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

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

A screening study for effects on fertility does not need to be conducted because a pre-natal developmental toxicity study is available.

An extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity.

Link to relevant study records

Reference

Reference 1

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because a pre-natal developmental toxicity study is available

Reason / purpose for cross-reference:

data waiving: supporting information

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

A study assessing developmental toxicity after oral administration of MWCNT which was performed similar to OECD TG 414 in rats, resulted in decreased maternal thymus weights 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 respective NOAELs were considered to be 1000 mg/kg bw/day for fetal development and 200 mg/kg bw/day for maternal toxicity.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

For details on endpoint specific justification please see read-across report in section 13 or find a link in cross-reference “assessment report”.

Reason / purpose for cross-reference:

assessment report

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: overall effects

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

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'.

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

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

This study assessing developmental toxicity after oral application 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:

The study used as source investigated developmental toxicity of MWCNT in rats. The study results of the source compound were considered applicable to the target compound. Justification and applicability of the read-across approach (structural analogue) is outlined in the read-across report in section 13 or find a link in cross reference “assessment report”.

Reference 2

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

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

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

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

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

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

40 mg/kg bw/day

Dose / conc.:

200 mg/kg bw/day

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.

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

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'.

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'.

Key result

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: overall effects

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

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'.

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

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

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

 

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.

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.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Experimental exposure time per week (hours/week):

168

Species:

rat

Quality of whole database:

High quality study comparable to guideline study with acceptable restrictions

Additional information

A study assessing developmental toxicity of MWCNT was conducted similar to OECD TG 414 in specific pathogen free Sprague-Dawley rats.

The test material (MWCNT Hanwha CM-95) 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). 12 female rats were assigned to each dosing group. The number of pregnant females in control, low dose, mid dose or high dose group was 11, 10, 12 or 11, respectively. Clinical signs, mortality, body weights, food consumption, serum biochemistry, kidney oxidation damage, gross findings, organ weights, and Caesarean section findings (Gravid uterus weight, number of corpora lutea, number of implantations, early resorptions, late resorptions, live and dead fetuses, external examinations, soft tissue examinations, skeletal examinations, fetal sex ratio, fetal and placental weights) 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.

Justification for classification or non-classification

The criteria for classification were not met.