Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2019-05-07 to 2019-05-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed to current OECD guidelines with no significant deviations and run in OECD GLP certified lab.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Vinyl ethylene carbonate
EC Number:
700-261-7
Cas Number:
4427-96-7
Molecular formula:
C5H6O3
IUPAC Name:
Vinyl ethylene carbonate
Test material form:
liquid
Specific details on test material used for the study:
Batch No.: A03-18-0081
Purity: ≥99.9%

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium, other: TA97a
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 102
Species / strain / cell type:
S. typhimurium TA 1535
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : The S9 was prepared from the livers of the rats induced with Aroclor1254, and the details are shown below: Male SD (Sprague-Dawley) rats were treated by a single intraperitoneal injection of Aroclor 1254 at the dose of 500 mg/kg body weight five days prior to the S9 preparation. The livers of the rats were taken out under aseptic conditions, homogenized in a 0.15 mol/L KCl solution (1g liver: 3 mL KCl solution) and centrifuged at 11000 rpm for 10 min with AllegraTM 64R Auto Freeze Centrifuge. The supernatant of S9 was stored in liquid nitrogen (-196 °C) not more than two years.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Before being used, this batch of S9 was tested for its sterility, its protein content (not higher than 40 mg/mL), and its capability to activate known mutagens in Ames test.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Distilled water (H2O)
Controls
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
other: Dexon; 2-Aminofluorene; 1, 8-Dihydroxyanthraquinone; 2-Aminoanthracene
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : two, the first and validation experiment

METHOD OF TREATMENT/ EXPOSURE:
- Plate-incorporation in the first test, pre-incubation method in the validation test.

TREATMENT AND HARVEST SCHEDULE:
- Plate-incorporation
- In the absence of S9 mix: 200 μL of histidine-biotin solution, 100 μL of fresh cultures of bacteria, 500 μl of PBS, 100 μL of the test item formulation or H2O or positive control solution and 2000 μL of molten top-layer agar medium kept in a water bath at 42.7 °C - 47.5 °C were added to a sterile test tube under aseptic conditions. After vortexing, the mixture was evenly overlaid on the surface of MGA plate, all dose levels and the controls were performed in triplicate. After the top-layer agar was solidified, the plates were inverted and incubated for about 48 hours at 36.0 - 38.7 °C. The test item precipitate was observed by naked eyes in three parallel plates of each dose group of TA98 before and after incubation. After incubation, the number of revertant colonies in each plate was counted. As counting the plates of the positive controls (except for TA1535), the back of a plate was divided into eight sectors on back, and two diagonal sectors were chosen randomly and counted, then the result was multiplied by 4 as the number of revertant colonies, and the signs of background lawn were observed microscopically.
- In the presence of S9 mix: All procedures were the same as those in the absence of S9 mix except for PBS replaced with S9 mix.
- Pre-incubation
- Preincubation period, if applicable: Fresh cultures of bacteria, PBS or S9 mix, and working solution of the test item or solvent or positive control solution were mixed in a sterile test tube and pre-incubated at 35.5 - 36.9 °C for a minimum of 20 min firstly.
- Exposure duration/duration of treatment: All tested plates were inverted and incubated for about 69 hours at 35.0 - 38.4 °C.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: the number of revertant colonies, the data on the background lawn and precipitate of the test item
Evaluation criteria:
CRITERIA OF CYTOTOXICITY
The test item is evaluated as cytotoxicity if one of the below criteria is met.
- Compared with the concurrent solvent control, the number of the revertant colonies has significant decrease or none;
- Compared with the concurrent solvent control, the sign of the background lawn has obvious thinning or clearing.
CRITERIA OF POSITIVE RESULT
- When there is a concentration-related increase over the range (equal to or greater than two times that of the concurrent solvent control in TA97a, TA98, TA100, TA102 and equal to or greater than three times that of the concurrent solvent control in TA1535) in the mean number of revertant colonies in at least one strain with or without metabolic activation system, the test item should be evaluated as positive.
- When there is a reproducible increase (equal to or greater than two times that of the concurrent solvent control in TA97a, TA98, TA100, TA102 and equal to or greater than three times that of the concurrent solvent control in TA1535) at one or more concentrations in the mean number of revertant colonies in at least one strain with or without metabolic activation system, the test item should be evaluated as positive.
CRITERIA OF NEGATIVE RESULT
The test item should be evaluated as negative if none of the above criteria is met.
CRITERIA OF EQUIVOCAL RESULT
Although most tests give clear positive or negative results, in some instances the data generated prohibit making a definite judgment about test item. Results of this type should be reported as equivocal.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium, other: TA97a
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle 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
Remarks:
Cytotoxicity was observed only at 5000 μg/plate dose level.
Vehicle 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
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity was observed only at 5000 μg/plate dose level.
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle 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
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH:
- Data on osmolality:
- Possibility of evaporation from medium:
- Water solubility:
- Precipitation and time of the determination:
- Definition of acceptable cells for analysis:
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES:
- Preliminary test: The standard plate incorporation method was performed at 3 dose levels, including 5000, 1000 and 200 μg/plate, in five tester strains of TA97a, TA98, TA100, TA102 and TA1535, with and without metabolic activation system. Solvent control (H2O) in each tester strain was performed at the same time. The dose volume of each dose group and solvent control group were 0.1ml/plate, in duplicate.
- Preliminary test results: About the precipitation of the test item, monitoring of TA98 showed that there was no precipitate at any designed dose level before and after incubation with and without metabolic activation system. Moreover, compared to the concurrent solvent controls, the test item was considered to be no cytotoxicity to all tester strain at all designed dose levels with and without metabolic activation.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : In the first test and the validation test, the mean number of revertant colonies in the solvent controls and positive controls were within the range of historical control data in this lab. In addition, the background lawn in the solvent controls and positive controls had grown as densely packed microcolonies which form a uniform layer observed with microscope. So the sensitivity of the assay and the efficacy of the S9 mix were validated.

