Carbon black

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

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

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

oxidative stress

Type of information:

experimental study

Adequacy of study:

weight of evidence

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

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline available

Principles of method if other than guideline:

To examine the role of oxidative stress in development of carcinomas in rats following chronic high-dose inhalation of carbon black, the formation of a known mutagenic lesion, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) was analysed in the lung DNA of female rats exposed for 13 weeks to increasing concentrations of carbon black; 1, 7, and 50 mg/m(3) of Printex-90 (16 nm; specific surface area 300 m(2)/g) and to 50 mg/m(3) of Sterling V CB (70 nm; surface area of 37 m(2)/g). Two different carbon blacks of different surface area were tested to study the effects of surface area 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 or test system 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)

Dose / conc.:

1 mg/L air (nominal)

Remarks:

Prinetx 90, measured 1.2 +/- 0.2

Dose / conc.:

7 mg/L air (nominal)

Remarks:

Printex 90, measured 7.1 +/- 1.8

Dose / conc.:

50 mg/m³ air (nominal)

Remarks:

Printex 90, measured 52.8 +/- 14.7 mg/m3

Dose / conc.:

50 mg/m³ air (nominal)

Remarks:

measured 48.2 mg/m3

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:

other: secondary genotoxicity

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 in the lung of the female F334 rats was seen after inhalation exposure at 7 mg/m3 and 50 mg/m3 high surface area carbon black (Printex-90) and 50 mg/m3 low surface area carbon black (Sterling V); but not at 1 mg/m3 (Printex-90). Consistent with these findings, a significant increase (p<0.05) in 8-oxo-dG induction was observed following 13 weeks of exposure to 50 mg/m3 (Printex-90) and after the 44-week recovery period in the group of animals exposed at 7 and 50 mg/m3 (Printex 90). Interestingly, no increase in 8-oxo-dG was observed for Sterling V at either time point despite lung particle overload. Although the retained mass dose of Sterling 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.

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.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

in vivo mammalian somatic cell study: gene mutation

Remarks:

secondary genotoxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

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

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 anti-inflammatory mediators underlying species specific mechanisms 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

Species:

other: rat, mouse, hamster

Strain:

other: F344, B6C3F1, F1B Syrian golden

Sex:

female

Details on test animals or test system 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:

other: 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.

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Effects of Subchronically Inhaled Carbon Black in Three Species. I. Retention Kinetics, Lung inflammation, and Histopathology

Author:

Elder A, Gelein R, Finkelstein JN, Driscoll KE, Harkema J, Oberdörster G

Year:

2005

Bibliographic source:

Toxicological Sciences 88(2), 614-629

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

The study was designed to test species differences and the influence of surface area. Particle retention kinetics, inflammation and histopathology were examined in female rats, mice and hamsters exposed for 13 weeks to high surface area carbon black (HSCb) at doses of 0, 1, 7, and 50 mg/m3. Rats were also exposed to 50 mg/m3 low surface area carbon black (LSCb). Groups of animals were sacrificed immediately after 13 weeks of exposure and after 3 and 11 months of recovery for bronchoalveolar lavage analysis as well as for measurements of lung burdens and lung histopathology

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Carbon black

EC Number:

215-609-9

EC Name:

Carbon black

Cas Number:

1333-86-4

Molecular formula:

C

IUPAC Name:

carbon

Test material form:

solid: nanoform, no surface treatment

Test animals

Species:

other: rat, mouse, hamster

Strain:

other: F344, B6C3F1, F1B Syrian golden hamster

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: rats from Harlan (Indianapolis, IN), B6C3F1 mice from Charles River (Wilmington, MA), F1B Syrian golden hamsters from BioBreeders (Watertown, MA)
- Age at study initiation: 5 weeks
- Weight at study initiation: not reported
- Housing: AAALAC-accredited barrier facility with 12-h light/dark cycle
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least two weeks

