Carbon black

Toxicity to reproduction: other studies

Administrative data

Endpoint:

toxicity to reproduction: other studies

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

not specified

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

Deviations were as follows:absorption of substance by dams unclear, therefore unclear if foetuses were actually exposed to the substance; bumber of examined litters too low; male were investigated only; one dose level only; treatment period too short; no examination of maternal animals (clinical signs, detailed clinical observations, body weight and food consumption); offspring not investigated for clinical signs, mortality, detailed clinical observations and body weight; historical control data and individual data missing

Data source

Reference

Reference Type:

publication

Title:

Carbon nanoparticles induce endoplasmic reticulum stress around blood vessels with accumulation of misfolded proteins in the fdeveloping brain of offspring

Author:

Onoda, A. et al.

Year:

2020

Bibliographic source:

Scientific Reports 10: 10028

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

In the present study a group of 10 pregnant ICR mice were administered carbon black nanoparticles (average primary particle size: 14 nm) in ultra pure water via intranasal inhalation at a concentration of 95 µg/mL kg bw on gestation days 5 and 9. A control group receiving distilled water was run concurrently. After birth one male offspring/dam was chosen and the brain was collected when the male offspring were 6 weeks old (n = 10 offspring/group). The purpose of the study was to investigate the endoplasmic reticulum (ER) stress along with accumulation of misfolded proteins and relationship between the ER stress and perivascular abnormalities in the brain of offspring mice maternally exposed to carbon black nanoparticles by using in situ FT-IR and immunofluorescence.

GLP compliance:

not specified

Remarks:

not specified in the publication

Type of method:

in vivo

Test material

Test material information

Constituent 1

Reference substance name:

Carbon black

EC Number:

215-609-9

EC Name:

Carbon black

Cas Number:

1333-86-4

Molecular formula:

C

IUPAC Name:

carbon

Test material form:

solid: nanoform

Details on test material:

- Analytical purity: > 99 % (carbon: > 99 wt%, nitrogen: 0.82 wt%, hydrogen: 0.01 wt%, and organic impurity contents: <1%wt).
- Average primary particle size: 14 nm
- Specific surface area: 295 - 338 m2/g
- Solubility: insoluble

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) of test material: Degussa Ltd (Frankfurt, Germany)

INFORMATION ON NANOMATERIALS
Carbon black nanoparticles were suspended at 5 mg/mL in ultra-pure water, sonicated for 30 minutes, and filtered through a 450-nm filter (S-2504) before intranasal instillation (Onoda et al, 2014)*. Hydrodynamic diameter of the test substance in suspension was determined by dynamic light scattering (NANO-ZS) with Rayleigh-Debye equation and transmission electron microscopy (JEM 1200EXII) on collodion-coated 200 Cu mesh (No. 6511). Filtered test item in suspension showed small agglomerated particles with a peak size of 84.2 nm and poly-dispersity index of 0.143 (Onoda et al, 2014)*. This size corresponds well with the typical small agglomerate sizes observed in test item samples (Onoda et al, 2014)*. The size distribution of the test item suspension was not altered until use for the intranasal instillation. Carbon black nanoparticles concentration in the suspension was 95 μg/mL by peak area of carbon signal (2.77 keV) obtained using a field emission scanning electron microscope (JSM-6500F) with an attached energy-dispersive X-ray analyzer (JSM-6500F) (Onoda et al, 2014)*.

*Reference:
- Onoda, A., Umezawa, M., Takeda, K., Ihara, T. & Sugamata, M. Effects of maternal exposure to ultrafine carbon black on brain perivascular macrophages and surrounding astrocytes in offspring mice. PLoS One. 9(4), e94336, https://doi.org/10.1371/journal. pone.0094336 (2014).

Test animals

Species:

mouse

Strain:

ICR

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SLC Inc. (Hamamatsu, Shizuoka, Japan)(pregnant females were purchased (gestation day 3))
- Age at study initiation: 11 weeks of age
- Housing:
Pregnant maternal animals: housed in cages (1 pregnant mouse/cage)
Pups: 3 pups/cage
- Diet (ad libitum)
- Water (ad libitum)

ENVIRONMENTAL CONDITIONS
- Temperature: 22 – 24 °C
- Humidity: 50 – 60%
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

inhalation

Type of inhalation exposure (if applicable):

other: intranasal instillation

Vehicle:

water

Remarks:

ultra pure

Details on exposure:

ADMINISTRAION
Carbon black nanoparticles were suspended at 5 mg/mL in ultra-pure water, sonicated for 30 minutes, and filtered through a 450-nm filter (S-2504) before intranasal instillation (Onoda et al, 2014)*. Pregnant mice were treated with intranasal instillation of carbon black nanoparticles suspension under anesthesia with isoflurane.

