Evaluating the evidence on genotoxicity and reproductive toxicity of carbon black: a critical review

Figures & data

Table 1. In vitro tests with carbon black related to mutagenicity/genotoxicity endpoints.

Test system	Method	Testing conditions	Test material/SSA	Result	Remark	References
Mutagenicity
Bacterial cell systems
Ames test with CB particles	OECD 471	As per GL	Printex 90, N339, Unipure Black, 100–300 m^2/g	Negative		(Kirwin et al. 1981; Degussa 1998; Hobson 2011)
Ames test with CB extracts	OECD 471	As per GL, Soxhlet extraction	Printex 70, Printex 90, N339	Negative		(Kirwin et al. 1981; Degussa 1997, 1998)
	Similar to OECD 471	Soxhlet extraction (benzene, 16 h (Agurell and Löfroth 1983, 1993) or toluene, 48 h (Rosenkranz et al. 1980))	N330, Black Pearls L	Positive; some negative	Mutagenic activities varied widely between different samples; positive findings in pre-1979 material were due to nitropyrene impurities	(Rosenkranz et al. 1980; Agurell and Löfroth 1983, 1993)
Mammalian cell systems
FE1-MML MutaMouse™ epithelial cell line	Transgenic cell line, Comet (Fpg)	75 µg/mL, cumulative CB dose 6 mg in 8 × 72 h incubation	Printex 90, 295 m^2/g	Small (1.2- to 1.4-fold) increases in transversion mutations at the cII and LacZ loci of a transgenic cell line (consistent with ROS damage); DNA strand breaks and oxidized DNA	The mutant frequency for the cII gene of the negative control cells increased 1.27-fold	(Jacobsen et al. 2007, 2011)
L5178Y Mouse Lymphoma	OECD 476, HPRT test	GLP, 3 h, up to 120 µg/mL	Carbon Black E3000281, containing Unipure Black LC 902, 200–260 m^2/g	Negative	The SCCS (2015) is of the opinion that 3 h exposure may have been too short for cellular uptake	(Lloyd 2011)
L5178Y Mouse Lymphoma	OECD 476	4 h (extended because of the difficulty of separating test material from cells), 10–40 mg/mL (-S-9); 5–15 mg/mL (+S-9)	N339, 100 m^2/g	Negative		(Kirwin et al. 1981)
Clastogenicity – Aneuploidy/micronucleus assays in vitro
A549 human lung cancer cell line	Micronuclei	0.02–200 mg/L (0.003–34 µg/cm^2); 6 h; no cytoB	Printex 90	Increase in micronuclei (max. 3.3%; controls: 0.7%) with plateau at 2 mg/L; cell growth reduced by 60% at the highest dose tested	Solvent control cultures (0.9% saline with 0.05% v/v Tween 80, serum) also showed an increase in micronucleus frequency after 48 h. No further data were reported on the solvent controls nor on cytotoxicity	(Totsuka et al. 2009)
RAW 264.7 mouse macrophage cell line	Micronuclei	0.01–100 mg/L, 48 h; 4 mg/L cytoB (28 h delayed co-treatment), 10% serum	Printex 90	Negative	Cytotoxicity: trypan blue exclusion, CBPI	(Migliore et al. 2010)
RAW 264.7 mouse macrophage cell line	Micronuclei	1, 3, and 10 µg/cm^2, 48 h; cytoB added after 44 h of incubation; after further 28 h of incubation, the cells were harvested	Degussa Huber NG90, diameter 200–250 nm	Positive (22, 36.5, and 50 micronuclei/1000 cells at 1, 3, and 10 µg/cm^2, controls: 14/1000 cells), 3 and 10 µg/cm^2 were cytotoxic (MTT assay); increased apoptosis		(Poma et al. 2006)
RAW 264.7 mouse macrophage cell line	Micronuclei, chromosome aberrations	1, 3, and 10 mg/L, 48 h; cytokinesis-block method for MNT, but no details reported; chromosome aberrations at 24, 48, and 72 h post-exposure; ROS production at 50 mg/L after 5 and 24 h	Printex 90	1 mg/L: MNT negative 3 and 10 mg/L: ↑MN, less than 2-fold); acentric chromosome fragments at all concentrations; accumulation in phagolysosomes, causing acute necrosis; ↑intracellular ROS production, similar at 5 and 24 h	Cytotoxicity: 50 and 100 mg/L (trypan blue exclusion, 72 h, MTS); CBPI; chromosomal effects: relevance uncertain (insufficient control data in RAW 264.7 cells)	(Di Giorgio et al. 2011)
Chinese hamster ovary (CHO)	OECD 479, sister chromatid exchange (SCE)	+/− metabolic activation 0.00032–1 mg/mL	N339, 100 m^2/g	Negative	OECD 479 was deleted in 2014	(Kirwin et al. 1981)