Ames test:
In two tests, the results of the viable count showed that the density of living bacteria in the cultures of each tester strain was within the acceptable range of 0.9 - 9×10^9 colony forming units (CFU)/mL.
In the first test, there was no precipitate at any dose level before and after the incubation, with and without metabolic activation system. In the validation test, the same result was obtained as in the first test.
In the first test, cytotoxicity was observed only at 5000 μg/plate dose level in some tester strains in two treatment conditions, including TA102 with S9mix and TA98 with and without S9mix. It was showed that the number of revertant colonies was significant decrease and/or the background lawn was thinner than the concurrent solvent controls. In the validation test, the same cytotoxicity results were observed as in the first test.
In the first test, with and without metabolic activation, the mean number of revertant colonies at each dose level in all tester strains was less than two times that of the concurrent solvent controls in TA97a, TA98, TA100, TA102 and less than three times that of the concurrent solvent controls in TA1535. In the validation experiment, the same mutagenic result was obtained as in the first test.

Applicant's summary and conclusion

Conclusions:
The results both in the first test and the validation test were negative. The test item considered to be non-mutagenic in the bacterial reverse mutation test using the histidine requiring tester strains of Salmonella typhimurium.
Executive summary:

The study was performed to evaluate the ability of the test item to induce reverse mutations in the genome of the histidine requiring tester strains of Salmonella typhimurium according to OECD Guideline 471 (Bacterial Reverse Mutation Assay).

Five histidine requiring (his-) mutant tester strains of Salmonella typhimurium including TA97a, TA98, TA100, TA102 and TA1535 were treated with Vinyl ethylene carbonate using the standard plate incorporation method and the pre-incubation method at six dose levels, in triplicate, with the solvent controls and positive controls, both in the presence and absence of the cofactor-supplemented S9 (S9 mix).Based on the results of the preliminary test (initial toxicity test) that had been performed in this lab before, six dose levels were selected in the first experiment, including 1500, 500, 150, 50, 15 and 5 g/plate with and without metabolic activation. Distilled water (H2O) was used as solvent. The validation experiment was conducted using the same dose levels and solvent as the first experiment.

 

In two tests, the results of the viable count showed that the density of living bacteria in the cultures of each tester strain was within the acceptable range of 0.9 - 9×10^9 colony forming units (CFU)/mL. At the same time, all results of positive and solvent controls met the requirements of this test, so the sensitivity of the test system and the efficacy of the S9 mix were validated.

In the first test, there was no precipitate at any dose level before and after the incubation, with and without metabolic activation system. In the validation test, the same result was obtained as in the first test.

In the first test, cytotoxicity was observed only at 5000 μg/plate dose level in some tester strains in two treatment conditions, including TA102 with S9mix and TA98 with and without S9mix. It was showed that the number of revertant colonies was significant decrease and/or the background lawn was thinner than the concurrent solvent controls. In the validation test, the same cytotoxicity results were observed as in the first test.

In the first test,with and without metabolic activation, the mean number of revertant colonies at each dose level in all tester strains was less than two times that of the concurrent solvent controls in TA97a, TA98, TA100, TA102 and less than three times that of the concurrent solvent controls in TA1535. In the validation experiment, the same mutagenic result was obtained as in the first test.

 

Under the conditions of this study, the results both in the first test and the validation test were negative. The test item considered to be non-mutagenic in the bacterial reverse mutation test using the histidine requiring tester strains of Salmonella typhimurium.