*Reference:
- Onoda, A., Umezawa, M., Takeda, K., Ihara, T. & Sugamata, M. Effects of maternal exposure to ultrafine carbon black on brain perivascular macrophages and surrounding astrocytes in offspring mice. PLoS One. 9(4), e94336, https://doi.org/10.1371/journal. pone.0094336 (2014).

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Gestation days 5 and 9

Frequency of treatment:

once (assumed, since it was not clearly stated)

Duration of test:

Approximately 61 days (from gestation day 3 up to 6 weeks after birth)

Doses / concentrations

Dose / conc.:

95 other: µg/mL/ kg bw

No. of animals per sex per dose:

10 female mice

Control animals:

yes

Details on study design:

EXAMINATIONS
After birth, the number of pups per dam was adjusted to 10 on postnatal day 1. One male offspring per one dam was randomly chosen and the brain was collected from the selected offspring at 6 weeks after birth (n = 10/group). All sample collection was performed under isoflurane anesthesia.

PREPARATION OF MOUSE BRAINS
Six-week-old mice anesthetized by isoflurane were transcardially perfusion with phosphate buffered saline (PBS) followed by perfusion-fixation with 4% paraformaldehyde in 0.1 M phosphate buffer. Brains were collected after perfusion-fixation, post-fixed in 4% paraformaldehyde in 0.1 M phosphate buffer for 20 hours, and subsequently cryoprotected in phosphate-buffered sucrose solutions (10% sucrose, 4–6 h; 20% sucrose, 4–6 h; 30% sucrose, 12 – 24 hours) containing 0.1% sodium azide. Sections were then embedded in Tissue-Tek OCT compound and frozen in Histo-Tek Hyfluid at −80 °C. Serial sections (10-μm thick) were prepared from the frozen blocks using a Tissue-Tek Polar instrument, and mounted onto an IR-reflective stainless-steel base for in situ FT-IR or a glass slide for immunofluorescence. Sections were air-dried for 30 hours before measurement and staining to prevent interference due to moisture.

IN SITU FT-IR

The detail method was described by Onoda et al. (2017)*. Briefly, for in situ FT-IR measurements, an IRT-7000 IR microscope was used combined with an FT/IR-6100 spectrometer. Spectra were acquired in reflection mode using a 16× Cassegrain lens and collected in the mid-IR range of 700–4000/cm at a resolution of 4/cm over 64 scans from 30 × 30-μm apertures. The reflection spectra were obtained from tissues around brain blood vessels in the cerebral cortex using lattice measurement (x-axis: 7 points, y-axis: 7 points, total of 49 spectra acquired). Sixty-four spectra were acquired from only embedding medium (OCT compound) regions and an average of these spectra was used as a common basal line for analysis of FT-IR spectral data. Smoothing and normalization of the obtained spectra were performed on the region containing the amide bands (1000–2000/cm) using Spectra Manager Software Ver. 2. The spectra for protein secondary structural analysis was deconvoluted and calculated ratios of secondary structure contents (α-helix, β-sheet, β-turn, and random coil) from peak intensities of the amide I bands (1600–1700/cm). The calculated ratios of secondary structures were visualized using the universal RGB code on the protein mapping analysis software (IR-SSE)(Sarver & Krueger, 1991)*.

