4-(2-acryloyloxy-ethoxy) benzophenone

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:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

The test method was based on MHLW Japan No. 7 (2011), MOL Japan Notice No. 67 (1997), and MOL Japan Notice No. 67 (1997).

Deviations:

no

Remarks:

No deviations ocurred that impacted the integrity of the study.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-(2-acryloyloxy-ethoxy) benzophenone

EC Number:

836-681-3

Cas Number:

22421-66-5

Molecular formula:

C18H16O4

IUPAC Name:

4-(2-acryloyloxy-ethoxy) benzophenone

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: 3M Company, Lot 10001
- Expiration date of the lot/batch: no data
- Purity test date: No data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at room temperature, shielded from light.
- Stability under storage conditions: Stable
- Stability under test conditions: Stable - Heat, discoloring and foam formation were not observed in preparation of the test substance and the stability of the test substance was confirmed after 2 hours.
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: The test article dissolved in DMSO at 50 mg/L

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test article was dissolved in DMSO.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Disolved in DMSO.

Method

Target gene:

Histidine operon, 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 : 7 week old male Sprague-Dawley rats treated with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

TA100 without S9: 39.1, 78.1, 156, 313, 625, 1250 ug/plate
TA100 with S9: 39.1, 78.1, 156, 313, 625, 1250, 2500, 5000 ug/plate
TA1535 without S9: 39.1, 78.1, 156, 313, 625, 1250, 2500, 5000 ug/plate
TA1535 with S9: 39.1, 78.1, 156, 313, 625, 1250, 2500, 5000 ug/plate
TA98 without S9: 9.77, 19.5, 39.1, 78.1, 156, 313, 625, 1250 ug/plate
TA98 with S9: 156, 313, 625, 1250, 2500, 5000 ug/plate
TA1537 without S9: 9.77, 19.5, 39.1, 78.1, 156, 313, 625, 1250 ug/plate
TA1537 with S9: 39.1, 78.1, 156, 313, 625, 1250, 2500, 5000 ug/plate
WP2uvrA without S9: 313, 625, 1250, 2500, 5000 ug/plate
WP2uvrA with S9: 313, 625, 1250, 2500, 5000 ug/plate

Vehicle / solvent:

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

- Justification for choice of solvent/vehicle: Test article solubility and test system compatibility.

- Justification for percentage of solvent in the final culture medium: Per the test guideline.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: 2-Aminoanthracene: All strains with S9 mix. 2-(2-Fury)-3-(5-nitro-2-furyl) acrylamide: TA100, TA98, and WP2uvrA without S9, 9-Aminoacridine hydrochloride hydrate: TA1537 without S9

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate
- Number of independent experiments : Two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): No data
- Test substance added in preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 minutes
- Exposure duration/duration of treatment: 48 hours
- Harvest time after the end of treatment (sampling/recovery times): 48 hours

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 48 hours
- Selection time (if incubation with a selective agent): 48 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 48 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: Histidine and tryptophan-minimal agar for 48 hours
- Criteria for small (slow growing) and large (fast growing) colonies: No data

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

METHODS FOR MEASUREMENTS OF GENOTOXICIY : A test article was considered mutagenic if it induced a dose-dependent increase in the number of revertant colonies to a level equal to or greater than 2-fold of the solvent control in any tester strain with or without metabolic activation.

Rationale for test conditions:

Per MHLW Japan No. 7 (2011), MOL Japan Notice No. 67 (1997), and MOL Japan Notice No. 67 (1997).

Evaluation criteria:

A test was considered valid if:
- The positive control substances produced clear positive results in their respective tester strains (see positive control detail section).
- There are more than 4 doses showing no microbial growth inhibition and more than 5 doses applicable to evaluation.
- The results of the sterility test indicate that there was no bacterial contamination.
- There are no plates that become invalid for measurement due to contamination or other unexpected reasons.

Statistics:

No statistical analysis was conducted.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

313 ug/plate and greater without S9, 5000 ug/plate with S9

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 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

625 ug/plate and greater without S9, 1250 ug/plate and greater with S9

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 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

625 ug/plate and greater without S9, 1250 ug/plate and greater with S9

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 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

313 ug/plate and greater without S9, 1250 ug/plate and greater with S9.