Chinese hamster ovary (CHO)	OECD 487 (2010), micronuclei	GLP, 10, 20, and 30 μg/mL for pulse treatments in the absence and presence of S9-mix; 10, 15, and 20 μg/ml for the continuous treatment in the absence of S9-mix; vehicle: DMSO	Carbon Black E3000281, containing Unipure Black LC 902, 200–260 m^2/g	Negative	CytB not used during the 3 h exposure, but added immediately afterwards (delayed co-treatment), SCCS questions the validity of the study (SCCS 2015)	(Lloyd 2012)
Undifferentiated hamster epithelial cell line M3E3/C3	Micronuclei	0.1, 1, and 2 µg/mL, 30 h	Carbon black, not specified	Inconclusive (<2-fold increase over controls)		(Riebe-Imre et al. 1994)
PAH-DNA adducts
A549 human lung epithelial cells	^32P post-labeling	Particles: 100 µg/cm^2, DMSO extracts: 30–300 µg/cm^2 for 24 h	Printex 90 (PAH: 0.039 ppm, 300 m^2/g), Sterling V (PAH: 8.8 ppm, 30–40 m^2/g), N330 (PAH: 2.4 ppm, 70–90 m^2/g), Lampblack 101 (PAH: 0.057 ppm, 20 m^2/g)	Negative (Printex 90, N330, Lampblack 101); some adducts were found with Sterling V extract. The identity of those could not be identified and they were not dose-dependent with regard to the spot intensity	The in vitro conditions showing the effect with Sterling V will not be encountered in vivo and renders this mechanism in particle-induced lung cancer at in vivo exposures highly unlikely	(Borm et al. 2005)
DNA damage & repair/comet assays
RAW 264.7 mouse macrophage cell line	Comet	1, 3, 10, and 50 mg/L, 24 h	Printex 90	1, 3, and 10 mg/L: negative 50 mg/L:↑DNA damage		(Di Giorgio et al. 2011)
RAW 264.7 mouse macrophage cell line	Comet +/− Fpg and Endo III	1, 10, and 100 mg/L, 24 h incubation for cytotox test, not reported for Comet assay;	Standard carbon black (particle size 500 nm) and N330 (20–50 nm)	Negative for standard carbon black; positive for N330 at 1 mg/L (in buffer, +EndoIII), 10 mg/L (+EndoIII), and negative at 100 mg/L; both products ↑ NFkappaB at 10 and 100 mg/L and (not dose-dependently) of TNF-alpha and COX-2		(Rim et al. 2011)
Primary mouse embryo fibroblasts	Comet	5 concentrations up to 100 mg/L, 24 h	CB from Nano-Innovation Co. China, C > 99.4%, 12 nm	Cytotoxicity and oxidative damage; induced fewer DNA single strand breaks than the other materials tested	Comparative study with other materials, authors state importance of further in vivo tests	(Yang et al. 2009)
Human A549 and monocytic T-cells (THP-1)	Comet	16, 160, and 1600 ng/mL for 48 h	Vulcan M, furnace black, 100 nm from Cabot	Positive at 1.6 µg/mL; dichloromethane extracts negative; no cytotoxicity (AlamarBlue assay)		(Don Porto Carero et al. 2001)
A549 cells	Comet (Fpg)	1, 20, and 40 µg/cm^2 for 4 h	Carbon powder, Sigma Aldrich, <30 nm	Negative	SCCS (2015) notes shortcomings in methodology	(Karlsson et al. 2008)
Human embryonic lung Hel 299 fibroblasts and Chinese hamster V79 cells	Comet	4 concentrations up to 138 µg/cm^2, 3 h	Carbon black, Cabot, 37 nm	Negative		(Zhong et al. 1997)
FE1-MML MutaMouse™ epithelial cell line	Comet (Fpg)	75 µg/mL, cumulative CB dose 6 mg in 8 × 72 h incubation	Printex 90, 295 m^2/g	↑Oxidized purines		(Jacobsen et al. 2007)
A549 cells	Comet	25 mg/m^2 (100 µg/mL), 0.5–24 h, suspensions in culture medium sonicated for 20 min	Huber 990, 260 nm, Printex 90, 14 nm	Positive at ≥3 h exposure Both particle types: NFkappaB activation, only Printex 90: DNA single strand breaks, activation of p53 and DNA repair	Preliminary work, only one concentration tested. No double strand breaks (however those were detected in parallel experiment with urban dust)	(Mroz et al. 2007, 2008)
Human umbilical vein endothelial cells (HUVEC)	Comet (Fpg)	100 mg/L for 3 h	Printex 90	Oxidative DNA damage		(Frikke-Schmidt et al. 2011)
HepG2 cells	Comet (Fpg, hOGG1)	25 mg/L for 3 h	Printex 90	Oxidative DNA damage		(Vesterdal et al. 2014)
Caco-2 human intestinal carcinoma cell line	Comet (Fpg)	20 µg/cm^2 for 4 h	Printex 90	Negative		(Gerloff et al. 2009)
Cell transformation assays
