Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-11-24 to 2018-03-20
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:
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
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
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: crystalline
Details on test material:
- State of aggregation:
- Particle size distribution:
- Mass median aerodynamic diameter (MMAD):
- Geometric standard deviation (GSD):
- Shape of particles:
- Surface area of particles:
- Crystal structure:
- Coating:
- Surface properties:
- Density:
- Moisture content:
- Residual solvent:
- Activation:
- Stabilisation:
- Other:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
- Expiration date of the lot/batch:
- Purity test date:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity:
- Specific activity:
- Locations of the label:
- Expiration date of radiochemical substance:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Store in a cool, dry and well ventilated place and away from foodstuffs. In darkness at room temperature.
- Stability under test conditions: Yes
- Solubility and stability of the test substance in the solvent/vehicle: Slighthly soluble in water 33.9 mg/L
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Reacts with strong acids

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- Preliminary purification step (if any): None
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

Remark: Environmental precautions: Do not allow to enter drains or waterways.
Do not discharge into the subsoil/soil
Specific details on test material used for the study:
Test Item and information as provided by the Sponsor.
Identification: 2-(2-undecyl-4,5-dihydro-1H-imidazol-1-yl)ethanol
CAS Number: 136-99-2
EC Number: 205-271-0
Batch Number: 7J13AA
Purity: 95.4%
Physical state/Appearance: Yellow solid
Expiry Date: 15 October 2018
Storage Conditions: Room temperature in the dark
Formulated concentrations were adjusted to allow for the stated water/impurity content
(4.6%) of the test item.

Method

Target gene:
prokaryotic cells,
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
N/A
Additional strain / cell type characteristics:
other: All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived from S. typhimurium strain LT2 through mutations in the histidine locus. Additionally due to the "deep rough" (rfa-) mutation they p
Remarks:
The plasmid also confers ampicillin resistance which acts as a convenient marker (Mortelmans and Zeiger, 2000). In addition to a mutation in the tryptophan operon, the E. coli tester strain contains a uvrA- DNA repair deficiency
Cytokinesis block (if used):
N/A
Metabolic activation:
with and without
Metabolic activation system:
Rat Liver Homogenate
Test concentrations with justification for top dose:
Test for Mutagenicity: Experiment 1 - Plate Incorporation Method:
Dose selection:
Eight concentrations of the test item
(1.5, 5, 15, 50, 150, 500, 1500 and 5000 g/plate) were assayed in triplicate against each
tester strain, using the direct plate incorporation method.


Test for Mutagenicity: Experiment 2 – Pre-Incubation Method
Dose selection:
The dose range used for Experiment 2 was determined by the results of Experiment 1 and
was 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500 μg/plate.
Nine 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:
- Vehicle(s)/solvent(s) used:acetone
- Justification for choice of solvent/vehicle:
Dose selection
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate.
Nine 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
Details on test system and experimental conditions:
Salmonella typhimurium
Strains Genotype Type of mutations indicated
TA1537 his C 3076; rfa-; uvrB-: frame shift mutations
TA98 his D 3052; rfa-; uvrB-;R-factor
TA1535 his G 46; rfa-; uvrB-: base-pair substitutions
TA100 his G 46; rfa-; uvrB-;R-factor

Escherichia coli
Strain Genotype Type of mutations indicated
WP2uvrA trp-; uvrA-: base-pair substitution
All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived from S. typhimurium strain LT2 through mutations in the histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess a faulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to larger molecules. A further mutation, through the deletion of the uvrB- bio gene, causes an inactivation of the excision repair system and a dependence on exogenous biotin. In the strains TA98 and TA100, the R factor plasmid pKM101 enhances chemical and UV-induced mutagenesis via an increase in the error prone repair pathway. The plasmid also confers ampicillin resistance which acts as a convenient marker (Mortelmans and Zeiger, 2000). In addition to a mutation in the tryptophan operon, the E. coli tester strain contains a uvrA- DNA repair deficiency which enhances its sensitivity to some mutagenic compounds. This deficiency allows the strain to show enhanced mutability as the uvrA repair system would normally act to remove and repair the damaged section of the DNA molecule (Green and Muriel, 1976 and Mortelmans and Riccio, 2000).
Evaluation criteria:
The reverse mutation assay may be considered valid if the following criteria are met:
All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al., (1975), Maron and Ames (1983), Mortelmans and Zeiger (2000), Green and Muriel (1976) and Mortelmans and Riccio (2000).
All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (negative controls). Acceptable ranges are presented as follows:
Salmonella typhimurium strain TA98 8 to 60
Salmonella typhimurium strain TA100 60 to 200
Salmonella typhimurium strain TA1535 7 to 40
Salmonella typhimurium strain TA1537 2 to 30
Escherichia coli strain WP2uvrA 10 to 60
All tester strain cultures should be in the range of 0.9 to 9 x 109 bacteria per mL.
Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivitiv

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.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.
Statistics:
3.6 Statistical Analysis
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. Values that the program concluded as statistically significant but were within the in-house historical profile were not reported.

Results and discussion

Test resultsopen allclose all
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:
other: All salmonella strains
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:
other: TA98 and WP2uvrA
Remarks:
In the second experiment (pre incubation method), the toxic limit of the test item was selected as the maximum concentration. The test item again induced a toxic response in the second mutation test (pre-incubation method), with weakened bacterial
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
Non mutagenic

Any other information on results incl. tables

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) in Experiment 1 (plate incorporation method).  Minor increases in TA98 revertant colony frequency were noted at 5 and 15 µg/plate in the presence of S9 mix only.  However, these responses were within the in-house historical vehicle/untreated control values for the tester strain and were, therefore considered of no biological relevance. 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:
2-(2-undecyl-4,5-dihydro-1H-imidazol-1-yl)ethanol was considered to be non-mutagenic under the conditions of this test.
Executive summary:

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.

1.2       Methods

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to nine dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). 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 0.05 to 500 µg/plate.  Nine 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.  

1.3       Results

The vehicle (acetone) control plates gave counts of revertant colonies within the normal range.  All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation.  Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

In the first experiment (plate incorporation method), the maximum dose level of the test item was selected as 5000 µg/plate (the maximum recommended concentration).  The test item induced toxicity as weakened bacterial background lawns to all of the tester strains in both the presence and absence of S9-mix, initially from 150 µg/plate (all Salmonella strains) and 500 µg/plate (WP2uvrA).  

In the second experiment (pre incubation method), the toxic limit of the test item was selected as the maximum concentration.  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 15 µg/plate (TA100), 50 µg/plate (TA1535 and TA1537) and 150 µg/plate (TA98 and WP2uvrA).  In the presence of S9-mix, weakened bacterial background lawns were noted from 150 µg/plate (all of the Salmonella strains) and at 500 µg/plate (WP2uvrA).  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.

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) in Experiment 1 (plate incorporation method).  Minor increases in TA98 revertant colony frequency were noted at 5 and 15 µg/plate in the presence of S9 mix only.  However, these responses were within the in-house historical vehicle/untreated control values for the tester strain and were, therefore considered of no biological relevance.

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).