Carbon black

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity effects are not expected to be stronger with the non-nano form. Carbon black, irrespective of form display a similar surface chemistry as the functional groups on surface of their particles are essentially the same but may differ in concentration. A relevant systemic exposure after oral, dermal or respiratory administration is not expected (see section on toxicokinetics for details). The lack of relevant systemic exposure is substantiated by the lack of systemic effect in acute toxicity tests with excessive high dosing of the substance up to the technically feasible concentrations. In addition, the particle sizes of the bulk forms are generally larger than for the nanoforms. On this basis, differences in toxicological outcome is not expected. Both bulk and nanoforms are insoluble in water, and biological fluids. As a consequence, toxicity if any, will be driven by the physical presence of the particles in the lung. Consequentially, they share a common mode of action that involves ROS generation and inflammation in lung. It is therefore justified to read-across the data between the different forms of carbon black forms to assess genotoxicity. Data generated with untreated forms are used to predict the properties of the bulk forms (see discussion on untreated nanoforms)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

Endpoint-specific read-across justification" attached under section 13.2

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Type of assay:

bacterial reverse mutation assay

Species / strain:

other: TA1535, TA1537, TA1538, TA98 and TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Carbon Black (N339)_Kirwin et al 1981)

Cytotoxicity / choice of top concentrations:

other: cytotoxicity in TA 100 viability was reduced by 27% at 7500 ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Species / strain:

other: Salmonella. typhimurium TA98, TA100, TA1537, TA1538,

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

Carbon Black (Black Pearl L)_Sanders et al 1981

Cytotoxicity / choice of top concentrations:

not specified

Species / strain:

other: Salmonella typhimurium: TA98, TA100, TA1535, TA1537, TA1538

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

(Carbon Black (Printex 35)_Degussa 1988

Cytotoxicity / choice of top concentrations:

cytotoxicity

Species / strain:

other: S. typhimurium: TA100, TA1537, TA98, E.coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

Carbon black (N-330)_Agurell and Loefroth

Cytotoxicity / choice of top concentrations:

not specified

Species / strain:

other: TA 1535, TA 1537, TA 98, TA 100, Escherichia coli WP2uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(Carbon Black (Printex 90)_Degussa 1998

Cytotoxicity / choice of top concentrations:

cytotoxicity

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(Carbon Black (Unipure Black LC902)_Hobson 2011

Cytotoxicity / choice of top concentrations:

cytotoxicity

Additional information on results:

Carbon Black (N-339)_Kirwin et al 1981: N-339 carbon black was tested at the highest practical concentration in the Ames test. Cellular toxicity was determined by comparing survival of an appropriately diluted TA100 culture plated on complete agar containing the solvent or increasing concentrations of a carbon black suspension. The viable count was reduced by 27% at 7500 ug/plate. The results show that N-339 carbon black did not cause a significant increase in the reversion index of any of the tester strains with or without metabolic activation by rat liver microsomes. The positive controls were functional.

Carbon black (Black Pearls L)_Sanders et al 1981: Carbon black (Black Pearls L) as is, and different HPLC-fractions of the extracts were tested. Particles were first suspended in DMSO for 5 hours and an aliquot containing 500 μg of carbon black was tested. S. Typhimurium TA98, TA100, TA1537, TA1538 were tested (without metabolic activation). The Carbon black particles elicited a negative response in the assay.

Carbon Black (Unipure Black LC902)_Hobson 2011.: Carbon Black (Unipure Black LC902) was not mutagenic in Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 either in the presence or absence of metabolic activation. Carbon Black (Printex 90). In order to investigate the potential of Printex 90 to induce gene mutations in bacteria, the plate incorporation test (experiment I) and the pre-incubation test (experiment II) were performed in Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and in E. coli WP2uvrA. Printex 90 was suspended in DMSO and tested with and without metabolic activation (S9 mix) in triplicate at the following concentrations: 33.3, 100.0, 333.3, 1000.0, 2500.0 and 5000.0 ug/plate. Cytotoxicity was only observed in experiment II with Salmonella typhimurium TA 100 and E. coli WP2 uvrA at concentrations equal or higher than 2500 ug/plate. No increases in revertant frequencies were detected in any of the tester strains at any dose level either with or without metabolic activation. (increases in revertant frequencies were always below a factor of 2 as compared to the controls). The positive controls were functional.
 