C3H/10T1/2 CL8 mouse embryo cells	EU method B.21	4 concentrations, 2–16 mg/mL	N339, 100 m^2/g	Negative		(Kirwin et al. 1981)
Hamster M3E3/C3 epithelial cell line	Similar to EU method B.21	100, 300, and 500 µg/mL	CB, not specified	Inconclusive	Non-validated cell line	(Riebe-Imre et al. 1994)
Cell-free systems
Supercoiled plasmid	–		Fine CB and Printex 90, 253.9 m^2/g	Single strand breaks in directly exposed DNA after treatment with Printex90		(Stone et al. 1998)
Oxidative stress
A549 cells	MTT, oxidative stress	Up to 0.78 µg/mm^2	Fine CB and Printex 90, 253.9 m^2/g	Printex 90 induced oxidative stress	These results suggest that ultrafine CB induces a greater oxidative stress than fine CB	(Stone et al. 1998)
Human primary bronchial epithelial cells	Western blot analysis	6.13–30.7 µg/cm^2	CB, 250 nm, 7.8 m^2/g; ultrafine CB, 11 nm, 457 m^2/g	30.7 µg/cm^2: Ultrafine CB stimulated proliferation of human airway epithelium via epidermal growth factor (EGF) receptor-mediated signaling pathway and oxidative stress; NOEC (DNA synthesis): 12.3 µg/cm^2	Stimulation was similar to that caused by 10% fetal bovine serum	(Tamaoki et al. 2004)
THP-1 monocytes, THP-1 derived macrophages (THP-1a), HUVECs	WST-1, trypan blue, ROS	2.5, 12.5, 25, and 100 mg/L	Printex 90	↑cytotoxicity at ≥12.5 mg/L (THP1, -1a) and all cell types at 100 mg/L; ↑LDH at 100 mg/L only in THP-1; no effect on trypan blue exclusion; ↑intracellular ROS, plateau at 12.5 mg/L		(Cao et al. 2014)

CB: carbon black; CBPI: cytokinesis-block proliferation index; DMSO: dimethylsulfoxide; Endo III: endonuclease III; Fpg: formamidopyrimidine DNA glycosylase; GL: guideline; GLP: Good Laboratory Practice; HPRT: hypoxanthine guanine phosphoribosyltransferase; i.t.: intratracheal; MN: micronuclei; MNT: micronucleus test; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; SCCS: Scientific Committee on Consumer Products, opinion on carbon black (nano-form), December 2015; SSA: specific surface area.

Table 2. In vivo tests with carbon black related to mutagenicity/genotoxicity endpoints.

Test system	Method	Testing conditions	Test material/PPS/SSA	Result	Remark	References
Mutagenicity
Germ cell test
Female germline mutations	ESTR mutations in oocytes	Mouse, i.t., 4 × 67 µg/animal during gestation	Printex 90	Negative		(Boisen et al. 2013)
Somatic cells
RLE-6TN rat alveolar epithelial cells (type II cells) + BAL cells (ex vivo)	HPRT test	BAL cells ex vivo from rats 15 months after single intratracheal instillation of saline or 10 or 100 mg/kg bw CB	Monarch 900, 15 nm, 230 m^2/g	10 mg/kg: NOEL 100 mg/kg: positive (factor 7.6) at 15 months after exposure	Supports threshold and secondary genotoxicity by ROS species	(Driscoll et al. 1997)
Alveolar epithelial cells (type II cells)	HPRT test	Rat, 90 days inhal., 6 h/day, 5 days/week; 1.1, 7.1, and 52.8 mg/m^3 and recovery	Monarch 880, 16 nm. 220 m^2/g, MMAD 0.88 µm	1.1 mg/m^3: NOAEL 7.1 mg/m^3: positive (factor 3.4); negative after recovery; 52.8 mg/m^3: positive (factor 4.3, 3.2, and 2.7 after exposure and 3- and 8-month recovery, respectively)	Mutations only at inflammatory doses, with epithelial hyperplasia	(Driscoll et al. 1996, 1997)
BAL	HPRT test	Rat, mouse, hamster (all female); 90-day inhalation, 1, 7, and 50 mg/m^3 + recovery	Printex 90	1 mg/m^3: NOEL 7.50 mg/m^3: ↑hprt in BAL cells, rat ≫ mouse; not in hamster		(Carter et al. 2006)
Lung, kidney	gpt (lung, kidney) and Spi (only lung) mutations	Male gpt transgenic mouse, 200 µg 1× or 4×, examined 8 or 12 weeks after exposure for mutations, 5/group	Printex 90	No gpt mutations were induced; (frequency of gpt and Spi-mutations/deletions in lungs slightly, but not significantly higher than in controls)	(Microscale kaolin was positive in both assays); rarely used assay with high standard deviation	(Totsuka et al. 2009)
Retina pigment epithelium	DNA deletions	Mouse, oral, 500 mg/kg bw	Not specified	No effects on DNA deletions in the retina pigment epithelium of offspring		(Reliene et al. 2005)
