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

< 0.000001Contains CBI Standard Review Risk Assessment for P09-416

August 11, 2009 2 of 53 Other General InformationContains CBI Standard Review Risk Assessment for P09-416

August 11, 2009 3 of 53 Health Hazard Assessment

Nanoparticle absorption/deposition/removal

For insoluble particles with size less than 10 micr ometers, 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 a nd terminal bronchioles is vi a phagocytosis (engulfment) by macrophages, a type of white blood cell, follow ed by migration of th e particle-c ontaining macrophage upward to the ciliar y escalator to exit the lung, followed either by swallowing or expectorating. Particle-containi ng macrophages are also capable of penetrating lung tissue and ultimately entering the blood or lymphatic syst em. In experimental animals exposed via inhalation, nano-particles have been found in the lymph nodes n ear the lung and other organs, presumably via transport in macrophages th rough the blood and/or lymphatic systems.

For particles with size greater than 10 micromet ers, deposition in the upper respiratory passages may occur and removal by the ciliary escalator is expected. These larg er 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 prol onged period of time resulting in pathology in a process informally referred to as “lung overloa d” and otherwise known as chronic obstructive pulmonary disease (COPD). In this process, pa rticles may build-up in the alveoli and terminal bronchioles. When taken up by macrophages, part icles 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 dest ruction of lung tissue. More lymphocytes may collect at particulate laden foci further exacerbating the in flammatory process. For any poorly soluble particle (PSP), there is potentia l concern for adverse lung effects via this process, sequentially leading to chronic lung infla mmation followed by progressive fi brosis (even sometimes after exposures are discontinued), emphysema, and po tentially 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 signifi cantly. The absorption, distribution, metabolism, and excretion of nanoparticles via the oral route of exposure, also is no t well understood at this

time. Neither dermal nor oral r outes of exposure are believed to pl ay a significant role in this risk assessment for worker or public exposures.Contains CBI Standard Review Risk Assessment for P09-416

August 11, 2009 4 of 53 Carcinogenicity (D. Lai – See attached report)

There are no data on the potenti al human health effects of itself (that comprise ). These aggregates/agglomerat es 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 l ung. Particle surface characteristics are considered key factors in free radical and react ive 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 at tached toxicity and carcinoge nicity hazard review).

In a subchronic inhalation toxicity study, (2.22 mg/m 3 , 55 nm in diameter) and microparticles (2.35 mg/m 3 , 0.93 μ m in diameter) were exposed to rats for 3 hr/day for 10 consecutive days. No gross or mi croscopic lesions were ob served in either group of treated rats, and only minimal changes in hematology and serum chemistry, and in bronchoalveolar lavage (BAL) flui d 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/m 3 for 0.05 um and 0, 5, 15, and 30 mg/m 3 for the 1 um There were no biologically si gnificant 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 studie s have been conducted with ultr afine 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 Leve l (NCEL). Evaluated st udies 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 material s, 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 tes ting of the PMNs is recommended at this time (see Attached Mutagenicity hazard review).Contains CBI Standard Review Risk Assessment for P09-416

August 11, 2009 5 of 53 Immunotoxicity (R. Ward)

Based on published scientific data, some nanopart icles appear to have immunotoxicity hazard potential. Pulmonary exposure to these PMNs ma y lead to toxicity to alveolar macrophage, impaired clearance of inhaled bacteria and pos sible systemic immunosuppression (see attached Immunotoxicity hazard review).

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

This report summarizes the potential a dverse developmental effects of

The three PMN substa nces 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 rout e of administration (i.p.) that is not an expected route of human exposure, this study does not appear acceptabl e for assessing the potential developmental toxicity of . This study was pr eviously reviewed in 2004 as part of a standard review for (see attached De velopmental 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 th e 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 ar e also available to generate

concern levels releva nt to .Contains CBI Standard Review Risk Assessment for P09-416

August 11, 2009 6 of 53 Some fraction of the PMN material is in the resp irable range. Therefor e, the concern for any poorly soluble particles (PSP) supports a health c oncern for these PMNs. In addition, there is a

concern for cardiovascular effects. Dermal ab sorption of particles over 1 micrometer is not considered significant thus the derm al route of exposure is not asse ssed in this risk assessment. Likewise, oral exposures are not consider ed relevant for workers or the public.

Table 1 indicates an inhalation exposure valu e (0.11 mg/day/individual) during processing for the PMN. This value of 0.11, divided by 10 m 3 inhaled by a worker each work day, yields an inhalation exposure con centration of 0.011 mg/m 3 . A NCEL of 0.025 mg/m 3 may be derived

from the on as explained in the footnote below. 1
This NCEL may be appropriate as it is deri ved from a chronic carcinogenesis study of

. As indicated above, a disc ussion of this study is

attached.

