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Genetic toxicity in vitro

Description of key information

Carbon black was not mutagenic in the Ames test, and the standard in vitro genotoxicity tests. Biological responses described after exposure to carbon black in in vitr oinvestigations tended to be non-specific effects such as cytotoxicity and DNA strand breaks due to often unrealistic and high exposure levels; in only a few instances, were assay interferences controlled or even considered. DNA strand breaks observed at sub-toxic doses in macrophage cell lines are considered to be due to particle clearance without significant ROS production, and therefore should not be considered as indicative of genotoxicity or as an adverse toxicological effect.

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Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
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:
comparable to guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
Target gene:
Mouse lymphoma L5178Y TK+/- gene
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: Fischer's medium supplemented with streptomycin and Pluronic S68
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not reported
- Periodically checked for karyotype stability: not reported
- Periodically "cleansed" against high spontaneous background: yes, according to the procedures of Clive and Spector (1975)
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
liver S-9 from Sprague-Dawley rats
Test concentrations with justification for top dose:
10-40 mg/mL without S9 and 5-15 mg/mL with S9; exposure time of 4 hours was extended because of the difficulty of separating the insoluble test material from the cells
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: not reported
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Remarks:
acetone
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
non-activated assay
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Remarks:
acetone
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
activated assay
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; the test material was sterilized by suspending it in a single glas centrifuge with 0.5 mL acetone for each dose level. The acetone was evaporated in a hood for 4 d. In this tube the carbon black were incubated to cell/cell medium.

DURATION
- Preincubation period: none
- Exposure duration: because of the difficulty of separating the insoluble test material from cells, the 4 h contact time was extended
- Expression time (cells in growth medium): 3 days
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): selective medium containing 1.0 ug/mL trifluorothymidine (TFT) and 0.35% noble agar.

NUMBER OF REPLICATIONS: two flasks per control and test concentration both for the activated and the non-activated assay; 1 flask was used for viability control, the other was treated with selective medium. Two independent assays were conducted.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth;
Evaluation criteria:
Not reported.
Statistics:
Not reported
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
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
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not reported
- Effects of osmolality: not reported
- Evaporation from medium: not reported
- Water solubility: not reported
- Precipitation: yes (suspensions)
- Other confounding effects: not reported

RANGE-FINDING/SCREENING STUDIES: not reported

COMPARISON WITH HISTORICAL CONTROL DATA: not reported

Table 1: Mutant frequencies and cytotoxicity, non-activated assay

  Acetone control  N-339, 10mg/mL   N-339, 15 mg/mL   N-339, 20 mg/mL   N-339, 25 mg/mL   N-339, 30 mg/mL  N-339, 35 mg/mL   N-339, 40 mg/mL  Acetone control  EMS 0.5 uL/mL  EMS 1.0 uL/mL 
Growth, percent 100  144  105  81  29  58  18  11  100  98  17 
Mutant frequency 0.7  0.7  0.6  0.6  0.7  0.7  0.6  0.4  1.4  10.9  33.1 

Table 2: Mutant frequencies and cytotoxicity, activated assay #1

   Acetone control N-339, 10 mg/mL  N-339, 15 mg/mL  Acetone control  DMBA, 2.5 ug/mL   DMBA, 5.0 ug/mL
Growth, percent  100 47  100  19  17 
Mutant frequency  0.6 0.6  0.7  1.4  3.5  4.9 

Because of the wide spread of doses in the frst evaluation, a second assay was conducted with a narrower range of doses. The results are presented in Table 3:

Table 3: Mutant frequencies and cytotoxicity, activated assay #2

   Acetone control N-339, 5 mg/mL  N-339, 7 mg/mL N-339, 9 mg/mL N-339, 11 mg/mL  N-339, 13 mg/mL  N-339, 15 mg/mL  Acetone control  DMBA, 5 ug/mL  DMBA, 7.5 ug/mL 
Growth, percent  100  175  211  210  198  138  63  100  82  43 
Mutant frequency   0.9 0.7  0.5  0.5  0.3  0.3  0.3 1.1 3.8  9.0 
Conclusions:
Interpretation of results:
negative with metabolic activation
negative without metabolic activation

N-339 carbon black did not induce any significant increase in mutant frequencies in the L5178Y mouse lymphoma TK+/- assay with and without metabolic activation. The mouse lymphoma L5178Y TK +/- assay can detect both point mutations and chromosomal alterations.
Executive summary:

Cellular toxicity was seen in the non-activated assay at 40 mg/mL N-339 carbon black. The activated assay exhibited cellular toxicity at 10 mg/mL. Over a wide range of cncentrations (15 -45 mg/mL), N-339 carbon black did not induce any significant increase in mutant frequencies in the non-activated assay. Because of the wide spread of doses in the first assay with metabolic activation (rat liver S-9), a second assay was conducted with a narrower range of doses. The results corroborated the negative findings of the first assay. The positive controls (EMS, DMBA) were functional.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
start of experiments: 30 June 1997, end of experiments: 14 July 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Genes for histidine and tryptophan synthesis
Species / strain / cell type:
other: S. typhimurium TA 1535, 1537, 98, 100 and E. coli WP2 and WP2 uvrA
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver-S9 mix (ICN Biomedicals GmbH/Germany)
Test concentrations with justification for top dose:
The extract was tested undiluted, and as 80, 60, 50, 40, and 10% dilutions
Vehicle / solvent:
- Vehicle/solvent: Dimethylsulphoxide (DMSO)
- Justification for choice of solvent/vehicle: not reported
Untreated negative controls:
yes
Remarks:
water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
yes
Remarks:
water
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
further positive control substances for assays without metabolic activation: 4-NOPD (10-40 µg, for TA 1537, 98), MMS (1 uL; for E. coli strains); for assays with metabolic activation: 2-AA (2.5 ug; for TA 1535, 1537, 98, 100 and E. coli strains). Migrat
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation; two independent experiments were performed. In experiment I, the plate incorporation method was used; in experiment II, the pre-incubation method was applied.
PREPARATION OF THE TEST SUBSTANCE: 40 grams of the test substance were extracted with 600 mL of toluene in a Soxhlet extractor (the toluene was replenished about 10 times per hour). The toluene was then evaporated and the residue was dried at 140 deg centigrade for two hours. The residue was then taken up in 40 g of dimethylsulfoxide (DMSO).

DURATION
- Preincubation period: 60 minutes at 37 deg C
- Exposure duration: at least 48 hours

NUMBER OF REPLICATIONS:for each strain and dose level, including the controls, three plates were used.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth;
Evaluation criteria:
A test substance was considered mutagenic in this assay, if
- a dose-related and reproducible increase in the number of revertants was found, or if - a substantial and reproducible increase for at least one test concentration was found.
A substantial increase was defined as
- a at least two-fold increase in the number of revertants in TA 100
- a at least three-fold increase in the number of revertants in strains TA1535, TA1537, TA98, WP2 and WP2uvrA.
Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of a potential mutagenic potential, regardless of whether the aforementioned increases were observed or not.
Statistics:
no statistics performed (not required y guideline)
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
maximum 1.4-fold increase in mutation frequency over control
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
maximum 1.7-fold increase in mutation frequency over control
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
maximum 1.2-fold increase in mutation frequency over control
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
maximum 1.2-fold increase in mutation frequency over control
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
maximum 1.3-fold increase in mutation frequency over control
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
maximum 1.4-fold increase in mutation frequency over control
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not reported
- Effects of osmolality: not reported
- Evaporation from medium: not reported
- Water solubility: not reported
- Precipitation: not reported
- Other confounding effects: not reported

RANGE-FINDING/SCREENING STUDIES: The toxicity of the test item extract was determined with strains TA98 and TA100 in a pre-experiment. 8 concentrations (5, 10, 20, 40, 50, 60, 80 and 100%) were tested for toxicity and mutation induction with each 3 plates both in the presence and the absence of metabolic activation. The experimental conditions in this pre-experiment were the same as for the main plate incorporation test. No toxicity was observed with the highest test concentration (100%, corresponding to an equivalent amount of 109 mg Printex 70 per plate).

COMPARISON WITH HISTORICAL CONTROL DATA: yes (spontaneous reversion frequencies for all used strains reported, i.e. TA1535 5-30, TA1537 4-32, TA98 18-63, TA100 79-197, E.coli WP2 23-69 and E. coli WP2uvrA 27-65).


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Remarks on result:
other: strain/cell type: S. typhimurium TA 1535
Remarks:
Migrated from field 'Test system'.