Lung carcinoma	K-ras, p53 mutations; hypermethylation of p16 gene	F344 rat, 24 months (16 h/day, 5 days/week), 0. 2.5, and 6.5 mg/m^3)	Diesel exhaust or CB (Elftex-12, 43 m^2/g)	No mutations in p53 were observed; a low frequency (3/50) and variable pattern of activating mutations were identified in K-ras independent of exposure	Small sample size; no significant differences between the yields of mutants recovered from diesel exhaust, carbon black, or sham-exposed rats	(Nikula et al. 1995; Swafford et al. 1995; Belinsky et al. 1997)
Clastogenicity
Peripheral human blood lymhocytes	Cytogenetic assay	28 workers (2–8 years exposed, tire industry), 15 controls; 100 metaphases analyzed	Not specified; co-exposure to substances in tire industry	Positive association (5.07% versus 2.27 in unexposed controls)	Only abstract available	(Babu et al. 1989)
DNA Adducts
Lung	^32P post-labeling	F344 rat (female), 13 week, 6 h/day, 5 days/week; Printex 90: 0, 1, 7, and 50 mg/m^3 Stering V: 50 mg/m^3 all in filtered air; 3/group	Printex 90 (PAH: 0.039 ppm, 300 m^2/g), Sterling V (PAH: 8.8 ppm, 30–40m^2/g)	No adduct formation		(Borm et al. 2005)
Lung, liver	^32P post-labeling, HPLC	F344 rat, 0.64 mg/kg, 1× p.o. (gavage) or i.t., 7–10/group	Printex 90	No adduct formation in lungs; ↑etheno-adducts in liver after gavage; no change after i.t.	Large variation in the levels in liver within the control and exposure groups	(Danielsen et al. 2010)
Lung	^32P post-labeling	Wistar rat (f), 11.3 mg/m^3, 18 h/day, 5 days/week, 20 months, whole-body	Printex 90; MMAD, 0.65 µm; 270 m^2/g;	No adduct formation		(Gallagher et al. 1994)
Lung	^32P post-labeling	F344 rat (m), 10 mg/m^3; 7 h/day, 5 days/week, 12 weeks; whole-body; 6/group	Elftex-12 furnace black; 2 μm MMAD (large mode); 0.1 μm MMAD (small mode); 43 m^2/g	No adduct formation	Significant localized inflammation in the rat lung was induced	(Wolff et al. 1990)
Lung alveolar epithelial type II cells	^32P post-labeling	F344 rats, 6.2 mg/m^3 (respirable fraction);16 h/day, 5 days/week, 12 weeks; whole-body	Elftex-12 furnace black; 2 μm MMAD (large mode); 0.1 μm MMDD (small mode); 43 m^2/g	25 and 5 adducts/10e9 in exposed and filtered-air controls, respectively		(Bond et al. 1990)
DNA damage and repair
Studies in rats
Lung, alveolar lining cells	PAR, γ-H2AX, 8-OH-dG, OGG1, 1 month after last instillation	Rat, i.t., 6 mg/rat 1×/month, 3 months (total dose 18 mg/rat); CB was dispersed in phys. saline with Tween 80 (conc. not reposted) before instillation	Printex 90	↑γ-H2AX- and 8-OH-dG-positive nuclei in lung tissue (2.1- and 1.8-fold, respectively); number of OGG1-positive nuclei (↓); frequency of OGG1-positive cytoplasm ↑(4-fold)	γ-H2AX is a pre-toxic lesion (Nikolova et al. 2014); alveolar lining cells displayed a granular pattern of OGG1 in the cytoplasm, probably reflecting mitochondrial expression of the enzyme	(Ziemann et al. 2011; Rittinghausen et al. 2013)
Lung	8-Oxo-dG	Rat (f), 13 weeks, 6 h/day, 5 days/week inhalation, 5/g (1, 7, and 50 mg/m^3 Printex90, 50 mg/m^3 Sterling V); 5/group, + recovery	Printex 90, 300 m^2/g, Sterling V, 30–40 m^2/g	1 mg/m^3: NOEL 7 mg/m^3: ↑oxidative damage after 44 weeks recovery; max. factor 1.5; 50 mg/m^3: ↑lung burden, particularly Sterling V, ↑oxidative damage (only Printex 90) at 0 and 44 weeks after exposure, lung load 1–7 mg/lung, PMNs↑		(Gallagher et al. 2003)
Liver, lung	8-Oxo-dG, εdG, εdA, OGG1, mRNA expression, 24 h after exposure	F344 rat, 0.64 mg/kg, 1× p.o (gavage). or i.t., 7–10/group	Printex 90	mRNA levels of genes related to oxidative stress unchanged after i.t., but ↑after p.o.; 8-oxo-dG increased by 23% after p.o.; no change in OGG1	Endotoxin content of test preparation: 25 mg/L	(Danielsen et al. 2010)
Comet assays – inhalation
BAL	Comet assay (± hOGG1), 1 day and 14 days post-exposure	Wistar rat (m), 6 mg/m^3 for 14 days (6 h/day, 5 days/week) + 14 d recovery, n = 5/group	Printex 90	No strand breaks, no oxidative DNA damage; positive controls (KBrO3, i.p.) were functional)		(Lindner et al. 2017)