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: The HSCb (Printex 90, surface area 300 m2/g) aggregate aerosols had aerodynamic diameters ranging from 1.2 - 2.4 µm (geometric standard deviations [GSD]: 2.0 - 3.1); the LSCb (Sterling V, surface area 37 m2/g) aggregate aerosols had aerodynamic diameters of 0.6 - 0.9 µm (GSD: 3.0 - 3.7)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: All exposures took place in compartmentalized, horizontal flow whole-body inhalation chambers (ca.. 300 L).
- Method of holding animals in test chamber: not reported. Each chamber can hold up to 32 rats or hamsters or up to 64 mice.
- Source and rate of air: not reported
- Method of conditioning air: not reported
- System of generating particulates/aerosols: the particle-containing exposure atmospheres were generated using a Wright dust feeder (low and mid dose carbon black) or a Venturi jet generator (high dose cabon black). Particles were deionized by passing the aerosols through an 83Kr source.
- Temperature, humidity, pressure in air chamber: not reported.
- Air flow rate: total flow through the chambers was ca 100 L/min.
- Air change rate: not reported.
- Method of particle size determination: mass concentration and particle size were periodically measured using gravimetric and impactor sampling, respectively.
- Treatment of exhaust air: not reported.

TEST ATMOSPHERE
- Brief description of analytical method used: Aerosol concentration was continuously monitored by a RAS-2 for the mid and high-dose or by a RAM-1 for the low dose (Monitoring Instruments for the Environment [MIE], Inc., Bedford, MA).

TEST CONCENTRATION ADAPTATION
5 weeks into the exposures of mice and hamsters, it was determined that the retained burden of HSCb was lower than was found for rats, which were exposed first. The concentrations for the mouse and hamster exposures were accordingly increased from 7 to 15 mg/m3 for the mid-dose and from 50 to 75 mg/m3 for the high-dose to produce equivalent predicted normalized lung burdens. For hamsters, the low dose also had to be increased from 1 to 1.1 mg/m3.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Aerosol concentrations were continuously monitored by a RAS-2 for the mid and high-dose or by a RAM-1 for the low dose (Monitoring Instruments for the Environment [MIE], Inc., Bedford, MA).

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours per day; 5 days per week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 mg/m³ air (nominal)

Remarks:

Printex 90

Doses / concentrations 2

Dose / conc.:

7 mg/m³ air (nominal)

Remarks:

Printex 90

Doses / concentrations 3

Dose / conc.:

50 mg/m³ air (nominal)

Remarks:

Printex 90

Doses / concentrations 4

Dose / conc.:

50 mg/m³ air (nominal)

Remarks:

Sterling V

No. of animals per sex per dose:

Groups of five to six females were exposed to filtered air, or the three dose levels of HSCb or 50 mg/m3 LSCb. For the histopathological and particle dosimetry analyses, groups of six animals were used; for all other endpoints, groups of five animals were used.

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: the doses were chosen to span a no observed adverse effects level (NOAEL) to particle overload
- Rationale for animal assignment (if not random): females were chosen because they were previously shown to be more sensitive to the induction of lung tumors by poorly soluble, low toxicity particles than males
- Section schedule rationale (if not random): additional sentinels were euthanized throughout the study to monitor pathogen status

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
DETAILED CLINICAL OBSERVATIONS: Yes
BODY WEIGHT: Yes
- Time schedule for examinations: body weights were obtained every 2 weeks.
HAEMATOLOGY: No
CLINICAL CHEMISTRY: Yes, in lavage fluid
Measurements were performed immediately after exposure and 3 and 11 months post-exposure; carbon black retention was also evaluated after 5 weeks of exposure.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: lungs

Other examinations:

- Determination of carbon black lung burden
- Measurement of cellular and biochemical parameters in lavage fluid
- Pulmonary morphometry
- Cell proliferation assessment (BrdU method)

Statistics:

