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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-04-16 and 2015-06-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH Guideline S2 (R1): Genotoxicity testing and data interpretation for pharmaceuticals intended for human use (June 2012)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
1,4-phenylenebis(nitrilo-2,2-dimethylprop-1-yl-3-ylidene) didodecanoate
Cas Number:
1154521-93-3
Molecular formula:
C40H68N2O4
IUPAC Name:
1,4-phenylenebis(nitrilo-2,2-dimethylprop-1-yl-3-ylidene) didodecanoate
Test material form:
liquid

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Trinova Biochem GmbH (Rathenau Str. 2 (earlier: Kerkrader Str. 10));
D-35394 Giessen, Germany
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction of Phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver
Test concentrations with justification for top dose:
5000, 1600, 500, 160, 50, 16 and 5 μg/plate (inital and cofirmatory mutation test)
Exception: TA 1537 (+ S9 mix): 1600, 500, 160, 50, 16, 5 and 1.6 μg/plate (confirmatory mutation test)
Vehicle / solvent:
acetone
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-1,2-phenylene-diamine (NPD)
Remarks:
Strain: TA 98, wihtout metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Strains: TA 100 and TA 1535, without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Strain: TA 1537, without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Strain: E.coli WP2 uvrA
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2AA)
Remarks:
All strains, with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min (preincubation method)
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: determination of the number of revertant colonies
Rationale for test conditions:
According to guidelines
Evaluation criteria:
A test item is considered mutagenic if:
- a dose-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase is considered biologically relevant if:
- in strain TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.

Criteria for a Negative Response:
A test article is considered non-mutagenic in this bacterial reverse mutation assay if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In the performed experiments the test item solution precipitated in a form of drops (“microdrops”) on the plates; in all examined strains at the concentrations of 5000 and 1600 μg/plate, without and with addition of metabolic activation (±S9 Mix) following the plate incorporation procedure (Initial Mutation Test) and at the concentration level of 5000 μg/plate, without metabolic activation (-S9 Mix) following the pre-incubation procedure (Confirmatory Mutation Test).

RANGE-FINDING/SCREENING STUDIES: Based on the pre-experiment study, the following concentrations were examined in the initial and confirmatory mutation test: 5000, 1600, 500, 160, 50, 16 and 5 µg/plate. In the confirmatory mutation test slight modification of the concentrations were done in Salmonella typhimurium TA 1537 (+ S9 mix), the following concentrations were examined: 1600, 500, 160, 50, 16, 5 and 1.6 µg/plate.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes; The spontaneous revertant colony numbers of the acetone vehicle control plates showed mostly characteristic mean numbers agreed with the actual historical control data ranges in the main experiments; however the spontaneous revertant colony numbers of the acetone vehicle control plates were slightly than the chracteristic mean numbers agreed with the actual historical data range in the initial mutation test in S. typhimurium TA100 (- S9 mix). The slightly higher counts were evaluated as acceptable without any influence on the final conclusion of the study.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Signs of cytotoxicity has been observed in all tested strains with and/or without metabolic activation. The 500 µg/plate was found to be the lowest cytotoxic concentration, observed in the case of Salmonella typhimurium strains, in the presence of exogenous metabolic activation (+S9 mix).

Any other information on results incl. tables

For summary of results see attached background material.

Applicant's summary and conclusion

Conclusions:
The reported data of this mutagenicity assay shows, that the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, the test item is considered non-mutagenic in this bacterial reverse mutation assay.
Executive summary:

To investigate the mutagenic potential of the test item, a Bacterial Reverse Mutation Assay (using Salmonella typhimurium TA 98, TA 1537, TA 1535, TA 100 and Escherichia coli WP2 uvrA) was conducted with and without metabolic activation system (S9 mix). The test item was dissolved (mixed with) in acetone. In the Initial (experiment I, plate incorporation test) and Confirmatory Mutation (experiment II, preincubation test) Tests the following concentrations were examined: 5000, 1600, 500, 160, 50, 16 and 5 μg/plate. In the Confirmatory Mutation Test slight modification of the concentrations was done in Salmonella typhimurium TA1537, in presence of metabolic activation (+S9 Mix), the originally planned concentration levels were lowered and the following concentrations were examined: 5000; 1600, 500, 160, 50, 16, 5 and 1.6 μg/plate. The concentrations, including the controls, were tested in triplicate. In the performed experiments positive and negative (vehicle) controls were run concurrently. No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. Sporadic increases in revertant colony numbers compared to the vehicle control values mostly within the actual historical control data ranges were observed in both independently performed main experiments. However, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments. In the performed experiments inhibitory effect of the test item (decreased number of revertant colony numbers and/or affected background lawn development) was observed in all examined bacterial strains. The 500 μg/plate was found to be the lowest cytotoxic concentration, observed in the case of Salmonella typhimurium strains, in the presence of exogenous metabolic activation (+S9 Mix). In conclusion, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used.

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