- 1 - TI: Sylvon VonDerPool RAD Disposition March 20, 2009

PM: Zofia Kosim

Addendum to P09-142-144 Background Information

P09-142/143/1/44 (P09-142C), submitted by the Nano-C, Inc.,is 3’HCyclopropa[1,9][5,6]fullerene-C60-1h-3 ’ -phenyl-, methyl ester. The PMN is expected to be used

Physicochemical Properties

P09-142 P09-143 P09-144

MW: 910.91 Physical State: neat solid

BP: > 400

VP: < 0.000001 1031.02

neat solid

> 400

< 0.000001 1031.02

neat solid

> 400

< 0.000001

Additional Chemical Info: P09-143/144 each consis ts of two enantiomers; however, the specific composition of the enantiomers is undefined. (C hemistry Report Attachment # 5 hard copy only).

Chemical Structure

O

O O

O

P09-142- 2 - O

O O

O

P09-143

O

O O

O

P09-144

General Information

The Submitter (Nano-C, Inc.), made no claim th at the PMN chemical contains any unique/novel properties.

Health Effects

For particles with size less than 100 nanometers, ab sorption is expected to be poor via all routes based on analogues. For particle s with size greater than 100 nanom eters, absorption is expected

to be nil via all routes based on physical/chemical properties. Th ere is concern for lung overload if respirable particles are inha led. For any poorly soluble par ticles (PSP), there is potential concern for adverse lung eff ects including cancer, lung fibros is, lung inflammation and induce systemic effects including imm unotoxicity if inhaled (Human Health Report 1/13/09).

Carcinogenicity

P09-142C is a fullerene (C60, buckyball) att ached with a 3’H-cy clopropyl-3’-butanoic acid, 3’-phenyl-, methyl ester group. They are in soluble particles with diameter ranging from

about 0.2-160 μ m. An ICB document for P09-142-144, posted in the last day or so listed the particle size of the PMN at 0.15 – 176 microns (based on three-r un analysis). There were no particles detected below 100 nanometers and appr oximately 90% of the particles were larger than 1,000 nanometers.- 3 - There are no data on the potential human health effects of the P09-142C. These aggregates/agglomerates mentioned above have health concerns of particles under the PSP category. A number of PSP such as crystallin e silica, TiO2 and carbon black have been shown to be carcinogenic. Particle su rface characteristics are considered to be the key factors in free radicals and reactive oxygen speci es (ROS) formation and in the development of fibrosis and cancer by quartz (crystalline sili ca). By analogy to these carcinoge nic mineral particles, there is a concern for the PMN for pulmonary fibrosis an d cancer if inhaled (Hea lth Hazard Review for P09-142C, see attachment # 1 hard copy only).

In addition, there is concern that the PMN may disperse/disa ggregate in the lung fluids to particles within the nanoscale. Hence, ther e would be a concern for systemic effects of nanoparticles beyond the lungs. For instance, Nurk iewcz et al. (2008) found that adverse cardiovascular effects were seen on exposure to ultraf ine TIO2 at inhalation doses below those that induced pulmonary effects.

Using energy-filtered transmission electron microscopy and electron tomography, uptake

of nC 60 (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 tetrahydron furan (THF) to prepar e water-soluble suspensions of nC 60, cytotoxicity of nC 60 to human cells or toxicity in the brain of juvenile largemouth ba ss due to lipid peroxidation and generation of reactive oxygen species (ROS) were reported. However, the use of nC 60 suspensions prepared by solvent exchange methods in toxicity studies has been suspected to be

due to the toxic effects of TH F; the toxicity data of nC 60 remains a topic of debate within the scientific community. In a subchr onic inhalation toxicity study, C 60 fullerene nanoparticles (2.22 mg/m 3 , 55 nm in diameter) a nd 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 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 90-day subchronic study, Wistar-Han rats and B6C3F1 mice were exposed by nose-only inhalation to two sizes of C60 (0.05 um or 1 um) 3 hours per day 5 days pe r week for 90 days. Exposure concentrations were 0, 0.5, 2.5 mg/m 3 for 0.05 um C60, and 0, 5, 15, and 30 mg/m 3 for the 1um C60. There were no biologically significant exposure-related changes in these parameters in rats or mice exposed to either 0.05 um or 1 um C60 (N.J.Walke r et al. 2009). No chr onic exposure inhalation

studies on fullerenes are availabl e (Health Hazard Review for P 09-142C see attachment # 1 hard copy only).

Mutagenicity

No mutagenicity data were provided with PMNs 09-142, -143 and -144. 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 nanotubule materials in humans. No tes ting of the PMNs is recommended at this time- 4 - (Mutagenicity Hazard Report see Attachment # 2 hard copy only).