BAL, liver	Comet, 5 and 24 days after exposure	Mouse, inhalation (whole body), 42 mg/m^3, 1 h/day, gd 8–18), 5–6/group	Printex 90	No effect on gestation and lactation; strand breaks in BAL not increased (5 and 24 days after exposure), but higher levels in liver (max 1.6-fold increase) of dams and offspring	Pregnancy usually affects liver function; no historical control data provided – questionable association with CB exposure; in inhalation study oral exposure of damns probable (whole-body chamber)	(Jackson et al. 2012a)
BAL	Comet, 1 h after last exposure	TNF+/+ and TNF−/− mice, 4 × 20 mg/m^3 for 1.5 h, inhalation, 4/group; nose-only	Printex 90, 295 m^2/g; diesel exhaust particles (DEP), filtered air	% neutrophils in BAL not different from air controls (but significant increase in DEP treated animals); ↑IL-6 mRNA in lung; ↑DNA strand breaks in the BAL cells of DEP- and CB-exposed TNF^−/− (by a factor of approximately 2 in carbon black exposed animals)	TNF induces neutrophil apoptosis as a mechanism for removal of neutrophils (Murray et al. 1997); this might explain the reduced DNA damage in TNF +/+ mice as compared to TNF−/− mice	(Saber et al. 2005)
Comet assays – intratracheal instillation
BAL cells, lung, liver	Comet, 1, 3, or 28 days after exposure, +/− fpg	C57BL/6 female mice, i.t. 18, 54, and 162 µg/mouse; suspensions in physiological saline with 10% added acellular BAL (or BAL +10% saline; both reported in publication); endotoxin 0.142 EU/mg Printex 90	Printex 90	BAL 18 µg: ↑DNA strand breaks at ayd1 and day 28; 54 µg: ↑DNA strand breaks at day 28; 162 µg: ↑ DNA strand breaks at day 1, day 3, and day 28; Lung 18 µg: ↑DNA strand breaks at day 1; 54 µg: ↑DNA strand breaks at day 3 and day 28; 162 µg: ↑ DNA strand breaks at day 1, day 3, and day 28; Liver 18, 54, and 162 µg: ↑DNA strand breaks at day 1 and day 28; not at day 3. At day 1, not dose-related Inflammation 18, 54, and 162 µg: significant and dose-dependent increase in BAL neutrophil count still present at day 28	Results indicate clearance and repair activity; maximum neutrophil influx at day 1, maximum recruitment of other cells at day 3; vehicle contained particles >20 nm; ↑FPG sensitive sites in lung at day 1 at all dose levels, and at day 3 only at 162 µg	(Bourdon et al. 2012b, 2012c)
BAL	Comet, 3 and 24 h after exposure	Mouse, wild-type (C57) and ApoE-/-, i.t., (18, 54 µg/mouse); inhal, 60 mg/m^3 (30, 90 min); 7 animals/group	Printex 90	54 µg: single strand DNA damage in BAL cells at 3 h after i.t. treatment; inh caused only marginal effects (increases in cytokine mRNA, distribution between neutrophils and macrophages)	Compared to normal mice, the ApoE-/- model is more sensitive to inflammatory effects; results of comet assay after inhalation not reported; no comet results either for the 24 h measurement after i.t. instillation; 60 mg/m^3 was selected as similar dose to highest it dose regarding inflammation	(Jacobsen et al. 2009)
BAL	Comet, 24 h after exposure	C57BL/6 mouse (f), i.t., 54 µg/mouse; in physiol. Saline containing 10% BAL from unexposed mice	Printex 90, Lampblack 101	Negative (examined 24 h post-instillation); macrophage depletion and increase in neutrophils with Printex 90 only	DNA damage may have been repaired by 24 h	(Saber et al. 2012)
BAL, lung	Comet, 3 h; 1, 2, 3, 4, 5, 14, and 42 days post-exposure; included also toxicogenomic analysis	C57BL/6 mouse (f), i.t. 162 µg/mouse, suspension in nanopore water	Printex 90	↑neutrophil at all time points; ↑DNA strand breaks in BAL cells 3 h and 3 days post-exposure, and in lung tissues 2–5 days post-exposure; ca. 2600 genes were differentially expressed (± 1.5 fold; p ≤ .05) across all time-points in the lungs; gene expression changes associated with inflammatory response followed a biphasic pattern, with initial changes at 3 h declining to base-levels by 3 days, increasing again at 14 days, and then persisting to 42 days post-exposure	Max. ↑neutrophils between day 1 and day 5, but still increased at day 42; max ↑macrophages between day 2 and day 5; altered transcript levels were associated with immune-inflammatory response and acute phase response pathways; genes involved in DNA repair, apoptosis, cell cycle regulation, and muscle contraction were also differentially expressed.	(Husain et al. 2015)
