Residues (petroleum), vacuum distn. residue hydrogenation

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Study period:

1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: This publication is classified as reliable with restrictions because it is an acceptable and a well-documented study report.

Data source

Reference

Reference Type:

publication

Title:

Induction of micronuclei in cultured mammalian cells by fume condensates of roofing asphalt

Author:

Qian, H.W., Ong, T., and Whong, W.Z.

Year:

1996

Bibliographic source:

American Journal of Industrial Medicine 29:554-559

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

In a mammalian micronuclei assay, Chinese hamster cell V79 cultures were exposed to Type I and Type III roofing oxidized asphalt fume condensates (generated at temperatures similar to actual roofing operation (316 ± 10°C)) in DMSO at concentrations of 0, 62.5, 125, 187.5, and 250 μg/mL for 24 hours.
 
Cells were subcultured at a concentration of 5 x 105 cells/ 60 mm dish for each treatment. Incubation Period: Cells were incubated at 37 degrees Celsius in a 5% carbon dioxide humidified atmosphere for 24 hours. The asphalt fume condensate dissolved in DMSO was added to each culture. Four different concentrations were tested for each sample. Cells were treated for 24 hours, then washed twice with PBS, and fresh medium was placed in each disk. After an additional 24-hr incubation, cells were detached by trypsinization, collected by centrifugation, and resuspended in culture medium at a density of 1 x 106 cells/mL.

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Roofing oxidized Asphalt fume condensates

IUPAC Name:

Roofing oxidized Asphalt fume condensates

Details on test material:

- Name of test material (as cited in study report): Asphalt Fume Condensates (Type I and Type III)
- Substance type: Bitumen
- Physical state: Brown-black cement like solid or semi-solid manufactured by vacuum distillation of crude petroleum

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: Minimum essential media supplemented with 10% bovine serum, 1 mM L-glutamine 100 units penicillin/mL, and 100 ug of streptomycin/mL
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: No data
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: No data

Additional strain / cell type characteristics:

not specified

Metabolic activation:

not specified

Test concentrations with justification for top dose:

0, 62.5, 125, 187.5, and 250 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Details on test system and experimental conditions:

Type I and Type III roofing asphalts were prepared by heating small pieces of the asphalt to 316 ± 10 degrees Celsius on an electric heating mantle. ( a method similar to that used in actual roofing operation.) The fumes generated were collected in glass impingers by cryotraps and organic solvents (50/50 mixture of cyclohexane/acteone).

METHOD OF APPLICATION: Cells were subcultured at a concentration of 5 x 10^5 cells/ 60 mm dish for each treatment.
Incubation Period: Cells were incubated at 37 degrees Celsius in a 5% carbondioxide humidified atmoshpere for 24 hours.
The asphalt fume condensate dissolved in DMSO was added to each culture.
Four different concentrations were tested for each sample. Cells were treated for 24 hours, then washed twice with PBS, and fresh medium was placed in each disk. After an additional 24-hr incubation, cells were detached by trypsinization, collected by centrifugation, and resuspended in culture medium at a density of 1 x 10^6 cells/ml.

SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays): Diff-Quik stain


NUMBER OF REPLICATIONS: not reported


NUMBER OF CELLS EVALUATED: 3,000 for each group


DETERMINATION OF CYTOTOXICITY
- Method: The results were expressed as the mean number of cells with micronucleus per 1000 cells

Evaluation criteria:

Frequency of micronucleated cells

Statistics:

Data were analyzed by trend and Chi-square tests.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: strain/cell type: V79

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive Both Type I and Type III caused a similar dose-related increase in the frequency of micronucleated cells.

Both types of Asphalt Fume Condensates were found capable of causing micronucleaus formation in mammalian cells in vitro. The genotoxic potential appears to be similar for both types of asphalt condensate. Both condensates caused a similar dose-related increase in the frequency of micronucleated cells. The increase was statistically significant for all four concentrations tested.

The results show that both types of roofing asphalt fume condensates caused a significant increase in the frequency of miicronucleated cells. These findings indicate that both Type I and Type IIl roofing asphalt fumes are capable of causing principally cytogenetic damage by spindle apparatus in cultured mammalian cells.

Executive summary:

A read across in vivo micronucleus test on oxidised bitumen was negative for genotoxicity. In addition chronic inhalation studies with oxidised bitumen, together with comparative fume compostion information, indicate that read across to the bitumen category, is appropriate.

In a mammalian micronuclei assay, Chinese hamster cell V79 cultures were exposed to Type I and Type III Asphalt Fume Condensates in DMSO at concentrations of 0, 62.5, 125, 187.5, and 250 μg/mL for 24 hours.

 

Cells were subcultured at a concentration of 5 x 10⁵ cells/ 60 mm dish for each treatment. Incubation Period: Cells were incubated at 37 degrees Celsius in a 5% carbon dioxide humidified atmosphere for 24 hours. The asphalt fume condensate dissolved in DMSO was added to each culture. Four different concentrations were tested for each sample. Cells were treated for 24 hours, then washed twice with PBS, and fresh medium was placed in each disk. After an additional 24-hr incubation, cells were detached by trypsinization, collected by centrifugation, and resuspended in culture medium at a density of 1 x 10⁶ cells/mL.

The results were expressed as the mean number of cells with micronucleus per 1000 cells. Both types of Asphalt Fume Condensates were found capable of causing micronucleaus formation in mammalian cells in vitro. The genotoxic potential appears to be similar for both types of asphalt condensate. Both condensates caused a similar dose-related increase in the frequency of micronucleated cells. The increase was statistically significant for all four concentrations tested. These findings indicate that both Type I and Type IIl roofing asphalt fumes are capable of causing principally cytogenetic damage by spindle apparatus in cultured mammalian cells.

This study received a Klimisch score of two and is classified as reliable with restrictions because it is an acceptable and a well-documented study report.