None

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation in all Salmonella and E. coli strains tested
negative without metabolic activation in all Salmonella and E. coli strains tested

Printex 70 toluene extract did not induce an increase in mutation frequency in any of the tester strains with and without metabolic activation.
Executive summary:

In order to investigate the potential of Printex 70 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 strains WP2 and WP2uvrA. Printex 70 was Soxhlet extracted with toluene and the residue taken up in DMSO. The extract was tested with and without metabolic activation (S9 mix) in triplicate at the following concentrations: 100, 80, 60, 50, 40 and 10%. No cytotoxicity was observed up to the highest concentration tested. 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.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
start of experiments: 13 October 1998, end of experiments: 03 November 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Genes for histidine and/or tryptophan synthesis
Species / strain / cell type:
other: Salmonella typhimurium TA 1535, TA 1537, TA98, TA100, E. coli WP2uvrA
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver S-9 (prepared at BSL Bioservice GmbH)
Test concentrations with justification for top dose:
The extract was tested undiluted, and as 80, 60, 40, 20 and 10% dilutions
Vehicle / solvent:
- Vehicle/solvent: Dimethylsulphoxide (DMSO)
- Justification for choice of solvent/vehicle: not reported
Untreated negative controls:
yes
Remarks:
water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
yes
Remarks:
water
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
further positive control substances for assays without metabolic activation: 4-NOPD (10-40 µg, for TA 1537, 98), MMS (1 uL; for E. coli WP2uvrA); for assays with metabolic activation: 2-AA (2.5 ug; for TA 1535, 1537, 98, 100 and E. coli WP2uvrA). Migrat
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation; two independent experiments were performed. In experiment I, the plate incorporation method was used; in experiment II, the pre-incubation method was applied.
PREPARATION OF THE TEST SUBSTANCE: 40 grams of the test substance were extracted with 600 mL of toluene in a Soxhlet apparatus. The toluene was then evaporated and the residue was dried at 140 deg centigrade for two hours. The residue was then taken up in 40 g of dimethylsulphoxide (DMSO).

DURATION
- Preincubation period: 60 minutes at 37 deg C
- Exposure duration: at least 48 hours

NUMBER OF REPLICATIONS:for each strain and dose level, including the controls, three plates were used.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth;

METABOLIC ACTIVATION: liver-S9 mix from phenobarbital and beta-naphtoflavone induced male Wistar rats.
Evaluation criteria:
A test substance was considered mutagenic in this assay, if
- a dose-related and reproducible increase in the number of revertants was found, or if
- a substantial and reproducible increase for at least one test concentration was found.
A substantial increase was defined as
- a at least two-fold increase in the number of revertants in TA 100
- a at least three-fold increase in the number of revertants in strains TA1535, TA1537, TA98, and WP2uvrA.
Statistics:
no statistics performed (not required by guideline)
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
maximum 1.4-fold increase in mutation frequency over control
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
maximum 1.4-fold increase in mutation frequency over control
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
maximum 1.5-fold increase in mutation frequency over control
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
maximum 1.3-fold increase in mutation frequency over control
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
maximum 1.3-fold increase in mutation frequency over control
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not reported
- Effects of osmolality: not reported
- Evaporation from medium: not reported
- Water solubility: not reported
- Precipitation: not reported
- Other confounding effects: not reported

RANGE-FINDING/SCREENING STUDIES: The toxicity of the test material extract was determined with strains TA98 and TA100 in a pre-experiment. 8 concentrations (5, 10, 20, 40, 50, 60, 80 and 100%) were tested for toxicity and mutation induction with each 3 plates both in the presence and the absence of metabolic activation. The experimental conditions in this pre-experiment were the same as for the main plate incorporation test. No toxicity was observed with the highest test concentration.

COMPARISON WITH HISTORICAL CONTROL DATA: yes (spontaneous reversion frequencies for all used strains reported, i.e. TA1535 5-30, TA1537 4-32, TA98 18-63, TA100 79-197, and E. coli WP2uvrA 27-65).
Remarks on result:
other: other: S. typhimurium TA 1535
Remarks:
Migrated from field 'Test system'.

None

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation in all tested Salmonella strains and in E. coli WP2 uvrA
negative without metabolic activation in all tested Salmonella strains and in E. coli WP2 uvrA

Printex 90 toluene extract did not induce an increase in mutation frequency in any of the tester strains with and without metabolic activation.
Executive summary:

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 Soxhlet extracted with toluene and the residue taken up in DMSO. The extract was tested with and without metabolic activation (S9 mix) in triplicate at the following concentrations: 100, 80, 60, 40, 20 and 10%. No cytotoxicity was observed up to the highest concentration tested. 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.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
start of experiments: 31 August 1998, end of experiments: 26 October 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Genes for histidine and/or tryptophan synthesis
Species / strain / cell type:
other: Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100, Escherichia coli WP2uvrA
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver S-9, prepared at BSL Bioservice GmbH
Test concentrations with justification for top dose:
The test material was suspended in dimethylsulphoxide (DMSO) and diluted prior to treatment to the following test concentrations: 33.3; 100.0; 333.3; 1,000.0; 2,500.0; 5,000.0 ug/plate.
Vehicle / solvent:
- Vehicle/solvent: Dimethylsulphoxide (DMSO)
- Justification for choice of solvent/vehicle: not reported
Untreated negative controls:
yes
Remarks:
water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
yes
Remarks:
water
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
further positive control substances for assays without metabolic activation: 4-NOPD (10-40 µg, for TA 1537, 98), MMS (1 uL; for E. coli WP2uvrA); for assays with metabolic activation: 2-AA (2.5 ug; for TA 1535, 1537, 98, 100 and E. coli WP2uvrA). Migrat
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation; two independent experiments were performed. In experiment I, the plate incorporation method was used; in experiment II, the pre-incubation method was applied.

PREPARATION OF THE TEST SUBSTANCE: The test substance was suspended in dimethylsulphoxide (DMSO) and diluted prior to treatment.

DURATION
- Preincubation period: 60 minutes at 37 deg C
- Exposure duration: at least 48 hours

NUMBER OF REPLICATIONS:for each strain and dose level, including the controls, three plates were used.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth;

METABOLIC ACTIVATION: liver-S9 mix from phenobarbital and beta-naphtoflavone induced male Wistar rats.
Evaluation criteria:
A test substance was considered mutagenic in this assay, if
- a dose-related and reproducible increase in the number of revertants was found, or if
- a substantial and reproducible increase for at least one test concentration was found.
A substantial increase was defined as
- a at least two-fold increase in the number of revertants in TA 100
- a at least three-fold increase in the number of revertants in strains TA1535, TA1537, TA98, and WP2uvrA.
Statistics:
no statistics performed (not required by guideline)
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
maximum 1.3-fold increase in mutation frequency over control
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
maximum 1.3-fold increase in mutation frequency over control
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
maximum 1.3-fold increase in mutation frequency over control
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
maximum 1.3-fold increase in mutation frequency over control
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in TA100 (without S-9, pre-incubation experiment) at concentrations equal or greater than 2,500 ug/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
maximum 1.4-fold increase in mutation frequency over control
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in E. coli WP2 uvr A (without S-9, pre-incubation experiment) at concentrations equal or greater than 2,500 ug/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not reported
- Effects of osmolality: not reported
- Evaporation from medium: not reported
- Water solubility: not reported
- Precipitation: the test material was suspended in the vehicle
- Other confounding effects: not reported

RANGE-FINDING/SCREENING STUDIES: The toxicity of the test material was determined with strains TA98 and TA100 in a pre-experiment. 8 concentrations (3.3, 10, 33.3, 100, 333, 1000, 2500, 5000 ug/plate) were tested for toxicity and mutation induction with each 3 plates both in the presence and the absence of metabolic activation. The experimental conditions in this pre-experiment were the same as for the main plate incorporation test. No toxicity was observed with the highest test concentration.

COMPARISON WITH HISTORICAL CONTROL DATA: yes (spontaneous reversion frequencies for all used strains reported, i.e. TA1535 5-30, TA1537 4-32, TA98 18-63, TA100 79-197, and E. coli WP2uvrA 27-65).
Remarks on result:
other: other: S. typhimurium TA 1535
Remarks:
Migrated from field 'Test system'.

None

Conclusions:
Interpretation of results (migrated information):
negative without metabolic activation in all tested Salmonella strains and in E. Coli WP2 uvrA
negative with metabolic activation in all tested Salmonella strains and in E. Coli WP2 uvrA

Printex 90 did not induce an increase in mutation frequency in any of the tester strains with and without metabolic activation (increases in mutation frequencies were always below a factor of 2 as compared to the controls). The positive controls were functional.
Executive summary:

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.

