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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:
experimental study
Adequacy of study:
key study
Study period:
1988 to 1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This study is classified as reliable with restrictions because it is an acceptable, well-documented study report.

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
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

Constituent 1
Reference substance name:
Blends of straight run gas oil and fluid catalytically cracked gas oil, diesel fuel (CAS# 68334-30-5)
IUPAC Name:
Blends of straight run gas oil and fluid catalytically cracked gas oil, diesel fuel (CAS# 68334-30-5)
Details on test material:
Name of test material (as cited in study report): Blend 55% straight run gas oil, 45% fluid catalytically cracked gas oil (MD 57389), Diesel fuel (MD57393), Blend 54% straight run gas oil, 46% cracked gas oil components (MD 57391), Blend 50% straight run gas oil, 50% cracked gas oil components (MD 57386), Straight run gas oil with 59% cracked gas oil (MD 57401), Straight run gas oil cracked gas oil blend (MD 57388)
- 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- MD 57389
- 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): Diesel fuel (MD 57393)
- 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- MD 57393
- 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): Blend 54% straight run gas oil, 46% cracked gas oil components (MD 57391)
- 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- MD 57391
- 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): Blend 50% straight run gas oil, 50% cracked gas oil components (MD 57386)
- 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

- Name of test material (as cited in study report): Straight run gas oil with 59% cracked gas oil (MD 57401)
- 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- MD 57401
- 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): Straight run gas oil cracked gas oil blend (MD 57388)
- 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- MD 57388
- 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


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
- 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:
other: benzo(a)pyrene 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 reported


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:
other: marginally mutagenic; MI- 1.7
Cytotoxicity / choice of top concentrations:
not specified
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
MI-2.0
Cytotoxicity / choice of top concentrations:
not specified
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
MI- 2.3
Cytotoxicity / choice of top concentrations:
not specified
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
MI-3.9
Cytotoxicity / choice of top concentrations:
not specified
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
MI-7.6
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
Mutagenicity index (MI)- 9.0
Cytotoxicity / choice of top concentrations:
not specified
Remarks on result:
other: other: Diesel fuel (MD 57393)
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 in the blend 55% straight run gas oil, 45% fluid catalytically cracked gas oil substance (MD 57389); the blend 54% straight run gas oil, 46% cracked gas oil components substance (MD 57391); the Blend 50% straight run gas oil, 50% cracked gas oil components substance (MD 57386); the straight run gas oil with 59% cracked gas oil (MD 57401); and the straight run gas oil cracked gas oil blend (MD 57388). The mutagenicity index for these substances ranged from 2.0 to 9.0. The diesel fuel (MD 57393) was marginally mutagenic with an MI of 1.7.

Table 1. Mutagenicity Indices of Test Materials

Test Material Type

Sample Number

Mutagenicity Index

Blend 55% straight run gas oil, 45% fluid catalytically cracked gas oil

MD 57389

9.0

Diesel fuel

MD 57393

1.7

Blend 54% straight run gas oil, 46% cracked gas oil components

MD 57391

2.3

Blend 50% straight run gas oil, 50% cracked gas oil components

 

MD 57386

7.6

Straight run gas oil with 59% cracked gas oil

MD 57401

3.9

Straight run gas oil cracked gas oil blend

MD 57388

2.0

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive based on the amount of cracked material present in the blend

A dose-related increase in revertants compared to the mean solvent control value occurred in the blend 55% straight run gas oil, 45% fluid catalytically cracked gas oil substance (MD 57389); the blend 54% straight run gas oil, 46% cracked gas oil components substance (MD 57391); the blend 50% straight run gas oil, 50% cracked gas oil components substance (MD 57386); the straight run gas oil with 59% cracked gas oil (MD 57401); and the straight run gas oil cracked gas oil blend (MD 57388). The mutagenicity index for these substances ranged from 2.0 to 9.0. These substances are considered genotoxic materials. The diesel fuel (MD 57393) was marginally mutagenic with an MI of 1.7.
Executive summary:

In a genetic toxicity study, a modified Ames test was conducted using six different types of petroleum-derived middle distillate test substances [blend 55% straight run gas oil, 45% fluid catalytically cracked gas oil substance (MD 57389); blend 54% straight run gas oil, 46% cracked gas oil components substance (MD 57391); blend 50% straight run gas oil, 50% cracked gas oil components substance (MD 57386); straight run gas oil with 59% cracked gas oil (MD 57401); straight run gas oil cracked gas oil blend (MD 57388); and diesel fuel (MD 57393)]. DMSO test article extracts were applied mixed with phosphate buffer (non-activating) or S-9 mix (activating) andSalmonella typhimuriumbroth culture, using the preincubation method. The pre-incubated mixture was overlayed on toVogel-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 in the blend 55% straight run gas oil, 45% fluid catalytically cracked gas oil substance (MD 57389); the blend 54% straight run gas oil, 46% cracked gas oil components substance (MD 57391); the blend 50% straight run gas oil, 50% cracked gas oil components substance (MD 57386); the straight run gas oil with 59% cracked gas oil (MD 57401); and the straight run gas oil cracked gas oil blend (MD 57388). The mutagenicity index for these substances ranged from 2.0 to 9.0. These substances are considered genotoxic materials. The diesel fuel (MD 57393) was marginally mutagenic with an MI of 1.7.

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

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