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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
fertility, other
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
- Principle of test and short description of test conditions:
The aim of the study was to investigate the effects of lung exposure to multiwalled carbon nanotubes (MWCNTs) on fertility and pregnancy following a single intratracheal instillation of 67 μg of NM-400 one day prior to cohabitation with a mature male.

- Parameters analysed / observed:
* Female: Delivery time, litter parameters, maternal inflammation and histopathology of lung and liver
* Male offsring: locomotor activity, startle response, and daily sperm production (DSP)
In the female animals, lung and liver bore evidence of MWCNT exposure when assessed 6 weeks and 4 months after exposure.
GLP compliance:
no
Limit test:
yes
Justification for study design:
Not applicable

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Key physico-chemical characteristics of NM-400 in this study

PARTICLE CHARACTERISTICS
Particle size: 295 ± 234 nm (geodesic)
Length: 295 ± 234 nm (geodesic)
Diameter: 10 ± 3nm
Aspect ratio: 31 ± 26

PHASE: highly bent mwcnt
SURFACE AREA (BET): not available
CHEMICAL COMPOSITION:
Al: 53,447 ± 136 ppm
Fe: 3658 ± 31 ppm
Co: 2067 ± 7 ppm
Na: below mdt (10 ppm), but higher concentrations unevenly distributed
THERMOGRAVIMETRIC ANALYSIS: weight loss 84 ± 3 wt% a. inhomogeneous sample, representative subsample>8mg (o2 atmosphere, 30–800 ◦ c, 10 ◦ c/min)
XRD (TGA RESIDUAL): mainly amorphous, however indications of alumina oxide

Test animals

Species:
mouse
Strain:
C57BL
Details on species / strain selection:
Justification for the species selection:
The C57BL/6J strain was used as test species, due to the widespread use of this species in nanotoxicology, including
our own previous studies on developmental nanotoxicology.

REFERENCES:
1. Jackson P, Hougaard KS, Vogel U, Wu D, Casavant L, Williams A, et al.
Exposure of pregnant mice to carbon black by intratracheal instillation: toxicogenomic
effects in dams and offspring. Mutation Research 2012;745(1/2):
73–83.

2.Jackson P, Hougaard KS, Boisen AM, Jacobsen NR, Jensen KA, Moller P, et al.
Pulmonary exposure to carbon black by inhalation or instillation in pregnant
mice: effects on liver DNA strand breaks in dams and offspring. Nanotoxicology
2011;6(June (5)):484–500.

3. Kyjovska ZO, Boisen AM, Jackson P, Wallin H, Vogel U, Hougaard KS. Daily
sperm production: application in studies of prenatal exposure to nanoparticles
in mice. Reproductive Toxicology 2013;36(January):88–97.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Animals: Naïve mice
- Number: 60 females and 60 males
- Species: C57BL/6JBomTac
- Source: Taconic Europe, Ejby, Denmark
- Age: were supplied at 7 (female) and 9 (male) weeks of age
- Housing: Upon arrival each cage hosted 5 animals. After 1 week female animals were assigned to two groups each of 30 animals, based on with similar weight distributions.
On Day 18, the female animals were instilled. On Day 19, they were transferred to male cages, one female to one male. When the conception was achieved (body weight gain indication), the male was removed from the cage, and the female housed alone.
- Bedding in female cages: soiled bedding from male cages was regularly added to female cages, to keep females cycling.

ENVIRONMETAL CONDITIONS:
Light: 12 h light from 6.00 a.m.
Food and water: access to food (Altromin 1324) and tap water ad libitum
Cages: polypropylene cages
Note: Procedures complied with EC Directive 86/609/EEC and Danish
regulations on experiments with animals (The Danish Ministry of Justice, Animal
Experiments Inspectorate, Permit 2006/561-1123).

Justification for the species selection: The C57BL/6J strain was used as
test species, due to the widespread use of this species in nanotoxicology, including
our own previous studies on developmental nanotoxicology.

REFERENCES:
Jackson P, Hougaard KS, Vogel U, Wu D, Casavant L, Williams A, et al.
Exposure of pregnant mice to carbon black by intratracheal instillation: toxicogenomic
effects in dams and offspring. Mutation Research 2012;745(1/2):
73–83.
Jackson P, Hougaard KS, Boisen AM, Jacobsen NR, Jensen KA, Moller P, et al.
Pulmonary exposure to carbon black by inhalation or instillation in pregnant
mice: effects on liver DNA strand breaks in dams and offspring. Nanotoxicology
2011;6(June (5)):484–500.
Kyjovska ZO, Boisen AM, Jackson P, Wallin H, Vogel U, Hougaard KS. Daily
sperm production: application in studies of prenatal exposure to nanoparticles
in mice. Reproductive Toxicology 2013;36(January):88–97.