Carbon Black (not treated_N330): The authors studied the variation in impurities of a furnace carbon black (ASTM N330) manufactured in Sweden over a 3-year period. The PAHs that were determined in benzene extracts and their ranges of concentration (mg/kg carbon black) were: phenanthrene, 0.9–15; fluoranthene, 4.5–72; pyrene, 26–240; benzo[ghi]fluoranthene, 7.2–72; cyclopenta[cd]pyrene, 6.6–188; chrysene, 0.1–1.3; benzo[b]fluoranthene, benzo[j]fluoranthene and benzo[k]fluoranthene, 0.4–18; benzo[e]pyrene, 0.9–19; benzo[a]pyrene, 0.9–28; perylene, 0.1–3.5; indeno[1,2,3- cd]pyrene, 2–43; benzo[ghi]perylene, 14–169; and coronene, 14–169.

Carbon Black (Printex 35)_Degussa 1988: Only in the presence of microsomal enzymes were increases in mutation frequencies found with tester strains TA98 (5.4-fold), TA100 (2.9-fold), TA1537 (13.9-fold), and TA1538 (5.2-fold) The laboratory`s criteria for a valid study were fulfilled (no details given). The report is incomplete, no information about method or treatment of the test substance., no negative controls reported. no conclusions can be drawn from this study (the report is incomplete, no information about method or treatment of the test substance, no negative controls reported)

Conclusions:

Ames Test is an unsuitable test system for all forms of carbon black.

Executive summary:

Mutagenicity – bacterial cell systems:

There are several studies available investigating point mutagenicity of carbon black. Six of the studies applied the non-treated carbon black nanoform (as is) for testing. In these studies, cellular uptake by the cells was not investigated. This hinders the interpretation of the results of these studies (Appendix R7-1 for nanomaterials applicable to Chapter R7a (Endpoint specific guidance) Version 2.0 – May 2017).

The remaining Ames studies have been performed with organic solvent extracts (following Soxhlet extraction) of the carbon black grade in question. These extracts have been prepared under rigorous conditions notably using solvents like DMSO, toluene, benzene or acetone, with extraction durations ranging from 16 hours up to 48 hours under high temperatures. Since these extractions were not performed under conditions considered physiologically relevant (e.g. using simulants and temperatures reflective of normal physiological conditions), the test samples are not representative of and do not reflect the substance as registered.

Considering the above, the Ames studies are considered not relevant for the purpose of hazard identification and risk assessment of mutagenic effects of carbon black.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

Endpoint-specific read-across justification" attached under section 13.2

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Mouse lymphoma L5178Y TK+/- gene
HPRT

Species / strain / cell type:

mouse lymphoma L5178Y cells

Species / strain / cell type:

mammalian cell line, other: RLE-6TN

Remarks:

Lung alveolar cells

Metabolic activation:

with and without

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(Carbon Black (N339)_Kirwin et al 1981

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5 mg/ml without S9, and at 10 mg/ml with S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(carbon Black (Unipure LC 902)

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

mammalian cell line, other: RLE-6TN

Remarks:

rat alveolar epithelial cell line

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

but negative when co-incubated with Catalase (Carbon Black (Monarch 990)_Driscoll et al 1997

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks:

quartz

Conclusions:

no direct mutagenicity in mutagenic in mammalian cells

Executive summary:

The results of studies with the carbon black (not treated nanoforms) are used to read across to the non-nano form as well as the treated nanoform because they share a common mechanism of action which involves the generation of oxidative species.

Mouse lymphoma cells were exposed for 3 hours to carbon black particles both in the presence and absence of metabolic activation in a GLP study performed according to OECD TG 476. No mutagenic activity was found (Lloyd 2011). It has been speculated that the exposure duration may have been too short for cellular uptake of the particles (which incidentally was not been investigated in the study) (EU Scientific Committee on Consumer Safety (SCCS 2015)). However, as separation of the insoluble carbon black particles from the cells after exposure is known to be difficult, if not impossible, sufficient exposure of cells is likely (see also (Kirwin et al. 1981)). Nonetheless, testing a furnace black (N-339, surface area 100 m²/g; DMSO suspension) at concentrations ranging from 5 mg/l to 40 mg/l and using a protocol similar to that of OECD Guideline 476, Kirwin et al. 1981 also reported negative effects following a longer exposure duration of 4 hours with and without metabolic activation (Kirwin et al. 1981).

