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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:
11 October 2016 - 02 November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
31 May 2008
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Reference substance 001
Cas Number:
210420-85-2
Constituent 2
Chemical structure
Reference substance name:
N-methyldidecylamine
EC Number:
230-990-1
EC Name:
N-methyldidecylamine
Cas Number:
7396-58-9
Molecular formula:
C21H45N
IUPAC Name:
N-decyl-N-methyldecan-1-amine
Constituent 3
Chemical structure
Reference substance name:
Water
EC Number:
231-791-2
EC Name:
Water
Cas Number:
7732-18-5
Molecular formula:
H2O
IUPAC Name:
Dihydrogen oxide
impurity 1
Reference substance name:
Unknown impurities
Molecular formula:
Not available
IUPAC Name:
Unknown impurities
Test material form:
solid
Details on test material:
- Appearance: Slightly yellow gel- Storage conditions: At room temperature; container flushed with nitrogen

Method

Target gene:
- S. typhimurium: Histidine gene- E. coli: Tryptophan gene
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by Aroclor 1254
Test concentrations with justification for top dose:
Experiment 1: Direct plate assayDose range finding test:TA100 and WP2uvrA without and with S9: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plateDirect plate assay: TA1535, TA1537, TA98 and TA100 without S9-mix: 0.052, 0.17, 0.51, 1.6, 5.0 and 16 µg/plateTA1535, TA1537 and TA98 with S9-mix: 0.17, 0.51, 1.6, 5.0, 16 and 48 µg/plateExperiment 2: Pre-incubation assayDose range finding test::TA100 and WP2uvrA without S9-mix: 0.016, 0.052, 0.17, 0.51, 1.6, 5.0 and 16 µg/plateTA100 and WP2uvrA with S9-mix: 0.052, 0.17, 0.51, 1.6, 5.0, 16 and 48 µg/platePre-incubation assay:TA100 and WP2uvrA without S9-mix: 0.016, 0.052, 0.17, 0.51, 1.6, 5.0 and 16 μg/plate.TA100 and WP2uvrA with S9-mix: 0.052, 0.17, 0.51, 1.6, 5.0, 16 and 48 μg/plate.TA1535, TA1537 and TA98 without S9-mix: 0.052, 0.17, 0.51, 1.6, 5.0 and 16 μg/plate.TA1535, TA1537 and TA98 with S9-mix: 0.17, 0.51, 1.6, 5.0, 16 and 48 μg/plate.
Vehicle / solvent:
- Vehicle used: Milli-Q water
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9, 5 µg/plate in saline for TA1535
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: ICR-191
Remarks:
without S9, 2.5 µg/plate in DMSO for TA1537 (direct plate)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-nitrofluorene
Remarks:
without S9, 10 µg/plate in DMSO for TA98 and 15 µg/plate in DMSO for TA98 (pre-incubation)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9, 650 µg/plate in DMSO for TA100
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9, 10 µg/plate in DMSO for WP2uvrA
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9, in DMSO, 2.5 μg/plate for TA1535 and TA1537, 1 μg/plate for TA98 and TA100 (direct plate), 5 μg for TA100 (pre-incubation), 15 μg for WP2uvrA
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)DURATION- Exposure duration: 48 hour- Pre-incubation period: 30 minutes (pre-incubation experiment only)NUMBER OF REPLICATIONS: - Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.NUMBER OF CELLS EVALUATED: 10E8 per plateDETERMINATION OF CYTOTOXICITY - Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.OTHER EXAMINATIONS: - The presence of precipitation of the test compound on the plates was determined.
Rationale for test conditions:
Concentrations were chosen based on dose range finding study
Evaluation criteria:
Acceptability of the assayThe assay is considered acceptable if it meets the following criteria:a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at Charles River Den Bosch.b) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.A test substance is considered negative (not mutagenic) in the test if:a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.b) The negative response should be reproducible in at least one independently repeated experiment.A test substance is considered positive if:a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.
Statistics:
No formal hypothesis testing was done.

Results and discussion

Test resultsopen allclose all
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
Positive controls validity:
valid
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
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
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
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
Remarks:
up to precipitating concentrations (with S9-mix)
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORSPrecipitation:Direct plate assay: For WP2uvrA precipitation was observed at single dose levels of 17 and 52 μg/plate in the absence and presence of S9-mix, respectively.Pre-incubation assay: Precipitation was observed in TA100 and WP2uvrA at concentrations of 5.0 μg/plate and upwards (absence of S9-mix) and 16 μg/plate and upwards (presence of S9-mix). Except in tester strain WP2uvrA,where no precipitation of the test item was observed at 16 μg/plate.RANGE-FINDING/SCREENING STUDIES:Direct plate assay:- In tester strain TA100, only two analysable dose levels could be used for the evaluation of the results in the absence of S9-mix. Therefore, this part of the study was again tested in the first mutation experiment. In tester strain WP2uvrA, toxicity was observed at dose levels of 52 μg/plate and above in the absence and 164 μg/plate and above in the presence of S9-mix.Pre-incubation assay:- In tester strain TA100, toxicity was observed at dose levels of 5.0 μg/plate and above in the absence and 48 μg/plate and above in the presence of S9-mix. In tester strain WP2uvrA, toxicity was observed at dose levels at 16 μg/plate in the absence of S9-mix and no cytotoxicity up to 48 μg/plate in the presence of S9-mix (slight precipitate).COMPARISON WITH HISTORICAL CONTROL DATA: - The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.ADDITIONAL INFORMATION ON CYTOTOXICITY:Direct plate assayTA1535: without S9: 16 µg/plate and above and with S9: 48 µg/plate and aboveTA1537: without S9: 16 µg/plate and above and with S9: 16 µg/plate and aboveTA98: without S9: 16 µg/plate and above and with S9: 48 µg/plate and aboveTA100: without S9: 16 µg/plate and above and with S9: 52 µg/plate and aboveWP2uvrA: without S9: 52 µg/plate and above and with S9: 164 µg/plate and abovepre-incubation assayTA1535: without S9: 1.6 µg/plate and above and with S9: 16 µg/plate and aboveTA1537: without S9: 1.6 µg/plate and above and with S9: 16 µg/plate and aboveTA98: without S9: 5.0 µg/plate and above and with S9: 48 µg/plate and aboveTA100: without S9: 1.6 µg/plate and above and with S9: 48 µg/plate and aboveWP2uvrA: without S9: 16 µg/plate and above and with S9: no cytotoxicity (Slight precipitate)

Applicant's summary and conclusion

Conclusions:
In an AMES test, performed according to OECD 471 guideline and in accordance with GLP principles, 1-Decanaminium, N-decyl-N,N-dimethyl-, hexanedioate (2:1) was found not to be mutagenic with or without metabolic activation.

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