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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
1988 to 1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This study is classified as reliable with restrictions because it is an acceptable and a well-documented study report.
Justification for type of information:
The standard OECD 471 test is not suitable to test petroleum UVCBs, because it has a tendency to produce false negatives for these substances. Therefore, the petroleum industry has developed a Modified Ames assay, optimized to accurately identify positive results for this endpoint. This deviation from the prescribed testing procedure requires some further explanation which is given in the attached document. The document gives a brief history of the development of the Modified Ames test and outlines Concawe’s proposed work (as part of a wider testing strategy, see Annex 13) to further support the use of this test for PS.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Modified Ames assay
Principles of method if other than guideline:
Modifications of the standard Ames assay include:
1. DMSO extractions of the test material dissolved in cyclohexane were used as opposed to the neat or solvent-diluted sample.
2. an eight-fold higher concentration of liver S-9 fraction from hamster rather than rat was used.
3. The NADP cofactor was increased from 4 to 8 mm.
GLP compliance:
not specified
Type of assay:
other: bacterial reverse mutation assay (Modified Ames test)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: low viscosity hydrocarbon liquid
Details on test material:
- Name of test material (as cited in study report): coker gas oil (MD 57396, N9)/(MD 57397, N10) Thermally cracked gas oil (MD 57394, N8)/(MD 57382, N12)
- Substance type: petroleum-derived middle distillate
- Physical state: liquid
- Analytical purity: not provided
- Impurities (identity and concentrations): not provided
- Composition of test material, percentage of components: not provided
- Isomers composition: not provided
- Purity test date: not provided
- Lot/batch No.: sample code- 57396
- Expiration date of the lot/batch: not provided
- Stability under test conditions: not provided
- Storage condition of test material: stored indoors in stainless steel containers at 18°C

- Name of test material (as cited in study report): coker gas (MD 57397, N10)
- Substance type: petroleum-derived middle distillate
- Physical state: liquid
- Analytical purity: not provided
- Impurities (identity and concentrations): not provided
- Composition of test material, percentage of components: not provided
- Isomers composition: not provided
- Purity test date: not provided
- Lot/batch No.: sample code- 57397
- Expiration date of the lot/batch: not provided
- Stability under test conditions: not provided
- Storage condition of test material: stored indoors in stainless steel containers at 18°C

- Name of test material (as cited in study report): thermally cracked gas (MD 57394, N8)
- Substance type: petroleum-derived middle distillate
- Physical state: liquid
- Analytical purity: not provided
- Impurities (identity and concentrations): not provided
- Composition of test material, percentage of components: not provided
- Isomers composition: not provided
- Purity test date: not provided
- Lot/batch No.: sample code- 57394
- Expiration date of the lot/batch: not provided
- Stability under test conditions: not provided
- Storage condition of test material: stored indoors in stainless steel containers at 18°C

- Name of test material (as cited in study report): thermally cracked gas (MD 57382, N12)
- Substance type: petroleum-derived middle distillate
- Physical state: liquid
- Analytical purity: not provided
- Impurities (identity and concentrations): not provided
- Composition of test material, percentage of components: not provided
- Isomers composition: not provided
- Purity test date: not provided
- Lot/batch No.: sample code- 57392
- Expiration date of the lot/batch: not provided
- Stability under test conditions: not provided
- Storage condition of test material: stored indoors in stainless steel containers at 18°C

See attachment for additional details

Method

Target gene:
gene for histadine synthesis
Species / strain
Species / strain / cell type:
S. typhimurium TA 98
Details on mammalian cell type (if applicable):
- Type and identity of media: Oxoid 32 medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not reported
- Periodically checked for karyotype stability: not reported
- Periodically "cleansed" against high spontaneous background: not reported
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix prepared with the livers of Syrian-Golden hamsters
Test concentrations with justification for top dose:
0, 1, 3, 5, 7, 10, 15, 20, 25, 40, 40, 50, 60 µl/plate
Vehicle / solvent:
cyclohaxane and dimethylsulfoxide (DMSO)
Controls
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Migrated to IUCLID6: 2-aminoanthracene and 2-nitrofluorene,
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: not reported
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): not reported
- Fixation time (start of exposure up to fixation or harvest of cells): 48 hours


NUMBER OF REPLICATIONS: not reported


NUMBER OF CELLS EVALUATED: not provided


Evaluation criteria:
Spontaneous and solvent control reversion rates for TA98 should fall between 30 and 55 revertants/plate for the assay to be considered acceptable.
The single dose of Stock 642-100'' CNN (standard reference material) at 50 µl must fall between 100-200 revertants/plate.
Statistics:
The mean number of revertants/plate for each dose was calculated. The Student-Newman Keuls Test was preformed if a dose-related doubling relative to the mean solvent control was not reached. Nonlinear regression, using the SAS NLIN procedure of Myers et al. was used to determine the slope of the dose response curve, if a doubling was reached.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
mutagenicity index (MI)- 4.0
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
MI- 9.3
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
MI- 2.1
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
MI- 3.1
Remarks on result:
other: other: coker gas oil (MD 57396)
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

A dose-related increase in revertants compared to the mean solvent control value occurred for all the test materials [coker gas oils (MD 57396 and MD 57397)] and thermally cracked gas oil (MD 57394 and MD 57382). The mutagenicity indexes ranged from 2.1 to 9.3.

Table 1. Mutagenicity Indices of Test Materials

Test Material Type

Sample Number

Mutagenicity Index

Coker gas oil

MD 57396

4.0

Coker gas oil

MD 57397

9.3

Thermally cracked gas oil

MD 57394

2.1

Thermally cracked gas oil

MD 57382

3.1

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive

The thermally cracked and coker gas oil samples showed positive results in the modified Ames test and are considered genotoxic materials.
Executive summary:

In a genetic toxicity study, a modified Ames test was conducted using four different types of petroleum-derived middle distillate test substances [coker gas oils (MD 57396 and MD 57397)] and thermally cracked gas oil (MD 57394 and MD 57382). DMSO extracts of the test materials were applied mixed with phosphate buffer (non-activating) or S-9 mix (activating) and Salmonella typhimuriumbroth culture, using the preincubation method. The pre-incubated mixture was overlayed on to Vogel-Bonner Minimal E medium plus 1.5% bacteriological grade agar and 2% glucose in Nunc Petri plates. The plates were allowed to incubate for 48 hours. After the incubation period, colonies of histidine protrophs in test and control plates were enumerated on a Artek automatic colony counter. The condition of the background lawn of bacterial growth was confirmed by visual observation with a Zeiss stereozoom microscope. The same procedures were used for the positive controls (2 -aminoanthracene, benzo (a) pyrene, and 2 -nitrofluorene).

A dose-related increase in revertants compared to the mean solvent control value occurred for all the test materials [coker gas oils (MD 57396 and MD 57397) and thermally cracked gas oil (MD 57394 and MD 57382)].The mutagenicity indexes ranged from 2.1 to 9.3. The positive Ames test results for the thermally cracked gas and coker gas oils indicate that these are considered genotoxic materials.

This study received a Klimisch score of 2. It is classified as reliable with restrictions because it is an acceptable and a well-documented study report.