Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 February 2018 to 19 February 2018 (Experimental initiation to Experimental completion)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
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:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Kyoeisha Chemical Co., Ltd
- Lot/batch No.of test material: 6033175
- Expiration date of the lot/batch: 17 Match 2019
- Purity test date: 3 October 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (Ambient) in original container as supplied by the Sponsor. Container kept tightly closed and away from heat or sunlight
- Stability under test conditions: Assumed stable for the duration of the study
- Solubility and stability of the test substance in the solvent/vehicle: The test item formed a homogeneous suspension (after sonication) in Dimethyl sulfoxide (DMSO) (Stock B 50000 µg/mL). No test item stability in the solvent was performed, assumed stable for the duration of the study.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: None

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Mixed with DMSO and sonicated to form a homogenous suspension acceptable for dosing

FORM AS APPLIED IN THE TEST (if different from that of starting material) Mixed with DMSO and sonicated to form a homogenous suspension acceptable for dosing

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Initial toxiicity mutation assay: 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/plate of N, N’-bis {6-[12-(hydroxy) octadecanoylamino] hexyl} decanediamide (+/- S9 mix)
Confirmatory mutation assay : 156.25. 312.5, 625, 1250, 2500, 5000 µg/plate of N, N’-bis {6-[12-(hydroxy) octadecanoylamino] hexyl} decanediamide (+/- S9 mix)

The test item formed a homogenous suspension in DMSO after sonication (Stock B 50000 µg/mL). DMSO was therefore selected as the vehicle for treatment. A volume of 100 µL of the test item from stock B (50000 µg/mL) was added to 2 mL of top agar to assess precipitation at the recommended maximum test concentration for soluble non-cytotoxic substances (i.e. 5 mg/plate). No precipitation was observed at 5000 µg/plate, hense 5000 µg/plate was selected as the highest concentration to be tested in the absence and presence (5 % v/v S9 mix) of metabolic activation.


Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was insoluble in distilled water. A homogeneous suspension was however formed in DMSO (Stock B 50000 µg/mL), acetone (Stock C 50000 µg/mL) and ethanol 95% (Stock D 50000 µg/mL), after sonication. From these vehivles, DMSO was selected as the vehicle for treatment.
Controls
Untreated negative controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-Aminoanthracene: TA1537, TA1535, Escherichia coli WP2uvrA, TA98 and TA100 (+S9) TA100 (-S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation);
- Cell density at seeding (if applicable): Cell densities (OD at 660 nm) of all tester strains were within the required range to produce cultures with approximately 1 - 2 x 109 bacteria/mL, demonstrating appropriate numbers of bacteria were plated.

DURATION
- Preincubation period: Not specified
- Exposure duration: Incubated at 37 ± 1 °C for 48 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity is detectable as decrease in the number of revertant colonies per plate and/or by a thinning of the bacterial background lawn.

Rationale for test conditions:
Study performed to standard test conditions in accordence woth OECD 471 and EU Method B.13/14.
Evaluation criteria:
Assay Evaluation Criteria
Once criteria for a valid assay had been met, responses observed in the assay were evaluated. The conditions necessary for determining a positive result were: there should be a dose related increase in the mean revertants per plate in at least one tester strain over the range tested and/or at one or more doses of the test item either in the absence or presence of the metabolic activation system.
The biological relevance of the results was considered:

Strains TA1535 and TA1537 and Escherichia coli WP2uvrA
Data sets were judged positive, when the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0 times the mean negative control value.

Strain TA98, TA100
Data sets were judged positive, when the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0 times the mean negative control value.
Statistical analysis was used as an aid in the evaluation of a dose response.
A response that did not meet all three of the above criteria (magnitude, concentration responsiveness, reproducibility) was not evaluated as positive.
Negative results obtained in the initial toxicity-mutation assay were confirmed by a confirmatory mutation assay, using the same method as specified above, with an alteration in concentration spacing.
Statistics:
Simple linear regression analysis was performed for tester strains of Salmonella typhimurium viz., TA1537, TA1535, TA98, TA100 and Escherichia coli WP2uvrA, separately, to assess the dose dependent nature of any increase in revertant colonies.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Untreated negative 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:
no cytotoxicity, but tested up to precipitating concentrations
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Untreated negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
From the results of this study, under the specified experimental conditions, N, N’-bis {6-[12-(hydroxy) octadecanoylamino] hexyl} decanediamide is concluded to be non-mutagenic in the Bacterial Reverse Mutation Assay using tester strains of Salmonella typhimurium viz., TA1537, TA1535, TA98, TA100 and Escherichia coli WP2uvrA.