Electronic Supplement Material
Aspect ratio has no effect on genotoxicity of multi-wall carbon nanotubes (MWCNTs)
1Jin Sik Kim, 2Kyu Lee, 2Young Hee Lee, 1Hyun Sun Cho, 1Ki Heon Kim, 3Kyung Hee Choi, 3Sang Hee Lee, 1Kyung Seuk Song, 4Chang Soo Kang, 1,4Il Je Yu*
1Biosafety Evaluation Headquarter, Korea Environment & Merchandise Testing Institute, Incheon, 2Department of Physics, Center for Nanotubes and Nanostructured Composites, Sungkyunkwan Advanced Institute of Nanotechnology, Sungkyunkwan University, Suwon,
3Risk Assessment Division, National Institute of Environmental Research, Incheon, 4Fusion Technology Research Institute, Hoseo University, Asan, Korea
*Corresponding Author: Il Je Yu, Fusion Technology Research Institute, Hoseo University, 165 Sechul-ri, Baebang-myun, Asan, 336-795, Korea. Tel: 82-41-540-9630, Fax: 82-41-540-9630
E-mail:
Supplement 1. Test results of Brunauer, Emmett, Teller (BET) surface area analysis for multi-wall carbon nanotubes (MWCNTs).
High-aspect-ratio / 177.57
Low-aspect-ratio / 195.29
Supplement 2. Degree of macro dispersion of multi-wall carbon nanotubes (MWCNTs) in D.W.
A (A.U.) / B (A.U.) / DOM (%) / A (A.U.) / B (A.U.) / DOM (%)
1% / 2.492±0.009 / 0.015±0.003 / 0.6±0.1 / 2.538±0.045 / 2.431±0.002 / 95.8±1.7
0.5% / 2.497±0.001 / 0.026±0.002 / 1.1±0.1 / 2.516±0.001 / 2.343±0.021 / 93.1±0.9
0.25% / 2.494±0.001 / 0.013±0.004 / 0.5±0.2 / 2.524±0.011 / 2.522±0.005 / 99.9±0.6
0.125% / 2.490±0.002 / 0.016±0.002 / 0.6±0.1 / 2.516±0.006 / 2.527±0.001 / 100.5±0.2
0.0625% / 2.473±0.010 / 0.018±0.003 / 0.7±0.1 / 2.522±0.001 / 2.522±0.001 / 100.0±0.0
0.03125% / 2.323±0.011 / 0.013±0.001 / 0.5±0.1 / 2.520±0.001 / 2.519±0.005 / 99.9±0.2
0.015625% / 1.594±0.027 / 0.042±0.007 / 2.7±0.5 / 2.484±0.001 / 2.315±0,005 / 93.2±0.2
0.0078125% / 1.012±0.006 / 0.067±0.003 / 6.7±0.3 / 2.178±0.015 / 1.518±0.039 / 69.7±1.7
A: Absorbance units of dispersed MWCNTs
B: Absorbance units of centrifuged supernatant
A.U.: Absorbance units
DOM: Degree of macro dispersion (B/AX100)
Supplement 3. Degree of macro dispersion of multi-wall carbon nanotubes (MWCNTs) in 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) solution.
A (A.U.) / B (A.U.) / DOM (%) / A (A.U.) / B (A.U.) / DOM (%)
1% / UD / 0.576±0.005 / UD / UD / 0.023±0.001 / UD
0.5% / UD / 0.557±0.008 / UD / UD / 0.026±0.001 / UD
0.25% / UD / 0.524±0.010 / UD / UD / 0.217±0.003 / UD
0.125% / UD / 0.219±0.002 / UD / UD / 0.912±0.004 / UD
0.0625% / UD / 0.212±0.002 / UD / UD / 1.127±0.001 / UD
0.03125% / 3.986±0.009 / 0.675±0.001 / 16.9±0.0 / 3.778±0.007 / 1.339±0.006 / 35.5±0.1
0.015625% / 2.464±0.001 / 0.446±0.001 / 18.1±0.0 / 1.951±0.001 / 0.574±0.006 / 29.4±0.3
0.0078125% / 1.386±0.001 / 0.239±0.002 / 17.2±0.1 / 1.480±0.002 / 0.452±0.002 / 30.4±0.1
A: Absorbance units of dispersed MWCNTs
B:Absorbance units of centrifuged supernatant
A.U.: Absorbance units
DOM: Degree of macro dispersion (B/AX100)
UD: Undetectable (A.U.>4)
Supplement 4. Number of revertant colonies for high-aspect-ratio (A) and low-aspect-ratio (B) multi-wall carbon nanotubes (MWCNTs) treatment in absence of S9 mix.