BAL cells, lung, liver	Comet, 1, 3, or 28 days after exposure	C57BL/6JBomTac mice, i.t., 0.67, 2, 6, 162 µg/mouse under 4% isoflurane anesthesia	Printex 90	0.67 µg: ↑neutrophils (day 1 and 3), eosinophils (3 days), lymphocytes (3 days); ↑DNA strand breaks (BAL, liver) 2 µg: ↑neutrophils (day 1), eosinophils (3 days) ↑DNA strand breaks (BAL, lung); 6 µg: ↑neutrophils (day 1); ↑DNA strand breaks (day 3, day 28; BAL, lung, liver) 162 µg: ↑neutrophils (day 1, day 3, and day 28), macrophages (day 1, day 28), ↓lymphocytes (day 1), ↑lymphocytes (day 28), total BAL cells (day 1, day 3, and day 28); ↑DNA strand breaks (only day 1, lung)	The study authors “interpret the increased DNA strand breaks occurring following these low exposure doses of nanoparticulate carbon black as DNA damage caused by primary genotoxicity in the absence of substantial inflammation, cell damage, and acute phase response”.	(Kyjovska et al. 2015)
BAL, liver	Comet, 5 and 24 days after exposure	Mouse, i.t. (4× up to 67 µg/animal), gd 8–18), 5–6/group	Printex 90	No effects in BAL In offspring 1.5-fold single strand breaks in liver	Pregnancy usually affects liver function; no historical control data provided – questionable association with CB exposure;	(Jackson et al. 2012a)
Lung	Comet assay, 3 h and 24 h after exposure	C57BL/6J mouse (m), 0.05 and 0.2 mg 1× i.t., examined 3 and 24 h after exposure; 5/group	Printex 90	0.05 mg: no effect 0.2 mg: positive (factor 1.1 to 2, 3 h after administration, no change in level of DNA damage at 24 h)		(Totsuka et al. 2009)
Other
Lung, BAL macrophages	Profibrotic growth factors and procollagen gene expression in lungs, 1 and 21 days post-exposure	Rat, single oropharyngeal aspiration, 2mg/kg bw	Raven 5000 Ultra II, 8 nm, 350–583 m^2/g	No induction of mRNA encoding for platelet-derived growth factor (PDGF)-A, -B, or –C, which are known to be associated with fibrosis	No fibrotic reaction in lung as evidenced by histopathology, no overt inflammatory response, >95% macrophages in BAL	(Mangum et al. 2006)
Lung	Toxicogenomic analysis	Mouse, i.t., 18, 54, and 162 µg/animal	Printex 90	162 µg: persistent pulmonary inflammation; gene expression changes similar to those typical for pulmonary injury and fibrosis; activation of the atherogenic HMG-CoA reductase pathway	Authors calculated BMDs for quantitative risk assessment based on adaptive responses (and not, as usually in risk assessment, based on adverse outcomes); all data based on i.t. mouse data; there is no concordance with human exposure routes and relevant gene expressions;	(Bourdon et al. 2012a, 2013)
Lung, liver, offspring	Toxicogenomic analysis	Mouse i.t., 0, 11, 54, or 268 μg/animal subdivided in four instillations on gestational days 7, 10, 15, and 18	Printex 90	11 µg: no effects; ≥54 µg: CB retention 268 µg: neutrophil-marked inflammation; altered expression of several cytokines and chemokines, both at the transcriptional and tissue protein levels; liver: female offspring more sensitive; cellular signaling, inflammation, cell cycle, and lipid metabolism pathways affected.	Probably secondary toxic effects in target and non-target tissues due to maternal toxicity	(Jackson et al. 2012b)

8-OH-dG: 8-hydroxyguanosine; BAL: broncho-alveolar lavage; ESTR: expanded simple tandem repeat; DE: diesel exhaust; γ-H2AX: phosphorylated H2AX; hOGG1: human 8-hydroxyguanine DNA-glycosylase; HPRT: hypoxanthine guanine phosphoribosyl transferase; i.t.: intratracheal; NOAEL: No observed adverse effect level; NOEL: No observed effect level; OGG1: 8-oxoguanine DNA glycosylase; PAR: poly(ADP-ribose); ROS: reactive oxygen species.

