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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:
2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid
Details on test material:
- Name of test material (as cited in study report): HCPE and Impurities
- Physical state: solid
- Expiration date of the lot/batch: 24 Jun 2015
- Storage condition of test material: room temperature

Method

Target gene:
- S. typhimurium: his-locus
- E. coli: trp-locus
Species / strainopen allclose all
Species / strain:
E. coli WP2 uvr A
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and β-naphthoflavone-induced rat liver S9 mix
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and β-naphthoflavone-induced rat liver S9 mix
Test concentrations with justification for top dose:
1st experiment: 0; 33; 100; 333; 1 000; 2 500 and 5 000 μg/plate
2nd experiment: 0; 500; 1 000; 2 000; 3 000; 4 000 and 5 000 μg/plate
Vehicle:
DMSO
Controlsopen allclose all
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
2.5 μg/plate, dissolved in DMSO for TA 1535, TA 100, TA 1537, TA 98; 60 μg/plate, dissolved in DMSO for E. coli WP2 uvrA
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
5 μg/plate, dissolved in DMSO for TA 1535, TA 100
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanidine
Remarks:
without S9
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
10 μg/plate, dissolved in DMSO - strain: TA 98
Positive control substance:
other: 4-nitro-o-phenylenediamine
Remarks:
without S9
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
100 μg/plate, dissolved in DMSO - strain: TA 1537
Positive control substance:
9-aminoacridine
Remarks:
without S9
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
5 μg/plate, dissolved in DMSO - strain: E. coli WP2 uvrA
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
other: demonstration of the efficacy of the S9 mix
Positive control substance:
benzo(a)pyrene
Remarks:
To demonstrate the efficacy of the S9 mix in this assay, the S9 batch was characterized with benzo(a)pyrene.
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48-72h

SELECTION AGENT (mutation assays): agar with minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin for Salmonella typhimurium strains and 0.5 mM tryptophan for E. Coli


DETERMINATION OF CYTOTOXICITY
- Method: his- or trp- background growth, number of his+ or trp+ revertants

Evaluation criteria:
Acceptance criteria
Generally, the experiment was considered valid if the following criteria were met:
 The number of revertant colonies in the negative controls was within the range of the
historical negative control data for each tester strain.
 The sterility controls revealed no indication of bacterial contamination.
 The positive control substances both with and without S9 mix induced a distinct increase in
the number of revertant colonies within the range of the historical positive control data or
above.
 Fresh bacterial culture containing approximately 109 cells per mL were used.

Assessment criteria
The test substance was considered positive in this assay if the following criteria were met:
 A dose-related and reproducible increase in the number of revertant colonies, i.e. at least
doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and
E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA
1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either
without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
 The number of revertants for all tester strains were within the historical negative control
data range under all experimental conditions in at least two experiments carried out
independently of each other.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
no
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
ambiguous
Remarks:
slight increases in the number of his+ or trp+ revertants
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
other: slight increases in the number of his+ or trp+ revertants
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
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:
other: slight increases in the number of his+ or trp+ revertants
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
A biologically relevant increase in the number of his+ revertants was not observed using the
tester strain TA 1537 and TA 98 in the standard plate test either without S9 mix or after the
addition of a metabolizing system.
A reproducible and partly dose-dependent increase in the number of his+ revertants
exceeding a factor of 3 compared to the concurrent vehicle control was observed in tester
strain TA 1535 without S9 mix: Increase at a concentration of 5 000 μg/plate (1st Exp.) and at
concentrations of 3 000; 4 000 and 5 000 μg/plate (2nd Exp.).
Furthermore, slight increases in the number of his+ or trp+ revertants were observed in tester
strain TA 1535, TA 100 and E.coli WP2 uvrA with and/or without S9 mix.

Any other information on results incl. tables

The positive findings were not confirmed by the preincubation test.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive

Thus, under the experimental conditions of this study, the test substance HCPE and
Impurities is mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation
assay in the absence and the presence of metabolic activation.
Executive summary:

The substance was tested for its mutagenic potential, in an OECD 471 guideline study (in compliance with GLP), based on the ability to induce point mutation in selected loci of several bacterial strains, i.e. Salmonella typhimurium (TA 1535, TA 100, TA 1537, TA98) and Escherichia coli (WP2 uvrA) in a reverse mutation assay. Bacteria were exposed to 33 -5000 µg substance/plate in a standard plate test in the presence and absence of a metabolizing system (rat liver S-9 mix). No precipitation of the test substance and no bacteriotoxic effect was found with and without S9 mix. A dose-dependent increase in the number of his+ revertants in the tester strain TA 1535 was observed in the standard plate test in the absence of a metabolizing system. Furthermore, slight increases in the number of his+ or trp+ revertants were observed in tester strain TA 1535, TA 100 and E.coli WP2 uvrA with and/or without S9 mix.Thus, under the experimental conditions of this study, the test substance HCPE and Impurities is mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and presence of metabolic activation.