Administration / exposure

Route of administration:
intratracheal
Vehicle:
other: Nanopure water with 2% mouse serum, a pooled preparation obtained from a normal mouse population (M5905, Sigma–Aldrich))
Details on exposure:
PARTICLE CHARACTERISATION
The test material NM-400 is a homogenized and sub-divided entangled MWCNTs powder prepared for the OECD Working Party on Manufactured Nanomaterials (JRC nanomaterials Repository, Ispra, Italy). The sample was also analytically characterized by Transmission Electron Micrography (TEM), Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES), Thermogravimetric Analysis (TGA) and X-ray Diffraction (XRD) and Raman Spectroscopy.
REF: JRC European Commission. List of materials in the JRC Nanomaterials (NM) Repository. JRC European Commission; 2011, November 27. Last update 27.10.11.

TEST MATERIAL PREPARATION (as described in the article):
“MWCNTs were sonicated for 16 min (10 s pulses and 10 s pauses, total sonication time 8 min) at a concentration of 1.675 mg/mL or 1.340 mg/mL (40 μL or 50 μL instillation, gestational and preconceptional exposure study, respectively) in 0.2 m filtered, Ɣ-irradiated Nanopure Diamond UV water (Pyrogens: <0.001 EU/mL, total organic carbon: 3.0 ppb) with 2% mouse serum using a 400 W Branson Sonifier S450D (Branson Ultrasonics Corp., Danbury, CT, USA) mounted with a disruptor horn and operated at 10% amplitude. Samples were continuously cooled by ice during the sonication procedure to prevent excessive sample heating”

OVERALL DESIGN OF THE GESTATIONAL AND PRECONCEPTION EXPOSURE STUDIES
Gestational exposure
Particles: NRCWE-006 and NM-400
Initial group size: 22
Time of exposure: GD 8, 11, 15 and 18
Dose: 4×67 μg/animal (total 268 μg)

Preconceptional exposure
Particles: NM-400
Initial group size: 30
Time of exposure: One day pre-mating
Dose: 67 μg/animal
Details on mating procedure:
- Housing and mating procedure: Upon arrival each cage hosted 5 animals. After 1 week female animals were assigned to two groups each of 30 animals, based on with similar weight distributions.
On Day 18, the female animals were instilled. On Day 19, they were transferred to male cages, one female to one male. When the conception was achieved (body weight gain indication), the male was removed from the cage, and the female housed alone.
- Bedding in female cages: soiled bedding from male cages was regularly added to female cages, to keep females cycling.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
ANALYTICAL DETERMINATION (as described in the article):
"The stability of NM-400 dispersions was assessed by DLS and we found (data not shown) that the dispersion was satisfactory when vortexed and then sonicated in ultrasound bath for 5 min every 20 min.
The apparent particle size distribution in the particles in exposure medium was determined with a 633 nm He–Ne Dynamic Laser Scatter (DLS) Zetasizer nano ZS
(Malvern Inc., UK). Data were analyzed using the Dispersion Technology Software v. 5.0 (Malvern Instruments Ltd.). Samples were measured at 25 ◦ C in 1 mL disposable polystyrene cuvettes. For calculations of hydrodynamic size, we used the optical refractive (Ri ) and absorption indices (Ra ) of 2.020 and 2.00, respectively for NM400, and standard viscosity and optical properties of H2 O for the 2% serum Nanopure water vehicle".
Duration of treatment / exposure:
TIME OF EXPOSURE:
Gestational exposure: GD 8, 11, 15 and 18
Preconceptional exposure: One day pre-mating
Frequency of treatment:
single exposure
Details on study schedule:
OVERALL DESIGN OF THE GESTATIONAL AND PRECONCEPTION EXPOSURE STUDIES
Gestational exposure
Particles: NRCWE-006 and NM-400
Initial group size: 22
Time of exposure: GD 8, 11, 15 and 18
Dose: 4×67 μg/animal (total 268 μg)

Preconceptional exposure
Particles: NM-400
Initial group size: 30
Time of exposure: One day pre-mating
Dose: 67 μg/animal
Doses / concentrations
Dose / conc.:
67 other: μg/animal
Remarks:
Time of exposure: GD 8, 11, 15 and 18
No. of animals per sex per dose:
60 male and 60 female
Control animals:
yes
Details on study design:
- Dose selection rationale:
Doses and sample selection were chosen based on previous experimental results cited in the current publication.