A. High-aspect-ratio MWCNTs
Concentration (μg/plate) / Types of Bacteria (Mean ± S.D.)TA98 / TA100 / TA1535 / TA1537 / WP2uvrA
0 / 16 ± 3.1 / 141 ± 8.7 / 5 ± 1.2 / 4 ± 1.7 / 44 ± 4.4
12 / 15 ± 2.5 / 158 ± 11.4 / 8 ± 2.1 / 4 ± 2.1 / 48 ± 1.0
37 / 15 ± 4.7 / 147 ± 6.1 / 9 ± 0.6 / 5 ± 0.6 / 43 ± 4.5
111 / 13 ± 4.6 / 167 ± 8.7 / 8 ± 2.9 / 7 ± 3.2 / 42 ± 5.0
333 † / 13 ± 3.5 / 154 ± 14.5 / 8 ± 3.1 / 4 ± 1.2 / 45 ± 0.0
1000 † / 9 ± 4.9 / 145 ± 2.6 / 7 ± 0.6 / 4 ± 2.1 / 41 ± 0.6
Positive control / 453 ± 66.51) / 546 ± 2.62) / 307 ± 15.43) / 3770 ± 68.44) / 271 ± 3.15)
B. Low-aspect-ratio MWCNTs
Concentration (μg/plate) / Types of Bacteria (Mean ± S.D.)TA98 / TA100 / TA1535 / TA1537 / WP2uvrA
0 / 16 ± 2.1 / 170 ± 14.9 / 10 ± 2.6 / 6 ± 4.4 / 32 ± 4.0
12 / 10 ± 3.5 / 165 ± 3.2 / 9 ± 2.5 / 6 ± 3.5 / 42 ± 3.5
37 / 10 ± 2.5 / 151 ± 7.8 / 7 ± 2.0 / 6 ± 2.6 / 35 ± 7.5
111 / 11 ± 2.5 / 145 ± 9.1 / 9 ±3.2 / 3 ± 2.1 / 31 ± 4.0
333 † / 12 ± 1.5 / 150 ± 3.5 / 9 ± 4.2 / 3 ± 2.3 / 28 ± 8.9
1000 † / 11 ± 4.7 / 102 ± 0.6 / 8 ± 5.0 / 6 ± 1.7 / 25 ± 0.6
Positive control / 507 ± 34.31) / 554 ± 26.22) / 339 ± 13.93) / 2474 ± 112.14) / 271 ± 0.65)
†: aggregation and precipitation of test substance
1): 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2) 0.1 μg/plate
2): 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2) 0.01 μg/plate
3): Sodium azide (NaN3) 0.5 μg/plate
4): 9-Aminoacridine hydrochloride hydrate (9AA) 80 μg/plate
5): 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2) 0.01 μg/plate
Supplement 5. Number of revertant colonies for high-aspect-ratio (A) and low-aspect-ratio (B) multi-wall carbon nanotubes (MWCNTs) treatment in presence of S9 mix.
A. High-aspect-ratio MWCNTs
Concentration (μg/plate) / Types of Bacteria (Mean ± S.D.)TA98 / TA100 / TA1535 / TA1537 / WP2uvrA
0 / 23 ± 3.8 / 155 ± 7.5 / 12 ± 2.9 / 8 ± 3.0 / 49 ± 7.8
12 / 27 ± 5.8 / 189 ± 13.0 / 10 ± 2.3 / 7 ± 3.5 / 51 ± 7.2
37 / 23 ± 5.6 / 170 ± 13.7 / 9 ± 1.5 / 7 ± 1.2 / 45 ± 2.1
111 / 14 ± 2.6 / 185 ± 10.1 / 12 ± 2.5 / 8 ± 3.0 / 46 ± 1.0
333 † / 19 ± 1.5 / 167 ± 7.8 / 7 ± 2.6 / 7 ± 4.0 / 83 ± 8.3
1000 † / 15 ± 2.3 / 172 ± 16.8 / 11 ± 4.0 / 5 ± 1.2 / 81 ± 14.2
Positive control / 319 ± 16.21) / 1155 ± 94.82) / 210 ± 21.53) / 178 ± 8.34) / 435 ± 16.85)
B. Low-aspect-ratio MWCNTs
Concentration (μg/plate) / Types of Bacteria (Mean ± S.D.)TA98 / TA100 / TA1535 / TA1537 / WP2uvrA
0 / 19 ± 8.6 / 168 ± 12.2 / 7 ± 0.6 / 10 ± 1.7 / 39± 11.1
12 / 21 ± 4.7 / 173 ± 5.3 / 10 ± 2.3 / 8 ± 0.6 / 44 ± 10.7
37 / 19 ± 7.4 / 172 ± 4.0 / 9 ± 2.1 / 8 ± 3.1 / 39 ± 3.6
111 / 17 ± 1.7 / 185 ± 21.5 / 13 ± 2.3 / 8 ± 3.2 / 39 ± 1.2
333 † / 18 ± 3.6 / 173 ± 7.8 / 12 ± 3.1 / 9 ± 2.5 / 41 ± 5.0
1000 † / 11 ± 2.0 / 153 ± 15.4 / 10 ± 2.3 / 9 ± 1.2 / 30 ± 7.8
Positive control / 444 ± 44.51) / 1006±147.52) / 234 ± 24.93) / 273 ± 40.84) / 413 ± 56.75)
†: aggregation and precipitation of test substance
1): 2-Aminoanthracene (2AA) 1.0 μg/plate
2): 2-Aminoanthracene (2AA) 1.0 μg/plate
3): 2-Aminoanthracene (2AA) 2.0 μg/plate
4): 2-Aminoanthracene (2AA) 2.0 μg/plate
5): 2-Aminoanthracene (2AA) 10.0 μg/plate
Supplement 6. The number of viable cells in absence and presence of S9 mix for iron oxide.