The potential contribution of lung inflammatory cells to mutagenic responses was evaluated by coculturing bronchoalveolar (BAL) cells from rats treated with 100mg/kg bw carbon black with the rat alveolar epithelial cell line, RLE-6TN for 24hours and RLE-6TN cells selected for 6TG resistance. In vitro exposure of RLE-6TN cells to BAL cells increased hypoxanthine-guaninephosphoribosyl transferase (hprt) mutant frequency. Both macrophage and neutrophil enriched BAL cell populations were mutagenic to RLE-6TNcells, however, the mutagenic activity appeared greatest for neutrophils. Addition of catalase to BAL cell-RLE-6TN co-cultures inhibited the increase in hprt mutation frequency. This inhibition of BAL cell induced mutations by catalase points to a role for cell-derived oxidants in this response (Driscoll et al. 1997). This implies that the mutational events are induced by a secondary mechanism. (see also IUCLID section on in vivo genotoxicity for further study details).

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

test was conducted before guideline was published

Principles of method if other than guideline:

after 44 h of incubation, cytochalasin B was added. After further 28 h of incubation, the cells were harvested.

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell micronucleus test

Cytokinesis block (if used):

cytochalasin B was added. After further 28 h of incubation, the cells were harvested.

Test concentrations with justification for top dose:

1, 3 and 10 µg/cm2 (2.2, 6.6 and 22 µg/mL)

Vehicle / solvent:

complete medium

Key result

Species / strain:

other: mouse RAW 264.7 macrophage cell line

Metabolic activation:

not specified

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

In mouse RAW 264.7 cells, fine carbon black particles were consistently less genotoxic in the in vitro micronucleus test than fine atmospheric particle.

Executive summary:

Mouse RAW 264.7 cells were incubated for 48 hours with 1, 3, or 10 µg/cm2 fine carbon black (diameter 200 -250nm). Cytochalasine B was added after 44 hours, and the cells harvested after a further 28 hours. Carbon black induced 22.0 +/-1.4, 36.5 +/- 0.7 and 50 +/- 2 micronuclei/1000 cells (controls: 14 +/- 11; positive control 100 +/-2). The mid- and high-doses were cytotoxic. The micronuclei frequency was higher with atmospheric particles, when tested at the same dose levels.

Reference 4

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

Endpoint-specific read-across justification" attached under section 13.2

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Type of assay:

in vitro mammalian cell micronucleus test

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

MNT

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Carbon Black (Unipure LC-902)

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

in vitro SCE

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Carbon Black (N339)

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

Thymidine Kinase Assay

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Carbon Black (N339)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxicity at 5 mg/ml without S9, and at 10 mg/ml with S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

mammalian cell line, other: RAW 264.7

Remarks:

MNT

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

Carbon Black (Printex 90)

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

mammalian cell line, other: RAW 264.7

Metabolic activation:

not specified

Genotoxicity:

positive

Remarks:

negative at 1 mg/L and positive at 3 and 10 mg/L

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

mammalian cell line, other: mouse RAW 264.7 macrophage cell line

Metabolic activation:

not specified

Genotoxicity:

positive

Remarks:

negative at 1 µg/cm2 and positive at 3 and 10 µg/cm2 Carbon Black (NG90)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Negative for causing clastogenic effects

Executive summary:

The chromosome-damaging potential of carbon black was explored in vitro using the micronucleus test (MNT) which detects both potential aneugens and clastogens. A GLP micronucleus test performed according to OECD guideline 487 with Chinese Hamster Ovary (CHO) cells was negative even at the highest tested concentration of 120 mg/L Unipure black (vehicle DMSO) and an incubation time of 3 hours (Lloyd 2012). It has been suggested that the incubation period of 3 hours may have been too short to allow for enough time for particle uptake

(SCCS, 2015). However, no evidence for chromosomal damage was reported by Migliore et al. 2010 who used a considerably longer incubation time of 48 hours (20 hours without CytoB and 28h with cyto B). In this study mouse RAW 264.7 macrophages were exposed to carbon black (Printex 90; without metabolic activation) at concentrations of up to 100 mg/L and harvested 28 h following cytokinesis block (Migliore et al. 2010). Another micronucleus test in RAW 264.7 macrophages evaluating chromosome aberrations at 24 hours, 48 hours and

72 hours post-exposure of 1, 3, 10 mg/L Printex 90 for 48h reported negative results at 1 mg/L, but slightly increased (less than 2-fold) micronuclei frequencies at test concentrations of 3 mg/L and 10 mg/L; there were acentric chromosome fragments present at all concentrations tested. However, due to insufficient historical control data provided by the authors, the relevance of the chromosomal effects was considered uncertain given how minimal the increases were in comparison to control samples (Di Giorgio et al. 2011). In a further study by Poma et al. 2006, Mouse RAW 264.7 cells were incubated for 48 hours with 1, 3, or 10 μg/cm2 fine carbon black (diameter 200 - 250 nm), corresponding to 2.2, 6.6 and 22 μg/mL. Cytochalasine B was added after 44 hours, and the cells harvested after a further 28 hours. Evidence of increased micronuclei was registered at mid and high concentrations; 22.0 +/-1.4, 36.5 +/- 0.7 and 50 +/- 2 micronuclei/1000 cells (controls: 14 +/- 11; positive control 100 +/-2) which incidentally also caused cytotoxicity. In support of the MNT results, no indications for an effect on chromosomes was recorded in the mouse lymphoma L5178Y TK+/- assay - this assay can detect both point mutations and chromosomal alterations (Kirwin et al 1981, Lloyd et al 2011), nor in a sister chromatid exchange (SCE) chromosomal aberration study in CHO cells exposed with and without metabolic activation to concentrations of carbon black N339 up to 1 mg/ ml.

