Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018
Reliability:
1 (reliable without restriction)

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
1H-Imidazole-1-ethanol, 4,5-dihydro-, 2-norcoco alkyl derivs.
EC Number:
263-170-7
EC Name:
1H-Imidazole-1-ethanol, 4,5-dihydro-, 2-norcoco alkyl derivs.
Cas Number:
61791-38-6
Molecular formula:
an exact molecular formula cannot be provided for an UVBC, most representative example: C16H32N2O
IUPAC Name:
2-(4,5-dihydro-1H-imidazol-1-yl)ethan-1-ol
Test material form:
liquid
Specific details on test material used for the study:
Identification: 1H-Imidazole-1-Ethanol, 4,5-Dihydro-2-NorcocoAlkyl Deriv.
CAS No: 61791-38-6
EC No: 263-170-7
Physical state/Appearance: Orange coloured viscous liquid
Batch Number: FPAC1717924
Purity: 100%
Expiry Date: 29 April 2021
Storage Conditions: Room temperature in the dark
No correction for purity was required.

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:
Test for Mutagenicity: Experiment 1 – Plate Incorporation Method
The test item was tested using the following method. The maximum concentration was 5000 µg/plate (the OECD TG 471 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.

Test for Mutagenicity: Experiment 2 – Pre-Incubation Method
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was as follows:
Salmonella strains (without S9): 0.15, 0.5, 1.5, 5, 15, 50, 150 µg/plate.
E.coli strain WP2uvrA (with and without S9) and all Salmonella strains (with S9): 0.5, 1.5, 5, 15, 50, 150, 500 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 from plate incorporation to pre-incubation.
Vehicle / solvent:
dimethyl formamide
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
other: 2-Aminoanthracene (2AA)

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:
Experiment 1 (plate incorporation)
The maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 μg/plate.
The test item induced a toxic response in the first mutation test with weakened bacterial background lawns initially noted in the absence of S9-mix from 150 μg/plate (all Salmonella strains) and 500 μg/plate (WP2uvrA). In the presence S9-mix, weakened bacterial background lawns were noted from 500 μg/plate (all tester strains).
No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix).
There were no biologically relevant 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). Small, statistically significant increases in revertant colony frequency were observed at 1.5 μg/plate (TA100 dosed in the absence of S9-mix) and 15 μg/plate (TA98 dosed in the presence of S9-mix). However these responses were withinthe in-house historical vehicle/untreated control ranges for the tester strains and were, therefore considered of no biological relevance.

Experiment 2 (pre-incubation)
Based on the results of Experiment 1, the toxic limit of the test item was employed as the maximum concentration in the second mutation test.
The test item induced a stronger toxic response in the second mutation test with weakened bacterial background lawns initially noted in the absence of S9-mix from 15 μg/plate (all Salmonella strains) and 150 μg/plate (WP2uvrA). In the presence of S9-mix, weakened bacterial background lawns were initially noted from 150 μg/plate (all Salmonella strains) and at 500 μg/plate (WP2uvrA).
No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix).
There were no significant 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).

Applicant's summary and conclusion

Conclusions:
In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) the test item 1H-Imidazole-1-Ethanol, 4,5-Dihydro-2-Norcoco Alkyl Deriv. did not induce an increase in the frequency of revertant colonies at any of the dose levels used either with or without metabolic activation (S9-mix). Under the conditions of this test 1H-Imidazole-1-Ethanol, 4,5-Dihydro-2-Norcoco Alkyl Deriv. was considered to be non-mutagenic.
Executive summary:

Under the conditions of this test 1H-Imidazole-1-Ethanol, 4,5-Dihydro-2-Norcoco Alkyl Deriv. was considered to be non-mutagenic.