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Genetic toxicity: in vivo

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in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of genotoxicity: micronucleus test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014.04.21 - 2014.07.18
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
In vivo data included although not mandatory at present tonnage level, as it was avalible prior this registration.

Data source

Materials and methods

Test guideline
according to
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
21st July 1997
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Test material form:
solid: fibres
Details on test material:
- Shape of particles: Alligned and entangled Muli-walled Carbon nanotubes
- Diameter of the individual fiber: 7-9 nm, length ~100-200nm
- Analytical purity: >= 98.5% based on themogravimetric analysis
- BET surface area of particles: >400 - 700 m2/g
- Total pore volume = 4.30 cm3/g
- Average pore size = 37.85nm
- Surface properties: Pristine, XPS analysis: 93.36% carbon and 6.64% oxygen (no surface functionalization).
- Crystal structure: Raman coefficient avg. = Ig/Id = 0.72
- Mass median aerodynamic diameter (MMAD) was not not applicable. Instead, CML was determined.
CML: Geometric mean (GM) of the cumulative median length (CML) wwas 2,119.83 nm. The geometric
Specific details on test material used for the study:
-Recived18 February 2014
-Lot no JC142
-Diameter: 8-10 nm (SEM)
-Analytical purity: 99.1 ~99.4 % (We assumed 100 % for test substance and carried out the test)
- Storage condition of test material: room temperature.
-Solubility:The test substance was dispersed in Destilled water

(Scanning Electron Microscopy = SEM)

Test animals

Details on species / strain selection:
ICR mice has been widely used in mammalian erythrocyte micronucleus test. It has been known for suitable experimental animal for general toxicity. Moreover, sufficient fundamental data have been accumulated in toxicological study using ICR mice. Such data will be useful for interpretation and evaluation of test results.
Details on test animals and environmental conditions:
-Animals: SPF (Specific Pathogenic Free), CrljOri:CD1(ICR) mice.
- Age at study initiation:
-Preliminary study: 8 weeks
-Main study: 8 weeks
- Weight at study initiation (day before administration):
-Main study: Controls (35.74 ± 0.99g),
MWCNT 25 mg/kg (35.84 ± 1.34g), 50 mg/kg (35.41 ± 0.97g), 100mg/kg (35.43 ± 0.89g),
MMC 2.0mg/kg (35.63 ± 0.90g).
- Assigned to test groups randomly: [yes]
- Fasting period before study: yes
- Housing: polycarbonate cage (170 W x 235 L x 125 H ㎜)
-Preliminary and main study: 5 animals per cage during quarantine and acclimation period and 3 animals per cage during administration and observation period

- Diet (ad libitum):
- Water (ad libitum):
- Acclimation period: 7 days.

- Temperature (°C):
-Preliminary study: 21.0 ± 1.2 C
-Main study: 20.9 ± 0.5 C
- Relative humidity (%):
-Preliminary study: 56.1 ± 6.9 %
-Main study: 58.8 ± 1.7 %
- Air changes (per hr): 10-15 times/hour
- Photoperiod (12 hrs dark / 12 hrs light):

Administration / exposure

Route of administration:
The test substance was dissolved in distilled water and serially diluted with distilled water. The administration level of prepared test substance is determined to 10 ㎖/㎏
based on body weight measured at the administration date. The stability test and the homogeneity test were not performed because the test solution was prepared fresh in
treatment day.
- Concentration in vehicle: up to 1%
- Justification for choice of vehicle:
Reason for selection : Prior to execution of this study, we performed to the solubility test for the test substance in solvents which have compatibility in the test
system was assessed. As a result, distilled water was chosen the solvent because the test substance was found to be dispersed at 1 % concentration

Details on exposure:
Intraperitoneal (IP)
Dosing volume: 10 ml/kg body weight
Duration of treatment / exposure:
A single dose in the morning at the day af treatment.
Frequency of treatment:
All animals dosed once
Post exposure period:
Preliminary study: 24 hours and 48 hours.
Main study: 24 hours.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
25 mg/kg bw (total dose)
Dose / conc.:
50 mg/kg bw (total dose)
Dose / conc.:
100 mg/kg bw (total dose)
No. of animals per sex per dose:
Control animals:
yes, concurrent vehicle
Positive control(s):
- Mitomycin C (MMC)
- Justification for choice of positive control(s): well know positive control for this type of study/assay in accordance to OECD TG 474.
- Route of administration: intraperitoneal injection
- Doses / concentrations: 2mg/kg


Tissues and cell types examined:
Bonemarrow derived erythrocytes
Details of tissue and slide preparation:
-Reason for determination of dosage:
To determined dosage, preliminary study was performed. In the preliminary test, test substance was administrated in dose of 50 mg/kg, 25 mg/kg and 12.5 mg/kg by oral. As the result, There was not observed dead animal and special abnormality compared to negative control group. When observing according to dose level (12.5 mg/kg, 25 mg/kg, 50 mg/kg) and harvest time after administration (24 and 48 hours), no significant decrease in PCE/(PCE+NCE) ratio compared to negative control group was observed (Annex 1). On the basis of preliminary test result, the dose levels in the main study were determined at 50 mg/kg (maximum dose), 25 mg/kg (middle dose) and 12.5 mg/kg (minimum dose) and harvest time was conducted at 24 hours after administration of test substance.

