoparticles preferentially mobilize to mitochondria. Since 

mitochondria are redox active organelles, nanoparticles may alter ROS production and interfere 

with anti-oxidant defenses. Nanoparticles have also been implicated in interfering with cell 

signaling via ROS-mediated activation of cytokine gene expression 

. Therefore, like particles, ROS release following 

exposure to nanoparticles may contribute to oxidative stress, DNA damage, and proliferation of 

preneoplastic cells leading to cancer. 

Recent studies have shown that nanoparticles injury can also proceed by nonoxidant 

paradigms that are specific to their novel properties. One example is the release of toxic ions 

when the thermodynamic properties of a material (including surface free energy) favor particle 

dissolution to a suspending medium or biological environment. nanoparticles dissolution 

can induce cytotoxicity and apoptosis in mammalian cells due to the release of ions under 

aqueous conditions Another paradigm ofnanoparticle 

toxicity is the ability of some nanoparticles to form a corona with proteins which leads to adverse 

biological effects through protein unfolding, fibrillation, thiol cross-linking and loss of enzyme 

activity 

The toxicity of has not been characterized. Using energy-filtered transmission 

electron microscopy and electron tomography, uptake of (60-270 nm in diameter) to 

the cytoplasm and nucleus of human macrophage cells has been reported In 

a number of in vitro and in vivo studies utilizing the organic solvent to 

prepare water-soluble suspensions of cytotoxicity of to human cells 

or toxicity in the brain of juvenile largemouth bass due to lipid 

peroxidation and generation of reactive oxygen species (ROS) were reported. It was shown that 

the cytotoxicity of is a sensitive function of surface derivation; in two different human cell 
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lines, the lethal dose of changes over 7 orders of magnitude with relatively minor 

alterations in structure, with the underivatized more than three orders of 

magnitude more toxic than Distinct cytotoxic mechanisms of pure 

versus hydroxylated have also been proposed . However, the 

use of suspensions prepared by solvent exchange methods in toxicity studies has been 

suspected to be due to the toxic effects of the toxicity data of remains a topic of 

debate within the scientific community Furthermore, in contrast to in 

vitro studies which showed that underivatized was more than three orders of magnitude 

more toxic than 624, no differences in lung toxicity between the two samples were found 

when instilled intratracheally in rats. Unlike quartz particles, exposures at 

0.2, 0.4, 1 .5 or 3.0 mg/kg to rats did not induce any adverse lung tissue effects indicative of 

fibrosis 3 months postinstillation into rats only transient inflammatory and 

cell injury effects were noted in rats exposed to both types of . These findings 

exemplify the difficulty in interpreting and extrapolating in vitro toxicity data to in vivo effects. 

In a subchronic inhalation toxicity study, (2.22 mg/rn3, 55 nm 

in diameter) and microparticles (2.35 mg/m3

, 0.93 μm in diameter) were exposed to rats for 3 

hr/day for 10 consecutive days. lung burden were greater in nanoparticles-exposed rats than 

in microparticle-exposed rats; the calculated lung deposition rate and deposition fraction were 41 

and 50% greater, respectively, in the exposed rats. Lung half-lives for 

nanoparticle and microparticles were 26 and 29 days, respectively. At necropsy, no gross or 

microscopic lesions were observed in either group of treated rats, and only minimal changes in 

hematology and serum chemistry, and in bronchoalveolar lavage (BAL) fluid parameters were 

noted Longer duration studies may be needed for further toxicological 

assessment of the potential toxicity of inhaled particles. 

However, a number ofmineral 

particles such as have been shown to be carcinogenic 

(see “poorly soluble particulates category” report). Studies of have 

demonstrated that the toxic and carcinogenic effects are related to the surface area and surface 

activity of inhaled particle

 Surface modification of silica affects its cytotoxicity, 

inflammation responses and fibrogenicity Particle surface 

characteristics therefore, are considered to be key factors in free radicals and reactive oxygen 

species (ROS) formation and in the development of fibrosis and cancer by quartz 

 By analogy to these carcinogenic mineral particles, there is concern of the 

PMN for pulmonary fibrosis and cancer if inhaled. 