Tukey t-tests uing SigmaStat (SPSS Science, Chicago, IL). The two factors for the ANOVAs were exposure dose and time. Data were appropriately transformed (e.g., base 10 logarithm, natural logarithm) if an analysis of residuals suggested deviations from the asumptions of normality and equal variance. Comparisons were considered statistically significant when p<= 0.05

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

not specified

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY
No rats died during exposure to carbon black. Nine control rats died in the post-exposure phase due to a blocked water line. One each in the 1 and 50 mg/m3 HSCb groups also died in the post-exposure phase. Thirteen mice died during exposure, seven of which were in the control group; two were in the low-dose gorup, three in the mid-dose group, and one in the high-dose group. In the post-exposure phase, six controls, one low-dose, two mid-dose, and six high-dose mice died. One hamster (high-dose group) died during exposuer. Two hamsters each from the control, low-dose, and high-dose groups and four from the mid-dose group died in the post-exposure phase of the study. These deaths were most likely due to a change in housing conditions from wire-bottom cages (during exposure) to plastic cages (post-exposure) and reflects the heightened sensitivity of hamsters to environmental changes. Screening of sentinels (all three species) did not reveal parasitic, bacterial, or viral pathogens that would explain the unscheduled deaths. The authors conclude that the unscheduled deaths were not related to exposure, i.e., not associated with carbon black dose or time after exposure. Out of the total number of animals exposed, ca. 4% of the animals per species died prematurely.

BODY WEIGHT AND WEIGHT GAIN
Significant decreases in body weight during exposure occurred only in hamsters exposed to 50 mg/m3 HSCb. Rats of the high-dose groups lost weight during the exposure (not statistically significant). Body weights returned to control values during the recovery period for both species.

ORGAN WEIGHTS
Lung weights were increased in high-dose exposed animals, but this persisted only in rats and mice up to the end of he study period.
For rats, significant elevations in lung weight (up to more than twice the control lung weight) were found for the high-dose HSCb at the end of exposure and at all post-exposure time points. The lung weights of rats exposed to LSCb were between those for the high- and mid-dose HSCb in magnitude and were significantl y elevated 1 day and 3 months after expsoure.
In mice, lung weights were found to be elevated 1 day and 3 months post-exposure in the high- and mid-dose groups; at the end of the recovery period, i.e., 11 onths post-exposure, significant elevations were found only for the high-dose group, and these changes persisted up to 3 months post-exposure; after 11 months of recovery, no significant difference were found.
Thus, the changes in lung weight were resolved most rapidly in hamsters.

HISTOPATHOLOGY: NON-NEOPLASTIC
Lung inflammation and histopathology were more severe and prolonged in rats than in mice and hamsters; both were similar in rats exposed to mid-dose HSCb and LSCb.
No adverse histological effects of exposure were found at any post-exposure time point in the lungs of rats, mice or hamsters exposed to low-dose HSCb. The lungs of these rats, mice or hamsters were similar to those of the control rats, with the exception that some alveolar macrophages containing small amounts of carbon black were widely scattered throughout the alveolar air spaces of the pulmonary parenchyma. However, the number and morphological character of these particle-containing macrophages were not different from those in the controls.