-Test method:
(1) Extraction of bone marrow and preparation of slides:
At the appropriate harvest time points, the thigh bone of the test animal was collected with care to avoid blood contamination in autopsy room after putting the cervical
vertebra out. The bone marrow was collected in a centrifuge tube by flushing the thigh bone inside with FBS (Hyclone, Lot No. AJY168755) The extracted
bone marrow was centrifuged at 1,000 rpm for 5 minutes and then resuspended with small aliquots of FBS after discarding the supernatant. The bone marrow was smeared on a slide glass and then dried at room temperature and fixed in methanol for 5 minutes. Three slides a bone marrow were prepared.

(2) Staining of specimen:
For the scoring the PCEs to NCEs ratio, the slides were stained in 4 % Giemsa staining solution. For the scoring the MNPCEs from PCEs, the slides were fixed and
then stained with acridine orange (40 ㎍/㎖) and covered with a cover glasses.

(3) Slide analysis:
Observation method: The observation of slides was performed with blind method and PCE to NCE ratio
was examined by optical microscope at 400× magnification. The observation of
MNPCEs was performed by fluorescence microscope equipped with FITC filter.

Evaluation criteria:
The PCE/NCE ratio was determined by scoring the number of PCEs and NCEs observed while scoring 200 erythrocytes per animal. The micronuclei frequency
(expressed as percent micronucleated cells) was determined by analyzing the number of MNPCEs from 2,000 PCEs per animal. In case of observation by fluorescence microscope, PCE was counted cells which appeared as red-fluorescence color without nucleus by acridine orange staining. NCE stained with acridine orange was appeared as only shadow entity without fluorescence. In case of observation by optical microscope, PCE stained with Giemsa staining solution was counted cells appeared as purple or blue color. NCE stained with Giemsa staining solution was counted cells appeared as pink color. According to criteia in micronucleus judgement, the largest size was defined as 1/2 size of erythrocyte diameter and the smallest size was defined as up to the limit of identification. Shape was Including circle, donut, semi-circle. Color was defined as same color as nucleus of cell.
Statistical analysis:
(1) Micronuclei frequency and PCE/(PCE+NCE) ratio
a) Comparison of the negative control and treated groups: ANOVA test
b) In case of p<0.05 at 1), test for dose-response : Linear logistic regression
c) The negative and positive control groups : ANOVA test
(2) Body weight
a) Body weight of test animal at necropsy : ANOVA test
b) In case of significant difference at body weight Dunnett T3 or Duncan's multiple range
test was used.

6) Criteria
In case PCE/(PCE+NCE) ratio (Mean±SD) is more than 0.2 at all the animals, test is judged to be reasonable. Determined as a positive result if there is dose-related increase in the number of micronucleated cells or clear increase in the number of micronucleated cells in the single dose group and statistical significant increase in frequency of MNPCE(MNPCE/2000PCEs, Mean±SD, %). The result of statistical evaluation was regarded as significant when the P value was less than 0.05. Statistical analysis was performed with SPSS 12.0 program.

Results and discussion

Test results
Key result
no effects
Vehicle controls validity:
Negative controls validity:
destilled water
Positive controls validity:
Additional information on results:
The frequencies of MNPCEs in 2,000 PCEs per animal were 0.31 %, 0.13 %, 0.19 %, 0.20 % and 12.54 % in order of negative control group, 25, 50, 100 mg/kg administration group and positive control group. There was no statistically increase in frequency of PCEs having micronuclei in administration group compared to negative control group. There was clear statistically significant increase in positive control group compared with negative control group in frequency of MNPCE (p<0.05).

The PCE ratio of 200 erythrocytes (PCE+NCE), as a index of cytotoxicity, was 0.46, 0.46, 0.47, 0.46 and 0.37 in order of negative control, 25, 50, 100 mg/kg group and
positive control. There was no significant decrease in PCE/(PCE+NCE) ratio compared to negative control group.

No changes in body weight.
No observation of any abnormal clinical signs.

Applicant's summary and conclusion

The mammalian in vivo micronucleus test in bone marrow of mice was performed with JENO TUBE 8 MWCNT in accordance with OECD TG 474 under GLP.
JENO TUBE 8 MWCNT did not induce micronucleus in bone marrow cells at doses up to 50mg/kg bw
Executive summary:

The test substance, JENO TUBE 8 MWCNT, was evaluated for its potential to induce micronucleus in bone marrow of the intraperitoneal administrated mouse.

Animals were 8 weeks old at administration of test substance. Test animals were dosed once intraperitoneally with 3 dosing levels (100 ㎎/㎏, 50 ㎎/㎏, 25 ㎎/㎏) and then frequency of MNPCEs and cytotoxicity (PCE ratio in the erythrocytes) was determined at 24 hours after administration of test substance. As a results of counting the frequency of MNPCEs of 2,000 PCEs, there was not statistically significant increase at any dose groups compare with negative control group. Also, there was statistically significant increase in positive control group compare with negative control group(p<0.05). The PCEs ratio of 200 erythrocytes (PCE/(PCE+NCE)), as a index of cytotoxicity, was not significantly decrease in PCE/(PCE+NCE) ratio compared to negative control group. It was concluded that, under the condition of this study, test substance MWCNT did not induce micronucleus in mice bone marrow.