Table 3. Reproductive toxicity tests with carbon black.

Test system	Test material	Material preparation	Exposure dose and timing	Findings	Remarks	References
Sexual maturation, fertility, and reproduction
Mouse (C57BL/6/BomTac); intratracheal instillation	Printex 90	Carbon black was suspended in filtered water and subsequently sonicated for 8 min	Total doses of 11, 54, and 268 µg/animal; administered on gd 7, 10, 15, and 18	No changes in gestational, litter parameters, or sexual development in offspring. Inflammation seen in 268 μg/animal (females): female offspring showed altered habituation pattern during open field test	Effect found only in females	(Jackson et al. 2011)
Mouse (C57BL/6J) Intratracheal instillation	Printex 90	Carbon black was suspended in filtered water	67 µg/animal administered on gd 7, 10, 15, and 18: total dose of 268 µg/animal	↓sperm production in F2 when F1 fathers were exposed in utero	Few animals studied; effect seen at high, non-physiologically administered dose. Statistically significant, but numerically modest reduction of sperm production: without historical controls, biological relevance not assessable	(Kyjovska et al. 2013)
Intratracheal instillation in male ICR mice	14 nm (Printex 90), 56 nm (Printex 25), and 95 nm (Flammruss 101)	Carbon black was suspended in a normal saline solution containing 0.05% Tween 80 for instillation (no information provided on sonication)	100 µg/mouse 10 times at weekly intervals, resulting in a total dose of 1000 µg.	Increased testosterone with 14 nm CB and 56 nm CB, changes in seminiferous tubules and decreased daily sperm production with all CB.	The high dose of CB may result in inflammation and oxidative stress, which could be responsible for the reproductive effects. However, other organs were not observed for adverse effects.	(Yoshida et al. 2009)
Intratracheal instillation in ICR mice	14 nm CB – source not provided	Carbon black was suspended in normal saline solution with 0.05% (v/v) Tween 80 and sonicated for 5 min.	200 µg/mouse on gd 7 and 14, resulting in a total dose of 400 µg.	Male offspring showed histological changes in the testes, as well as decreases in daily sperm production. There was no marked effect on body weight, testicle weight, epididymis weight, or serum testosterone concentration.	Only one high dose was tested	(Yoshida et al. 2010)
Developmental effects
Rat, oral gavage, OECD 414 (Prenatal developmental toxicity study)	Unipure BlackLC 902	Carbon black was suspended in 0.5% aqueous sodium carboxymethylcellulose and given at 10 ml/kg bw.	Daily oral gavage at 0, 100, 300, or 1000 mg/kg/day during the sensitive period of organogenesis [gd 5 through 19].	No adverse maternal effects on embryo-fetal development. NOAEL was 1000 mg/kg/day for both maternal and developmental effects		(Ramesh 2012); as cited in SCCS 2015
Mouse (C57BL/6/BomTac) inhalation	Printex 90		42 mg/m^3, 1 h/day, between gd 8–18; whole body exposure	No increase in gestational or developmental toxicity. Liver: Increased DNA strand breaks in liver of dams and in liver of in utero exposed offspring – ↑max. 1.5-fold. Lungs: persistent inflammation; DNA strand breakage in BAL comparable to controls		(Jackson et al. 2012a)
Mouse (C57BL/6/BomTac); intratracheal instillation	Printex 90	Carbon black was suspended in filtered water and subsequently sonicated for 8 min	Total doses of 11, 54, and 268 µg/animal; administered on gd 7, 10, 15, and 18	No increase in gestational or developmental toxicity. All doses: No effects on DNA strand breaks in BAL and liver of dams and in liver of offspring CB retention in lungs of animals dosed with 54 and 268 µg/animal 27 days post-exposure Persistent lung inflammation only in high dose animals (268 µg/animal) High dose: altered expression of cytokine and chemokines in dams. Altered expression of hepatic genes in offspring: female offspring more sensitive.	Hepatic gene profiling in offspring: statistical threshold applied to identify significant change was less stringent p = .1 versus conventional p = .05 or higher	(Jackson et al. 2012a, 2012b)
