Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020-03-05 to 2020-04-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997-07-21
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008-05-30
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
1998-08
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2-(allyloxy)ethanol
EC Number:
203-871-7
EC Name:
2-(allyloxy)ethanol
Cas Number:
111-45-5
Molecular formula:
C5H10O2
IUPAC Name:
2-(allyloxy)ethanol
Test material form:
liquid

Method

Target gene:
The Salmonella typhimurium histidine (his) reversion system measures his- → his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA1535, TA100) and frameshift (TA1537, TA98) mutations. The Escherichia coli WP2 uvrA (trp) reversion system measures trp– → trp+ reversions. The Escherichia coli WP2 uvrA strain detects mutagens that cause other base-pair substitutions (AT to GC).
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- Source of S9: S9 fraction of phenobarbital- and β-naphthoflavone induced rat liver (Supplier: Trinova Biochem GmbH, Rathenau Str. 2., D-35394 Giessen, Germany; Manufacturer: MOLTOX INC. P.O. BOX 1189 BOONE, NC 28607 USA)
- Method of preparation of S9 mix: 400 mL of sterile salt solution (4 mM β-NADP Na, 5 mM D-Glucose 6-phosphate Na, 8 mM MgCl2, 33 mM KCl), 100 mL rat liver homogenate (S9) and 500 mL ice cold 0.2 M sodium phosphate-buffer, pH 7.4.
- Concentration or volume of S9 mix and S9 in the final culture medium: 500 µL of S9 mix (containing 50 µL S9) in a final volume of 2700 µL culture medium
- Quality controls of S9: Each batch of the S9 fractions used in this test had the appropriate biological activity (according to the provided certificate) and was active in the applied system (2AA treatments).
Test concentrations with justification for top dose:
±S9 Mix: 5000, 3200, 1600, 500, 160, 50, 16 µg/plate;
A maximum concentration of 5000 µg/plate was selected based on preliminary solubility testing, concentration range finding testing and as the maximum recommended concentration according to current regulatory guidelines (OECD, 1997).
Vehicle / solvent:
- Vehicle used: DMSO (NPD, 9AA, 2AA) and ultrapure water (test item, SAZ, MMS)
- Justification for choice of vehicle: The test item solutions was prepared in ultrapure water (ASTM Type I) and diluted prior to treatment. This vehicle is compatible with the survival of the bacteria and the S9 activity and was chosen based on the results of the preliminary solubility test.
- Justification for percentage of solvent in the final culture medium: Percentage of vehicle is compatible with the survival of the bacteria and the S9 activity as determined in a preliminary solubility test.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
ultrapure water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
ultrapure water
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-1,2-phenylene-diamine
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE
Experiment I: Plate incorporation method
Experiment II: Pre-incubation method

TREATMENT AND HARVEST SCHEDULE
- Preincubation period: 20 min
- Exposure duration: 48 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The colony numbers on the untreated, vehicle and positive controls and the test item treated plates were determined (counted manually, evaluated by unaided eye), the mean values, standard deviations and the mutation rates were calculated.
Evaluation criteria:
A test item is considered mutagenic if:
- A dose-related increase in the number of revertants occurs and/or;
- A reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.;

An increase is considered biologically relevant if:
- In strain TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control;
- In strain TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.;

Criteria for a Negative Response:
A test item is considered non-mutagenic in this bacterial reverse mutation assay if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.
Statistics:
According to the guidelines, the biological relevance of the results was the criterion for the interpretation of results, a statistical evaluation of the results was not regarded as necessary.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Completely soluble up to 50 mg/mL
- Precipitation and time of the determination: No precipitation was observed.

RANGE-FINDING/SCREENING STUDIES:
In a preliminary solubility test, a test item concentration of 50 mg/mL was chosen as a stock concentration. In a preliminary concentration range finding test with TA98 and TA100, seven different concentrations (5000; 1600; 500; 160, 50, 16 and 5 μg/plate) were tested for toxicity and mutation in the presence and absence of metabolic activation using 3 plates each. The colony and background lawn development remained within the corresponding historical control data ranges.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: In the performed experiments the revertant colony numbers of the untreated and dimethyl sulfoxide (DMSO) control plates in the different experimental phases were slightly higher or lower than the ultrapure water vehicle control plates. Most of the higher or lower revertant counts of these controls remained in the corresponding historical control data ranges; however, in the initial mutation test in the case of S. typhimurium TA100 (+S9) and in the case of E. coli WP2 uvrA (+S9), the revertant colony numbers of the respective DMSO control, in the confirmatory mutation test in the case of E. coli WP2 uvrA (+S9), the revertant colony numbers of the untreated control were slightly above the corresponding historical control data ranges. These changes were considered as acceptable without any effect on the results and conclusion of the study.

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship: A concentration-response relationship was seen for both positive results (TA1535 and TA100, without metabolic activation)

Ames test:
- Signs of toxicity: No.
- Individual plate counts: See "Attached background material".
- Mean number of revertant colonies per plate and standard deviation: See "Attached background material".

HISTORICAL CONTROL DATA
- Positive historical control data: See "Attached background material".
- Negative historical control data: See "Attached background material".

Applicant's summary and conclusion

Conclusions:
The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item induced gene mutations by base-pair substitution in the genome of the strains of Salmonella typhimurium TA100 and TA1535. In conclusion, the test item is considered mutagenic in this bacterial reverse mutation assay.
Executive summary:

Five bacterial strains were used to investigate the mutagenic potential of the test item in two independent experiments, in a plate incorporation test (experiment I, initial mutation test) and in a pre-incubation test (experiment II, confirmatory mutation test). The test item was dissolved in ultrapure water (ASTM Type I) and the following concentrations were investigated in the initial and confirmatory mutation tests: ±S9: 5000; 3200; 1600; 500; 160; 50 and 16 μg/plate. In the initial and confirmatory mutation tests Salmonella typhimurium TA98, TA100, TA1537, TA1535 and Escherichia coli WP2 uvrA were investigated.  Each assay was conducted with and without metabolic activation (±S9). The concentrations, including the controls, were tested in triplicate. In the performed experiments positive and negative (vehicle) controls were run concurrently. The two fully independent main experiments were performed at the same time, with modified methodology (different methods for treatment), in parallel, in accordance with the referred guidelines. In the performed experiments all of the validity criteria, regarding the investigated strains, negative (vehicle) and positive controls, S9 activity and number of investigated analysable concentration levels were fulfilled.In the initial mutation test, following treatment with the test item significant, biological relevant, dose-related changed revertant colony number increases, positive results were noticed in S. typhimurium TA1535 at the concentrations of 5000 and 3200 μg/plate, in the absence and also in the presence of exogenous metabolic activation (±S9). The mutagenic effect of the test item was unequivocal, characteristic and intensive. The positive results were adequately repeated, confirmed in the confirmatory mutation test in the absence of metabolic activation (-S9). Additionally, unequivocal positive results were obtained in S. typhimurium TA1535 at the concentration of 1600 μg/plate (-S9), and in S. typhimurium TA100 at the concentrations of 5000 and 3200 μg/plate (-S9) (at strains whose auxotrophy was caused by base-pair substitution). While the biologically relevant revertant colony number increases were adequately confirmed, repeated in the case of Salmonella typhimurium TA1535 strain the positive results obtained at the Salmonella typhimurium TA100 were not further investigated, confirmed. In the performed experiments inhibitory effect of the test item (decreased number of revertant colony numbers and/or affected background lawn development) was not observed in any case. No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9) throughout the study.