Overall, the standard in vitro genotoxicity studies in mouse lymphoma and CHO cells were all negative. Chromosomal effects in phagocytic cells at low carbon black doses may have been the result of the phagocytic activity and efficiency of these cells

Reference 5

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline available

Principles of method if other than guideline:

PAH-Adducts to DNA were determined by 32P post-labelling according to the method previously described (Van Schooten et al., 1997). Original particles, extracted particles (Soxhlet extraction with toluene) and the extracts (in DMSO) were investigated.

GLP compliance:

not specified

Type of assay:

other: DNA adducts

Species / strain / cell type:

mammalian cell line, other: A549 human lung epithelial cells (American Type Culture Collection)

Details on mammalian cell type (if applicable):

- Type and identity of media: DMEM culture medium supplemented with 10% heat-inactivated fetal calf serum (Sigma-Aldrich), l-glutamine, and 30 IU/ml penicillin–streptomycin at 37 -C and 5% CO2.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not reported
- Periodically checked for karyotype stability: not reported

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

without

Test concentrations with justification for top dose:

30 - 300 µg/cm2 (DMSO extracts), 100 µg/m2 (particles, original or washed). The particle concentration of 100 µg/cm2 was used as the maximum concentration because of the absence of in vitro cytotoxicity as determined in MTT and LDH assays

Vehicle / solvent:

Carbon black suspensions were made in HBSS, sonicated for 5 min in a water bath, and diluted into the culture dishes at the indicated final concentrations using two replicate wells per treatment or in 75-cm2 culture plates for extraction of DNA.

Untreated negative controls:

yes

Remarks:

TiO2

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: Diesel-SRM-2975, EPA PHA standard mix

Details on test system and experimental conditions:

The current study was designed to test the possible release and bioavailability of polycyclic aromatic hydrocarbons (PAHs) from a set of commercial carbon blacks (CBs) as well as the ability of these PAHs to form bulky DNA adducts. In four commercial CBs (Printex 90, Sterling V, N330, Lampblack 101), leaching of PAH was examined through (1) release of parent PAHs in saline with or without surfactant, and (2) PAH adducts in lung epithelial cells (A549). In vitro experiments were done with original and extracted particles, as well as organic extracts of carbon blacks in DMSO. As positive controls, B[a]P (0.03 µM) and a mixture of 16 PAHs (0.1 µM) were used.

Rationale for test conditions:

Original and extracted particles were tested at 100µg/cm2,

Evaluation criteria:

Statistical comparison (see below)

Statistics:

Statistical analysis was performed using Student’s t test using SPSS v.9 for Windows. A difference was considered significant at P < 0.05.

Key result

Species / strain:

mammalian cell line, other: human lung epithelial cells (A549)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No measurable release of PAHs from the 4 tested carbon blacks (1mg/mL) was found into aqueous dipalmytoyl phosphatidylcholine (DPPC) solutions containing 100–1000 mcg/mL DPPC. An increase in DPPC concentration up to 10 mg/mL also showed no measurable PAH release. To avoid contamination of the column by remains of the DPPC and to improve chromatographic resolution, leachates were treated with phospholipase enzymatic digestion. Again, the PAH concentrations in the digestion solutions were without exception below the detection limit. These experiments thus confirmed the results that were obtained without digestion of the DPPC.

PAH levels [mg/kg] after Soxhlet extraction: 0.057 phenantrene, 0.002 anthracene, 0.05 fluoranthene, 0.813 pyrene, 0.011 benzo(a)pyrene, 0.172 benzo(ghi)perylene.

Conclusions:

PAHs were not released from lampblack. No PAH adducts were found in lung epithelial cells (A549) after exposure to either original carbon black particles or their DMSO extracts.