Immunotoxicity

Based on published scientific data, some na noparticles appear to have immunotoxicity hazard potential. Pulmonary e xposure to these PMNs may lead to toxicity to alveolar macrophage, impaired clearance of inhaled bact eria and possible systemic immunosuppression (Immunotoxicity Hazard Report, see atta chment # 3 hard copy only).

Developmental Toxicity This report summarizes the potential a dverse developmental effects of PMN P-09-

0142/0143/0144. The three PMN substa nces are carbon-60 fullerene derivatives. No data were available on the developmental toxicity of the PMN substances themselves. There are limited data (one study, described below) for C60-fu llerene, 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 P-09-0142/0143/0144. This study was pr eviously reviewed in 2004 as part of a standard review for (Developmental Toxi city Hazard Report see Attachment # 4 hard copy only).

Environmental Effects: Ecotoxicity concerns for the PMN chemicals are mitigated based on no

releases to water.

Occupational Exposure TABLE 1 (P09-142) Scenario # Sites # Workers Inhal (mg/d) Derm (mg/d) Manufacturing 0.64 (particle) 3100 (solid) Processing 0.0064 “ 3100 “ 18 (liquid)

Use negligible 87 “

TABLE 2 (P09-143/144) Scenario # Sites # Workers Inhal (mg/d) Derm (mg/d) Manufacturing 0.32 (particle) 3100 (solid) Processing 0.032 “ 3100 “ 18 (liquid)

Use Negligible 87 “

Risk Assessment

Rationale: No to minor effects were seen with pur e C60 fullerenes in the two subchronic studies. However, it is not possible to draw conclusions about these derivitized PMN fullerenes based on these subchronic studies with pure C60 fullerenes. Some fraction of the PMN material is in the respirable range. Therefore, the concern for a ny poorly soluble particles (PSP) support a health concern for these PMNS. In addition, there is a concern for cardiovascular effects.- 5 - A comparison of Tables 1 and 2 above indica te a greater inhalati on exposure value (0.64 mg/day) during manufacturing for P09-142. Th erefore, since P09- 142/143/144 are close analogues, the inhalation exposure value of 0.64 mg/day (0.064 mg/m 3 ) with the NCEL of 0.05 mg/m 3 for crystalline silica will be used for the risk assessment calculations.

Therefore, the worst case worker inhalation exposure of 0.064 mg/m 3 for P09-142C during manufacturing when compar ed to the NCEL of 0.05 mg/m 3 for crystalline silica poses a potential carcinogenic risk to workers expos ed via the inhalation route.

Testing Recommendations

Inhalation is the pref erred method of exposure of th e respiratory tract for hazard identification and to obtain dose-response data fo r use in quantitative risk assessment. A 90- day inhalation study by nose-only admi nistration in male or female rats with up to 3 months observation post exposure is recommended fo r evaluating the toxi city of nanoscale substances (CFR 870.3465). There are several ad ditional considerations for using the 90-day subchronic protocol at CFR 870.3465:

i. Evaluation includes markers of damage, oxidant stress, cell proliferation, the degree/intensity and duration of pulmonary inflammation, fibrosis, and cytotoxic effects in the broncoalveolar lavage fluid (BALF) and histopathology of pulmonary and ex-pulmonary organs/tissues (cardiovascular, CNS, liver, kidney, etc., )

. It is also recommended that the potential for cardi ovascular toxicity be determined through monitoring

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

cardiovascular effects.

ii. Data on pulmonary deposition, clear ance half-life (biopersistence) and

translocation of the test material are de sirable to aid in interpreting the test

results

. iii. Size distributions should be de termined prior to, and during, the

inhalation tests.

iv. Differences in the physicochemical properties of the as-administered material – relative to the as-produc ed material -- should be provided Techniques used to produce the as-adm inistered material should also be

described.

v. The proposed protocol for testing shoul d be provided to EPA for review and comment prior to th e initiation of testing.

vi. The results of the subchronic inha lation study may support the need to conduct a 2-year inhalation study bioassa y in rats with the PMN nanoscale

substance.

File:TSCA_PMN-90-day_InhalationTesting_12032008Sayre.doc References

1. Timothy R Nurkiewicz, Dale W Porter, A nn F Hubbs, Jared L Cumpston, Bean T Chen, David G Frazer, and Vincent Castranova , Particle and Fibre Toxicology, 2008.- 6 - 2. N.J.Walker, G.L. Baker, J.A. Dill, D.R. Germolec, K.L. White. J.H. Roycroft, Evaluation of the effect of particle size on the toxixity and to xicokinetics of fullere ne C60 in rats and mice following nose-only inhalation exposure .. Abstract No. 233. 2009 Itinery Planner. Baltimore, MD: Society of Toxicvology.

.