Supplement 7. Degree of macro dispersion of multi-wall carbon nanotubes (MWCNTs) in F-12 medium.
Concentration (μg/ml) / High-Aspect-Ratio / Low-Aspect-RatioA (A.U.) / B (A.U.) / DOM (%) / A (A.U.) / B (A.U.) / DOM (%)
200 / 1.432±0.006 / 0.688±0.003 / 6.1±0.2 / 1.170±0.006 / 0.083±0.001 / 7.1±0.0
100 / 0.711±0.002 / 0.086±0.006 / 12.0±0.9 / 0.656±0.002 / 0.095±0.002 / 14.4±0.2
50 / 0.590±0.005 / 0.080±0.001 / 13.6±0.2 / 0.513±0.001 / 0.075±0.001 / 14.6±0.1
25 / 0.270±0.000 / 0.045±0.001 / 16.8±0.4 / 0.291±0.001 / 0.045±0.001 / 15.4±0.4
12.5 / 0.166±0.001 / 0.025±0.001 / 15.1±0.6 / 0.158±0.001 / 0.046±0.002 / 29.4±1.0
6.25 / 0.092±0.002 / 0.016±0.002 / 17.8±1.9 / 0.081±0.000 / 0.032±0.001 / 39.1±0.7
3.125 / 0.053±0.001 / 0.011±0.000 / 20.9±0.2 / 0.042±0.001 / 0.017±0.001 / 41.0±1.7
1.5625 / 0.029±0.001 / 0.006±0.001 / 19.5±1.5 / 0.030±0.001 / 0.013±0.001 / 41.8±1.7
A: Absorbance units of dispersed MWCNTs
B:Absorbance units of centrifuged supernatant
A.U.: Absorbance units
DOM: Degree of macro dispersion (B/AX100)
Supplement 8. Comparison of body weights for test animals exposed to high-aspect-ratio (A) and low-aspect-ratio (B) multi-wall carbon nanotubes (MWCNTs).
A. High-aspect-ratio MWCNTs
Sampling time (hrs) / Dose(mg/kg) / Animal
No. / Body weights (gram, Mean±S.D.)
At time of administration / At time of sacrifice
24 / U.C. / 6 / 37.39 ± 1.88 / 37.05 ± 1.62
V.C. / 6 / 37.54 ± 1.84 / 32.02 ± 2.00
12.5 / 6 / 37.06 ± 1.62 / 35.91 ± 2.17
25 / 6 / 37.62 ± 1.56 / 36.14 ± 1.74
50 / 6 / 37.55 ± 1.65 / 36.02 ± 2.02
MMC / 6 / 37.68 ± 1.33 / 37.03 ± 1.39
B. Low-aspect-ratio MWCNTs
Sampling time (hrs) / Dose(mg/kg) / Animal
No. / Body weights (gram, Mean±S.D.)
At time of administration / At time of sacrifice
24 / U.C. / 6 / 34.54±1.76 / 34.47±1.65
V.C. / 6 / 34.88±1.33 / 34.37±1.24
12.5 / 6 / 35.27±1.19 / 33.73±1.30
25 / 6 / 34.92±1.29 / 33.05±1.43
50 / 6 / 35.00±0.80 / 32.04±0.89
MMC / 6 / 35.45±0.86 / 34.73±0.95
U.C.; Untreated control, V.C.; Vehicle control, MMC; Mitomycin C (2.0 mg/ml).
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