The worst case worker inhalation exposure of 0.011 mg/m 3 for P09-416 during manufacturing is slightly less than the NCEL of 0.025 mg/m 3 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 th e 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 st ructurally similar to the PMN material, the NTP study found that there were no statistically signif icant adverse effects following a 90-day inhalation study. On the ot her hand, the DS study by found adverse effects in a longer-term study, but DS is more distantly re lated 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 suppor ted by: 1) a low NCEL indicating significant toxicity for these particles; 2) the proximity of estimated e xposure to the NCEL; and, 3) the possibility that exposures to respirable par ticulate are likely higher than estimated due to deficiencies in particle-sizing me thods that under-estimate the sma ller, 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 ad ministration 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 seve ral additional considerations for using the 90-day subchronic protocol at CFR 870.3465:

a. Evaluation includes markers of damage, oxi dant stress, cell proliferation, the degree/intensity and duration of pulmonary in flammation, fibrosis, a nd cytotoxic effectsContains CBI Standard Review Risk Assessment for P09-416

August 11, 2009 7 of 53 in the bronchoalveolar lavage fluid (BALF) and histopathology ofpulmonary and expulmonary organs/tissues (cardiovascular, CN S, liver, kidney, etc ., ). It is also recommended that the potential for cardi ovascular toxicity be determined through monitoring of the most se nsitive blood and/or plas ma endpoints indicative of

cardiovascular effects.

b. Data on pulmonary deposition, clearance half-lif e (biopersistence) a nd 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 shoul d 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 st udy may support the need to conduct a 2-year inhalation study bioassay in rats wi th 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 re garding 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 solubili zing the stirred pure

underivatized with ster ile water for 2 months in the presence of sunlight, indicating that concentrations of 35 ppm were reached resu lting in 10-200 nm aggregates with on the outside of the aggregates a nd underivatized on the inside. The solutions containing the aggregat es were further processed by centrifugation to remove larger aggregates and the resulting supernatant was used fo r testing with aquatic species. In salt water,

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

In acute toxicity studies, could not determine EC/L C50 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 reprodu ction and molting. In th e 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 bi omarkers at 0.5 ppm concentration. Likewise with Medaka, no effects were det ected clinically or with biomarkers.

The fate and effects of are reviewed in another relevant paper by This review discusses several studies employi ng to help solubilizeContains CBI Standard Review Risk Assessment for P09-416

August 11, 2009 8 of 53 It is suspected that may contri bute to the toxicity found since tests without it,

show less or n o toxicity. This overv iew of the literature should not be considered comprehensive. More data is being generated in this new and devel oping 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 exp ected to have very low water solubility (personal communication: D. Darling). As a result, its measured concentration in the aqueous environment would be expected to b e 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 ma terials to aquatic organisms in the environment cannot be determined quantitatively at this time by comparing predicted surface

water concentrations with a concentration of con cern. Qualitatively, risk to aquatic organisms is considered to be low because of the extremely lo w 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.Contains CBI Standard Review Risk Assessment for P09-416

August 11, 2009 9 of 53Contains CBI Standard Review Risk Assessment for P09-416

August 11, 2009 10 of 53 

## 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 49Contains CBI Standard Review Risk Assessment for P09-416

August 11, 2009 11 of 53 cases: I:Contains CBI Standard Review Risk Assessment for P09-416

August 11, 2009 12 of 53Contains CBI Standard Review Risk Assessment for P09-416

August 11, 2009 13 of 53Contains CBI Standard Review Risk Assessment for P09-416

August 11, 2009 14 of 53 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 que stions regarding this review.

This memo contains Confidential Business Information.

Attachment

cc: Bob MorcockContains CBI Standard Review Risk Assessment for P09-416

August 11, 2009 15 of 53 Health Hazard Review of

I. CONCLUSION

There are no data on the poten tial human health effects of the . Using energyfiltered transmission electron microsc opy and electron tomography, uptake of to the cytoplasm and nucleus of human macrophage cells has been reported. In a

numb er of in vitro and in vivo studies utilizing the organic so lvent 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 reporte d. However, the use of suspensions prepared by solvent exchange methods in toxicity stud ies has been suspected to be due to the toxic effects of

the toxi city data of remains a to pic of debate within the scientific community. In a

subchronic inhalation toxicity study, (2.22 mg/rn 3 , 55 nm in diameter) and microparticles (2.35 mg/m 3 , 0.93 μ m in diameter) were exposed to rats for 3 hr/day for 10 consecutive days. No gross or mi croscopic 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 chroni c exposure inhalation studies on are

available.