III. TEST RECOMMENDATION 
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A 90-day inhalation study in rats to evaluate the pulmonary toxicity and carcinogenicity 

potential of is recommended. Since bronchoalveolar lavage fluid (BALF) analysis can 

provide sensitive biochemical markers for toxic responses and information on early signs of 

cytotoxicity, inflammation and fibrosis - important mechanistic 

information for particle toxicity/carcinogenicity, it is recommended to include BALF analysis in 

the 90-day inhalation study. 
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. 
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02/23/09

MEMORANDUM

SUBJECT: : Immunotoxicity Standard Review 

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

RAD/SSB (7403M) 

THRU: Donald Rodier, Branch Chief 

RAD/SSB (7403M) 

TO: Sylvon Von-Der-Pool Ph.D., Technical Integrator 

NCSAB (7403M) 

 TSCA CONFIDENTIAL BUSINESS INFORMATION 

Conclusion: Based on published scientific data analogs, 

would be 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: 

 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). 
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However, these PMNs consists of nano-sized 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 

have the potential to cause pulmonary toxicity. 

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

damage leading to effects like granulomas, fibrosis and wall thickening. The review by Stern et 

al.(6) lists 7 published rodent pulmonary 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 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 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 

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

monocytogenes (103

 bacteria/mouse). Exposure to both and Listeria monocytogenes 

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

pre-exposure significantly decreased the pulmonary clearance of Listeria 

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

bacterial clearance in 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 exposure may lead to increased susceptibility to lung 

infections in exposed populations. 

Systemic immunotoxicity to inhaled 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 

(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 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, 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, 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 
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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 in the published 

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

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

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

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

and production of ROS. 

Collectively, these results support the idea that 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

 
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STANDARD REVIEW OF DEVELOPMENTAL EFFECTS FOR 

I. INTRODUCTION

This report summarizes the potential adverse developmental effects of PMN 

 No data were 

available on the developmental toxicity of the PMN substances themselves. There are limited 

data (one study, described below) for , an analogue of the PMN substance; 

however, this study had several deficiencies (see section IV of this report). Due to these 

deficiencies and the use of a route of administration (i.p.) that is not an expected route of human 

exposure, this study does not appear acceptable for assessing the potential developmental 

toxicity of . This study was previously reviewed in 2004 as part of a 

standard review for 

 A comprehensive literature search of online databases, including (but not limited to) 

Medline, TOXLINE, TSCATS, and HSDB, was conducted to identify health effects data for the 

PMN substances and related carbon compounds. Search terms that were used in the literature 

search included No developmental toxicity data were 

found. 

II. CONCLUSIONS

 The study on described below reported 

that 18 hours after intraperitoneal administration to pregnant mice, was clearly distributed 

into the embryos based on the characteristic color development of .” The study also reported 

that intraperitoneal administration of a single dose of 25 mg/kg (lowest dose tested) to pregnant 

mice was the lowest dose at which treatment-related effects were observed, but effects at this 

dose level were equivocal (one of the pregnant mice had one embryo with an abnormal 

enlargement of the head; the other pregnant mouse had all normal embryos). Therefore, it 

appears more appropriate to designate 50 mg/kg as the lowest dose at which a treatment-related 

effect was observed (based on head and tail region abnormalities in half of the embryos 

examined and adverse effects on the yolk sac) and 25 mg/kg as the dose at which no treatmentrelated effects were observed. Maternal toxicity was not evaluated in this study. This study had 

several deficiencies (see section IV of this report). 

III. BASIS FOR CONCLUSIONS

in vivo

exposed groups of 2 pregnant SLC-ICR mice to 

 (>99.9% purity) solubilized with polyvinylpyrrolidone (PVP) via intraperitoneal (i.p.) 

administration at 0 (distilled water control), 0 (PVP control), or 137 mg/kg on day 10 of 

gestation. Embryos were examined at 18 hours post-administration. The number of embryos 

examined per dose level and per female was not indicated. 

 At 137 mg/kg, all embryos died, and showed severe abnormalities such as abnormal 

flexion. It was not indicated in the study report whether embryos at this dose level showed 
Contains CBI Standard Review Risk Assessment for P09-416 

August 11, 2009 25 of 53 

abnormal enlargement of the head (as seen at 50 and 25 mg/kg, described below). Embryos in 

the control groups appeared normal. 

 In an effort to determine the lowest effective dose, the authors exposed further groups of 

2 pregnant SLC-ICR mice to solubilized with PVP via i.p. administration at 0 (sodium 

chloride solution control), 0 (3.3 g/kg PVP + sodium chloride solution control), 0 (6.6 g/kg PVP 

+ sodium chloride solution control), 25, or 50 mg/kg on day 10 of gestation. Embryos were 

examined at 18 hours post-administration. Maternal toxicity was not evaluated. 

