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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

DiBrORMA has been tested in two in vitro genotoxicity/mutagenicity studies. The results of the studies were:

Negative when tested according to OECD 471 in the presence and absence of metabolic activation.

Positive when tested according to OECD 490 in the presence of metabolic activation.

Link to relevant study records

Reference

Reference 1

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

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

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

Target gene:

Salmonella typhimurium: histidine operon, Escherichia coli: tryptophan operon

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

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

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

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

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

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

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.

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

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: SHBH7951
- Purity, including information on contaminants, isomers, etc.: 99.96%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At 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:
Stable for the duration of the study.
- 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.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): Prior to use in the assay, L5178Y/TK+/- cells were cleansed to reduce the frequency of
spontaneously occurring TK-/- cells. Using the procedure described by Clive and Spector (1975), L5178Y cells were cultured for 24 hours in the presence of thymidine, hypoxanthine, methotrexate and glycine to poison the TK-/- cells. L5178Y/TK+/- cells were prepared in 50% conditioned F0P supplemented with 10% horse serum and 2 mM L-glutamine (F10P) and 50% Fischer's Media for Leukemic Cells of Mice with 0.1% Pluronics F-68 (F0P). All media contained antibiotics.

Target gene:

thymidine kinase locus in L5178Y mouse lymphoma cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: L5178Y/TK+/- mouse lymphoma cells, the American Type Culture Collection (repository number CRL-9518), Manassas, VA.
- Suitability of cells: No data.

For cell lines:
- Absence of Mycoplasma contamination: Yes.
- Number of passages if applicable: No data.
- Methods for maintenance in cell culture: No data.
- Cell cycle length, doubling time or proliferation index : No data.
- Modal number of chromosomes: No data.
- Periodically checked for karyotype stability: No data.
- Periodically ‘cleansed’ of spontaneous mutants: Yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: 50% conditioned F0P supplemented with 10% horse serum and 2 mM L-glutamine (F10P) and 50% Fischer's Media for Leukemic Cells of Mice with 0.1% Pluronics F-68 (F0P). All media contained antibiotics.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : purchased commercially from Moltox (Boone, NC).
- 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 µL/mL
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Each lot of
S9 was assayed for sterility and its ability to metabolize at least two pro-mutagens to forms
mutagenic to Salmonella typhimurium TA100.

Test concentrations with justification for top dose:

In the preliminary toxicity assay, the concentrations tested were 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 µg/mL. The maximum concentration evaluated approximated the limit dose for this assay.

Vehicle / solvent:

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

- Justification for choice of solvent/vehicle: OECD 490.

- Justification for percentage of solvent in the final culture medium:OECD 490.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

other:

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): 1 x 106 cells/100 mm plate
- Test substance added in cloning medium containing 0.22 to 0.24% agar. For estimation of
cloning efficiency at the time of selection of those same cultures, 200 cells/100 mm plate were cultured in triplicate in cloning medium without TFT (viable cell (VC) plate). Cultures were incubated under standard conditions (37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air) for 11 days.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: No data.
- Exposure duration/duration of treatment: 4 hour treatment – 1 and 2 days after treatment.
24 hour treatment – immediately after test article removal, and 2 and 3 days after
treatment.

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 2 days
- Selection time (if incubation with a selective agent): 11 days
- Fixation time (start of exposure up to fixation or harvest of cells):
- Method used: agar or microwell plates for the mouse lymphoma assay: agar.
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure. Trifluorothymidine-resistant colonies for the positive and vehicle control cultures, as well as the
test article-treated cultures at concentrations ≥31.3 µg/mL using a 4 hour treatment with S9, were sized according to diameter over a range from approximately 0.2 to 1.1 mm
- Number of cells seeded and method to enumerate numbers of viable and mutants cells:
- Criteria for small (slow growing) and large (fast growing) colonies: Small: ≤0.63 mm, Large: >0.63 mm

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: The cytotoxic effects of each treatment condition were expressed relative to the vehicle-treated control for suspension growth over two days post-treatment and for total growth (suspension growth corrected for plating efficiency at the time of selection). The mutant frequency for each treatment condition was calculated by dividing the mean number of colonies on the TFT-plates by the mean number of colonies on the VC-plates and multiplying by the dilution factor (2 x 10-4), and was expressed as TFT-resistant mutants/106 surviving cells. The induced mutant frequency (IMF) was defined as the mutant frequency of the treated culture minus the mutant frequency of the vehicle control cultures.