A number of mineral particles such as have been shown to be carcinogenic. Particle surface characteri stics are considered to be key factors in free radicals and reactive oxygen speci es (ROS) formation and in the development of fibrosis and cancer by quartz By analogy to these carcinogeni c mineral particle s, 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 larg er), shapes (facetted to spherical), surface charges and crystallinity, depending on the preparation m ethods

Because of their nanoscale and unique physicoc hemical properties, th ere are concerns of for human health effects. Many of th e special properties of nanoparticles are due to the nano-size and an extremely large surface-to-v olume ratio relative to bulk materials. As aContains CBI Standard Review Risk Assessment for P09-416

August 11, 2009 16 of 53 particle decreases in size, th e surface area increases and a grea ter proportion of atoms/molecules are found at the surface compared to those inside. Thus nanoparticle s have a much larger surface area per unit mass and a higher po tential for biological interaction compared with larger particles. The increase in the surf ace-to-volume ratio results in the increase of the particle surface energy which may become reactive. Therefore, as materials reach the na no-scale, they often display different chemical and electronic prope rties/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 nanoparticle s produced enhanced toxicity responses when compared with larger-s ized particles of sim ilar chemical composition. For example, even a typically inert bulk compou nd, such as can elic it a biological response

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

μ m (surface area: 3.99 x 10 2 cm/g) to 23.5 nm (surface area: 2.95 x 10 5 cm 2 /g), the toxicity (LD 50 ) of particles sharply increased from >5000 mg/kg to 413 mg/kg in mice

In a subchronic inhalati on study in rats, particles (20 nm) have been shown to elicit a persistently higher inflammatory reaction in the lungs co mpared to the largersizes (250 nm)

Nanoparticles of various chemical compositions have been s hown to generate reactive oxygen species (ROS) in both in vivo and in vitro studies

. demonstrated that nanoparticle s preferentially mobilize to mitochondria. Since mitochondria are redox active orga nelles, nanoparticles may alte r ROS production and interfere with anti-oxidant defenses. Nanoparticles have al so 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 oxid ative stress, DNA damage, and proliferation of preneoplastic cells leading to cancer.

Recent studies have shown that nanopartic les injury can also proceed by nonoxidant paradigms that are specific to their novel properties. One exampl e 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 Anot her paradigm ofnanoparticle toxicity is the ability of some nanoparticles to form a corona with proteins which leads to adverse biological effects through protein unfolding, fibr illation, thiol cross-linking and loss of enzyme activity

The toxicity of has not been characterized. Using energy-filtered transmission electron microscopy and elec tron tomography, uptake of (60-270 nm in diameter) to the cytoplasm and nucleus of hum an macrophage cel ls has been reported In

a number of in vitro and in vivo studies utilizing the organic so lvent to prepare water-soluble suspensions of cytotoxicity of to human cells or toxicity in the brai n of juvenile largemouth bass due to lipid peroxidation and generation of reactive oxygen speci es (ROS) were reported. It was shown that the cytotoxicity of is a sensitive function of surface deri vation; in two different human cellContains CBI Standard Review Risk Assessment for P09-416

August 11, 2009 17 of 53 lines, the lethal dose of changes over 7 orders of magnitude with relatively minor

alterations in struct ure, with the underivatized more than three orders of magnitude more toxic than Di stinct cytotoxic mechanisms of pure versus hydroxylated ha ve 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 eff ects 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 e ffects 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/rn 3 , 55 nm in diameter) and microparticles (2.35 mg/m 3 , 0.93 μ m in diameter) were exposed to rats for 3 hr/day for 10 consecutive days. lung burden were greater in na noparticles-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 mi nimal changes in hematology and serum chemistry, and in bronchoal veolar lavage (BAL) fluid parameters were

noted Longer duration stud ies 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” re port). Studies of have demonstrated that the toxic and carcinogenic e ffects are related to th e surface area and surface activity of inhaled particle

Surface modi fication of silica aff ects its cytotoxicity, inflammation responses and fibroge nicity Particle surface characteristics therefore, are c onsidered to be key factors in free radicals an d reactive oxygen species (ROS) formation and in the development of fibrosis and cancer by quartz By analogy to these carcinogenic mineral partic les, there is concern of the PMN for pulmonary fibrosis and cancer if inhaled.

