Reaction mass of (((1,3-phenylenebis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl) bis(2-methylacrylate) and (1,3-phenylenebis(oxy))bis(propane-3,1-diyl) bis(2-methylacrylate) and 3-(3-(2-(2-(methacryloyloxy)ethoxy)ethoxy)phenoxy)propyl methacrylaterate

Genetic toxicity: in vitro

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)

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 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

Test material information

Constituent 1

Reference substance name:

Reaction mass of (((1,3-phenylenebis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl) bis(2-methylacrylate) and (1,3-phenylenebis(oxy))bis(propane-3,1-diyl) bis(2-methylacrylate) and 3-(3-(2-(2-(methacryloyloxy)ethoxy)ethoxy)phenoxy)propyl methacrylaterate

EC Number:

944-271-1

Cas Number:

2305048-54-6

Molecular formula:

not applicable for multi-constituent.

IUPAC Name:

Reaction mass of (((1,3-phenylenebis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl) bis(2-methylacrylate) and (1,3-phenylenebis(oxy))bis(propane-3,1-diyl) bis(2-methylacrylate) and 3-(3-(2-(2-(methacryloyloxy)ethoxy)ethoxy)phenoxy)propyl methacrylaterate

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: 3M Compan, Lot 653940
- Purity, including information on contaminants, isomers, etc.: 100% (per Protocol)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from light
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Test article dilutions were prepared immediately before use and delivered to the test system at room temperature under filtered light.
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: No data
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: No data
- Reactivity of the test material with the incubation material used (e.g. plastic ware): No data, but not expected

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: test article was solubilized in DMSO
- Preliminary purification step (if any): None

FORM AS APPLIED IN THE TEST: test article was solubilized in DMSO

Method

Target gene:

Salmonella typhimurium: histidine operon, Escherichia coli: tryptophan operon

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:

Type and composition of metabolic activation system:
- source of S9 : Aroclor 1254-induced rat liver S9
- method of preparation of S9 mix : The S9 was prepared from male Sprague-Dawley rats that were injected intraperitoneally with Aroclor™ 1254 (200 mg/mL in corn oil) at a dose of 500 mg/kg, five days before sacrifice.
- concentration or volume of S9 mix and S9 in the final culture medium : 10% v/v
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Each bulk preparation of S9 was assayed for its ability to metabolize benzo(a)pyrene and 2-aminoanthracene to forms mutagenic to Salmonella typhimurium TA100.

Test concentrations with justification for top dose:

50.0, 150, 500, 1500 and 5000 μg per plate, highest dose recommended by OECD 471

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: Solvent chosen based on test article solubility and test system compatibility

- Justification for percentage of solvent in the final culture medium: Solvent levels were within OECD 471 guidelines

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthrocene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : 1

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 0.3x109 cells per milliliter
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: None
- Exposure duration/duration of treatment: 48-72 hours
- Harvest time after the end of treatment (sampling/recovery times): No data

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 48-72 hours
- Selection time (if incubation with a selective agent): 48-72 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 48-72 hours
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure.: tryptophan and histidine minimal agar

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method.: background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICIY
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.
For the test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article as specified below:
Strains TA1535 and TA1537: Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.
Strains TA98, TA100 and WP2 uvrA: Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response was evaluated as negative if it was neither positive nor equivocal.

- OTHER:

Rationale for test conditions:

Per OECD 471.

Evaluation criteria:

For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.
For the test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article as specified below:
Strains TA1535 and TA1537
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.
Strains TA98, TA100 and WP2 uvrA
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response was evaluated as negative if it was neither positive nor equivocal.

Statistics:

No data

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Precipitate was observed beginning at 1500 μg per plate with all conditions.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Precipitate was observed beginning at 1500 μg per plate with all conditions.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Precipitate was observed beginning at 1500 μg per plate with all conditions.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Precipitate was observed beginning at 1500 μg per plate with all conditions.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Precipitate was observed beginning at 1500 μg per plate with all conditions.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No data
- Data on osmolality: No data
- Possibility of evaporation from medium: No data
- Water solubility: DMSO was the vehicle of choice based on the solubility of the test article and compatibility with the target cells. The test article formed a clear solution in DMSO
- Precipitation and time of the determination: Precipitate was evaluated after the incubation period by visual examination without magnification. Precipitate was observed beginning at 1500 ìg per plate with all conditions.
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES (if applicable):
In the preliminary toxicity assay, the dose levels tested were 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 μg per plate. No toxicity was observed. Precipitate was observed beginning at 1000 μg per plate with all conditions. Based upon these results, the maximum dose tested in the mutagenicity assay was 5000 μg per plate.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : control articles performed as expected

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible : No data
- Statistical analysis; p-value if any : No data

Ames test:
- Signs of toxicity : test article must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article
- Individual plate counts : See results.
- Mean number of revertant colonies per plate and standard deviation : No increase in revertant colonies was observed in treated plates.

- Genotoxicity results:
o Number of cells treated and sub-cultures for each cultures : 0.3x109 cells/mL
o Number of cells plated in selective and non-selective medium : 0.3x109 cells/mL
o Number of colonies in non-selective medium and number of resistant colonies in selective medium, and related mutant frequency

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: Contract research lab mantains records of historical positive control data for reference.
- Negative (solvent/vehicle) historical control data: Contract research lab mantains records of historical negetive/solvent control data for reference.

Applicant's summary and conclusion

Conclusions:

Based on the results of the study, DiBrorma is negative in the bacterial reverse mutation assay (Ames assay) in the presence and absence of metabolic activation.

Executive summary:

DiBrorma was tested in a GLP-compliant, OECD 471 (1997) bacterial reverse mutation assay.Salmonella typhimuriumTA98, TA1535, TA100, and TA1537 andEscherichia colistrain WP2uvrA were exposed to dibrorma in DMSO at 50.0, 150, 500, 1500 and 5000 μg per plate with and without an exogenous metabolic activation system, Aroclor 1254-induced rat liver S9. No toxicity was observed. Positive and negative controls behaved as expected. Precipitate was observed beginning at 1500 μg per plate with all conditions. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Based on the results of the study, DiBrorma is negative in the bacterial reverse mutation assay (Ames assay) in the presence and absence of metabolic activation.