Multi-Walled Carbon Nanotubes (MWCNT), synthetic graphite in tubular shape

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

published in 2011

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Evaluation of maternal toxicity in rats exposed to multiwall carbon nanotubes during pregnancy.

Author:

Jeong-Hyeon Lim, Sung-Hwan Kim, In-Chul Lee, Changjong Moon, Sung-Ho Kim, Dong-Ho Shin, Hyoung-Chin Kim, Jong-Choon Kim

Year:

2011

Bibliographic source:

Environmental Health and Toxicology, vol. 26, Article ID: e2011006, published online: Apr.14, 2011.

Reference 2

Reference Type:

publication

Title:

Maternal exposure to multi-wall carbon nanotubes dose not induce embryo-fetal developmental toxicity in rats.

Author:

Jeong-Hyeon Lim, Sung-Hwan Kim, In-Sik Shin, Na-Hyeong Park, Changjong Moon,
Seong-Soo Kang, Sung-Ho Kim, Seung-Chun Park, Jong-Choon Kim

Year:

2011

Bibliographic source:

Birth Defects Research (Part B), 92, 69-76.

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

fewer number of dams per group tested

GLP compliance:

not specified

Remarks:

Published study performed by University staff in the Republic of Korea

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

carbon

EC Number:

936-414-1

Molecular formula:

C

IUPAC Name:

carbon

Test material form:

solid: nanoform, no surface treatment

Details on test material:

Hanwha CM-100 related substance;

Batch / Lot : NA
Date : NA
Appearance : black powder
Purity (wt%) : ca. 95 (carbon)
Bundle length : ca. 200 µm
Tube diameter : 10 nm – 15 nm
BET surface area (m²/g) : NA
Shape category : elongated
Shape of particles : tube
Crystal structure : crystalline
Surface treatment/functionalisation : no
State of aggregation : agglomerated

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Hanwha Nanotech, Incheon Korea
- Purity: 95% carbon, 5% iron

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: ultrasonication after suspension in vehicle for 3 minutes
- Final concentration of a dissolved solid: 2, 10, or 50 mg/ml

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Specific pathogen free

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Orient Bio, Seoul, Korea
- Age at study initiation: 10 weeks
- Housing: clear polycarbonate cages with stainless steel wire lids. Mating: two females were cohabited with one male in the same cage overnight. Mated females: individually housed
- Diet (e.g. ad libitum): commercial rodent chow (Samyang Feed, Wonju, Republic of Korea), ad libitum
- Water (e.g. ad libitum): UV-irradiation sterilized tap water, ad libitum
- Acclimation period: 1 week (including quarantine)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 +/- 3°C
- Humidity (%): 50 +/- 10%
- Air changes (per hr): 13 - 18
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

1% sodium carboxymethylcellulose solution

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on the report 'Takagi A, Hirose A, Nishimura T, Fukumori N, Ogata A, Ohashi N, et al. Induction of mesothelioma in p53+/- mouse by intraperitoneal application of multi-wall carbon nanotube. J Toxicol Sci 2008; 33(1): 105-116.'
- Amount of vehicle (if gavage): 20 ml/kg/day

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- Impregnation procedure: [cohoused]
- If cohoused:
- M/F ratio per cage: 1 : 2
- Length of cohabitation: overnight
- Proof of pregnancy: [sperm in vaginal smear] referred to as [day 0 ] of gestation

Duration of treatment / exposure:

Gestation day (GD) 6 - GD19

Frequency of treatment:

once daily

Duration of test:

14 days from GD 6 through GD 19

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

40 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

12 mated females per dosing group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Preliminary study with 5 females per group for 10 days, no maternal toxicity or developmental toxicity observed. Therefore, the doses of 40, 200 and 1000 mg/kg bw/day were selected using a scaling factor of five.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked: mortality, morbidity, general appearance, behaviour

BODY WEIGHT: Yes
- Time schedule for examinations: on GD 0, 6, 9, 12, 15, and 20

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: complete gross postmortem examination, absolute and relative (organ-to-body weight ratio) weights of
lung, adrenal glands, liver, spleen, kidneys, thymus, heart, and ovaries measured