Endpoint:
genetic toxicity in vitro, other
Remarks:
sister chromatid exchange 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:
guideline study without detailed documentation
Qualifier:
according to
Guideline:
OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)
Version / remarks:
Guideline deleted in 2014
GLP compliance:
not specified
Type of assay:
sister chromatid exchange assay in mammalian cells
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Ham's F12 medium, supplemented with 10% fetal calf serum;
- 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:
with and without
Metabolic activation system:
S-9 mix of Aroclor 1254 treated Fischer 344 rats
Test concentrations with justification for top dose:
0.00032-1 mg/ml
Vehicle / solvent:
Carbon black was prepared in dimethylsulphoxide (DMSO) as a stock suspension of 0.1 g/ml (time and temperature unspecified) and then diluted in culture medium, (one. 0.32 µg/ml - 1000 µg/ml) exposure time 2 hrs.
Untreated negative controls:
yes
Remarks:
culture medium
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
non-activated experiment Migrated to IUCLID6: 0.5 uL/mL
Untreated negative controls:
yes
Remarks:
culture medium
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
N-dimethylnitrosamine
Remarks:
activated experiment Migrated to IUCLID6: 0.3 uL/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium;
DURATION
- Exposure duration: 2 hours
- Expression time (cells in growth medium: 24 hours

STAIN (for cytogenetic assays): BrdU
NUMBER OF REPLICATIONS: 4 replicate cultures/dose
DETERMINATION OF CYTOTOXICITY
- Method: not reported
Statistics:
Results for the solvent control and the test material or positive ocntrol were compared by Student's t-test and the standard error (SE) of the mean.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
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
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not reported
- Effects of osmolality: not reported
- Evaporation from medium: not reported
- Water solubility: not reported
- Precipitation: not reported
- Other confounding effects: not reported

RANGE-FINDING/SCREENING STUDIES: not reported

COMPARISON WITH HISTORICAL CONTROL DATA:not reported

Table 1 CHO Sister Chromatid Exchange (SCE) frequencies, non-activated experiment

   Negative control DMSO control  N-339, 0.32 ug/mL  N-339, 1.60 ug/mL  N-339, 8.00 ug/mL  N-339, 40.00 ug/mL  N-339, 200.00 ug/mL  N-339, 1000.00 ug/mL  EMS, 0.5 uL/mL  
Number of chromosomes  399 481  383  388  367  401  393  405  424 
 No. of SCE 328  303  317  297  298  309  342  252  1736 
SCE per chromosome +/- Standard Error 0.822 +/- 0.045 0.630 +/- 0.036 0.828 +/- 0.046* 0.765 +/- 0.044   0.812 +/- 0.047* 0.771 +/- 0.044   0.870  +/- 0.047* 0.622  +/- 0.036 4.094 +/-0.098*  
SCE per cell 16.49  12.60  16.55*  15.30  16.24*  15.42  17.40*  12.44  81.88* 

* significantly greater than solvent control value, p < 0.01

Table 2 CHO Sister Chromatid Exchange (SCE) frequencies, activated experiment

  Negative control DMSO control  N-339, 0.32 ug/mL  N-339, 1.60 ug/mL  N-339, 8.00 ug/mL  N-339, 40.00 ug/mL  N-339, 200.00 ug/mL  N-339, 1000.00 ug/mL  DMN, 0.3 uL/mL  
Number of chromosomes  383 412  376  412  400  502  382  389  495 
No. of SCE 334  291  383  355  345  373  265  363  774 
SCE per chromosome +/- Standard Error 0.872 +/- 0.048 0.706 +/-0.041 1.019 +/- 0.052* 0.862 +/- 0.046   0.863 +/- 0.046 0.743 +/- 0.038   0.694  +/- 0.043* 0.933  +/- 0.049* 1.564 +/-0.056*  
SCE per cell 17.44 14.12  20.38*  17 -24  17.26*  14.86  13.88*  18.66*  31.28* 

* significantly greater than solvent control value, p 0.01

Conclusions:
Interpretation of results:
negative with metabolic activation
negative without metabolic activation

N-339, a rubber grade furnace black did not induce sister chromatid exchanges in Chinese Hamster Ovary cells.
Executive summary:

N-339 carbon black did not induce any significant increase in sister chromatid exchange frequencies in Chinese Hamster Ovary (CHO) cells, both in the absence and presence of metabolic activation. The highest tested concentration was 1000 ug/mL (suspension). The positive controls (EMS, DMN) were functional.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
not reported
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:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
- co-cultures of rat alveolar epithelial cells with BAL cells were used
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT gene
Species / strain / cell type:
other: rat alveolar epithelial cell line, RLE-6TN
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
without
Test concentrations with justification for top dose:
RLE-6TN cells were exposed in vitro to BAL cells from rats 15 months after intratracheal instillation of saline or 10 and 100 mg/kg carbon black;
Vehicle / solvent:
saline
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Remarks:
saline
Positive controls:
yes
Positive control substance:
other: alpha-quartz
Key result
Species / strain:
mammalian cell line, other: rat alveolar epithelial cell line, RLE-6TN
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Hprt mutation frequency was increased in alveolar type II cells from rats exposed to 10 and 100 mg/kg of alpha-quartz, 100 mg/kg carbon black (but not at 10 mg/kg carbon black) and 100 mg/kg titanium dioxide. Neutrophilic inflammation was detected in all rats exposed to 10 and 100 mg/kg of alpha-quartz, and 100 mg/kg carbon black. In vitro exposure of RLE-6TN cells to BAL cells from rats treated with 10 and 100 mg/g of alpha-quartz or 100 mg/kg carbon black increased hprt mutant frequency. Both macrophage and neutrophil enriched BAL cell populations were mutagenic to RLE-6TN cells. The mutagenic activity, however, appeared greatest for neutrophils. Addition of catalase to BAL cell:RLE-6TN co-cultures inhibited the increase in hprt mutation frequency. The inhibition of BAL cell-induced mutations by catalase implies a role for cell-derived oxidants in this response.

HPRT mutation frequencies in rat alveolar epithelial cells exposed in vitro to BAL cells from rats 15 months after treatment

Treatment  Ratio of BAL cells to epithelial cells
   10:1  50:1
 Saline 3.0 +/- 1.3 (9)  4.0 +/- 1.2 (9)  
 alpha-Quartz, 10 mg/kg 2.7 +/- 1.1 (6)   12.1 +/- 1.5* (6) 
 alpha-Quartz, 100 mg/kg 2.7 +/- 1.1 (6)    34.6 +/- 4.4* (6) 
 Carbon black, 10 mg/kg 2.0 +/- 1.3 (6)   3.9 +/- 0.3 (6) 
 Carbon black, 100 mg'/kg 0.8 +/- 0.1 (6)  17.0 +/- 3.1* (6) 
 * p < 0.05  
Conclusions:
Interpretation of results:
negative in cultures treated with catalase
positive (co-culture with ex vivo neutrophils from animals treated with 100 mg/kg bw)
negative (co-culture with ex vivo neutrophils from animals treated with 10 mg/kg bw)

In vitro exposure of RLE-6TN cells to BAL cells from rats treated intratracheally with 100 mg/kg carbon black increased hprt mutant frequency. Both macrophage and neutrophil enriched BAL cell populations were mutagenic to RE-6TN cells. The inhibition of BAL cell-induced mutations by catalase implies a role for cell-derived oxidants in the mutagenic response.
Executive summary:

The potential contribution of lung inflammatory cells to the mutagenic responses was evaluated by co-culturing bronchoalveolar (BAL) cells with the rat alveolar epithelial cell line, RLE-6TN for 24 hours and the RLE-6TN cells selected for 6TG resistance. In vitro exposure of RLE-6TN cells to BAL cells from rats treated intratracheally with 100 mg/kg carbon black increased hprt mutant frequency. Both macrophage and neutrophil enriched BAL cell populations were mutagenic to RE-6TN cells.

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented publication which meets basic scientific principles
Reason / purpose:
reference to same study
Qualifier:
no guideline available
Principles of method if other than guideline:
PAH-Adducts to DNA were determined by 32P postlabeling according to the method previously described (Van Schooten et al., 1997). Orignial 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 maximumm 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) or in rat lungs after exposure to two CBs (Printex 90, Sterling V) for 13 weeks (50 mg/m3). [The in vivo study is described further in the next chapter of this IUCLID.] 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.
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
Remarks:
Printex 90 (PAH: 0.03 ppm), N330 (PAH: 2.4 ppm), Lampblack 101 (PAH: 0.057 ppm)
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
mammalian cell line, other: human lung epithelial cells (A549)
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
Stering V (PAH: 8.8 ppm)
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 standard diesel or carbon blacks (1 mg/mL) in aqueous dipalmytoyl phosphatidylcholine (DPPC) solutions containing 100–1000 Ag/mL DPPC was found. An increase in DPPC concentration up to 10 mg/mL also showed no measurable PAH release. Based on the detection limit for HPLC determination of PAH, the amount of a single PAH that possibly leaches from diesel particles under these circumstances is less than 1.2% for phenanthrene, <0.4% for pyrene, <1.0% for anthracene, <1.3% for chrysene, and <1.3% for fluoranthene. 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.
Conclusions:
IThe study was performed in order to test the possible release of PAHs from commercial carbon black and their ability to form PAH adducts, PAH adducts were analyzed in lung epithelial cells (A549) after exposure to either original carbon black particles with surface areas between 20 and 300 m2/g (Lampblack 101, Sterling V, N330 and Printex 90), or to their DMSO extracts. Adduct spots were found with Sterling V only. However, there was no dose-response relationship and the spot remained unidentified
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) or in rat lungs after exposure to two CBs (Printex 90, Sterling V) for 13 weeks (50 mg/m3). 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 measured in saline or surfactant-containing saline. In vitro incubations with CB particles (30–300 ug/cm2) revealed no adduct spots except for Sterling V. However, the spot was not concentration dependent and remains unidentified. Lung DNA from rats after inhalation of Printex 90 or Sterling V showed no spots related to PAH–DNA adduct formation compared to sham-exposed rats.