 At 50 mg/kg, abnormal shape (e.g., head and tail region abnormalities) was observed in 

50% of the embryos (number of abnormal embryos per pregnant female not indicated); yolk sacs 

were also adversely affected (exhibited shrunken membrane, narrow blood vessels). The authors 

reported that “At 50 mg/kg, was clearly distributed into the embryos based on the 

characteristic color development of ” The study report did not indicate whether this was 

also observed at the other tested doses of 25 mg/kg and 137 mg/kg. At 25 mg/kg, one of the 

pregnant mice had one embryo with an abnormal enlargement of the head; the other pregnant 

mouse had all normal embryos. Embryos in the control groups appeared normal. The authors 

reported a LOAEL of 25 mg/kg for developmental effects on embryos after in vivo

treatment of dams. However, since the effect seen at 25 mg/kg was equivocal (one of the 

pregnant mice had one embryo with an abnormal enlargement of the head; the other pregnant 

mouse had all normal embryos), it appears more appropriate to designate 50 mg/kg as the lowest 

dose at which a treatment-related effect was observed and 25 mg/kg as the dose at which no 

treatment-related effects were observed. 

 The study authors indicated that proliferation and differentiation of midbrain (MB) cells 

was adversely affected by in vivo exposure to a derivative (not described further), but 

these data were not provided. 

in vitro

removed MB tissues from the embryos of pregnant SLC-ICR mice 

on day 11 of gestation, dissociated the tissues into individual cells using trypsin, and plated the 

cell suspension. The MB cells were exposed in vitro to + PVP or PVP alone for 6 days. 

Individual exposure concentrations were not stated, but appeared to range from 40 to 600 

μg/mL. Cell differentiation and cell proliferation were inhibited in -treated cells (treatment 

with PVP alone also resulted in inhibition of cell differentiation and proliferation, but at much 

higher concentrations than . This effect appeared to be concentration-dependent. 

Furthermore, Figure 2(A) in the study report indicates that inhibition of cell differentiation as a 

result of treatment occurred at a concentration (approximately 300 μg/ml) at which no 

inhibition of cell proliferation, an indicator of cytotoxicity, was seen. For the authors 

reported an IC50 value of 430 μg/mL for inhibition of MB cell differentiation, and an IC50 value 

of 470 μg/mL for inhibition of MB cell proliferation. 

IV. ASSESSMENT OF STUDY QUALITY

No statistical analysis was performed in the study. Additionally, the in vivo

administration route was intraperitoneal, and only 2 mice per dose level were tested. Yolk sacs 

were reportedly examined at the 50 mg/kg dose, but it is not clear if yolk sacs were examined at 
Contains CBI Standard Review Risk Assessment for P09-416 

August 11, 2009 26 of 53 

the 137 mg/kg dose, and whether effects were seen in them. Additionally, the number of 

embryos examined per dose level and per female was not indicated. Some of the individual test 

concentrations for the in vitro experiment were not given; the reader is required to deduce the 

test concentrations from the figures. Also, in the in vitro experiment, the authors appear to use 

cell proliferation as indicator of cytotoxicity, but this was not plainly stated in the study report. 

V. REFERENCES
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February 23, 2009 

MEMORANDUM

SUBJECT: Mutagenicity Hazard Review of 

FROM: Michael C. Cimino, Ph.D. 

 Biologist 

 Science Support Branch 

 Risk Assessment Division (7403M) 

 

TO: Sylvon vonderPool 

 Technical Integrator 

 New Chemical Screening and 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 Based upon 

their nanotech properties, the PMNs may induce gene mutation in bacterial cells and in 

mammalian cells in culture and in vivo, chromosomal effects in mammalian cells in culture and 

in vivo, and DNA effects in mammalian cells in culture and in vivo. They may not induce gene 

mutation and DNA effects in lower eukaryotes in vivo or cell transformation in mammalian cells 

in culture. 

 Due to uncertainty about their ability to penetrate cells, and to caveats based upon the 

nonstandard methodologies employed to obtain the majority of the positive results so far 

demonstrated in nanotech materials, testing of these materials in genotoxicity assays remains 

problematic, and negative responses would not conclusively rule out the potential mutagenicity 

hazard of materials in humans. No testing of the PMNs is recommended at this time. 
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III. BASIS FOR THE CONCLUSIONS

 No mutagenicity data were provided with the Premanufacturing Notices on: 

 

Structures of the three 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 

. A recently published review of genotoxicity data on nanomaterials 

has also appeared and its data and conclusions are discussed below. 

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, 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 and 

mitochondrial DNA damage in vivo in mice in the form of reduction in mitochondrial DNA 

ampliWcation visualized by a quantitative polymerase chain reaction 

induced gene mutations in mouse 

embryonic stem cells in vitro and chromosome mutation in the form on 
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micronuclei in vivo in rats exposed intratracheally and also in vitro by the cytokinesis-block 

method 

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. 