Rationale for test conditions:

Per OECD Guideline 490.

Evaluation criteria:

A result was considered positive if a concentration-related increase in mutant frequency was observed in the treated cultures and one or more treatment conditions with 10% or greater total growth exhibited induced mutant frequencies of ≥90 mutants/106 clonable cells (based on the average mutant frequency of duplicate cultures). If the average vehicle control mutant frequency was >90 mutants/106 clonable cells, a doubling of mutant frequency over the vehicle would also be required.
A result was considered negative if the treated cultures exhibited induced mutant frequencies of less than 90 mutants/106 clonable cells (based on the average mutant frequency of duplicate cultures) and there was no concentration-related increase in
mutant frequency.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: The test article did not have an adverse impact on the pH of the cultures (pH 7.5 at the top dose).
- Data on osmolality: The osmolality of the cultures was acceptable as it did not exceed the osmolality of the vehiclecontrol by more than 120%.
- Possibility of evaporation from medium: no data.
- Water solubility: no data.
- Precipitation and time of the determination: Visible precipitate was observed at concentrations ≥125 µg/mL at the beginning of treatment and at concentrations ≥250µg/mL by the end of treatment with 4-hour treatment without S9.
- Definition of acceptable cells for analysis:
- Other confounding effects:

STUDY RESULTS
- Concurrent vehicle negative and positive control data

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.
- Any other criteria: GEF

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
o Relative total growth (RTG) or relative survival (RS) and cloning efficiency: Relative
total growth of the cloned cultures ranged from 12 to 46% (4-hour treatment with S9), 30 to 103% (4-hour treatment without S9) and 10 to 97% (24-hour treatment without S9). One
replicate at 50 µg/mL for 24-hour treatment without S9 was too toxic with RSG value less than 10%, therefore, they were excluded from counting.

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: The contract lab that conducted the study keeps records of historical control ranges to determine if a study is within historical values.

Conclusions:

These results indicate DiBrORMA was positive for the ability to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells, in the presence of an exogenous metabolic activation system.

Executive summary:

An in vitro mammalian cell gene mutation test was conducted on DiBrORMA according to OECD Guideline 490 and GLP in mouse lymphoma L5178Y cells. The test article was evaluated for its ability to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells in the presence and absence of an exogenous metabolic activation system. Dimethyl sulfoxide (DMSO) was used as the vehicle. In the preliminary toxicity assay, the concentrations tested were 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 µg/mL. The maximum concentration evaluated approximated the limit dose for this assay. Relative suspension growth (RSG) was 17, 30 and 34% at concentrations of 62.5 µg/mL (4-hour treatment with S9), 250 µg/mL (4-hour treatment without S9) and 31.3 µg/mL (24-hour treatment without S9), respectively. RSG was 0% at all higher concentrations using all treatment conditions. Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 3.91, 7.81, 15.6, 31.3, 62.5 and 100 µg/mL (4-hour treatment with S9), 15.6, 31.3, 62.5, 125 and 250 µg/mL (4-hour treatment without S9) and 3.91, 7.81, 15.6, 31.3, 50 and 62.5 µg/mL (24-hour treatment without S9). In the definitive mutagenicity assay, visible precipitate was observed at concentrations ≥125 µg/mL at the beginning of treatment and at concentrations ≥250µg/mL by the end of treatment with 4-hour treatment without S9. Cultures treated at concentrations of 3.91, 7.81, 15.6, 31.3 and 62.5 µg/mL (4-hour treatment with S9), 15.6, 31.3, 62.5, 125 and 250 µg/mL (4-hour treatment without S9) and 3.91, 7.81, 15.6, 31.3 and 50 µg/mL (24-hour treatment without S9) exhibited 17 to 38%, 28 to 94% and 11 to 106% RSG, respectively, and were cloned. Relative total growth of the cloned cultures ranged from 12 to 46% (4-hour treatment with S9), 30 to 103% (4-hour treatment without S9) and 10 to 97% (24-hour treatment without S9). One replicate at 50 µg/mL for 24-hour treatment without S9 was too toxic with RSG value less than 10%, therefore, they were excluded from counting. No increases in induced mutant frequency ≥90 mutants/106 cloneable cells were observed under treatment condition without S9. Increases in induced mutant frequency ≥90 mutants/106 cloneable cells were observed at concentrations ≥31.3 µg/mL using a 4-hour treatment with S9. One replicate at 31.3 µg/mL using a 4-hour treatment with S9 was ≤ 90 mutants/106 cloneable cells however the other replicate and the average was ≥90 mutants/106 cloneable cells, therefore the dose level 31.3 µg/mL is also considered as positive. Trifluorothymidine-resistant colonies for the positive and vehicle control cultures, as well as the test article-treated cultures at concentrations ≥31.3 µg/mL using a 4 hour treatment with S9, were sized according to diameter over a range from approximately 0.2 to 1.1 mm. The data on colony size distributions showed both an increase in the frequency of small (≤0.63 mm) and large colonies (>0.63 mm) when the test article-treated cultures were compared to the vehicle control cultures. An increase in the frequency of small colonies is consistent with damage to multiple loci on chromosome 11 in addition to functional loss of the TK locus. An increase in large colony mutants is indicative of localized damage in the form of a point mutation or small deletion within the TK locus. The colony sizing for the MMS and DMBA positive controls yielded the expected increase in small colonies (verifying the adequacy of the methods used to detect small colony mutants) and large colonies. These results indicate DiBrORMA was positive for the ability to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells, in the presence of an exogenous metabolic activation system.