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:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
GLP compliance:
not specified
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
Printex 90
Species / strain / cell type:
other: mouse peritoneal macrophages
Details on mammalian cell type (if applicable):
RAW 264.7 mouse macrophage cell line
Cytokinesis block (if used):
yes
Test concentrations with justification for top dose:
1,3,10 mg/L, 48h; cytokinesis-block method for MNT, but no details reported; chromosome aberrations at 24, 48 and 72h post-exposure; ROS production at 50mg/L after 5 and 24h
Key result
Species / strain:
other: RAW 264.7
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Additional information on results:
1mg/L: MNT negative
3, 10mg/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 24h
Remarks on result:
other: at 1 mg/L negative
Conclusions:
A micronucleus test in RAW 264.7 mouse macrophages was negative at 1 mg/L, but slightly increased (less than 2-fold) micronuclei frequencies were found at test concentrations of 3 and 10 mg/L; there were acentric chromosome fragments present at all concentrations tested. However, due to insufficient historical control data, the relevance of the chromosomal effects was considered uncertain.
Executive summary:

The cyto- and genotoxic effects of single and multi-walled CNTs (SWCNTs, MWCNTs) and carbon black (CB) on the mouse macrophage cell line RAW 264.7 was investigated, including inflammatory response, release of tumor necrosis factor- (TNF-), intracellular reactive oxygen species (ROS) production, cell death (both necrosis and apoptosis), chromosomal aberrations and cellular ultrastructural alteration. Micronuclei frequencies were slightly increased at test concentrations of 3 and 10 mg/L; there were acentric chromosome fragments present at all concentrations tested. However, due to insufficient historical control data, the relevance of the chromosomal effects was considered uncertain. The effects might be related to the phagocytic activity of this cell type.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
August – October 2011
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
2010
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Cytokinesis block (if used):
Cytochalasin B was not present during the 3h treatment, but added immediately after
Metabolic activation:
with and without
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid

The cells were exposed for 3 hours. This might have been too short for particle uptake (SCCS comment)

Conclusions:
Carbon black did not induce micronuclei in CHO cells either in the absence or presence of metabolic activation. The EU Scientific Committee on Consumer Safety (SCCS) speculate that the 3 hour exposure period may have been too short.
Executive summary:

A study according to OECD guideline 487 was performed with carbon black. This study is summarised and assessed by the EU Scientific Committee on Consumer Safety (SCCS) in their opinion on carbon black of December 2015. Though the study was performed according to the OECD protocol, the SCCS speculate that a 3h exposure period may have been too short. However, as separation of the insoluble carbon black particles from the cells after exposure is difficult, if not impossible, sufficient exposure of cells is likely (see also Kirwin et al. 1981).

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:
guideline study without detailed documentation
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
GLP compliance:
not specified
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
mammalian cell line, other: RAW 264.7 mouse macrophage cell line
Cytokinesis block (if used):
28h delayed co-treatment with 4 mg/L
Test concentrations with justification for top dose:
0.01-100mg/L, 48h; 4mg/L cytoB (28h delayed co-treatment),
Vehicle / solvent:
contained 10% serum
Key result
Species / strain:
mammalian cell line, other: RAW 267.4 mouse macrophage cell line
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Positive controls validity:
valid
Conclusions:
No increase in micronucleus frequency was found with carbon black
Executive summary:

No increase in micronucleus frequency was found with carbon black

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:
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
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.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
T
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:
no guideline available
Principles of method if other than guideline:
The cells were incubated with or without the test article in a total of eight exposure rounds making the total exposure time (8x72) 576 h. Similar exposures were also performed with quartz particles (cumulative dose 8 mg specific surface area of 2.9 m2/g, mean particle size of 1.59 μm). After the fifth incubation with BaP, the positive control cells began to grow markedly slower than all other cells and only yielded 25% confluence compared with about 80% for all other samples. Therefore, they received pure medium for the remaining three exposure rounds. After the eight treatments the plates were washed thoroughly with PBS to remove the excess of particles. To further reduce the load of particles, and thereby easing mutation analysis, the
cells were trypsinized and reseeded without test substance for 72 h, then harvested and stored at -800C.
Frozen cell pellets with 1.5–3 million cells were suspended in lysis buffer, and DNA was purified. The phage preparation was used to infect Escherichia coli G1250. Phages with mutations that inactivate the cII locus were identified by plaque formation under selective growth conditions. The total number of infective phages was determined by plaque formation under nonselective growth conditions.
All DNA used for the lacZ analysis was purified by phenol/chloroform extraction before the mutation analysis. Briefly, copies of the lacZ transgene were rescued from the FE1 MML cells using the TranspackTM lambda packing system, and the mutant frequency was determined using the P-gal-positive selection assay.
Oxidatively damaged DNA. The level of DNA damage measured by the Comet assay was assessed in five sets of experiments each conducted on a different day. The level of strand breaks and formamidopyrimidine [fapy]-DNA glycosylase (FPG) sensitive sites in FE1 MML cells were analyzed by single cell gel electrophoresis (Comet) assay. FPG sensitive sites were detected by incubation of the agarose-embedded nuclei with FPG protein. The level of
DNA damage was determined by a visual scoring system.
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
Target gene:
cll and lacZ
Species / strain / cell type:
mammalian cell line, other: FE1 MML mouse epithelial cell line
Test concentrations with justification for top dose:
75µg/ml; the cells were incubated with or without carbon black in a total of eight exposure rounds making the total exposure time (8 x 72 hr)
576 hr. The cumulative dose added was 6 mg for carbon black
Vehicle / solvent:
none; DMSO for positive control
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
other: Benzo[a]pyrene (BaP)
Remarks:
Cell medium used as solvent for carbon black
Key result
Species / strain:
mammalian cell line, other: FE1 MML mouse epithelial cell line
Genotoxicity:
ambiguous
Remarks:
increase in mutant frequency 1.4-fold (cII), and 1.2-fold (lacZ)
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks on result:
other: The mutant frequency for the cII gene of the negative control cells increased 1.27-fold

For carbon black exposed cells, the mutant frequency was 1.40-fold (95% CI: 1.22–1.58) for cII and 1.23-fold (95% CI: 1.10–1.37) for lacZ compared with identically passaged untreated cells. Quartz did not significantly affect the mutant frequency. Carbon black also induced DNA strand breaks (P = 0.02) and oxidized purines (P = 0.008), as measured by the Comet assay. Quartz induced marginally more oxidized purines, but no change in strand breaks.more oxidized purines, but no change in strand break

Conclusions:
Interpretation of results: study consistent with secondary mutations by ROS under conditions of excessive carbon black dose.
The study authors concluded that carbon black was weakly mutagenic in vitro at the cII and lacZ loci. It also induced DNA strand breaks and oxidized DNA bases. In a follow-up study by the same authors, it was concluded that the largest increases in base substitutions were observed in G:CtoT:A, G:C to C:G, and A:T to T:A transversion mutations; this is in keeping with a genetic finger print of ROS and is further substantiated by the observations that Printex 90 generates ROS and oxidatively damages DNA.
Executive summary:

Cells were incubated with or without the test article in a total of eight exposure rounds resulting in a total exposure of 576 h (8x72h) and a total dose of 6 mg. Similar exposures were also performed with quartz particles (cumulative dose 8 mg, specific surface area of 2.9 m2/g, mean particle size of 1.59 μm). After the fifth incubation with BaP, the positive control, cells began to grow markedly slower than all other cells and only yielded 25% confluence compared with about 80% for all other samples. Therefore, they received pure medium for the remaining three exposure rounds. After the eight treatments the plates were washed thoroughly with PBS to remove the excess of particles. To further reduce the load of particles, and thereby easing mutation analysis, the cells were trypsinized and reseeded without test substance for 72 h, then harvested and stored at -80°C. Frozen cell pellets with 1.5–3 million cells were suspended in lysis buffer, and DNA was purified. The phage preparation was used to infect Escherichia coli G1250. Phages with mutations that inactivate the cII locus were identified by plaque formation under selective growth conditions. The total number of infective phages was determined by plaque formation under nonselective growth conditions. All DNA used for the lacZ analysis was purified by phenol/chloroform extraction before the mutation analysis. Briefly, copies of the lacZ transgene were rescued from the FE1 MML cells using the TranspackTM lambda packing system, and the mutant frequency was determined using the P-gal-positive selection assay. Oxidatively damaged DNA. The level of DNA damage measured by the Comet assay was assessed in five sets of experiments each conducted on a different day. The level of strand breaks and formamidopyrimidine [fapy]-DNA glycosylase (FPG) sensitive sites in FE1 MML cells were analyzed by single cell gel electrophoresis (Comet) assay. FPG sensitive sites were detected by incubation of the agarose-embedded nuclei with FPG protein. The level of DNA damage was determined by a visual scoring system.The study authors concluded that carbon black was weakly mutagenic in vitro at the cII and lacZ loci. It also induced DNA strand breaks and oxidized DNA bases. In a follow-up study by the same authors, it was conclude that the largest increases in base substitutions were observed in G:CtoT:A, G:C to C:G, and A:T to T:A transversion mutations; this is in keeping with a genetic finger print of ROS and is further substantiated by the observations that Printex 90 generates ROS and oxidatively damages DNA.