OTHER:
- Analysis of Kidney Oxidation Damage
Kidney preparation: crushing of the kidney tissue (glass-teflon homogenous grinder, few seconds), centrifuging homogenized at 4°C, at 800 g for 10 minutes. Protein quantification according to Lowry method. Measurement of activity of antioxidant enzymes (catalase, glutathione reductase, glutathione peroxidase, glutathione-S-transferase), glutathione concentration, degree of lipid
peroxidation

- Serum Biochemical Examination
Blood samples were taken from posterior vena cava. Parameters examined: aspartate aminotransferase, alanine aminotransferase, total cholesterol, triglyceride, alkaline phosphatase, blood urea nitrogen, creatinine, glucose, total bilirubin, albumin, total protein

Animals were sacrificed by ether inhalation and exsanguination via the aorta

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: live and dead fetuses

Blood sampling:

Yes, please refer to 'Maternal Examinations'

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations:No
- fetal sex ratio calculated
- fetal weight
- placental weight

Statistics:

Data are presented as means ± S.D.
The unit of statistical measurement is the pregnant female or the litter. Quantitative data (maternal body weight, food consumption, fetal body weight, placental weight): one-way analysis of variance followed by Scheffe's multiple comparison test when the differences were significant. Number of corpora lutea, total implantations, live and dead fetuses, fetal alterations: Kruskal-Wallis nonparametric analysis of variance, followed by Mann-Whitney U-test where applicable. Gender ratio and proportions of litters with malformations and developmental variations: chi-square tests and Fischer's exact probability tests. Computer program: GraphPad InStat version 3.0 (GraphPad Software Inc., CA, USA). P-value

Indices:

Pre-implantation loss (%) = ([No. of corpora lutea - No. of implantation sites]/No. of corpora lutea) * 100.

Post-implantation loss (%) = ([No. of implantation sites - No. of live embryos]/No. of implantation sites) * 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

1 of 10 dams in the 40 mg/kg group and 3 of 12 dams in the 1000 mg/kg group showed decreased locomotor activity and depression after treatment. These clinical signs were transient in nature, and not dose-dependent in either incidence or severity.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Please refer to table 1 in section 'any other information on results incl. tables'.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Please refer to table 2 in section 'any other information on results incl. tables'.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Significant decrease in absolute and relative thymus weights in the 1000 mg/kg group compared to the control group.
Please refer to table 3 in section 'any other information on results incl. tables'.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

No effects observed in the analysis of kidney oxidation damage.
Please refer to table 4 in section 'any other information on results incl. tables'.

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

Number of pregnant females out of twelve (control, low dose, mid dose, high dose, respectively):
11, 10, 12, 11

Details on maternal toxic effects:

For more details please refer to table 5 in section 'any other information on results incl. tables'.

Effect levels (maternal animals)

1

Key result

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: overall effects

2

Key result

Dose descriptor:

LOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

organ weights and organ / body weight ratios

Maternal abnormalities

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

other: thymus

Description (incidence and severity):

Significant decrease in absolute and relative thymus weights in the 1000 mg/kg group compared to the control group.
Please refer to table 3 in section 'any other information on results incl. tables'.

Results (fetuses)

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

 

Table 1: Body Weight Changes in Pregnant Rats Treated With Multi-Wall Carbon Nanotubes During Gestational Days 6–19

	Multi-wall carbon nanotubes (mg/kg/day)
Parameters	0	40	200	1000
No. of rats mated	12	12	12	12
No. of pregnant rats	11	10	12	11
Gestational day 0	246.5+11.14^a	247.1+17.08	243.7+16.96	242.0+19.65
Gestational day 6	283.7+12.60	286.1+19.65	280.4+16.70	283.4+25.12
Gestational day 9	296.2+15.63	297.3+20.35	289.4+18.08	290.6+27.92
Gestational day 12	309.8+15.56	312.5+20.66	303.2+19.52	303.9+28.66
Gestational day 15	323.0+16.30	328.6+21.05	318.6+20.59	320.3+31.07
Gestational day 20	381.3+25.97	387.4+28.61	367.2+28.13	377.4+39.56
Body weight gain during pregnancy	134.8+17.12	140.3+21.11	123.6+23.42	135.4+26.26
Corrected body weight^b	319.9+16.77	323.4+23.07	306.3+28.76	303.4+39.51
Gravid uterine weight	61.4+16.72	63.9+21.99	60.9+20.35	74.0+9.79

^aValues are presented as mean+SD (g).