References:
1. Takagi A, Hirose A, Nishimura T, Fukumori N, Ogata A, Ohashi N, et al. Induction of mesothelioma in p53+/− mouse by intraperitoneal application of multiwall carbon nanotube. Journal of Toxicological Sciences 2008;33(February (1)):105–16.
2. Jackson P, Hougaard KS, Vogel U, Wu D, Casavant L, Williams A, et al. Exposure of pregnant mice to carbon black by intratracheal instillation: toxicogenomic effects in dams and offspring. Mutation Research 2012;745(1/2): 73–83.
3. Jackson P, Hougaard KS, Boisen AM, Jacobsen NR, Jensen KA, Moller P, et al. Pulmonary exposure to carbon black by inhalation or instillation in pregnant mice: effects on liver DNA strand breaks in dams and offspring. Nanotoxicology 2011;6(June (5)):484–500.
4. Erdely A, Liston A, Salmen-Muniz R, Hulderman T, Young SH, Zeidler-Erdely PC, et al. Identification of systemic markers from a pulmonary carbon nanotube exposure. Journal of Occupational and Environmental Medicine 2011;53(June (6 Suppl.)):S80–6.

- Rationale for animal assignment (if not random): Animals were divided in two goups, 30 animals each, based on their body weight.

Examinations

Parental animals: Observations and examinations:
Maternal lung and liver histology
Partuition and lactation
Sperm parameters (parental animals):
Sperm production
Litter observations:
Behavioural testing
- Acoustic startle reaction (ASR) and prepulse inhibition (PPI) were tested one week after weaning (4 weeks of age).
Postmortem examinations (parental animals):
SACRIFICE: Dams were anaesthetized by subcutaneous injection of Hypnorm–Dormicum and euthanised by withdrawal of heart blood, 6–7 weeks or 4 months after the single exposure

ORGANS: Lungs and livers. The tissues were than preserved in paraffin, sectioned and stained with hematoxylin and eosin for histological examination.
Statistics:
In the preconceptional exposure study, gestational parameters were analyzed by Mann–Whitney U-test.
Delivery time was analyzed by log rank test. ANOVA was used for the remaining data.
ANCOVA was selected to controlled the litter size in both overall and pair wise comparisons, birth weights, and pre-weaning pup weights parameters.
PPI was analyzed separately for each prepulse intensity, as described in Ref 1.
SYSTAT Software Package 9 and SAS 9.2 were used to perform the analyses.

Ref 1 (cited in the article):
Hougaard KS, Andersen MB, Hansen AM, Hass U, Werge T, Lund SP. Effects of prenatal exposure to chronic mild stress and toluene in rats. Neurotoxicology and Teratology 2005;27(January (1)):153–67.
Reproductive indices:
Not specified
Offspring viability indices:
Not specified

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality:
not specified
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
LUNG
Six weeks after exposure:
• Histopathological changes in the lungs (2/3 treated animals)
• Bronchiolar subepithelial edema (2/3 treated animals – 0/3 control animals)
• Perivascular edema of connective tissue (1/3 treated animals – 0/3 control animals)
• Hyperplasia of bronchiolar epithelial cells in the lung (2/3 treated animals – 0/3 control animals)
• Single macrophages occurred sporadically (2/3 treated animals – 0/3 control animals).
Four months after exposure:
• Infiltration of mononuclear cells close to bronchiolar and arterial walls (6/6 treated animals – 0/6 control animals)
• and of macrophages (aggregates of several cells) (3/6 treated animals – 0/6 control animals).
• Edema of supportive tissue surrounding bronchioles and/or blood vessels was also seen (2/6 treated animals – 0/6 control animals)
• Desquamation of the bronchiolar epithelium was recorded in the lungs from one exposed (1/6 treated animals – 2/6 control animals)

Cellular composition in BAL fluid was determined 6–7 weeks after exposure. No significant differences were measured apart from relatively more dead cells in BAL from exposed females [F = (1,11) = 6.945, p = 0.023, ANOVA].





LIVER
Six weeks after exposure:
• Microfoci of necrosis (3/3 treated animals – 1/3 control animals)
• Enlargement of single hepatocytes (2/3 treated animals – 0/3 control animals)
• Increased number of Kupffer cells and binucleate hepatocytes exposed compared to control livers (3/3 treated animals – 0/3 control animals)
Four months after exposure:
• Focal hyperplasia and hypertrophy of Kupffer cells (3/6 and 4/6 treated animals – 0/6 control animals)
• Foci of inflammatory cells (1/6 treated animals – 0/6 control animals)
• Edematous endothelial cells (1/6 treated animals – 0/6 control animals)
• Microfoci of necrosis (4/6 treated animals – 3/6 control animals)
Histopathological findings: neoplastic:
not specified

Reproductive function / performance (P0)

Reproductive performance:
no effects observed
Description (incidence and severity):
Numbers of litters: 24 of 27 exposed females - 26 of 28 controls
Delivery time: delivery of the first litter was delayed on average 5 days (p < 0.01, log rank test) for exposed females (difference statistically significant)
Gestational and litter parameters: similar in control and exposed groups

Results: F1 generation

General toxicity (F1)

Clinical signs:
not specified
Description (incidence and severity):
Open field test and the acoustic startle response: determined for 4 or 13–14 week old male offspring, respectively. No differences were recorded.
Dermal irritation (if dermal study):
not specified
Mortality / viability:
not specified
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Description (incidence and severity):
Daily sperm production and testis weight in male offspring at age 125 days: unaffected by the maternal exposure.