IMMUNOFLUORESCENCE

Serial sections were stained using immunofluorescent antibodies following standard methodology to evaluate protein expression of glial fibrillary acidic protein (GFAP) as an astrocyte activation marker, macrophage mannose receptor (MMR/CD206) as a selective marker of perivascular macrophages (PVMs), activating transcription factor 6 (ATF6) and C/EBP-homologous protein (CHOP/GADD153/DDIT3) as endoplasmic reticulum stress markers (Yoshikawa et al., 2015; Ito et al, 2009)*. Sections were submerged in PBS to remove the Tissue-Tek OCT compound and then blocked by 10% normal donkey serum (IHR-8135) for 1 hour at room temperature. Sections were then incubated with primary goat polyclonal anti-GFAP antibody (1:500 in PBS) or goat polyclonal anti-MMR antibody (1:200 in PBS) for 16 hours at 4 °C. After rinsing 3 times for 5 minutes per a rinse with PBS, sections were incubated with secondary Dylight 488-conjugated donkey anti-goat IgG (1:1000 in PBS) for 120 minutes at room temperature. After rinsing 3 times for 5 minutes per a rinse with PBS, sections were incubated with primary rabbit polyclonal anti-ATF6 antibody (1:100 in PBS) or rabbit polyclonal anti-CHOP antibody (1:100 in PBS) for 16 hours at 4 °C. After rinsing 3 times for 5 minutes per a rinse with PBS, sections were further incubated with secondary Dylight 649-conjugated donkey anti-rabbit IgG (1:1000 in PBS) for 120 minutes at room temperature. Sections were then rinsed three times for 5 minutes per rinse with PBS and twice for 5 minutes per rinse with distilled water. Nuclei were counterstained using Hoechst 33342.

CELL COUNTING

The expression of GFAP, MMR, ATF6, and CHOP was evaluated using a fluorescent microscope (BZ-9000) to detect and quantify ATF6-positive astrocytes, CHOP-positive astrocytes, ATF6-positive PVMs, and CHOP-positive PVMs in the cerebral cortex. For each brain obtained from offspring mice, one hundred sections (total 1,000 μm) were prepared from bregma of cerebrum to olfactory bulb along the coronal plane. One of every five sections (twenty sections per mouse; about 20 mm2/mouse at 50-μm intervals) was chosen for the quantitative analysis of cells positive for GFAP, MMR, ATF6, and CHOP. Cell counting was done under fluorescence microscopy (BZ-9000) at 200× magnification and calculated per mm2 area.


*Reference:
- Onoda, A., Kawasaki, T., Tsukiyama, K., Takeda, K. & Umezawa, M. Perivascular Accumulation of β-Sheet-Rich Proteins in Offspring Brain following Maternal Exposure to Carbon Black Nanoparticles. Front Cell Neurosci. 11, 92, https://doi.org/10.3389/fncel.2017.00092 (2017).
- Sarver, R. W. Jr & Krueger, W. C. Protein secondary structure from Fourier transform infrared spectroscopy: a data base analysis. Anal Biochem. 194(1), 89–100 (1991).
- Yoshikawa, A. et al. Deletion of Atf6α impairs astroglial activation and enhances neuronal death following brain ischemia in mice. J Neurochem. 132(3), 342–353, https://doi.org/10.1111/jnc.12981 (2015).
- Ito, Y. et al. Involvement of CHOP, an ER-stress apoptotic mediator, in both human sporadic ALS and ALS model mice. Neurobiol Dis. 36(3), 470–476, https://doi.org/10.1016/j.nbd.2009.08.013 (2009).

Statistics:

All data are expressed as means in the form of box-and-whisker diagrams. The numbers of activating transcription factor 6 (ATF6)-positive astrocytes, ATF6-positive perivascular macrophages, and CHOP-positive perivascular macrophages were analysed using Mann-Whitney’s U test. Proportions of secondary structure contents were evaluated using Kruskal-Wallis test followed by Steel-Dwass post hoc test. The level of significance was set at p < 0.05. Statistical analyses were carried out using R version 3.5.3 (https://www.r-project.org/).

Results and discussion

Effect levels

Remarks on result:

other: see "Remarks"

Remarks:

Maternal exposure to carbon black nanoparticle induces endoplasmic reticulum (ER) stress associated with accumulation of misfolded proteins. Notably, offspring specifically showed high induction of ER stress in perivascular macrophages (PVMs) and reactive astrocytes only around brain blood vessels, along with accumulation of β-sheet-rich proteins regarded as misfolded proteins.

Observed effects

The expression levels of activating transcription factor 6 (ATF6) and CHOP as endoplasmic reticulum (ER) stress markers of astrocytes and macrophages were investigated and found that maternal carbon black nanoparticles (CB-NP) exposure specifically upregulate the ER stress markers around brain blood vessels, but not within parenchyma far from blood vessels. The expression level of glial fibrillary acidic protein (GFAP) increased in the CB-NP group compared to the control group. Thus, to evaluate the expression of the ER stress markers in the reactive astrocytes highly expressing GFAP, the marker proteins were visualized by double-immunofluorescence with GFAP. High ATF6 expression was particularly observed in the reactive astrocytes around brain blood vessels in the exposure group. In contrast, no expression of ATF6 was observed in the astrocytes of the control group, even in the case of GFAP-positive astrocytes. The ATF6 upregulation was also observed in perivascular macrophages (PVMs) (macrophage-mannose-receptor positive cells). Furthermore, PVMs also expressed CHOP in the exposure group. The number of ATF6-positive astrocytes, ATF6-positive PVMs, and CHOP-positive PVMs was significantly increased after maternal exposure to low doses of CB-NP. In contrast, high expression of ATF6 and CHOP were not observed in neurons and other glial cells in the brains of control and CB-NP exposure groups.