III. TEST RECOMMENDATIONContains CBI Standard Review Risk Assessment for P09-416

August 11, 2009 18 of 53 A 90-day inhalation study in rats to evaluate the pulmonary toxicity and carcinogenicity potential of is recommen ded. Since bronchoalveolar lavage fluid (BALF) analysis can provide sensitive biochemical markers for toxic responses and information on early signs of cytotoxicity, inflammation and fi brosis - important mechanistic information for particle toxicity /carcinogenicity, it is recommended to include BALF analysis in the 90-day inhalation study.Contains CBI Standard Review Risk Assessment for P09-416

August 11, 2009 19 of 53 .Contains CBI Standard Review Risk Assessment for P09-416

August 11, 2009 20 of 53Contains CBI Standard Review Risk Assessment for P09-416

August 11, 2009 21 of 53 02/23/09

MEMORANDUM

SUBJECT: : Immunotoxicity Standard Review

FROM: Ronald E. Ward Ph.D., Microbiol ogist/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 asso ciated 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).Contains CBI Standard Review Risk Assessment for P09-416

August 11, 2009 22 of 53 However, these PMNs consists of nano-sized particles. There is a concern that nano-sized particles, having an increased surface ar ea 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

have the potential to cause pulmonary toxicity. Once taken up by humans, may cause oxidative stress, in flammation, and cell damage leading to effects like granulomas, fibros is and wall thickening. The review by Stern et al.(6) lists 7 published rodent pul monary toxicity studies. All but one showed evidence of lung toxicity.

Studies in the literature al so support the idea that both lo cal pulmonary immunity and systemic immunity can be impacted followi ng 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 coworkers (7) showed that intratracheal instillation of 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 (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 and Listeria monocytogenes amplified lung inflammation and collagen form ation. Despite this inflammatory response, 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 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 exposure may lead to in creased susceptib ility 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/m 3 respirable aggregates of (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 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 this study showed that particulat e 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, 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, 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 ofContains CBI Standard Review Risk Assessment for P09-416

August 11, 2009 23 of 53 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 role s in inducing immunosuppression.

There seems to be no direct immunotoxicity testing of in the published literature (9). In addition, there are only a fe w experimental repeat-dose studies of (reviewed in 10). Interestingly, existing data show that have a low acute toxicity (reviewed in 10). and co lleagues (10) postulate that, at low doses, may have

beneficial in vivo effects; whereas, at high doses, inha lation 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.

ReferencesContains CBI Standard Review Risk Assessment for P09-416

August 11, 2009 24 of 53 STANDARD REVIEW OF DEVELOPM ENTAL EFFECTS FO R

I. INTRODUCTION

This report summarizes the potential a dverse 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 rout e of administration (i.p.) that is not an expected route of human exposure, this study does not appear acceptabl e for assessing the potential developmental toxicity of . This study was pr eviously reviewed in 2004 as part of a

standard review for

A comprehensive literature search of onlin e 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 mi ce, was clearly distributed into the embryos based on the ch aracteristic color development of .” The study also reported that intraperitoneal administration of a single dos e of 25 mg/kg (lowest dose tested) to pregnant

mice was the lowest dose at which treatment-relate d effects were observed, but effects at this dose level were equivocal (one of the pre gnant 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 sect ion IV of this report).

III. BASIS FOR CONCLUSIONS

in vivo exposed groups of 2 pregna nt SLC-ICR mice to (>99.9% purity) solubilized with polyvin ylpyrrolidone (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 hour s 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 embr yos at this dose level showedContains 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 effec tive dose, the authors exposed further groups of 2 pregnant SLC-ICR mice to solubilized wi th PVP via i.p. administration at 0 (sodium chloride solution control), 0 (3.3 g/kg PVP + sodi um chloride solution co ntrol), 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, narro w blood vessels). The authors reported that “At 50 mg/kg, was clearly distributed into the embryos based on the characteristic color development of ” Th e 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 de velopmental 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 a ppropriate 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 indi vidual cells using tr ypsin, and plated the cell suspension. The MB cells were exposed in vitro to + PVP or PVP alone for 6 days. Individual exposure concentrat ions were not stated, but appe ared 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 ce ll differentiation and proliferation, but at much higher concentrations than . This effect appeared to be concentration-dependent. Furthermore, Figure 2(A) in the study report indi cates that inhibition of cell differentiation as a

result of treatment occurred at a con centration (approximately 300 μg/ml) at which no inhibition of cell proliferation, an indicator of cytotoxicity, was seen. For the authors reported an IC 50 value of 430 μg/mL for inhibition of MB cell differentiation, and an IC 50 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 s acs were examined atContains 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 wa s 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 plai nly stated in the study report.