Endpoint:
in vitro gene mutation study in mammalian cells
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:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
Target gene:
Mouse lymphoma L5178Y TK+/- gene
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: Fischer's medium supplemented with streptomycin and Pluronic S68
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not reported
- Periodically checked for karyotype stability: not reported
- Periodically "cleansed" against high spontaneous background: yes, according to the procedures of Clive and Spector (1975)
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
liver S-9 from Sprague-Dawley rats
Test concentrations with justification for top dose:
10-40 mg/mL without S9 and 5-15 mg/mL with S9; exposure time of 4 hours was extended because of the difficulty of separating the insoluble test material from the cells
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: not reported
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Remarks:
acetone
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
non-activated assay
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Remarks:
acetone
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
activated assay
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; the test material was sterilized by suspending it in a single glas centrifuge with 0.5 mL acetone for each dose level. The acetone was evaporated in a hood for 4 d. In this tube the carbon black were incubated to cell/cell medium.

DURATION
- Preincubation period: none
- Exposure duration: because of the difficulty of separating the insoluble test material from cells, the 4 h contact time was extended
- Expression time (cells in growth medium): 3 days
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): selective medium containing 1.0 ug/mL trifluorothymidine (TFT) and 0.35% noble agar.

NUMBER OF REPLICATIONS: two flasks per control and test concentration both for the activated and the non-activated assay; 1 flask was used for viability control, the other was treated with selective medium. Two independent assays were conducted.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth;
Evaluation criteria:
Not reported.
Statistics:
Not reported
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
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
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not reported
- Effects of osmolality: not reported
- Evaporation from medium: not reported
- Water solubility: not reported
- Precipitation: yes (suspensions)
- Other confounding effects: not reported

RANGE-FINDING/SCREENING STUDIES: not reported

COMPARISON WITH HISTORICAL CONTROL DATA: not reported

Table 1: Mutant frequencies and cytotoxicity, non-activated assay

  Acetone control  N-339, 10mg/mL   N-339, 15 mg/mL   N-339, 20 mg/mL   N-339, 25 mg/mL   N-339, 30 mg/mL  N-339, 35 mg/mL   N-339, 40 mg/mL  Acetone control  EMS 0.5 uL/mL  EMS 1.0 uL/mL 
Growth, percent 100  144  105  81  29  58  18  11  100  98  17 
Mutant frequency 0.7  0.7  0.6  0.6  0.7  0.7  0.6  0.4  1.4  10.9  33.1 

Table 2: Mutant frequencies and cytotoxicity, activated assay #1

   Acetone control N-339, 10 mg/mL  N-339, 15 mg/mL  Acetone control  DMBA, 2.5 ug/mL   DMBA, 5.0 ug/mL
Growth, percent  100 47  100  19  17 
Mutant frequency  0.6 0.6  0.7  1.4  3.5  4.9 

Because of the wide spread of doses in the frst evaluation, a second assay was conducted with a narrower range of doses. The results are presented in Table 3:

Table 3: Mutant frequencies and cytotoxicity, activated assay #2

   Acetone control N-339, 5 mg/mL  N-339, 7 mg/mL N-339, 9 mg/mL N-339, 11 mg/mL  N-339, 13 mg/mL  N-339, 15 mg/mL  Acetone control  DMBA, 5 ug/mL  DMBA, 7.5 ug/mL 
Growth, percent  100  175  211  210  198  138  63  100  82  43 
Mutant frequency   0.9 0.7  0.5  0.5  0.3  0.3  0.3 1.1 3.8  9.0 
Conclusions:
Interpretation of results:
negative with metabolic activation
negative without metabolic activation

N-339 carbon black did not induce any significant increase in mutant frequencies in the L5178Y mouse lymphoma TK+/- assay with and without metabolic activation. The mouse lymphoma L5178Y TK +/- assay can detect both point mutations and chromosomal alterations.
Executive summary:

Cellular toxicity was seen in the non-activated assay at 40 mg/mL N-339 carbon black. The activated assay exhibited cellular toxicity at 10 mg/mL. Over a wide range of cncentrations (15 -45 mg/mL), N-339 carbon black did not induce any significant increase in mutant frequencies in the non-activated assay. Because of the wide spread of doses in the first assay with metabolic activation (rat liver S-9), a second assay was conducted with a narrower range of doses. The results corroborated the negative findings of the first assay. The positive controls (EMS, DMBA) were functional.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
August – October 2011
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
tested up to solubility limits

The cells were exposed for 3 hours. This might have been too short for particle uptake (SCCS comment)

Conclusions:
Carbon black was not mutagenic at the hprt locus of L5178Y mouse lymphoma cells. According to the EU Scientific Committee on Consumer Safety (SCCS) the exposure period may have been too short.
Executive summary:

A study according to OECD guideline 476 was performed with carbon black. This study is summarised and assessed by the EU Scientific Committee on Consumer Safety (SCCS) in their opinion on carbon black of December 2015. Though the study was performed according to the OECD protocol, the SCCS speculates that "a 3h exposure time [...] is not always sufficient for detection of mutagenicity using the hprt forward mutation assay. Additionally, no uptake studies were performed to confirm cellular uptake. Thus, although the study did not identify mutagenicity, an appropriate exposure time might not have been applied." However, as separation of the insoluble carbon black particles from the cells after exposure is difficult, if not impossible, sufficient exposure of cells is likely (see also Kirwin et al. 1981).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

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 evidence supporting 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;Danielsen et al. 2010;Gallagher et al. 1994;Wolff et al. 1990). Clearly, genetic damage might occur by ROS generated 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 subchronic 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).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA adducts
Type of information:
experimental study
Adequacy of study:
key study
Study period:
not reported
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented publication which meets basic scientific principles
Qualifier:
no guideline available
Principles of method if other than guideline:
F344 rats were exposed by inhalation for 13 weeks to 0, 1, 7, 50 mg/m3 (Printex 90) or 50 mg/m3 (Sterling V). Then DNA was extracted from whole lung DNA immediately after exposure. The lungs of the rats for DNA analysis were not lavaged but the vascular system was perfused. DNA was extracted and used to determine oxidative DNA damage. To determine whether PAHs were available and subsequently transformed into DNA-binding metabolites, lungs of three animals from each exposure group were analysed for DNA adducts, immediately after exposure.
GLP compliance:
not specified
Type of assay:
other: DNA adducts, 32P postlabelling assay
Specific details on test material used for the study:
Printex 90, Sterling V
Species:
rat
Strain:
Fischer 344
Sex:
female
Details on test animals and environmental conditions:
All animal exposures took place at the University of Rochester, Department of Environmental Medicine.

TEST ANIMALS
- Source: F-344 rats were purchased from Harlan (Indianapolis, IN),
- Age at study initiation: not reported
- Weight at study initiation: not reported
- Fasting period before study: not reported
- Housing: not reported
- Diet (e.g. ad libitum): Purina rodent chow ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: not reported

ENVIRONMENTAL CONDITIONS
- Temperature (°C): not reported
- Humidity (%): not reported
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): not reported

IN-LIFE DATES: From: not reported To: not reported
Route of administration:
inhalation
Details on exposure:
All animal exposures took place at the University of Rochester, Department of Environmental Medicine.
Three particle exposure levels (1, 7, and 50 mg/m3) were used forPrintex-90 and one concentration (50 mg/m3) for Sterling V,
All animals were exposed for 13 weeks and DNAwas extracted from whole lung DNA from rats immediately after exposure.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours/day; 5 days/week
Post exposure period:
not reported
Remarks:
Doses / Concentrations:
1, 7, 50 mg/m3 of Printex 90; 50 mg/m3 of Sterling V, as well as a sham exposure group
Basis:

No. of animals per sex per dose:
To study whether PAHs are available and subsequently transformed into DNA-binding metabolites, lungs of three animals from every exposure group immediately after exposure were analyzed for DNA adducts. For these samples, 32P postlabeling was performed using either nuclease P1 enrichment or butanol extraction to enhance DNA adduct detection. Both procedures are suitable to detect PAH-related DNA adducts with its own specificities
Control animals:
yes, sham-exposed
Positive control(s):
B[a]P-diol-epoxide modified DNA adduct standard
Tissues and cell types examined:
To study whether PAHs are available and subsequently transformed into DNA-binding metabolites, lungs of three animals from every exposure
group immediately after exposure were analyzed for DNA adducts.
Details of tissue and slide preparation:
32P postlabeling was performed using either nuclease P1 enrichment or butanol extraction to enhance DNA adduct detection. Both procedures are suitable to detect PAH-related DNA adducts with its own specificities.
Note: The lungs of rats for DNA analysis were not lavaged but the vascular system was perfused. DNA was extracted by Jane Gallagher (US-EPA)
who used this DNA for determination of oxidative DNA damage (see Gallagher et al., 2003, IUCLID section 7.6.2).
Evaluation criteria:
Statistical comparison (details see below)
Statistics:
Statistical analysis was performed using Student’s t test using SPSS v.9 for Windows. A difference was considered significant at a level of p < 0.05.
Key result
Sex:
female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: A series of in vitro experiments were conducted alongside this in vivo study and showed that PAHs are very tightly bound to carbon black
Additional information on results:
A series of in vitro experiments were conducted alongside this in vivo study and showed that PAHs are very tightly bound to carbon black
Conclusions:
Interpretation of results (migrated information): negative (DNA adducts)
No adducts were found in DNA from lung homogenates isolated immediately after 13 weeks of inhalation of up to 50 mg/m3 of Printex 90 and Sterling V, which resulted in lung burdens of 4.9 mg and 7.6 mg, respectively. Although the lung burden was significantly lower than lung burden following a six or 24 month inhalation period, Sterling V contains at least 1000-fold the amount of PAHs compared with Printex 90. Lung DNA from rats following inhalation of carbon black showed no spots relating to PAH-DNA adduct formation compared to sham-exposed animals.
Endpoint:
genetic toxicity in vivo, other
Remarks:
secondary genotoxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
before 2007
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:
equivalent or similar to
Guideline:
other: OECD Test No. 476: In Vitro Mammalian Cell Gene Mutation Tests using the Hprt and xprt genes
Version / remarks:
ex vivo hprt test using bronchoalveolar lavage cells co-cultured with rat alveolar lining cells
Principles of method if other than guideline:
This study investigated pro- and antiinflammatory mediators underlying species specific mechaisms in particle-induced lung inflammation using ex vivo mutational analysis of inflammatory cells co-incubated with lung epithelial cells
GLP compliance:
not specified
Type of assay:
other: ex vivo HPRT assay
Specific details on test material used for the study:
Printex 90 particles with a surface area of 300 m2/g; the aerosol aerodynamic diameters ranged during exposure from 1.2 to 1.6 µm (GSD 2.2–2.8). The polyaromatic hydrocarbon content of Printex 90 was 0.16 ppm
Species:
other: rat, mouse, hamster
Strain:
other: F344, B6C3F1, F1B Syrian golden
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan, Indianapolis, USA (rats), Carles River, Wilmington, MA, USA (mice), BioBreeders, Watertown, MA, USA (hamsters)
- Age at study initiation: 5 weeks of age
- Weight at study initiation: not reported
- Fasting period before study: not reported
- Housing:AAALAC accredited barrier facility
- Diet (e.g. ad libitum): Purina chow diet ad lib.; the hamster diet was changed during the study to increase fiber content;
- Water (e.g. ad libitum): ad lib.
- Acclimation period: at least 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): not reported
- Humidity (%): not reported
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12-hour cycles