Executive summary:

This study was designed to test the possible release and bioavailability of polycyclic aromatic hydrocarbons (PAHs) from a set of commercial carbon blacks (CBs) as well as the ability of these PAHs to form bulky DNA adducts. In four commercial CBs (Printex 90, Sterling V, N330, Lampblack 101), leaching of PAH was examined through (1) release of parent PAHs in saline with or without surfactant, and (2) PAH adducts in lung epithelial cells (A549). In vitro experiments were done with original and extracted particles, as well as organic extracts of CB in DMSO. As positive controls, B[a]P (0.03 AM) and a mixture of 16 PAHs (0.1 AM) were used. No leaching of PAHs was found. In vitro incubations with Lampblack revealed no adduct spots.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Genetic toxicity effects are not expected to be stronger with the non-nano form. Carbon black, irrespective of form display a similar surface chemistry as the functional groups on surface of their particles are essentially the same but may differ in concentration. A relevant systemic exposure after oral, dermal or respiratory administration is not expected (see section on toxicokinetics for details). The lack of relevant systemic exposure is substantiated by the lack of systemic effect in acute toxicity tests with excessive high dosing of the substance up to the technically feasible concentrations. In addition, the particle sizes of the bulk forms are generally larger than for the nanoforms. On this basis, differences in toxicological outcome is not expected. Both bulk and nanoforms are insoluble in water, and biological fluids. As a consequence, toxicity if any, will be driven by the physical presence of the particles in the lung. Consequentially, they share a common mode of action that involves ROS generation and inflammation in lung. It is therefore justified to read-across the data between the different forms of carbon black forms to assess genotoxicity. Data generated with untreated forms are used to predict the properties of the bulk forms (see discussion for untreated nanoforms).

Link to relevant study records

Reference

Reference 1

Endpoint:

genetic toxicity in vivo, other

Remarks:

in vivo mammalian somatic cell study: cytogenicity / lymphocyte micronucleus

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

Endpoint-specific read-across justification" attached under section 13.2

Reason / purpose for cross-reference:

read-across source

Qualifier:

no guideline followed

GLP compliance:

no

Type of assay:

mammalian erythrocyte micronucleus test

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Route of administration:

inhalation: aerosol

Frequency of treatment:

16 hours/day, 5 days/week

Tissues and cell types examined:

lymphocytes

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not specified

Remarks on result:

other:

Remarks:

No treatment-related differences in chromosome damage were found in blood lymphocytes from rats in the control group compared with rats exposed to carbon black and killed after three months of exposure.

Conclusions:

negative for causing clatogenicity in vivo

Executive summary:

Peripheral blood was isolated from rats exposed to 6.5 mg carbon black (Elftex 12)/m3 for 3 months at an exposure frequency of 16 hour/day, 5 days a week (Mauderly et al. 1994). Blood lymphocytes were extracted and tested for micronuclei. No treatment-related differences in chromosome damage were found in blood lymphocytes from rats in the treated groups in comparison to the control group.

Reference 2

Endpoint:

in vivo mammalian somatic and germ cell study: gene mutation

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

Endpoint-specific read-across justification" attached under section 13.2

Reason / purpose for cross-reference:

read-across source

Type of assay:

other: ESTR mutations in oocytes

Species:

mouse

Strain:

C57BL

Sex:

female

Route of administration:

intratracheal

Key result

Sex:

female

Genotoxicity:

negative

Toxicity:

not specified

Conclusions:

No effects were observed: the expanded simple tandem repeat (ESTR) mutation rates in CB-exposed F2 female offspring were not statistically different from those of F2 female control offspring.
The ESTR assay belongs to the array of rodent assays developed for monitoring for germline mutation. In their 2006 publication comparing the performance of traditional assays (e.g. MSLand heritable translocation assay) for monitoring germline mutation with ESTR, Singer et al 2006 conclude that “induced ESTR instability represents a relatively sensitive method of identifying agents causing germline mutation in rodents…”. However, they allow that their conclusions were based on the limited amount of experimental data. A more robust and extensive characterisation of assay performance, requiring substantially more data, is needed before the ESTR assay can be eligible for use within the context of regulatory testing. Singer's et al's determination is echoed in the 2008 review article by Verhofsrad et al.2008, who also highlight that the biological relevance of mutations, determined at non-coding DNA sites, as are tandem repetitive sequences, for the offspring are not apparent. There is also an issue of untargeted mutations, seen to occur at frequencies much higher than predicted that require elucidation. A full comprehension of the origin of the untargeted mutations is a prerequisite for the use of ESTR in formal risk assessment. Somers CM Mutat Res.2006 Jun 25;598(1-2):35-49. Epub 2006 Feb 28, Singer TM, Lambert IB, Williams A, Douglas GR, Yauk CL. Detection of induced male germline mutation: correlations and comparisons between traditional germline mutation assays, transgenic rodent assays and expanded simple tandem repeat instability assays. Mutat. Res. 2006;598:164-193Nicole Verhofstad, Joost O. Linschooten, Jan van Benthem, Yuri E.Dubrova, Harry van Steeg, Frederik J. van Schooten and Roger W. L. Godschalk. New methods for assessing male germ line mutations in humans and genetic risks in their offspring. Mutagenesis (2008) 23 (4): 241-247.Somers CM1. Expanded simple tandem repeat (ESTR) mutation induction in the male germline: lessons learned from lab mice. Mutat Res. 2006 Jun 25;598(1-2):35-49. Epub 2006 Feb 28

