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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Remarks:
Genetic Laboratory, JBS, Inc., Japan
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): ammonium Perfluoro-3,6-dioxaoctanate (EEA-NH4)
- Physical state: white solid
- Analytical purity: 99.5%
- Lot/batch No.: RS4-56
- Storage condition of test material: room temperature, shield from light

Method

Target gene:
his
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell lines (if applicable):
Not applicable
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/5,6-benzoflavone-induced rat liver S9
Species / strain:
E. coli WP2 uvr A
Details on mammalian cell lines (if applicable):
Not applicable
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/5,6-benzoflavone-induced rat liver S9
Test concentrations with justification for top dose:
156, 313, 625, 1250, 2500, 5000 µg/plate, both in absence and presence of metabolic activation
Vehicle:
distilled water
Controlsopen allclose all
Negative controls:
no
Solvent controls:
yes
Remarks:
distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
Remarks:
strains TA98, TA100, WP2uvrA

Migrated to IUCLID6: in absence of S9-mix
Negative controls:
no
Solvent controls:
yes
Remarks:
distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
strain TA1535

Migrated to IUCLID6: in absence of S9-mix
Negative controls:
no
Solvent controls:
yes
Remarks:
distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
strain TA1537

Migrated to IUCLID6: in absence of S9-mix
Negative controls:
no
Solvent controls:
yes
Remarks:
distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
strains TA98, TA100, TA1537

Migrated to IUCLID6: in presence of S9-mix
Negative controls:
no
Solvent controls:
yes
Remarks:
distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
Remarks:
strains TA1535, WP2uvrA

Migrated to IUCLID6: in presence of S9-mix
Details on test system and conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes at 37 °C
- Expression time (cells in growth medium): 48 hours at 37 °C

The test was performed in triplicate. A confirmatory test was also performed in triplicate.
Evaluation criteria:
The number of the revertant colonies of the negative control and positive control in all bacterial strains was compared with the values of the historical data in our laboratory. The values of the negative control and the positive control were judged to satisfy acceptance criteria when either of the next two criteria were filled. One of the criteria is that the number of the revertant colonies is within the range of the standard value (mean ± 2SD) of the historical data. Another criterion is although the number of the revertant colonies is out of the range in the standard value, it is judged to be appropriate value because it occurs by chance from the comparison with the historical data.

When the experiment satisfies the following criteria, it was judged that the test result was obtained from the test of the appropriate procedure.
1) The growth of the contaminant in the test materials, such as Nutrient broth, the test substance solution of the highest concentration, S9Mix, sodium phosphate buffer are not detected from the result of the sterility test.
2) The negative control values satisfy acceptance criteria.
3) The positive control values satisfy acceptance criteria. And the number of the revertant colonies of the positive control increases two-fold or more compared with the negative control.

When the negative control and positive control values satisfy acceptance criteria and the test results satisfy acceptance criteria, the test substance was judged positive for mutagenic activity when biologically significant increases such as significant increases in the number of the revertant colonies with clear dose-relativity and reappearance was obtained.
Statistics:
Two statistical analyses of Dunnett's multiple comparison method (one-side test) and linear regression method were used in this test. The number of the revertant colonies of each bacterial strain of each dose was compared with the negative control in both of the presence and the absence of metabolic activation, and statistically significant difference in the number of the revertant colonies between those two groups was analyzed first by the multiple comparison method (p<0.01). When the statistical significant difference was obtained by the multiple comparison method, the specific dose-relativity was analysed by linear regression method (p<0.01).

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
at 5000 µg/plate
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
at 5000 µg/plate
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
The test substance did not show a statistically significant dose-related increase in the number of revertant colonies compared with the negative control in any bacterial strains regardless of the presence or the absence of metabolic activation. In addition, the reappearance of the test results between the main and the confirmatory test was confirmed. Therefore, it is concluded that the mutagenic activity of the test substance is considered negative under the test conditions employed.

Applicant's summary and conclusion