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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:
1998-02-10 to 1998-02-13
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This study is classified as reliable with restrictions because no GLP statement was provided, but the study was otherwise well-conducted.
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.
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Modified ames assay as described by Blackburn et al. (1986) and ATSM Standard Method Number E 1687-95
Principles of method if other than guideline:
Modified ames assay as described by Blackburn et al. (1986) and ATSM Standard Method Number E 1687-95.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Mobil CRU#s 98039, 97125, 97084
- Substance type: Residual aromatic extract
- Physical state: liquid

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with
Metabolic activation system:
hamster S9
Test concentrations with justification for top dose:
0, 12, 24, 36, 48, 60 μL test material/plate; 0, 3, 6, 9, 12, 15 μL positive control oil/plate
Vehicle / solvent:
Dimethylsuphoxide
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Details on test system and experimental conditions:
The study was carried out according to ASTM standard method number E 1687-95, which is based on a modified Ames assay described by Blackburn et al. (1986). The three test substances (Mobil CRU#s 98039, 97125, 97084) were diluted (1:5) in DMSO in a glass tube at 45 degress Celsius, vortexed for 30 seconds every five minutes for 30 minutes, and separated by centrifugation. Finally, the lower extract phase harvested and stored until testing. The reference oil was diluted 1:4 with DMSO prior to testing.

The extracted oil sample, tester bacteria (TA98), and hamster S9 metabolizing mixture were combined in suspension, incubated for 20 minutes at 37 degrees celsius, poured into a petri plate, incubated for 48 hours at 37 degrees Celsius, and revertant colonies were counted.
Evaluation criteria:
Evaluation criteria were not explicitly stated in the study report, but ASTM standard method number E1687-97 considers substances to be a potential dermal carcinogens in mice if the mutagenicity index (MI) is greater than 2.0 revertants per microliter of DMSO extract, equivocal if the MI is ≥1.0 but ≤2.0, and unlikely to be carcinogenic if the MI <1.0. Further testing for equivocal substances is needed to determine carcinogenic potential.
Statistics:
Mutagenicity index (MI) was calculated from the linear portion of the initial dose-response curve using linear regression analysis.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
ambiguous
Remarks:
CRU 97125 – mutagenicity index = 0.43
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
negative
Remarks:
CRU 98039 – mutagenicity index = 0.1; CRU 97084 – mutagenicity index = 0.34
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: other: CRU 97125
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Results of bacterial mutagenicity assays (mean revertants ± standard deviation)

DOSE (μL/plate)

POSITIVE CONTROL Reference Oil

(Assay #1)

POSITIVE CONTROL Reference Oil

(Assay #2)

Mobil CRU# 98039

Mobil CRU# 97125

Mobil CRU# 97084

0

43 ± 1

42 ± 2

43 ± 5

43 ± 5

42 ± 4

12

51 ± 3

50 ± 4

46 ± 3

48 ± 5

47 ± 6

24

64 ± 3

63 ± 3

45 ± 5

55 ± 6

49 ± 7

36

76 ± 2

81 ± 1

49 ± 5

60 ± 5

56 ± 5

48

89 ± 2

91 ± 6

48 ± 7

65 ± 5

57 ± 5

60

98 ± 2

100 ± 5

49 ± 7

68 ± 6

63 ± 6

 

 

 

 

 

 

Mutagenicity Index (MI)

3.8

4.1

0.1

0.43

0.34

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation CRU 98039 – mutagenicity index = 0.1; CRU 97084 – mutagenicity index = 0.34
positive with metabolic activation CRU 97125 – mutagenicity index = 0.43

Two of the three residual aromatic extract samples (Mobil CRU#s 98039 and 97084) are considered not mutagenic in an in vitro genotoxicity test (modified Ames assay, preincubation method) in the presence of hamster S9 fraction. The mutagenicity index for the test substances are 0.1 and 0.34 respectively. Mobil CRU# 97125 showed a low level of acticity with a mutagenicity index of 0.43. Authors concluded that the three residual aromatic extracts are unlikely to have dermal carcinogenic potential in mouse skin.
Executive summary:

In a modified bacterial reverse mutation assay (assay), three samples of residual aromatic extract (Mobil CRU#s 98039, 97125, 97084) were diluted (1:5) in DMSO in a glass tube at 45 degress Celsius, vortexed for 30 seconds every five minutes for 30 minutes, and separated by centrifugation. The lower extract phase harvested and stored until testing. A positive control reference oil was diluted (1:4) with DMSO prior to testing. The extracted oil samples ( tested at concentrations of 0, 12, 24, 36, 48, or 60 μl test material/plate), tester bacteria (TA98), and hamster S9 metabolizing mixture were combined in suspension, incubated for 20 minutes at 37 degrees Celsius, poured into a petri plate, incubated for 48 hours at 37 degrees Celsius, and revertant colonies were counted. The mutagenicity index was calculated from the intial slope of the linear portion of the dose-response curve.

 

Under conditions of the study, two of the three residual aromatic extract samples (Mobil CRU#s 98039 and 97084) are considered not mutagenic in an in vitro genotoxicity test (modified Ames assay, preincubation method) in the presence of hamster S9 fraction. The mutagenicity index for the test substances are 0.1 and 0.34 respectively. Mobil CRU# 97125 showed a low level of activity with a mutagenicity index of 0.43. Authors concluded that the three residual aromatic extracts are unlikely to have dermal carcinogenic potential in mouse skin. This study received a Klimisch score of 2 and is classified as reliable with restrictions because it did not contain a GLP statement but is otherwise a well-conducted study.