In situ FT-IR analysis combined with immunofluorescence was performed to investigate protein structures on serial sections of the brain. First, brain perivascular tissues where ER stress was induced in the maternal CB-NP exposure group displayed high in situ FT-IR signals derived from β-sheet structures. Second, β-sheet structures were specifically and significantly increased in the areas around blood vessels with ATF6-positive astrocytes, ATF6-positive PVMs, and CHOP-positive PVMs compared with other areas around blood vessels. Additionally, α-helix structures were significantly decreased around these blood vessels. In the control group, there was no difference between the ratios of these secondary structures around brain blood vessels with and without ER stress.

Please aols refer to the field "Attached background material" below.

Applicant's summary and conclusion

Conclusions:

In the present study a group of 10 pregnant ICR mice were administered carbon black nanoparticles (average primary particle size: 14 nm) in ultra pure water via intranasal inhalation at a concentration of 95 µg/mL kg bw on gestation days 5 and 9. A control group receiving distilled water was run concurrently. After birth one male offspring/dam was chosen and the brain was collected when the male offspring were 6 weeks old (n = 10 offspring/group). The purpose of the study was to investigate the endoplasmic reticulum (ER) stress along with accumulation of misfolded proteins and relationship between the ER stress and perivascular abnormalities in the brain of offspring mice maternally exposed to carbon black nanoparticles by using in situ FT-IR and immunofluorescence.

Maternal exposure to carbon black nanoparticle induces endoplasmic reticulum (ER) stress associated with accumulation of misfolded proteins. Notably, offspring specifically showed high induction of ER stress in perivascular macrophages (PVMs) and reactive astrocytes only around brain blood vessels, along with accumulation of β-sheet-rich proteins regarded as misfolded proteins.

The reference exhibits major reporting and experimental deficiencies, which do not allow an independent review about the exposure-effects correlation:

First, it was not determine by the authors, if the substance actually reached the foetuses after the dams were treated with it. It is even unclear, if the substance was absorbed by the dams and reach blood circulation and, then, that the substance was able to cross the placenta, which is a prerequisite in order to reach the foetus. In conclusion, it can be determine for certainty that the substance caused the changes observed in the foetuses.

In addition, the number of pregnant animals is too (n = 10/group), which causes a low number of litters available. From each litter one male offspring was chosen for further investigation. The guideline OECD 426 foresees that 20 litters should be investigated as foreseen by the guideline OECD 426. This low number of animals significantly reduces the statistical power and reduces the meaningful evaluation of the effect of the test item. Furthermore, males were investigated only, which makes it impossible to draw any conclusion on females. In addition, the guideline foresees that at least three dose level and a concurrent control should be used. In this study, only one dose level was tested. The usage of one dose level precludes the possibility to demonstrate any dose-related response and the determination of a No-Observed-Adverse Effect level (NOAEL). Also, the duration of treatment was too short. The pregnant females were exposed on gestation days 5 and 9 instead of daily from time of implantation (gestation day) throughout lactation (PNDT 21), so that the pups were exposed to the test substance during pre- and postnatal neurological development, as foreseen by the guideline. Using a shorter duration of treatment as recommended by the guideline precludes the full exposure of pre- and postnatal neurological development. Moreover, the maternal animals were not investigated for clinical signs, detailed clinical observations, body weight and food consumption. Therefore, it is impossible to determine, if the dams showed signs of toxicity and if those causes a secondary effects in the offspring. The offspring themselves were also not observed for clinical signs, mortality, detailed clinical observations and body weight. Since clinical signs were not observed, no conclusion can be draw, if the effects observed in this study had any effect on the neurological functions of the offspring. Lastly, historical control data or individual data were not provided. Since the historical control data was not provided, it is not possible to determine, if the findings were within or outside the range of normal biological variation of the rat strain. In addition, individual data would be helpful in order to determine, if the results contain outliners, which might influence the outcome of the results.