II) reportedly is a gene mutagen in Salmonella strains TA102, TA104 and YG3003 in the 

presence of activation when irradiated with visible light but it 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. 

is reported as positive for gene mutations in 

Salmonella, in mouse lymphoma cells in culture in alveolar lung cells in vivo in rats 

exposed via trachea and in the in vivo transgenic rodent assay in 

MutaTMMouse It induces DNA effects in the form of sister chromatid 

exchanges (SCEs) in mammalian cells in vitro Small particles of 

 (14-15nm) induce DNA effects in the Comet assay , 

but medium-sized particles (37nm) do not

 These have also previously been reviewed and are not further 

discussed here. 

 The data on are summarized in the Table on the following page: 
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TABLE: MUTAGENICITY DATA ON PRODUCTS 

(Most results as reported in abstracts or a review) 

Gene mutations Chromosome 

mutations 

Chemical DNA Other 

SAL lower 

eukar

yotes 

mam

mali

an 

vit 

mam

mal 

viv 

CA 

vitro

MN 

vit 

M

N 

vi

v 

SC

E 

come

t 

other 

viv 

Cell 

transfor

mation 

 neg neg 

 neg neg 

 neg neg 

 neg neg pos 

vit 

pos 

mito 

 pos 

ES 

 pos po

s 

IT 

 

 neg 

BFU 

 neg neg 

vit 

 

pos 

& 

neg 

neg 

SMA

RT 

 neg 

SMAR

T 

neg 

BALBc 

pos pos 

L51 

pos 

hprt 

viv 

IT; 

pos 

TGR 

pos 

 pos 

vit 

pos 

(2 in 

SP), 

neg 

(1 in 

LP) 

vit 

 

Abbreviations: BFU = body fluid urine; CA = chromosome aberrations; ES = mouse embryonic stem cells: hprt = hypoxanthine-guanine; 

phosphoribosyltransferase locus; IT = intratracheal administration; L51 = mouse lymphoma cells; LP = large particles; mito = mitochondrial DNA damage; 

MN = micronucleus; neg = negative; pos = positive; SAL = Salmonella; SCE = sister chromatid exchange; SP = small particles; TGR = transgenic rodent 

assay; vit = in vitro; vv = in vivo
By analogy to other may induce 

gene mutation in bacterial cells and mammalian cells in culture and in vivo, chromosomal effects 

in mammalian cells in culture and in vivo, and DNA effects in mammalian cells in culture and in 

vivo. They may not induce gene mutation and DNA effects in lower eukaryotes in vivo or cell 

transformation in mammalian cells in culture. 

 Unfortunately, a large proportion of the positive data on materials has 

been obtained with assays for which no standardized (i.e., OPPTS or OECD) guidelines exist, or 

with assays employing unusual strains, cell lines or routes of administration. As examples, a test 

guideline for the in vitro micronucleus assay is currently being considered by the OECD but has 

not yet been approved. The transgenic rodent assay is under consideration for development of a 

test guideline by OECD, and the Comet assay is undergoing validation by ICCVAM, ECVAM 

and JaCVAM (respectively, the Interagency Coordination Committee on the Validation of 

Alternative Methods, the European Center for the Validation of Alternative Methods, and the 

Japanese Centre for the Validation of Alternative Methods). The strains used in the Salmonella

assay (other than on materials that were PMNs) were often not standard ones found in the test 

guideline for the assay. The intratracheal route of administration is not usually employed for in 

vivo studies. 

 Several national and international bodies (e.g, OECD; EU Expert Group) are attempting 

to develop appropriate mutagenicity testing schemes for nanotech materials. A recent extensive 

overview of the state of genotoxicity testing of nanotech materials has 

emphasized the desirability of using standardized methods. 

 If mutagenicity testing were to be conducted, an in vitro micronucleus assay by the draft 

OECD guideline 487 using the cytokinesis-block method might be employed. Alternatively, an 

in vivo micronucleus assay (OPPTS 870.5395; OECD 474) by the intratracheal route might be 

useful, with sampling of the alveolar cells rather than the usual bone marrow and/or peripheral 

blood. It is acknowledged that such testing would be in the nature of a research project, and that 

finding a laboratory qualified to conduct the test would be difficult. 

 Due to uncertainty about their ability to penetrate cells, and to the caveats expressed in 

the preceding three paragraphs, testing of nanomaterials in genotoxicity assays remains 

problematic, and negative responses would not conclusively rule out the potential mutagenicity 

hazard of materials in humans. No testing of the PMNs is recommended at this time. 
Contains CBI Standard Review Risk Assessment for P09-416 

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 9

 

 

 

 
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Mode: 
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STANDARD REVIEW ENGINEERING REPORT CBI: Yes 

Focus Ready Draft 4/16/2009 Standard Review Report 6/22/2009 

 

PV (kg/yr): 180 Binding PV Import Only 

SUBMITTER: Konarka Technologies Inc. 

t 

an

 solar cells. CA 149:108336 - Component of active layers in 

solar cells. 