IN-LIFE DATES: From: not reported To: not reported
Route of administration:
inhalation: aerosol
Vehicle:
air
Details on exposure:
Animal studies were performed at the University of Rochester.
Aerosol aerodynamic diameter ranged during exposure from 1.2 to 1.6 um (GSD = 2.2 - 2.8)
BAL cells in Ham’s medium were added to the RLE-6TN cultures at 50:1 BAL cell:epithelial cell ratios. The cocultures were incubated overnight and then washed two times with phosphate-buffered saline (PBS) to remove BAL cells. The RLE-6TN cells were maintained for 7 days in RLuE media to allow for phenotypic expression of 6TG resistance. After the 7-day expression period, the RLE-6TN cells were harvested and cell viability determined by trypan blue exclusion.
Cells were reseeded in RLuE medium (six dishes seeded for each BAL cell-RLE-6TN coculture). After overnight incubation, plating efficiency was determined with one of the six dishes. The remaining dishes were fed every 2 to 3 days with Ham’s F-12 medium without hypoxanthine, supplemented with BPE, FBS, IGF, insulin, EGF, and 40 uM 6-thioguanine (6TG) to select for mutation in the hprt gene. After 14 days in culture, the cells were fixed and stained for cytokeratin and 6TG-resistant colonies were counted. Mutation frequencies were calculated.
Duration of treatment / exposure:
13 weeks with or without post-exposure periods of 3 and 11 months. Bronchoalveolar lavage (BAL) was performed and fluid and cells were collected. Lavage cells were pelleted and the supernatant was used for biochemical and immunologic analyses.
Frequency of treatment:
6 hours/day, 5 days/week
Post exposure period:
3 and 11 months
Dose / conc.:
1 mg/m³ air (nominal)
Dose / conc.:
7 mg/m³ air (nominal)
Dose / conc.:
50 mg/m³ air (nominal)
No. of animals per sex per dose:
5 females / group
Control animals:
yes, sham-exposed
Positive control(s):
No
Tissues and cell types examined:
Bronchoalveolar lavage (BAL) fluid was used for cellular and biochemical analysis (superoxide, hydrogen peroxide, nitric oxide, TNF-alpha, macrophage inflammatory protein-2 (MIP-2), IL-10); superoxide dismutase, glutathione reductase, and glutathione peroxidase levels in BAL fluid and lung tissue, gamma-glutamylcysteine synthetase and manganese superoxide dismutase mRNA expression. Ex vivo mutational analysis of inflammatory cells was evaluated by co-incubating BAL cells with RLE-6TN lung epithelial cells. Lung tissue was evaluated for gene expression of various antiinflammatory mediators.
Statistics:
one-way analysis of variance (ANOVA) and Tukey multiple comparisons; significance level set at 0.05
Key result
Sex:
female
Genotoxicity:
positive
Remarks:
secondary genotoxicity
Toxicity:
yes
Remarks:
(chronic inflammation)
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
not examined
Key result
Sex:
female
Genotoxicity:
negative
Remarks:
(non-inflammatory dose)
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
not examined
Additional information on results:
The ex vivo hprt mutation frequency of BAL cells from rats, mice and hamster exposed to 1 mg/m3 was not increased; significant increases were found at 7 mg/m3 (rat, mouse) and 50 mg/m3 (rat, mouse).

Percent of PMNs (neutrophils) in lavage fluid was used as an indicator of lung inflammation. PMNs were elevated in the mid- and high-dose groups in rats through 3 months of recovery. PMNs remained elevated in rats in the high-dose group through the end of the study. At the end of exposure in mice and hamsters, the mid- and high-dose groups were also elevated compared with the controls and the low-dose group; the magnitudes of response were similar for the two species. During the recovery period, the percent of PMNs from the mid- and high-dose mice remained different from each other and the rest of the exposure groups. In hamsters, the percent of PMNs decreased between 3 and 11 months of recovery and only the high-dose group remained significantly elevated throughout the entire study. In summary, the PMN response was the highest and the response more protracted in the rat versus the mouse or hamster and increased during recovery for the mid- and high-dose groups.

Conclusions:
The differences in pro- and antiinflammatory responses between rat, mouse and hamster may contribute to species differences in inflammation, genotoxicity and tumorigenesis. The inflammatory response (PMN/neutrophil count in bronchoalveolar lavage fluid (BALF)) was the highest and the response more protracted in the rat versus the mouse or hamster and increased during recovery for the mid- and high-dose groups..Mice and hamsters demonstrated an increased anti-inflammatory response. Hamster bronchoalveolar lavage (BAL) cells did not significantly increase the mutation frequency in the hprt gene for any dose at any time point. Both the rat and mouse showed dose-related effects in the hprt mutation frequency. Although the mouse response significantly decreased by the 11-month postexposure time point, the rat response was sustained throughout the course of the study. The authors conclude that secondary indirect genotoxic response exists at levels at which chronic inflammation exists; at levels below this, no hazard should be anticipated.
Executive summary:

This study compared inflammatory responses and ex vivo hprt mutation frequencies in rat, mouse and hamster after subchronic inhalation of carbon black (1, 7 or 50 mg/m3). Rats demonstrated greater propensity for generating a proinflammatory response and hprt mutations, whereas mice and hamsters demonstrated an increased anti-inflammatory response. No effects on hprt mutation frequencies were found at a dose level of 1 mg/m3, indicating a secondary indirect genotoxic response at levels at which chronic inflammation exists.

Endpoint:
genetic toxicity in vivo
Type of information:
experimental study
Adequacy of study:
key study
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:
Rats were exposed for up to 13 weeks and the effects on the lung were characterized after 6.5 and 13 weeks of exposure and 3 and 8 months of recovery. Endpoints characterized after carbon black exposure included mutation in the hprt gene of alveolar epithelial cells, changes in bronchoalveolar lavage fluid markers of lung injury and inflammation, expression of mRNA for the chemokines, MIP-2 and MCP-1, and lung histopathology. Lung burdens of carbon black were also determined.
GLP compliance:
not specified
Type of assay:
other: hypoxanthine-guanine phosphoribosyl transferase gene (hprt) mutations in alveolar epithelial cells
Species:
rat
Strain:
Fischer 344
Sex:
male
Route of administration:
inhalation
Duration of treatment / exposure:
13 weeks (6 hours/day, 5 days/week)
Remarks:
Doses / Concentrations:
0; 1.1; 7.1; 52.8 mg/m3 carbon black
Basis:

