Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium 2-heptyl-2,3-dihydro-3-(2-hydroxyethyl)-1H-imidazole-1-propionate
EC Number:
271-928-3
EC Name:
Sodium 2-heptyl-2,3-dihydro-3-(2-hydroxyethyl)-1H-imidazole-1-propionate
Cas Number:
68630-95-5
Molecular formula:
C15H28N2O3.Na
IUPAC Name:
sodium 3-[2-heptyl-3-(2-hydroxyethyl)-2,3-dihydro-1H-imidazol-1-yl]propanoate
Test material form:
solid
Details on test material:
Product name: Crodateric CYAP
Batch Number: PS-196-555
Appearance: The sample has been produced from drying of the aqueous solution of the material as sold commercially. It is observed to be a brown sticky solid
Specific details on test material used for the study:
Identification: Crodateric CYAP
Batch Number: 145-1150
Purity: 98.5%
Physical state/Appearance: Solid
Expiry Date: 18 September 2018
Storage Conditions: Room temperature in the dark
Formulated concentrations were adjusted to allow for the stated water/impurity content (1.5%) of the test item.

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors).
Test concentrations with justification for top dose:
The dose range for Experiment 1 was predetermined and was 1.5 to 5000 g/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 5 to 5000 µg/plate. Seven test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both four non toxic dose levels and the toxic limit of the test item following the change in test methodology
Vehicle / solvent:
dimethyl formamide
Controls
Untreated negative controls:
yes
Remarks:
untreated control
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-Aminoanthracene (2AA)
Details on test system and experimental conditions:
The test item was tested using the following method. The maximum concentration was 5000 g/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 microg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
not determined
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:
not determined
Cytotoxicity / choice of top concentrations:
not determined
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:
not determined
Cytotoxicity / choice of top concentrations:
not determined
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:
not determined
Cytotoxicity / choice of top concentrations:
not determined
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:
not determined
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. In the first mutation test (plate incorporation method), the test item caused a visible reduction in the growth of the bacterial background lawns and/or substantial reductions in the revertant colony frequency ofSalmonellastrains 5000 µg/plate in the absence of S9-mix and toSalmonellastrains TA100, TA98 and TA1537 in the presence of S9. No toxicity was noted toEscherichia colistrain WP2uvrAat any test item dose level in both the absence and presence S9-mix or to TA1535 in the presence of S9. Consequently the maximum recommended dose level of the test item was employed as the maximum dose in the second mutation test. The test item again induced a toxic response in the second mutation test (pre‑incubation method) with weakened bacterial background lawns noted in the absence of S9-mix from 500 µg/plate (TA1537), 1500 µg/plate (TA100 and TA1535) and at 5000 µg/plate (TA98) and to all of theSalmonellastrains at 5000 µg/plate in the presence S9-mix. As in the first mutation test no toxicity was noted toEscherichia colistrain WP2uvrAat any test item dose level in both the absence and presence S9-mix. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology. 

No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix in both experiments.

There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre‑incubation method). 

Applicant's summary and conclusion

Conclusions:
No evidence of mutagenic potential. Crodateric CYAP was considered to be non-mutagenic under the conditions of this test.