Genetic toxicity in vivo

Description of key information

DiBrORMA was tested in an in vivo genotoxicity/mutagenicity study. The result of the study was:

Negative when tested according to OECD 474.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

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 Company, Batch 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: No data
- 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

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): None, test article was administered unchanged.
- Preliminary purification step (if any): No data

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

Species:

mouse

Strain:

CD-1

Details on species / strain selection:

Test system recommended per OECD 474.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS, Inc., Frederick, MD
- Age at study initiation: 7 weeks
- Weight at study initiation: 30.6-35.3 g
- Assigned to test groups randomly: Yes, randomization procedure within Microsoft Excel
- Fasting period before study: No data
- Housing: See below
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 72°F
- Humidity (%): 50
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): No data

Route of administration:

oral: gavage

Vehicle:

None

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: None, dosed neat.

Duration of treatment / exposure:

Exposed once

Frequency of treatment:

Exposed once

Dose / conc.:

0 mg/kg diet

Dose / conc.:

500 mg/kg diet

Remarks:

equivalent to 0.35 mL/kg

Dose / conc.:

1 000 mg/kg diet

Remarks:

equivalent to 0.69 mL/kg

Dose / conc.:

2 000 mg/kg diet

Remarks:

equivalent to 1.39 mL/kg

No. of animals per sex per dose:

6

Control animals:

yes, concurrent no treatment

Positive control(s):

Cyclophosphamide (CP)
- Justification for choice of positive control(s): Per OECD 474
- Route of administration: Oral gavage
- Doses / concentrations: 50 mg/kg

Tissues and cell types examined:

Polychromatic erythrocytes (PCE)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Based on a dose range-finding study.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): 24 and 48 hours after treatment

DETAILS OF SLIDE PREPARATION: Cells were re-suspended and a small drop of the bone marrow suspension was spread onto a clean glass slide. Four slides were prepared from each animal, air dried and fixed by dipping in methanol. One set of two slides (including 5 positive control slides) were stained with acridine orange for microscopic evaluation. The other set of slides was kept as backup and were archived. Each slide was identified by the harvest date, study number, and animal number (or slide number for positive control slides). Slides were coded using a random number table by an individual not involved with the scoring process.

METHOD OF ANALYSIS: The criteria for the identification of micronuclei are those of Schmid (1975). Micronuclei are brightly stained bodies that generally are round and that generally are between 1/20 and 1/5 the size of the PCE. Scoring was based upon the micronucleated cell, not the micronucleus; thus, occasional cells with more than one micronucleus were counted as one micronucleated PCE (MnPCE), not two (or more) micronuclei.

OTHER: 4000 PCE/animal

Evaluation criteria:

A test article was considered to have induced a positive response if:
a) at least one of the test article doses exhibited a statistically significant increase when compared with the concurrent negative control (p ≤ 0.05), and
b) when multiple doses were examined at a particular sampling time, the increase was dose-related (p ≤ 0.01 and R2≥70%), and
c) results of the group mean or of the individual animals in at least one group were outside the 95% control limit of the historical negative control data.
A test article was considered to have induced a clear negative response if none of the criteria for a positive response were met and there was evidence that the bone marrow was exposed to the test article (unless intravenous administration was used).