Control animals:
yes, sham-exposed
Sex:
male
Genotoxicity:
positive
Toxicity:
yes
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Additional information on results:
After 13 weeks of exposure to 1.1, 7.1 and 52.8 mg/m3 carbon black, lung burdens were 354, 1826, and 7861 ug carbon black, respectively. The lung clearance of carbon black appeared impaired after exposure to 7.1 and 52.8 mg/m3. 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 carbon black, with the effects being more pronounced at the higher exposure level. Subchronic inhalation of 1.1 mg/m3 carbon black did not elicit any detectable adverse lung effects. A significant increase in hprt mutation frequency in alveolar epithelial cells was detected immediately after 13 weeks of exposure to 7.1 and 52.8 mg/m3 carbon black as well as after 3- and 8-month recovery periods for the groups exposed to 52.8 mg/m3. The hprt mutation frequencies ranged from 5.2 to 8.2 mutants per 10e6 cells in the air control animals. 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 after exposure, respectively. A significant increase in the frequency of hprt mutations was detected immediately 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.
Conclusions:
Interpretation of results (migrated information): positive Study showed evidence for secondary genotoxicity caused by Reactive oxygen species as a result of inflammation, no primary genotoxicity was demonstrated by particles
The lung clearance of carbon black was impaired after exposure to 7.1 and 52.8 mg/m3. 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 carbon black, with the effects being more pronounced at the higher exposure level. Subchronic inhalation of 1.1 mg/m3 carbon black did not elicit any detectable adverse lung effects. A significant increase in hprt mutation frequency in alveolar epithelial cells was detected immediately after 13 weeks of exposure to 7.1 and 52.8 mg/m3 carbon black as well as after 3- and 8-month recovery periods for the groups exposed to 52.8 mg/m3. The hprt mutation frequencies ranged from 5.2 to 8.2 mutants per 10e6 cells in the air control animals. 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 after exposure, respectively. A significant increase in the frequency of hprt mutations was detected immediately 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.
Endpoint:
in vivo mammalian germ cell study: gene mutation
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
not reported
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:
Fifteen months after a single intratracheal exposure, bronchoalveolar lavage (BAL) cells were characterized as to number and type and lung histopathology performed. The alveolar type II cells were isolated and cultured in 6-thioguanine (6TG) containing media to select for mutation in the hprt gene. The potential contribution of lung inflammatory cells to in vivo mutagenic responses, were evaluated by co-culturing BAL cells with the rat alveolar epithelial cell line, RLE-6TN for 24 hours and the RLE-6TN cells selected for 6TG resistance.
GLP compliance:
not specified
Type of assay:
other: hypoxanthine-guanine phosphoribosyl transferase gene (hprt) mutations in alveolar epithelial cells
Species:
rat
Strain:
Fischer 344
Sex:
female
Route of administration:
other: intratracheal
Duration of treatment / exposure:
single exposure, animals sacrificed after 15 months
Remarks:
Doses / Concentrations:
0; 10; 100 mg/kg bw
Basis:

No. of animals per sex per dose:
6 rats/group (9 in the control group) were exposed to saline or saline suspensions of 10 and 100 mg/kg of alpha-quartz, carbon black or titanium dioxide by intratracheal instillation
Control animals:
yes, sham-exposed
Sex:
female
Genotoxicity:
positive
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Hprt mutation frequency was increased in alveolar type II cells from rats exposed to 10 and 100 mg/kg of alpha-quartz, 100 mg/kg carbon black and 100 mg/kg titanium dioxide. Hprt mutation frequencies per 10e6 epithelial cells: saline 4.0 +/- 1.2 (5 replicates), carbon black 10 mg/kg 3.9 +/- 0.3 (4 replicates), carbon black 100 mg/kg 17.0 +/- 3.1 (6 replicates). Neutrophilic inflammation was detected in all rats exposed to 10 and 100 mg/kg of alpha-quartz, and 100 mg/kg carbon black. In vitro exposure of RLE-6TN cells to BAL cells from rats treated with 10 and 100 mg/g of alpha-quartz or 100 mg/kg carbon black increased hprt mutant frequency. Both macrphage and neutrophil enriched BAL cell populations were mutagenic to RLE-6TN cells. Howecer, the mutagenic activity appeared greatest for neutrophils. Addition of catalase to BAL cell:RLE-6TN co-cultures inhibited the increase in hprt mutation frequency.
Conclusions:
Interpretation of results (migrated information): positive Study showed evidence for secondary genotoxicity caused by Reactive oxygen species as a result of inflammation, no primary genotoxicity was demonstrated by particles
Exposure of rats to doses of particles producing significant neutrophilic inflammation was associated with increased mutation in rat alveolar type II cells. The ability of particle-elicited macrophages and neutrophils to exert a mutagenic effect on epithelial cells in vitro supports a role for these inflammatory cells in the in vivo mutagenic effects of particle exposure. The inhibition of BAL cell-induced mutations by catalase implies a role for cell-derived oxidants in this response.
Endpoint:
genetic toxicity in vivo
Remarks:
Type of genotoxicity: other: oxidative stress
Type of information:
experimental study
Adequacy of study:
key study
Study period:
not reported
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented publication which meets basic scientific principles
Qualifier:
no guideline available
Principles of method if other than guideline:
To examine the role of oxidative stress, the formation of a known mutagenic lesion, 8-oxo-dG, was analysed in the lung DNA of rats. The exposure concentration of Sterling V (50 mg/m3) was selected to be equivalent in terms of retained mass in the lung to the high dose Printex 90 (50 mg/m3) 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.
GLP compliance:
not specified
Type of assay:
other: formation of 8-oxo-dG in lung
Species:
rat
Strain:
Fischer 344
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: F-344 rats were purchased from Harlan (Indianapolis, IN)
- Age at study initiation: not reported
- Weight at study initiation: between 200 and 250 g
- Fasting period before study: not reported
- Housing: not reported
- Diet (e.g. ad libitum): Purina rodent chow ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: not reported

ENVIRONMENTAL CONDITIONS
- Temperature (°C): not reported
- Humidity (%): not reported
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): not reported

IN-LIFE DATES: From: not reported To: not reported.
Route of administration:
inhalation
Details on exposure:
animals were exposed in 300-liter horizontal laminar flow whole-body chambers
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours/day; 5 days/week
Post exposure period:
An additional group of 5 female animals was held for 44 weeks in clean air (recovery group)
Remarks:
Doses / Concentrations:
1, 7 and 50 mg/m³ of Printex-90; 50 mg/m³ Sterling V
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
The aerosol concentrations (mean +/- SD) for the low, medium, and high Printex 90 exposure groups were 1.2 +/- 0.2, 7.1 +/- 1.8, and 52.8 +/- 14.7 mg/m3. The measured concentration of Sterling V was 48.2 mg/m3 over a 3-month period.
Basis:
analytical conc.
No. of animals per sex per dose:
5 female animals per group;
Control animals:
yes, sham-exposed
Tissues and cell types examined:
Lung DNA was extracted from animals after the end of the 13 week exposure period and from animals after a 44 week recovery period in clean air; further were analysed: lung burden and bronchoalveolar fluid (BAL).
Details of tissue and slide preparation:
The formation of 8-oxo-dG in the lung DNA was assessed using a reverse phase HPLC system coupled with UV and electrochemical (EC) detection.
Evaluation criteria:
Statistical comparision between groups (see below)
Statistics:
ANOVA and Tuckey, significance level at p less than or equal 0.05
Key result
Sex:
female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
not applicable
Remarks on result:
other: This design allows comparison of results on the basis of retained particle mass as well as retained particle surface area between the two Carbon Black products.
Key result
Sex:
female
Genotoxicity:
positive
Remarks:
increase in 8-oxo-dG in lung DNA only with Printex 90 at highest concentration (50 mg/m3) and at 7 mg/m3 (reversible after 44 weeks of recovery)
Toxicity:
yes
Remarks:
conditions of chronic inflammation and lung overload
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
not applicable
Remarks on result:
other:
Remarks:
-
Additional information on results:
Lung particle overload was achieved after exposure to 7 and 50 mg/m3 (Printex-90) and 50 mg/m3 (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 (Printesx-90) and at 7 and 50 mg/m3 after the 44-week recovery period. Interestingly, no increase in 8-oxo-dG was observed for Sterling V at either time point despite lung particle overload. Although the retained mass dose of Sterlinjg V at the end of exposure was even higher than for Printex-90 (50 mg/m3 exposure group; ca. 7.6 vs 4.8 mg), the surface area of the retained Sterling V was similar to that of the retained Printex-90 of the mid-dose exposures (7 mg/m3; ca. 0.2 m2 in both groups). 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 finding indicates that a retained large particle mass is not always correlated with similar adverse effects but that particle surface area is a better dose parameter. An increase (p<0.05) in lung lavage neutrophils was observed at 7 mg/m3 (Printex-90) and 50 mg/m3 (Printex-90 and Sterling V) at the 13-week exposure period and again at 50 mg/m3 (Printex-90 and Sterling V, 44-week recovery period).
Conclusions:
Interpretation of results (migrated information): other: evidence of secondary genotoxic mechanism under conditions of chronic inflammation
According to the study authors, 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 may play a role in the pathogenesis of rat lung tumours following long-term, high-dose exposure to Carbon Black in rats.
Executive summary:

The formation of 8-oxo-dG in the lung DNA was assessed after 13 weeks of exposure, and a 44-week recovery period in clean air. Lung burdens of CB were determined at both time points as well as differential cell populations from bronchoalveolar lavage

fluid (BAL). The results indicate that lung particle overload was achieved after exposure to 7 and 50 mg/m3 (Printex-90) and 50 mg/m3 (Sterling V) but not at 1 mg/m3 (Printex-90). Consistent with these results, a significant increase in 8-oxo-dG induction was observed following 13 weeks of exposure to 50 mg/m3 Printex-90 and at 7 and 50 mg/m3 after the 44-week recovery period. Interestingly, no increase in 8-oxo-dG was observed for Sterling V CB at either time point despite lung particle overload.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

Intake and inhalation of foreign particulate material stimulates its removal by phagocytosis, usually by macrophages and neutrophils. Depending on the amount of particulate matter, this may be an adaptive reaction or lead to an accumulation and activation of alveolar macrophages, and the recruitment of inflammatory cells such as neutrophils and lymphocytes from the circulating blood. In the absence of inflammation or any other adverse effect, the mere increase in alveolar macrophages and neutrophils should not be considered as adverse (ECETOC 2013). Particle clearance is characterised by reversiblechanges of biomarkers in BAL fluid (increases and corrective depletion of macrophages, neutrophils, lymphocytes and in glutathione reductase). After phagocytosis of particles, the then activated phagocytes, especially neutrophils are highly effective at generating and releasing reactive oxygen species (ROS), an important function in the degradation and removal of foreign material, including, e.g., bacteria.

