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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:
31 January, 2018 - 15 February, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
1-Propanaminium, N-(3-aminopropyl)-2-hydroxy-N,N-dimethyl-3-sulfo-, N-coco acyl derivs., hydroxides, inner salts
EC Number:
268-761-3
EC Name:
1-Propanaminium, N-(3-aminopropyl)-2-hydroxy-N,N-dimethyl-3-sulfo-, N-coco acyl derivs., hydroxides, inner salts
Cas Number:
68139-30-0
Molecular formula:
C9H19N2O5S(CnH(2n+1)), (n=7,9,11,13,15,17)
IUPAC Name:
1-Propanaminium, N-(3-aminopropyl)-2-hydroxy-N,N-dimethyl-3-sulfo-, N-coco acyl derivs., hydroxides, inner salts
Test material form:
solid
Specific details on test material used for the study:
Batch: DSP-001.
Purity: 97%.
Expiry Date: 15 August 2018.

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1537
Remarks:
his C 3076; rfa-; uvrB-:
Details on mammalian cell type (if applicable):
Obtained from Trinova Biochem GmbH on 27 June 2017.
Species / strain / cell type:
S. typhimurium TA 98
Remarks:
his D 3052; rfa-; uvrB-;R-factor
Details on mammalian cell type (if applicable):
Obtained from Trinova Biochem GmbH on 27 June 2017.
Species / strain / cell type:
S. typhimurium TA 1535
Remarks:
his G 46; rfa-; uvrB-:
Details on mammalian cell type (if applicable):
Obtained from Trinova Biochem GmbH on 27 June 2017.
Species / strain / cell type:
S. typhimurium TA 100
Remarks:
his G 46; rfa-; uvrB-;R-factor
Details on mammalian cell type (if applicable):
Obtained from Trinova Biochem GmbH on 27 June 2017.
Species / strain / cell type:
E. coli WP2 uvr A
Remarks:
trp-; uvrA-:
Details on mammalian cell type (if applicable):
Obtained from British Industrial Biological Research Association on 17 August 1987.
Metabolic activation:
with and without
Test concentrations with justification for top dose:
The maximum concentration was 5000 µg/plate (the maximum recommended dose level).
Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Vehicle / solvent:
The vehicle control used was as follows:
Identity: Sterile distilled water.
Supplier: Baxter.
Batch number (purity): 17H09BA1A (N/A), Expiry: 07/2020.
Controls
Untreated negative controls:
yes
Remarks:
triplicate
Negative solvent / vehicle controls:
yes
Remarks:
triplicate
Positive controls:
yes
Remarks:
triplicate
Positive control substance:
4-nitroquinoline-N-oxide
benzo(a)pyrene
other: N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG); 9-Aminoacridine (9AA); 2-Aminoanthracene (2AA).
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation
- Number of cells (x 10 9 /mL) for each treated and control culture:
TA100: Experiment 1 = 2.7; Experiment 2 = 1.5
TA1535: Experiment 1 = 1.2; Experiment 2 = 1.9
WP2uvrA: Experiment 1 = 3.2; Experiment 2 = 2.3
TA198: Experiment 1 = 3.1; Experiment 2 = 1.9
TA1537: Experiment 1 = 1.3; Experiment 2 = 1.5

DURATION
- Preincubation period:
- Exposure duration:
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):
Evaluation criteria:
There are several criteria for determining a positive result.
Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was as follows:
Salmonella strains TA100, TA1535 and TA1537 (with and without S9): 0.5, 1.5, 5, 15, 50, 150, 500, 1500 µg/plate;
Salmonella strain TA98 (with and without S9): 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/plate;
E.coli strain WP2uvrA (with and without S9): 5, 15, 50, 150, 500, 1500, 5000 µg/plate.

HISTORICAL CONTROL DATA
- Positive historical control data,Year: 2017
Range across all strains with and without S9: 95-4357.
Mean across all strains with and without S9: 186-2379.

Applicant's summary and conclusion

Conclusions:
1-Propanaminium, N-(3-aminopropyl)-2-hydroxy-N,N-dimethyl-3-sulfo-, N-coco acyl derivs., hydroxides, inner salts was considered to be non-mutagenic under the conditions of this test.
Executive summary:

A GLP compliant bacterial reverse mutation test was performed on the substance in accordance with OECD Test Guideline 471.

Several strains of bacteria were treated with the test item using both Ames plate incoporation and pre-incubation methods at doses ranging from 0.5 to 5000 µg/plate, with and without S9 -mix.

Sterile distilled water was used as the vehicle as the test item is soluble in water.

There were no increases in the frequency of revertant colonies observed for any of the bacterial strains, at any of the doses, with or without metabolic activation in the plate incorporation or pre-incubation experiments. All controls were valid.

Under the conditions of this test, 1-Propanaminium, N-(3-aminopropyl)-2-hydroxy-N,N-dimethyl-3-sulfo-, N-coco acyl derivs., hydroxides, inner salts was found to be non-mutagenic.

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