OTHER USES: CA 150:45934 - Component of electrochemical thin-film 

. 

 o s. 

30 po e 

 m and 

 1

 sed 

d capacitors and improve 

the mechanical properties of lubricants and plastics. 

MSDS: Yes Label: No 

th skin 

e 

cause eye irritation; may cause skin irritation; may be harmful 

 

LVE PPE: safety glasses, gloves, local ventilation 

CRSS : 

al Name: 3'H-Cyclopropa[1,9][5,6]fullerene-C60-Ih-3'-butanoic acid, 3'-

 opa[8,25][5,6]fullerene-C70-D5h(6)-3'-

ic 3' yl-,methyl ester 

20 /L @ 

 t 

 %<1000 

si at M SS Info: 

erial 

e LV

 5% of 

er , C8 ed 

Data: MP = 258 °C (DSC); WS = 0 g/L; Solubility in Toluene = 1.0 g/L. 

a 

 
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Consumer Use: Yes 

Related Cases and Related Cases and Misc SAT Info: 

 Unknown 

ion 

Eco: 1 No releases to Water 

OCCUPATIONAL EXPOSURE RATING: 1-2A 

NOTES & KEY ASSUMPTIONS: 

 

me s 

 Th l 

 

at

 a 

in in the 

 ses to 

st r

 re to both 

nh

 cells; 

 

er f 

 . 

ub ency 

1. 

 Al uring 

is 

d releases 

 a

 

nd assessed dust release and inhalation exposure (consistent with 

this IRER). 

POLLUTION PREVENTION CONSIDERATIONS: 

 be used in a solar module that will have a 

net benefit on the environment. 

EXPOSURE-BASED REVIEW: No 

INITIAL REVIEW ENGINEERING REPORT CBI: Yes 

PMN: 09-0416 

Processing: Encapsulated Photovoltaic Modules 

Number of Sites/ Location: 1 

Te ologies Inc. New Bedford MA 02745 

 

 
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August 11, 2009 Page 40 of 53 

days/yr to 271 by assuming 2 lines at the site. The LVE is imported in 1/2 L 

bottles. 

Process Description: Imported LVE (solid, 100%) --> added to 30 gallon 

nd mixed with other raw materials --> sample, filter, dispense 

lid, <3%) --> laminating and finishing coated product, LVE 

n solar cells encapsulated with SiO2 coater PET (solid; 

0.0000249%) 

 m th 

 

rated off-site using approved hazardous 

waste incinerator with 99% efficiency. 

2 kg/site-day over 271 days/yr from 1 site 

es 

 sion estimates 

009 kg/batch of LVE from filter media may be discarded as 

solid waste (2.7%). 

6E-2 kg/site-day over 271 days/yr from 1 site 

quipment Cleaning Losses of Solids from Process 

 

8k 0. 333kg batch may be 

g tank and coating equipment (5.4%). These 

wastes are incinerated off-site. 

2 kg/site-day over 271 days/yr from 1 site 

cineration (submission) 

del. Waste Stream 5: 

ion estimates 0.009 kg/batch LVE (2.7%) may be disposed as coating 

scraps. 
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August 11, 2009 Page 41 of 53 

Number of workers (all sites) with inhalation exposure: 3 

PA

 ma er 

64kg). Maximum number of exposure days 

respirator class is: I. Particulate (including solid or liquid 

droplets). 

INHALATION MONITORING DATA REVIEW 

1) el, regulator imit 

 dustry): s 

2)a) posure level > 1 mg/day? No 

 

 or reater? 1-2 No 

Inhalation Monitoring Data Desired? No 

Dermal: 

oncentration 

with dermal exposure: 3 

 Exposure to Carbon Nanotube Material, J. Toxicol. 

Environ. Health, 67:87-107. 

r 

Wastes; EPA/OPPT 2-Hand Dermal Contact with Liquids Model. 

MEMORANDUM of CBI TELEPHONE CONVERSATION 

 el u 

Organization: ERG 

 id 

iz narka Technologies Inc 

 3/2009 

 1 

 

ni hat TSCA CBI? 

PMN: 09-0416 

Q1: The mass balance parameters do not add up to 180kg/yr. 

 ou expect these 

process 

 t 

 

 a l solids (both 

ri
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Remarks: 
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