Executive summary:

Boisen et al 2013 investigated the effects of in utero exposure to Printex 90 carbon black (solid: nanoform, no surface treatment) on induction of mutations in the female mouse germline. In an exposure window spanning oogenesis, time mated pregnant C57BL/6J mice were instilled 4 times, on gestation days 7, 10, 15 and 18, with 67 µg carbon black per animal per instillation time point. At maturity, female offspring of carbon black treated dams were mated with unexposed mice of the CBA strain to produce an F2 generation. One hundred and seventy eight F2 progeny of in utero treated F1 females were analysed for ESTR mutations. Two hundred and fifty eight F2 offspring from the controls were analysed. The observed mutation rate in germ cells of CB-exposed F1 females was not significantly different from that of controls.

Reference 3

Endpoint:

genetic toxicity in vivo, other

Remarks:

in vivo mammalian somatic cell study: gene mutation/cytogenicity chromosomal aberration

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

Endpoint-specific read-across justification" attached under section 13.2

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

other: WoE assessment on data indicate a secondary mechanism of action related with ROS formation and associated with inflammation

Conclusions:

In summary, increases in hprt mutation frequencies were noted at concentrations that were clearly associated with marked pulmonary inflammation (Driscoll et al. 1996; Driscoll et al. 1997). Further empirical evidence supporting the conclusion that these mutations are due to a secondary mechanism and not due to a direct interaction of carbon black with DNA can be obtained by the negative results in DNA adduct studies. These studies have demonstrated the inability of carbon black to produce DNA adducts in the lungs of rats and in human lung epithelial cells (Borm et al. 2005; Gallagher et al. 1994; Wolff et al. 1990). In a recent Comet assay performed in accordance with OECD test guideline 489, no DNA strand breaks or oxidative DNA damage were found in BAL cells of rats after nose-only inhalation exposure to 6 mg/m3 Printex 90 for 14 days (6h/d, 5d/wk) (Lindner et al., 2017, nanoCOLT project).

Clearly, genetic damage can occur by generated ROS as a consequence of impaired particle clearance, i.e. under lung and macrophage overload conditions leading to pulmonary inflammation. In the rat, the most sensitive species with regard to lung overload, a threshold below which no genetic damage is expected to occur has been derived from sub-chronic inhalation studies at 1 mg/m3. This value was the NOAEL for any inflammatory effects including any increases in pro- or anti-inflammatory markers in well-conducted 90-day inhalation studies (Driscoll et al. 1996; Elder et al. 2005, Carter et al. 2006).

Executive summary:

Somatic cell mutations

Peripheral blood was isolated from rats exposed to 6.5 mg carbon black (Elftex 12)/m3 by inhalation for 3 months at an exposure frequency of 16 hour/day, 5 days a week. Blood lymphocytes were extracted and tested for micronuclei. No treatment-related differences in chromosome damage were found in blood lymphocytes from rats in the treated groups in comparison to the control group (Mauderly et al. 1994).