^bBody weight on gestational day 20—gravid uterine weight.

 

Table 2: Food Consumption by Pregnant Rats Treated With Multi-Wall Carbon Nanotubes During Gestational Days 6–19

	Multi-wall carbon nanotubes (mg/kg/day)
Parameters	0	40	200	1000
No. of dams	11	10	12	11
Gestational day 0	19.6+3.06^a	19.4+3.14	18.2+1.63	19.5+3.78
Gestational day 6	23.9+3.61	24.3+2.87	22.7+3.21	22.2+2.82
Gestational day 9	22.1+2.65	23.0+3.81	21.4+2.54	21.8+4.33
Gestational day 12	22.2+3.87	25.8+3.78	22.8+6.32	23.0+4.94
Gestational day 15	23.3+3.04	24.4+2.66	22.8+2.23	23.4+5.15
Gestational day 19	23.7+2.70	29.8+8.30^*	22.9+4.48	23.7+6.04

^aValues are presented as mean+SD (g).

 

Table 3: Absolute and Relative Organ Weights of Pregnant Rats Treated With Multi-Wall Carbon Nanotubes During Gestational Days 6–19

	Multi-wall carbon nanotubes (mg/kg/day)
Parameters	0	40	200	1000
No. of dams	11	10	12	11
Body weight at term	381.3+25.97^a	387.4+28.61	367.2+28.13	377.4+39.56
Lung (g)	1.25+0.122	1.24+0.083	1.24+0.106	1.20+0.090
Per body weight (%)	0.33+0.029	0.32+0.021	0.34+0.035	0.32+0.021
Adrenal glands (g)	0.087+0.0078	0.076+0.0127	0.089+0.0142	0.087+0.0143
Per body weight (%)	0.023+0.0017	0.020+0.0028	0.024+0.0048	0.023+0.0056
Liver (g)	15.39+1.673	15.50+1.661	15.08+1.964	15.05+1.855
Per body weight (%)	4.03+0.245	4.00+0.260	4.10+0.337	3.98+0.208
Spleen (g)	0.71+0.101	0.64+0.058	0.62+0.084	0.63+0.068
Per body weight (%)	0.19+0.032	0.17+0.015	0.17+0.020	0.17+0.013
Kidneys (g)	2.15+0.185	2.15+0.216	2.09+0.232	1.98+0.207
Per body weight (%)	0.56+0.050	0.56+0.048	0.57+0.045	0.53+0.042
Thymus (g)	0.49+0.063	0.51+0.081	0.44+0.107	0.36+0.101^*
Per body weight (%)	0.13+0.021	0.13+0.021	0.12+0.022	0.10+0.022*
Heart (g)	1.11+0.105	1.10+0.081	1.15+0.151	1.08+0.119
Per body weight (%)	0.29+0.022	0.28+0.021	0.31+0.035	0.29+0.024
Ovary (g)	0.15+0.021	0.15+0.015	0.15+0.033	0.13+0.013
Per body weight (%)	0.04+0.006	0.04+0.005	0.04+0.010	0.03+0.006

^aValues are presented as mean+SD.

^*Significant difference at P < 0.05 level when compared with the control group.