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:

Precipitation was observed at 625 ug/plate and greater.

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: Not expected based on the phys-chem properties of the test article.
- Water solubility: The test article did not dissolve or suspend in water at 50 mg/mL
- Precipitation and time of the determination: Precipitation was determined at colony counting. See "Test Results" section for data on precipitation in individual strains.

RANGE-FINDING/SCREENING STUDIES: A range-finding study was conducted up to 5000 ug/plate to determine appropriate dose levels. Microbial growth inhibition was observed at the following dose levels:

Without S9: 1250 ug/plate or greater (TA100, TA1535)
313 ug/plate or greater (TA98, TA1537)

With S9: 1250 ug/plate or greater (TA100, TA1535, TA1537)
5000 ug/plate (TA98)

Precipation of the test article was observed at the the following dose levels:
WIthout S9 at 1250 ug/plate or greater in all strains.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : Solvent and positive controls performed as expected, producing negative and clear positive results, respectively.

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible : A dose-dependent increase in revertant colonies of greater than 2-fold the solvent control is required for a positive genotoxicity conclusion.
- Statistical analysis; p-value if any : Statistical analysis was not perfomed on the results.


Ames test:
- Signs of toxicity : See cytotoxicity results in "Test Results" section.
- Individual plate counts : See attached results tables.
- Mean number of revertant colonies per plate and standard deviation : See attached results tables

- Genotoxicity results: See attached results tables

HISTORICAL CONTROL DATA: See attached results tables.

Applicant's summary and conclusion

Conclusions:

Based on the results of the study, AEBP is not mutagenic in the bacterial reverse mutation (Ames) assay in the presence or absence of metabolic activation (S9).

Executive summary:

The mutagenic potential of AEBP was evaluated in the Bacterial Reverse Mutation Assay with S. typhimurium strains TA98, TA100, TA1535, and TA1537 and E. coli strain WP2 uvrA in the presence and absence of a metabolic activation system (S9 mix). This study was performed in compliance with MHLW Japan GLP Notification 0331 No. 8 PFSB (2011), MOL Japan Notice No. 76 (1988), and MOL Japan Notice No. 13 (2000). The test method was based on MHLW Japan No. 7 (2011), MOL Japan Notice No. 67 (1997), and MOL Japan Notice No. 67 (1997). AEBP was prepared in DMSO at a ratio of 50 mg/mL. The solution was then diluted with DMSO to form the test substance concentrations used in the test. Strain specific positive controls (AF-2, NaN3, 9-AA, and 2-AA) along with a negative solvent control (DMSO) were also prepared. A dose-finding test was performed prior to the main study with all strains with and without metabolic activation at doses of 1.22, 4.88, 19.5, 78.1, 313, 1250 and 5000 ug/plate. From the results of the dose-finding test, all strains were tested in the main study at the following doses: TA100 without S9: 39.1, 78.1, 156, 313, 625, 1250 ug/plate. TA100 with S9: 39.1, 78.1, 156, 313, 625, 1250, 2500, 5000 ug/plate. TA1535 without S9: 39.1, 78.1, 156, 313, 625, 1250, 2500, 5000 ug/plate. TA1535 with S9: 39.1, 78.1, 156, 313, 625, 1250, 2500, 5000 ug/plate. TA98 without S9: 9.77, 19.5, 39.1, 78.1, 156, 313, 625, 1250 ug/plate. TA98 with S9: 156, 313, 625, 1250, 2500, 5000 ug/plate. TA1537 without S9: 9.77, 19.5, 39.1, 78.1, 156, 313, 625, 1250 ug/plate. TA1537 with S9: 39.1, 78.1, 156, 313, 625, 1250, 2500, 5000 ug/plate. WP2uvrA without S9: 313, 625, 1250, 2500, 5000 ug/plate. WP2uvrA with S9: 313, 625, 1250, 2500, 5000 ug/plate. There was no increase in the number of revertant colonies. The number of revertant colonies in the test substance treated groups was less than twice that of the corresponding negative control in all strains at all doses with or without metabolic activation. The results were confirmed based on the reproducibility of the test. Based on the results of the study, AEBP is not mutagenic in the bacterial reverse mutation (Ames) assay in the presence or absence of metabolic activation (S9).