ROS are, however, also continuously generated during normal cellular processes, e.g., involving mitochondrial respiration. Intracellular ROS are therefore well-controlled by physiological anti-oxidant mechanisms such as the intracellular GSH system to prevent intracellular oxidative damage, and, if present, any DNA damage is usually quickly, and error-free repaired. 

In accordance with their clearance function, macrophages are particularly sensitive to carbon black particles in terms of ROS production(Caoet al.,2014), cell signaling (e.g. calcium and NF-KB) and cytokine production(Brownet al.,2004;Stoneet al.,2009), although in other studies (with mouse peritoneal macrophages) carbon black did not trigger any pro-inflammatory reaction (measured as TNF-alpha), and seemed to reduce oxidative stress at concentration between 15 and 120mg/L(Figarolet al.,2015). Thein vitroNOEL for ROS generation by activated macrophages was at1µg/cm2after 24 hours of incubation(Aam and Fonnum 2007;Krollet al.,2011)and at 2.5µg/cm2in the human lung bronchial epithelial cell line 16HBE14o(Valet al.,2009). Pro-inflammatory effects (increases in CXCL8, -10 and -11 gene expression) were found in the bronchial epithelial BEAS-2B cell line after incubation with 10µg Printex 90/cm2for 10 hours(Ovreviket al.,2009). At a high dose (30µg/cm2; 6 hours) carbon black particles activated the NLRP3 inflammasome (caspase-1 activation) in mouse peritoneal and human alveolar macrophages(Reisetteret al.,2011). This carbon black concentration is however much higher than carbon black concentrations in occupational settings(Levyet al.,2011). In 16HBE14o cells it was shown that pro-inflammatory effects (GM-CSF mRNA expression) were dose-dependent and correlated with endocytosis (evidenced by an increase in cellular granularity). This shows the importance of the internalised amount for the cellular response. The inflammatory effects were inhibited by catalase suggesting that they were mediated by oxidative stress. Furthermore the magnitude of the effects was dependent on particle surface area(Hussainet al.,2009).

If, however, particle clearance is overburdened (a situation which is usually referred to as lung or particle overload), further recruitment of inflammatory cells and a persistent and prolonged inflammatory state may develop from incomplete particle removal, leading to further generation of ROS and possibly oxidative bursts of inflammatory cells, and in consequence oxidative DNA damage and impaired DNA-repair. This inflammatory-driven uncontrolled production of ROS is considered as hallmark of secondary genotoxicity associated with particle induced lung carcinomas in rats(Schins 2002). 

Persistent pulmonary inflammation is characterised by a prolonged increase in the number of polymorphonuclear leukocytes (measured in BAL fluid) and by an increased number of inflammatory cells in interstitial lung tissue (detected by histopathology). This also involves a cascade of oxidative stress, activation of redox-responsive transcription regulating proteins, histone acetylation and inflammatory gene transcription resulting in the release of a broad array of mediators such as ROS and reactive nitrogen species, cytokines (e.g. IL-1, TNF-alpha), arachidonic acid metabolites (e.g. PGE2, LTB4), chemokines (e.g. IL-8, MIP-2), growth factors (e.g. PDGF, EGF, IGF, FGF, VEGF), as well as proteases (e.g. elastases, collagenases, matrix metalloproteases) which injure the pulmonary architecture, leading to reparative proliferation of targets cells (e.g. epithelial and/or mesenchymal cells) and tissue remodeling. These processes favour the proliferation of mutated cells and the progression of the pre-neoplastic lesions.

Particle exposures that do not overwhelm host defense mechanisms and hence do not elicit inflammatory and proliferative responses would not be expected to pose an increased risk of genotoxicity and of lung tumours in humans. A NOEL of 1.0 mg/m3 respirable carbon black has been established in subchronic rat studies, with signs of mild inflammation found at the next higher tested dose level of 7 mg/m3(LOEL)(Carteret al.,2006;Driscoll 1996;Driscollet al.,1996;Driscollet al.,1997;Elderet al.,2005;Gallagheret al.,2003;Oberdorster 2002).

Consistent with this mode of action, oxidative DNA lesions(8-oxo-deoxyguanosine adducts) in carbon black exposed rats were only found at inflammogenic exposure levels and were mainly related to neutrophil influx; similarly, persistent lung inflammation was necessary to inducehprtmutations in lung epithelial cells of carbon black exposed rats. The mutationscould be prevented by treatment with the antioxidant catalase, supporting the role of ROS, and thus secondary genotoxicity, in the generation of mutations. Interestingly, low surface area carbon Black (37m2/g) induced less severe inflammatory changes on a mass-based dose when compared to high surface area carbon black(Driscoll 1996;Driscollet al.,1996;Driscollet al.,1997;Elderet al.,2005;Rittinghausenet al.,2013;Ziemannet al.,2011).

With regard to a possible direct mutagenic effect of PAH contaminants, all recentin vitroandin vivodata(Boisenet al.,2013;Bormet al.,2005;Danielsenet al.,2010;Totsukaet al.,2009)are consistent with the workshop conclusion of 2001(H. Greim 2001):“studies to date have notdemonstrated primary genotoxicity of carbon black with low PAH contamination using appropriatein vitro assays. DNA adducts related to associated organic compounds so far have not been foundin lung tissue from rats exposed chronically to carbon black, although in the same studies adductswere found in diesel exhaust-exposed rats.”

In summary, genotoxic effects are not expected to occur under conditions that do not induce persistent and prolonged inflammation. From the availablein vivodata in the most sensitive species (rat), lung overload would not be compromised up to concentrations of 1 mg/m3(respirable fraction).

Dose-response, temporal concordance, biological plausibility, coherence and consistency

The effects reported in animal studies (inflammation, lung epithelial cell injury, lung lesions, oxidative DNA damage, mutations) were dose-dependent. The MOA information for the rat, involving particle overload, persistent inflammation and cell proliferation, supports a nonlinear dose-response for lung cancer in the rat,i.e., a threshold mechanism.

All key events (inflammation, ROS generation, oxidative DNA damage) precede the observed genotoxic effect (mutation), and are consistent temporally with the hypothesized MOA. The association is consistent with currently accepted understanding of biological and pathological processes (plausibility) and is compatible with existing theory and knowledge about the chemical under investigation (coherence).

Particular sensitivity of the rat

In subchronic inhalation studies with carbon black, rats demonstrated greater propensity for generating a pro-inflammatory response, whereas mice and hamsters demonstrated an increased anti-inflammatory response. These differences in pro- and anti-inflammatory responses may contribute to the apparent species differences in inflammation and tumorigenesis(Carteret al.,2006).

The particular sensitivity of rats to poorly soluble particles such as carbon black may be explained a) by the distribution pattern of the retained particles within the lung compartment, which is different between species and b) the intrinsic differences between species in their oxidant-generating capacities and repair capacities after oxidant injury or DNA damage. During chronic inhalation exposure particles are retained to a greater degree in interstitial locations in lungs of non-human primates and dogs than in lungs of rats and it was hypothesized that the interspecies differences in particle location might contribute to corresponding differences in tissue response(Snipes 1996). This difference combined with the fact that human macrophages have about four times the volume of rat macrophages(Krombachet al.,1997)may contribute to the tendency of rats to respond to poorly soluble particles with more chronic inflammation and epithelial responses compared to humans.

Len to add Nikula studies

Epigenetics

Epigenetic alterations in genome function can be caused by heritable changes in gene expression occurring without changes in the DNA sequence. Several epigenetic mechanisms are known, including DNA methylation, histone modifications, and microRNA (miRNA) expression(Baccarelli and Bollati 2009)and ROS have been suggested to participate in epigenetic alterations and to modulate cellular signaling pathways(Ziechet al.,2011). These mechanismsare potentially relevant to understanding the activity of ROS in silencing various tumor suppressor genes and in subsequent tumor progression and metastasis.

Silencing the p16 tumour suppressor gene by methylation was described in 59 and 46% of diesel exhaust and carbon black induced rat tumour samples taken from the study performed by Nikulaet al.,(1995). In contrast, the estrogen receptor alpha (ER) gene was inactivated in only 14% of the diesel exhaust and carbon black tumours(Belinskyet al.,2002). The results suggest a role of ROS in an epigenetic mechanism of gene inactivation with aberrant promoter hypermethylation of regulatory genes. These results are however not indicative of a primary genetic or epigenetic activity of carbon black

Additional information

Justification for classification or non-classification

Justification for 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). 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.