Applicant's summary and conclusion

Executive summary:

The aim of this study was to determine the effects of the test material NM-400 on the establishment of pregnancy and the course of gestation of mature female C57BL/6J mice. Animals were treated with the 67 μg of NM-400 by intratracheal installation.

Animals were supplied at 7 (female) and 9 (male) weeks of age and acclimatised for 18 days. They were divided in two groups (30 animals each) based on weight distribution. The day after the installation, dams were co-housed with males until conception indicated by female weight increase. Males were removed from the cage after 8 weeks of cohabitation. The female mice were instilled once with 50μL of solution of vehicle with or without the test material NM-400 followed by 150μL of air, i.e. control and NM-400 groups, respectively. Mouse serum was prepared in-house. The stability of the test material NM-400 dispersions was assessed by DLS. It was confirmed to be satisfactory.

Studies have shown that inflammation as a result of exposure to endotoxin, might interfere with several steps in the preovulatory chain of endocrine events. For this reason, the endotoxin concentration was determined. The result showed a concentration below the detection limit of 0.05 EU/mL for NM-400. BAL cell composition and neutrophil granulocyte influx were also determined to assess lung inflammation after pulmonary exposure to the test material. No significant differences in BAL cell composition at about six weeks after the single intratracheal instillation of NM-400 MWCNT was recorded. The histopathology examination results of the lungs and livers of the female dams are as follow:

 

LUNG

Six weeks after exposure:

- Histopathological changes in the lungs (2/3 treated animals)

- Bronchiolar subepithelial edema (2/3 treated animals – 0/3 control animals)

- erivascular edema of connective tissue (1/3 treated animals – 0/3 control animals)

- Hyperplasia of bronchiolar epithelial cells in the lung (2/3 treated animals – 0/3 control animals)

- Single macrophages occurred sporadically (2/3 treated animals – 0/3 control animals).

Four months after exposure:

- Infiltration of mononuclear cells close to bronchiolar and arterial walls (6/6 treated animals – 0/6 control animals)

- Macrophages (aggregates of several cells) (3/6 treated animals – 0/6 control animals)

- Edema of supportive tissue surrounding bronchioles and/or blood vessels (2/6 treated animals – 0/6 control animals)

- Desquamation of the bronchiolar epithelium (1/6 treated animals – 2/6 control animals)

LIVER

Six weeks after exposure:

- Microfoci of necrosis (3/3 treated animals – 1/3 control animals)

- Enlargement of single hepatocytes (2/3 treated animals – 0/3 control animals)

- Increased number of Kupffer cells and binucleate hepatocytes exposed compared to control livers (3/3 treated animals – 0/3 control animals)

Four months after exposure:

- Focal hyperplasia and hypertrophy of Kupffer cells (3/6 and 4/6 treated animals – 0/6 control animals

- Foci of inflammatory cells (1/6 treated animals – 0/6 control animals)

- Edematous endothelial cells (1/6 treated animals – 0/6 control animals)

- Microfoci of necrosis (4/6 treated animals – 3/6 control animals)

 

The number of litters of the exposed dams did not differ from the control group: 24 of 27 exposed females - 26 of 28 controls.

The delivery of the first litter was delayed on average 5 days (p < 0.01, log rank test) for exposed females (difference statistically significant). Furthermore, the gestational and litter parameters were of the exposed groups were similar to the controls.

Daily sperm production and testis weight in male offspring at age 125 days, results show that they were unaffected by the maternal exposure. Open field test and the acoustic startle response were also determined for 4 or 13–14 week old male offspring, respectively. No differences were recorded.

According to the results of this study, an installation of a single dose of MWCNT lead to pathological changes in lungs and liver, which were observed 4 months after the exposure day. There was a slight delay in the delivery of the first litter. However, the test material did not interfere with reproductive functionality and sperm quality. Measurements on the oestrous cycle length, vaginal smearing post exposure have been not taken into consideration. These determinations could have added information on mating performance, copulation and conceiving time. 

In conclusion, the study gives important information about the possible effects of pre-conceptional exposure to MWCNT. However, the study design limitations such as lack of specific measurements and investigation of co-factors in pathological pathways, do not allow making firm conclusions.