A significant and dose-related increase in the hprt mutation frequency in rat alveolar epithelial cells was detected immediately after 13 weeks of inhalation exposure to 7.1 and 52.8 mg/m3 carbon black (Monarch 880, 220 m2/g) as well as after 3- and 8-month recovery periods for the groups exposed to 52.8 mg/m3. No effect was found in the epithelial cells of rats exposed to 1.1 mg/m3 (which was the no observed adverse effect level (NOAEL)). Exposure to 52.8 mg/m3 carbon black resulted in hprt mutation frequencies which were 4.3, 3.2, and 2.7-fold greater than the air control group, immediately and after 3- and 8-months post- exposure, respectively. A significant increase in the frequency of hprt mutations was detected after 13 weeks of exposure to 7.1 mg/m3 carbon black but not after 3 or 8 months of recovery. No increase in hprt mutation frequency was observed for epithelial cells obtained from rats exposed to 1.1 mg/m3 carbon black. Lung tissue injury and inflammation, increased chemokine expression, epithelial hyperplasia, and pulmonary fibrosis were observed after exposure to 7.1 and 52.8 mg/m3, with these effects being more pronounced at the higher exposure level (Driscoll et al. 1996). In a subsequent study (Driscoll et al. 1997), the relationship between the severity of inflammation and mutations was confirmed by co-incubating lung lavage inflammatory cells from carbon black exposed rats with lung epithelial cells from unexposed rats. Bronchoalveolar lavage (BAL) cells were isolated from the lung of rats 15 months after intratracheal instillation of saline or saline suspensions of carbon black (Monarch 900, 230 m2/g) at 10 and 100 mg/kg bw. When the percentage of neutrophils in the lavage fluid was ≥50%, the hprt mutation rate increased significantly, possibly related to the generation and release of reactive oxygen species (ROS) and/or depletion in antioxidants. The mutation spectrum was compatible with mutations being caused by ROS. Importantly, mutations in the hprt gene occurred only at the high-dose exposure concomitantly with inflammation and epithelial hyperplasia. Driscoll et al. 1996 & 1997 provide evidence for secondary genotoxicity caused by reactive oxygen species as a result of inflammation; no primary genotoxicity was demonstrated by particles.

In a study comparing inflammatory responses and ex vivo hprt mutation frequencies in rats, mice and hamsters after sub-chronic inhalation exposure to carbon black (1, 7 or 50 mg/m3), rats demonstrated greater propensity for generating a pro-inflammatory response and hprt mutations at the higher doses of 7 and 50 mg/m3. No effects on hprt mutation frequencies were found at a dose level of 1 mg/m3, which was also a dose at which inflammatory effects were not observed. These results show that chronic inflammation at higher exposures may lead to a secondary indirect genotoxic response (Carter et al. 2006). Importantly, it is noted that the increases in hprt mutation frequencies were seen only at concentrations that were clearly associated with marked pulmonary inflammation.

DNA adducts:

Further evidence supporting that these mutations are due to a secondary mode of action and not due to a direct genotoxic effect of carbon black demonatrated in the negative results from DNA adduct studies. Carbon black did not induce DNA adduct formation in the lungs or livers of rats (Borm et al. 2005; Danielsen et al. 2010; Gallagher et al. 1994;Wolff et al. 1990;Randerath et al 1996). Borm et al. (2005) analysed DNA obtained from lung homogenates isolated immediately after 13 weeks of inhalation exposure to up to 50 mg/m3 of Printex 90 and Sterling V resulting in lung burdens of 4.9 mg and 7.6 mg, respectively. 32P-postlabeling of lung DNA showed no spots relating to PAH-DNA adduct formation when compared to sham-exposed animals. An increase of adducts in rat alveolar type II cells when compared to the filtered-air controls was reported by Bond et al. (1990), however, the same low-surface area carbon black (Elftex 12) tested negative in another study (Wolff et al. 1990). The evidence shows that carbon black does not cause adduct DNA formation. This conclusion is consistent with the interpretation of these investigations by IARC that carbon black does not cause DNA adduct formation (IARC 2010).

DNA damage and DNA repair:

To examine the role of oxidative stress in the development of carcinomas in rats following chronic high-dose inhalation of carbon black, Gallagher et al. (2003) analysed the formation of the mutagenic lesion, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) in the lung DNA of female F334 rats exposed for 6 hours/day, 5 days/week for 13 weeks to increasing concentrations of carbon black; 1, 7, and 50 mg/m(3) of Printex-90 (16 nm; specific surface area 300 m(2)/g) and to 50 mg/m(3) of Sterling V (70 nm; surface area of 37 m(2)/g). The formation of 8-oxo-dG in the lung DNA was assessed at the end of a 13 week-exposure duration, and after a 44-week recovery period in clean air. Lung particle overload in the lung of the rats was seen after inhalation exposure at 7 mg/m3 and 50 mg/m3 high surface area carbon black (Printex-90) and 50 mg/m3 low surface area carbon black (Sterling V); but not at 1 mg/m3 (Printex-90). Consistent with these findings, a significant increase (p<0.05) in 8-oxo-dG induction was observed following 13 weeks of exposure to 50 mg/m3 (Printex-90) and after a 44-week recovery period in the groups of animals exposed at 7 and 50 mg/m3 (Printex 90). Interestingly, no increase in 8-oxo-dG was observed for Sterling V at either time point despite lung particle overload. It follows then that a No Observed Effect Level (NOEL) for oxidative damage (8-oxo-dG in lung DNA of 1 mg/m3 and a Lowest Observed Effect Level (LOEL)) for oxidativedamage (8-oxo-dG) in lung DNA at 7 mg/m3 can be derived for high-surface area carbon black (Printex 90, 300 m2/g). A No Observed Effect Level (NOEL) for oxidative damage at ≥50 mg/m3 can be derived for low surface area carbon black (Sterling V, 50 m2/g)(Gallagher et al. 2003). The exposure concentration of Sterling V was selected to be equivalent in terms of retained mass in the lung to the high dose Printex 90 at the end of exposure. However, in terms of retained particle surface area, the retained lung dose of Sterling V was equivalent to the mid-dose of Printex 90. This design allowed comparison of results on the basis of retained particle mass as well as retained particle surface area between the two types of carbon black particles. Since both Sterling V (50 mg/m³) and Printex 90 (7 mg/m³) did not induce significant increases in 8-oxo-dG in the lung at the end of the 13-week exposure, this indicates that a retained large particle mass is not always correlated with similar adverse effects, but that particle surface area is a better metric for toxicity. Further, these findings suggest that prolonged high dose exposure to carbon black can promote oxidative DNA damage that is consistent with the hypothesis that inflammatory cell-derived oxidants play a predominant role in the pathogenesis of rat lung