V. REFERENCESContains CBI Standard Review Risk Assessment for P09-416

August 11, 2009 27 of 53 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 material s, 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 testi ng of the PMNs is recomm ended at this time.Contains CBI Standard Review Risk Assessment for P09-416

August 11, 2009 28 of 53Contains CBI Standard Review Risk Assessment for P09-416

August 11, 2009 29 of 53 III. BASIS FOR THE CONCLUSIONS

No mutagenicity data were provided with the Prem anufacturing 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 WP2 uvrA , and not chromosome mutagens in mammalian CHL cells, in

both cases without and with meta bolic 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 without a nd with metabolic activ ation

.

Another analogue, was not a gene mutagen in

Salmonella in strains YG1024 and YG1029 without activ ation, 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 ampli W cation visualized by a quantitative polyme rase chain reaction

induced gene mutations in mouse embryonic stem cells in vitro and chromoso me mutation in the form onContains CBI Standard Review Risk Assessment for P09-416

August 11, 2009 30 of 53 micronuclei in vivo in rats exposed intr atracheally 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 visibl e 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 abstra cts and summaries, and were not

reviewed in detail.

is reported as positive for gene mutations in

Salmonella , in mouse lymphoma cells in cultu re in alveolar lung cells in vivo in rats exposed via trachea and in the in vivo transgenic rodent assay in

Muta TM Mouse 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 a ssay , but medium-sized particles ( 37nm) do not

These have also previ ously been reviewed and are not further

discussed here.

The data on are summa rized in the Table on the following page:Contains CBI Standard Review Risk Assessment for P09-416

August 11, 2009 31 of 53 TABLE: MUTAGENICITY DATA ON PRODUCTS (Most results as reported in abstracts or a review)

Gene mutations Chromosome

mutations DNA Other Chemical

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 aberrati ons; ES = mouse embryonic stem cells: hprt = hypoxanthine-guanin e; phosphoribosyltransferase locus; IT = intratracheal administration; L51 = mouse lymphoma cells; LP = large particles; mito = mi tochondrial DNA damage; MN = micronucleus; neg = negative; pos = positive; SAL = Salmonella; SCE = sister chromatid exchange; SP = smal l particles; TGR = transgenic rodent assay; vit = in vitro; vv = in vivoBy 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 posit ive data on materials has been obtained with assays for which no standardized (i.e., OPPTS or OECD) guidelines exist, or with assays employing unusual strain s, cell lines or routes of admi nistration. 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 as say is under consideratio n for development of a test guideline by OECD, and th e Comet assay is undergoing va lidation by ICCVAM, ECVAM and JaCVAM (respectively, the Interagency Coordination Co mmittee on the Validation of Alternative Methods, the European Center for the Validation of Alternative Methods, and the Japanese Centre for the Valid ation 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 in tratracheal route of administra tion is not usually employed for in

vivo studies.

Several national and international bodies (e.g, OECD; EU Expert Group) are attempting to develop appropriate mutagenic ity testing schemes for nanotech ma terials. A recent extensive overview of the state of genotoxic ity 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 alve olar cells rather than the us ual 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 cond uct the test would be difficult.

Due to uncertainty about their ability to pe netrate 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 testi ng of the PMNs is recomm ended at this time.Contains CBI Standard Review Risk Assessment for P09-416

August 11, 2009 Page 33 of 53Contains CBI Standard Review Risk Assessment for P09-416

August 11, 2009 Page 34 of 53Contains CBI Standard Review Risk Assessment for P09-416

August 11, 2009 Page 35 of 53 9Contains CBI Standard Review Risk Assessment for P09-416

August 11, 2009 Page 36 of 53Contains CBI Standard Review Risk Assessment for P09-416

August 11, 2009 Page 37 of 53 Mode:Contains CBI Standard Review Risk Assessment for P09-416

August 11, 2009 Page 38 of 53 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.

aContains CBI Standard Review Risk Assessment for P09-416

August 11, 2009 Page 39 of 53 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 02745Contains CBI Standard Review Risk Assessment for P09-416

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.Contains CBI Standard Review Risk Assessment for P09-416

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

riContains CBI Standard Review Risk Assessment for P09-416

August 11, 2009 Page 42 of 53Contains CBI Standard Review Risk Assessment for P09-416

August 11, 2009 Page 43 of 53Contains CBI Standard Review Risk Assessment for P09-416

August 11, 2009 Page 44 of 53 Remarks:Contains CBI Standard Review Risk Assessment for P09-416Contains CBI Standard Review Risk Assessment for P09-416

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