[(2,4-dibromophenoxy)methyl]oxirane

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:

February - August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

[(2,4-dibromophenoxy)methyl]oxirane

EC Number:

243-606-2

EC Name:

[(2,4-dibromophenoxy)methyl]oxirane

Cas Number:

20217-01-0

Molecular formula:

C9H8Br2O2

IUPAC Name:

2-[(2,4-dibromophenoxy)methyl]oxirane

impurity 1

Reference substance name:

2,4-dibromophenol

EC Number:

210-436-5

EC Name:

2,4-dibromophenol

Cas Number:

615-58-7

Molecular formula:

C6H4Br2O

IUPAC Name:

2,4-dibromophenol

impurity 2

Reference substance name:

[(p-bromophenoxy)methyl]oxirane

EC Number:

218-656-3

EC Name:

[(p-bromophenoxy)methyl]oxirane

Cas Number:

2212-06-8

Molecular formula:

C9H9BrO2

IUPAC Name:

2-[(4-bromophenoxy)methyl]oxirane

impurity 3

Reference substance name:

[(2,4,6-tribromophenoxy)methyl]oxirane

EC Number:

226-140-4

EC Name:

[(2,4,6-tribromophenoxy)methyl]oxirane

Cas Number:

5296-40-2

Molecular formula:

C9H7Br3O2

IUPAC Name:

2-[(2,4,6-tribromophenoxy)methyl]oxirane

Test material form:

liquid

Details on test material:

sample used for testing,
no further details

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: CG272
- Expiration date of the lot/batch: 11. July 2018
- Purity test date: not stated

RADIOLABELLING INFORMATION (if applicable)
not applicable

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: tested in pretest
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: assumed non reactive

Method

Target gene:

his-, trp-

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 100

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 102

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain 5

Species / strain / cell type:

other: S. typhimurium TA 97a

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9

Test concentrations with justification for top dose:

experiment 1a: 5 μL/plate, 1.5 μL/plate, 0.5 μL/plate, 0.15 μL/plate and 0.05 μL/plate
1b:
TA97a with and without metabolic activation: 0.5 μL/plate
TA98 with and without metabolic activation: 0.5 μL/plate
TA100 with metabolic activation: 0. 5 μL/plate
TA100 without metabolic activation: 0.15 μL/plate
TA102 with and without metabolic activation: 0.5 μL/plate
TA1535 with metabolic activation: 1.5 μL/plate
TA1535 without metabolic activation: 0.5 μL/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was chosen as vehicle, because the test item was sufficiently soluble, and this solvent
does not have any effects on the viability of the bacteria or the number of spontaneous
revertants in the tested concentrations.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 4-Nitro-1,2-phenylene diamine, 2-Nitro-Anthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: iin agar (plate incorporation); preincubation;k
- Cell density at seeding (if applicable): not applicable

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable

SELECTION AGENT (mutation assays): not applicable

SPINDLE INHIBITOR (cytogenetic assays): not applicable

STAIN (for cytogenetic assays): not applicable

NUMBER OF REPLICATIONS: 3

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: not applicable

NUMBER OF CELLS EVALUATED: not applicable

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): not applicable

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: not applicable

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth visual as bacterial background lawn and colonies
- Any supplementary information relevant to cytotoxicity:

OTHER EXAMINATIONS:
- Determination of polyploidy: not applicable
- Determination of endoreplication: not applicable
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): not applicable

- OTHER: not applicable

Rationale for test conditions:

according to guideline

Evaluation criteria:

The colonies were counted visually and the numbers were recorded. A validated spreadsheet
software (Microsoft Excel®) was used to calculate mean values and standard deviations
of each treatment, solvent control and positive control.
The mean values and standard deviations of each threefold determination were calculated
as well as the increase factor f(l) of revertant induction (mean revertants divided by mean
spontaneous revertants) of the test item solutions and the positive controls. Additionally, the
absolute number of revertants (Rev. Abs.) (mean revertants minus mean spontaneous revertants)
was given.
A substance is considered to have mutagenic potential, if a reproducible increase of revertant
colonies per plate exceeding an increase factor of 2 in at least one strain can be
observed. A concentration-related increase over the range tested is also taken as a sign of
mutagenic activity.

Statistics:

except for calculation of mean and standard deviation, no statistics applied.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: 97a

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 7

Key result

Species / strain:

S. typhimurium, other: TA97a

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 8

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Experiment 1a

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it is concluded that DENACOL EX-147 is mutagenic in the
Salmonella typhimurium test strains TA100 and TA1535 in the absence and presence of
metabolic activation under the experimental conditions in the present study.

Executive summary:

Two valid experiments were performed.

The study procedures described in this report were based on the most recent OECD and

EC guidelines.

The test item DENACOL EX-147 was tested in the Salmonella typhimurium reverse mutation

assay with five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and

TA1535). The test was performed in two experiments in the presence and absence of S9-

mix (rat liver S9-mix induced by Aroclor 1254).

In experiment 1a, DENACOL EX-147 (dissolved in DMSO) was tested up to concentrations

of 5 μL/plate in the absence and presence of S9-mix in the strains TA97a, TA98, TA100,

TA102 and TA1535 using the plate incorporation method.

DENACOL EX-147 showed no precipitates on the plates at any of the concentrations.

The bacterial background lawn was not present at the two highest concentrations (5 and

1.5 μL/plate) at all bacteria strains and no bacteria growth was observed.

The test item DENACOL EX-147 showed signs of toxicity towards all the bacteria strains in

both the absence and presence of metabolic activation in the following concentrations:

5, 1.5 and 0.5 μL/plate.

In the two lower concentrations (0.15 and 0.05 μL/plate), bacteria growth was observed.

The results of this experiment showed that three of the tested concentrations (0.5, 0.15 and

0.05 μL/plate) showed a significant increase in the number of revertants towards the tested

bacteria strain TA1535 in the presence and the absence of metabolic activation.

Towards the bacteria strain TA100, only in the lowest concentration 0.05 μl/plate in the treatment

without metabolic activation, a significant increase in the number of revertants was

observed.

Based on the results of experiment 1a, DENACOL EX-147 was tested up to the following

concentrations:

TA97a with and without metabolic activation: 0.5 μL/plate

TA98 with and without metabolic activation: 0.5 μL/plate

TA100 with metabolic activation: 0.5 μL/plate

TA100 without metabolic activation: 0.15 μL/plate

TA102 with and without metabolic activation: 0.5 μL/plate

TA1535 with metabolic activation: 1.5 μL/plate

TA1535 without metabolic activation: 0.5 μL/plate

In this experiment, the pre-incubation method was used.

DENACOL EX-147 showed no precipitates on the plates at any of the concentrations.

The bacterial background lawn was not present in the highest concentration (1.5 μL/plate).

In the next lower concentration (0.5 μL/plate) a decrease in the number of revertants was

observed in all tested bacteria strains, except by TA1535.

The results of this experiments showed that the test item DENACOL EX-147 caused an

increase in the number of revertants in the following bacteria strains compared to the solvent

control:

TA100: with and without metabolic activation

TA1535: with and without metabolic activation

The test item DENACOL EX-147 induced an increase in the number of revertants colonies

in these two bacteria strains, in the presence and absence of metabolic activation.

Based on the results of this study it is concluded that DENACOL EX-147 is mutagenic

in the Salmonella typhimurium strains TA100 and TA1535 in the absence and presence

of metabolic activation under the experimental conditions in this study.