Statistics:

Statistical analysis was performed on the micronucleus frequency (%MnPCE) and %PCE
using the animal as the unit. The mean and standard deviation of %MnPCE and %PCE were
presented for each treatment group.
The use of parametric or non-parametric statistical methods in the evaluation of data was
based on the variation between groups. The group variances for micronucleus frequency for
the untreated and test article groups at the respective sampling time were compared using
Levene’s test (significant level of p less than or equal to 0.05). Since the variation between groups was found
not to be significant; a parametric one-way ANOVA was performed followed by a Dunnett’s
post-hoc analysis to compare each dose group to the concurrent untreated control.
A linear regression analysis was conducted to assess dose responsiveness in the test article
treated groups (p less than or equal to 0.01 and R2 ≥ 70%).
A pair-wise comparison (Student’s T-test; p less than or equal to 0.05) was used to compare the positive control
group to the concurrent untreated control group.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not applicable

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg
- Rationale for exposure: limit dose as recommended by guideline

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): No statistically significant increase in the incidence of MnPCEs was observed in the test article treated groups relative to the untreated group at either time point (24 or 48 hours).
- Ratio of PCE/NCE (for Micronucleus assay): No appreciable reductions in the PCE/EC ratio were observed in the test article groups compared to the untreated group, indicating the test article did not induce cytotoxicity.
- Appropriateness of dose levels and route: As recommended by guideline
- Statistical evaluation: Statistical analysis was performed on the micronucleus frequency (%MnPCE) and %PCE using the animal as the unit. The mean and standard deviation of %MnPCE and %PCE were presented for each treatment group. The use of parametric or non-parametric statistical methods in the evaluation of data was based on the variation between groups. The group variances for micronucleus frequency for the untreated and test article groups at the respective sampling time were compared using Levene’s test (significant level of p < 0.05). Since the variation between groups was found not to be significant; a parametric one-way ANOVA was performed followed by a Dunnett’s post-hoc analysis to compare each dose group to the concurrent untreated control. A linear regression analysis was conducted to assess dose responsiveness in the test article treated groups (p< 0.01 and R2 ≥ 70%). A pair-wise comparison (Student’s T-test; p < 0.05) was used to compare the positive control group to the concurrent untreated control group.

Conclusions:

Based on the increased plasma bromine concentrations following dosing, the test article was absorbed and distributed to the body and therefore the bone marrow was properly exposed. No significant increase in the presence of micronuleated polychromatic erythrocytes (MnPCEs) was observed in any group indicating that DiBrORMA is negative in the mouse micronucleus assay.

Executive summary:

The test article, DiBrorma, was evaluated for its clastogenic activity and/or disruption of the mitotic apparatus by detecting micronuclei in polychromatic erythrocytes (PCEs) in mouse bone marrow in a GLP-compliant study conducted according to OECD Guideline 474 (2016). CD-1 mice (6 males/group at 24 hoursand an additional 6 males in the high dose and control groups at 48 hours) received 0 (untreated), 500, 1000, or 2000 mg/kg of undiluted DiBrORMA via single oral gavage. Blood was collected from 3 animals/timepoint at 1 hour (control only), 2, 4, 8, and 24 hours post-dose.The mice were euthanized at 24 hours or 48 hours post-dose as indicated above.The collected plasma samples were evaluated for the presence of bromine following a digestion step to release any covalently bound bromine (i.e., bromine bound in the test article compound or its metabolites). No significant increase in the presence of micronuleated polychromatic erythrocytes (MnPCEs) was observed in any group. Plasma bromine concentrations were increased following treatment with DiBrORMA while the concentration in the control group was approximately 2.4 ug/mL.The highest individual peak concentrations (Cmax) were 36.2, 58.9, and 57.6 ug/mL for the 500, 1000, and 2000 mg/kg groups, respectively, at 2, 4, and 2 hours post-dose, respectively (Tmax). At 24 hours post-dose,the mean plasma bromine concentrations had decreased to 6.1, 5.1, and 10.4 ug/mL for the 500, 1000, and 2000 mg/kg groups, respectively. Based on the increased plasma bromine concentrations following dosing, the test article was absorbed and distributed to the body and therefore the bone marrow was properly exposed.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on the results of the negative in vivo study, DiBrORMA does not meet the criteria for classification according to GHS.