tumours following long-term, high-dose exposure to carbon black in rats.

In rats exposed to 6 mg/m3 Printex 90 by nose-only inhalation for 14 days (6h/d, 5d/wk), no DNA strand breaks or oxidative DNA damage was found in BAL cells with the comet assay, measured at day 1 and day 14 post-exposure (Lindner et al. 2017). Also, in mice, no DNA damage was found in BAL cells of dams and their offspring after inhalation exposure (whole-body) of the pregnant mice to 42 mg/m3 (1h/d, gd8-18); however, an increase in DNA lesions/106 base pairs was found in livers of both dams and offspring. The level of oxidatively-generated DNA damage in the liver was not increased (only determined in the offspring by the level of formamidopyrimidine DNA glycosylase (fpg) enzyme sensitive sites). Analysis of BAL fluid cell composition demonstrated the presence of inflammation in the lungs. The observed effects in the liver of dams were most likely due to fur grooming and therefore due to gastrointestinal particle exposure, whereas the effect in the offspring could have been mediated by maternally induced inflammatory cytokines (Jackson et al. 2012). However, these observed effects could however also have been within the normal physiological variation (historical controls were not reported). Increased DNA strand breaks in BAL were found 1 hour after inhalation exposure of TNF-deficient mice to 4 x 20 mg/m3 (for 1.5 hours each); less damage was found in TNF +/+ mice, which might be explained by the fact that TNF induces neutrophil apoptosis as a mechanism for removal of neutrophils (Murray et al. 1997; Saber et al. 2005).

Germ cell mutations

The effects of carbon black (Printex 90) on female germ cell mutagenesis were studied in pregnant mice intratracheally instilled 4 times with 67 μg per animal, given during the critical developmental stages of foetal oogenesis (gestation days 7, 10, 15 and 18) (Boisen et al. 2013). The dose induced persistent pulmonary inflammation in the animals. Female offspring were raised to maturity and mated with unexposed males. Expanded simple tandem repeat (ESTR) germline mutation rates in the resulting F2 generation were determined from full pedigrees (mother, father, offspring) of F1 female mice. ESTR mutation rates in carbon black-exposed F2 female offspring were not statistically different from those of F2 female control offspring. The observed mutation rate in germ cells of carbon black-exposed F1 females was not significantly different from that of controls. Although the study protocol has not been internationally validated, the sensitivity of the model and the high dose employed gives reasonable confidence that carbon black did not induce mutations in oocytes.

Reference 4

Endpoint:

in vivo mammalian somatic cell study: gene mutation

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

Endpoint-specific read-across justification" attached under section 13.2

Reason / purpose for cross-reference:

read-across source

Type of assay:

transgenic rodent mutagenicity assay

Species:

mouse

Sex:

male

Route of administration:

intratracheal

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

test substance was phagocytized by macrophages; granuloma formation

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

other: lung overload condition

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

see set_not treated

Justification for classification or non-classification

It has been shown in several good quality experimental studies that carbon black is not directly mutagenic. Genotoxic effects are induced by secondary mechanisms such as oxidative stress; for these effects, triggered by inflammatory processes, there is a threshold which has been shown to be at 1 mg /m³ respirable for high-surface Carbon Black (e.g., Printex 90). The threshold for low-surface Carbon Blacks is higher. Carbon black is not classifiable according to the criteria as laid down in the CLP regulation.