CELLULAR AND BIOCHEMICAL PARAMETERS IN LAVAGE FLUID
Total bronchoalveolar lavage (BAL) cell numbers were relevated at 1 day post-exposure and all through the recovery period in the high-dose groups for all three species in comparison to all other exposure groups. Whereas total cell number gradually decreased over time in mice andhamsters, it stayed elevated in the rats exposued to high-dose HSCb and LSCb. The presence of polymorphonuclear leukocytes (PMN) in lavage fluid was used as a sensitive indicator of lung inflammation. PMN were elevated in the mid- and high-dose HSCb and LSCb groups of rats at the end of exposure and throughout the recovery period. In rats, the PMN responses to high-dose HSCb and LSCb were similar. The PMN remained elevated in rats from the high-dose HSCb and LSCb groups 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 to the ocntrols and low-dose groups. The magnitudes of response were similar for the two species. During the recovery period, the percentage of PMN from the mid- and high-dose mice remained different from each other and the rest of the exposure groups. In hamsters, the PMN response decreased between 3 and 11 months post-exposure such that only the high-dose group retained a signifcant response at the termination of the study. The magnitude and duration of the PMN response was the highest in rats.. There were no significant elevations in cellular or biochemical parameters for any of the animals exposed to low-dose HSCb.
OTHER FINDINGS
Equivalent or similar mass burdens were achieved in rats exposed to high-dose HSCb and LSCb. Surface area burdens were equivalent for 7 mg/m3 HSCBc and 50 mg/m3 LSCb.
Prolonged retention was found in rats exposed to mid- and high-dose HSCb and to LSCb, but LSCb was cleared faster than HSCb. Retention was also prolonged in mice exposed to mid- and high-dose HSCb, and in hamsters exposed to high-dose HSCb.

Effect levels

1

Key result

Dose descriptor:

NOEC

Remarks:

(rat, mouse)

Effect level:

1 mg/m³ air

Based on:

test mat.

Sex:

female

Basis for effect level:

other: pulmonary inflammation

2

Dose descriptor:

LOEC

Remarks:

(rat, mouse)

Effect level:

7 mg/m³ air

Based on:

test mat.

Sex:

female

Basis for effect level:

other: histopathology; lung inflammation; prolonged carbon black retention in lungs

3

Key result

Dose descriptor:

NOEC

Remarks:

(hamster)

Effect level:

7 mg/m³ air

Based on:

test mat.

Sex:

female

Basis for effect level:

other: pulmonary inflammation

4

Dose descriptor:

LOAEC

Remarks:

(hamster)

Effect level:

50 mg/m³ air

Based on:

test mat.

Sex:

female

Basis for effect level:

other: histopathology; prolonged carbon black retention in lung; lung inflammation

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

7 mg/m³ air

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

The results show that hamsters have the most efficient clearance mechanisms and least severe responses of the threee species tested. The results from rats also showe that particle surfae area is an important determinant of target tissue dose and, therefore, effects. From these results, a subchronic NOAEL of 1 mg/m3 respirable HSCb (Printex 90) can be assigned to female rats, mice, and hamsters.

Executive summary:

Particle retention kinetics, inflammation, and histopathology were examined in female rats, mice, and hamsters exposed for 13 weeks to high surface area Cb (HSCb) at doses chosen to span a no observable adverse effects level (NOAEL) to particle overload (0, 1, 7, 50 mg/m3, nominal concentrations). Rats were also exposed to low surface area Cb (50 mg/m3, nominal; LSCb). Retention and effects measurements were performed immediately after exposure and 3 and 11 months post-exposure; retention was also evaluated after 5 weeks of exposure. Significant decreases in body weight during exposure occurred only in hamsters exposed to high-dose HSCb. Lung weights were increased in high-dose Cb-exposed animals, but this persisted only in rats and mice up to the end of the study period. Equivalent or similar mass burdens were achieved in rats exposed to high-dose HSCb and LSCb, whereas surface area burdens were equivalent for mid-dose HSCb and LSCb. Prolonged retention was found in rats exposed to mid- and high-dose HSCb and to LSCb, but LSCb was cleared faster than HSCb. Retention was also prolonged in mice exposed to mid- and high-dose HSCb, and in hamsters exposed to high-dose HSCb. Lung inflammation and histopathology were more severe and prolonged in rats than in mice and hamsters, and both were similar in rats exposed to mid-dose HSCb and LSCb. The results show that hamsters have the most efficient clearance mechanisms and least severe responses of the three species. The results from rats also show that particle surface area is an important determinant of target tissue dose and, therefore, effects. From these results, a subchronic NOAEL of 1 mg/m3 respirable HSCb (Printex 90) can be assigned to female rats, mice, and hamsters.