Intranasal instillation in ICR mice	Printex 90	Suspended in distilled water, sonicated for 30 min and then filtered through a 450-nm filter	95 µg/kg/day on gd 5 and 9; resulting in approx. 6 µg/mouse	Brains collected from offspring at 6 and 12 weeks after birth, showed enlargement of granules of perivascular macrophages, decrease in number of perivascular macrophages, change in astrocyte phenotypes	The authors state that these changes indicate increased risk of dysfunction and disorder in the offspring brain, although no further studies were conducted to support this claim.	(Onoda et al. 2014)
Intradermal injection in pregnant rhesus macaques	Printex 90	Test material was suspended in 0.1% Tween 80- PBS at dose of 10 mg/ml, and sonicated for 10 min just before administration	Intradermal injection of test material suspension 4–6 times at intervals of 10–12 days. Total dose is not clear as the amount injected is not listed	Brains collected from new-born infants showed higher levels of hemoglobin.	The authors state that higher levels of hemoglobin are reportedly neurotoxic; however, the level of increase necessary to cause neurotoxicity is not clear.	(Mitsunaga et al. 2016)
Intranasal instillation in ICR mice	Printex 90	Carbon black was suspended at 1 mg/ml in saline with 0.05% (v/v) Tween 80 and sonicated for more than 30 min just before administration.	50 µg/mouse on gd 5 and 9; resulting in a total dose of 100 µg/mouse	Increased expression of collagen type VIII in the kidney of 12-week-old offspring mice but not in 3-week-old offspring mice. There was no difference in levels of serum creatinine or blood urea nitrogen, which are indicators of renal function.	Relevance of these findings is not clear as changes were noted in one endpoint of kidney function, and the change was seen at only one time-point	(Umezawa et al. 2011)
Mouse, (C57BL/6JP^un/P^un), Oral	Unclear	Test material was suspended in PBS	500 mg/kg bw; from gd 10.5–15.5 post-coitum	CB exposure did not increase the number of eye-spots.		(Reliene et al. 2005)
Intranasal instillation in ICR mice	Printex 90	Suspended in distilled water, sonicated for 30 min and then filtered through a 450-nm filter	95 µg/kg/day on gd 9 and 15; resulting in approx. 6 µg/mouse^a	Increased total thymocyte and lymphocytes in thymus and spleen of offspring.	Relevance of findings is unclear; different outcomes between two similar studies (see below).	(El-Sayed et al. 2015)
Intranasal instillation in ICR mice	Printex 90	Suspended in distilled water, sonicated for 30 min and then filtered through a 450-nm filter	95 µg/kg/day on gd 5 and 9; resulting in approx. 6 µg/mouse	Partial suppression of immune system development in offspring. Decrease in splenic T cells in offspring on post-natal days 1 through 5. Splenic T cells recovered on post-natal day 14.	Relevance of findings is unclear; different outcomes between two similar studies (see above).	(Shimizu et al. 2014)
Mouse (Balb C), intranasal	CB received from EPA or Harvard; no further description	Test material was suspended in PBS	Single dose application of 50 µg/animal on Day 14 of gestation	↑susceptibility to allergy in offspring of CB treated mothers. Authors conclude CB causes enhanced immune response in pregnancy and increased allergic susceptibility in offspring	Test material identity not apparent. Methodology to determine airway responsiveness controversial	(Fedulov et al. 2008)

Gd: gestation day; PBS: phosphate buffered saline.

^a Assuming 12-week female ICR mouse weight of 33 g (https://animals.ekmd.huji.ac.il/He/home/animalOrder/Documents/ICR.pdf), the dose of 95 μg/kg translates to 3 µg. This amount was given twice.

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