 

Table 4: Antioxidant Enzymes, Glutathione and Lipid Peroxidation Levels in the Livers of Pregnant Rats Treated With Multi-Wall Carbon Nanotubes During Gestational Days 6–19

	Multi-wall carbon nanotubes (mg/kg/day)
Parameters	0	40	200	1000
No. of dams	11	10	12	11
Catalase (unit/mg protein)	26.5 ± 3.87^a	25.5 ± 4.49	27.6 ± 5.11	22.8 ± 6.34
Glutathione reductase (unit/mg protein)	199.3 ± 10.43	231.3 ± 16.89	251.5 ± 15.90	228.9 ± 44.63
Glutathione-S-transferase (mmol/mg protein)	6.8 ± 0.63	6.0 ± 0.68	6.3 ± 0.24	5.7 ± 0.41
Glutathione peroxidase (nmol/mg protein)	2.6 ± 0.48	2.6 ± 0.35	2.4 ± 0.31	2.6 ± 0.51
Glutathione (nmol/mg protein)	264.2 ± 28.74	315.8 ± 63.2	230.8 ± 68.03	375.1 ± 29.78
Malondialdehyde (mmol/mg protein)	64.6 ± 11.12	71.4 ± 26.66	72.7 ± 12.25	72.4 ± 10.24

^aValues are presented as mean+SD.

Note: For the endpoints in table 4 different values are reported in the second reference.

 

 

Table 5: Cesarean Section Data in Pregnant Rats Treated With Multi-Wall Carbon Nanotubes During Gestational Days 6–19

	Multi-wall carbon nanotubes (mg/kg/day)
Parameters	0	40	200	1000
No. of dams	11	10	12	11
No. of corpora lutea	14.7+3.26^a	14.6+1.84	13.8+2.12	14.8+1.40
No. of implantation sites	11.6+3.70	11.9+4.18	11.5+3.63	14.0+1.61
Pre-implantation loss (%)^b	20.0+20.49	18.7+25.95	19.1+24.97	5.5+7.02
Fetal deaths	0.5+0.82	0.5+0.53	0.4+0.67	0.5+0.52
Resorption: Early	0.5+0.82	0.4+0.52	0.4+0.67	0.5+0.52
Late	0	0.1+0.32	0	0
Dead fetuses	0	0	0	0
Post-implantation loss (%)^c	3.1+5.65	3.6+3.81	4.7+6.30	3.2+3.67
Litter size	11.2+3.34	11.4+3.92	11.1+3.70	13.5+1.51
Male/female	70/53	59/55	73/60	78/71
Gender ratio	1.32	1.07	1.22	1.10
Fetal weight (g): Male	3.62+0.194	3.65+0.219	3.42+0.150	3.61+0.181
Female	3.39+0.233	3.50+0.287	3.28+0.150	3.41+0.204
Placental weight (g)	0.53+0.050	0.58+0.081	0.53+0.057	0.51+0.047

^aValues are presented as mean+SD.

^bPre-implantation loss (%) = ([No. of corpora lutea-No. of implantation sites]/No. of corpora lutea) x 100.

^cPost-implantation loss (%) = ([No. of implantation sites-No. of live embryos]/No. of implantation sites) x 100.

Applicant's summary and conclusion

Conclusions:

This study assessing developmental toxicity of MWCNT and which was performed similar to OECD TG 414 in rats, showed decreased maternal thymus weight at the highest dose tested (1000 mg/kg bw/day). No effect on pregnancy outcome, fetal growth, viability, or morphological development was observed. Therefore, the repective NOAELs are considered to be 1000 mg/kg bw/day for fetal development and 200 mg/kg bw/day for maternal toxicity.

Executive summary:

This study assessing developmental toxicity of MWCNT was conducted similar to OECD TG 414 in specific pathogen free Sprague-Dawley rats with the deviation that fewer dams (n = 12) were tested than required in the guideline.

The test material was administered in doses of 0, 40, 200 and 1000 mg/kg bw/day orally per gavage. Control animals received the same volume of pure vehicle (CMC solution).

Clinical signs, mortality, body weights, food consumption, serum biochemistry, kidney oxidation damage, gross findings, organ weights, and Caesarean section findings were examined.

In the high dose group, dams showed decreased thymus weights (absolute and body weight relative) when compared to control dams. Therefore, the maternal NOAEL was set to 200 mg/kg bw/day.

There were no effects on fetal development obsereved. Thus, the according NOAEL was set to 1000 mg/kg bw/day.