[(2,4-dibromophenoxy)methyl]oxirane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

one Guideline study according to OECD 471 available, resulting in positive genotoxicity under test conditions.

one Guideline study according to OECD 476 available, resulting in positive genotoxicity under test conditions.

one Guideline study according to OECD 487 available, resulting in inconclusive genotoxicity results under test conditions.

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:

February - August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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

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

Target gene:

his-, trp-

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

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.

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.

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

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

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

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

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

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

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

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

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18. August 2017 - 23. May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

Name DENACOL EX-147
Batch no. CG272
Appearance yellow liquid
Composition Mono Constituent, [(2,4-Dibromophenoxy)methyl]oxirane
Purity ≥ 90% (GC)
Homogeneity homogeneous liquid
Expiry date 11. Jul. 2018
Storage Room Temperature (20 ± 5°C)

Target gene:

Hprt

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

The V79 cell line has been used successfully in in vitro experiments for many years because
of its sensitivity to chemical mutagens. Especially the high proliferation rate (doubling
time 12 – 16 h in stock cultures) and a high cloning efficiency of untreated cells both
necessary for the appropriate performance of the study, recommend the use of this cell
line. The cells have a stable karyotype with a modal chromosome number of 22 (Bradley
et al., 1981). The cells were purchased by CLS (Eppelheim, Germany) and were sold under
the name V79-4. The modal chromosome number was analysed and confirmed by the
supplier of the cells.
Prior to use in the experiments, the cell cultures were cleansed of pre-existing mutant cells
by culturing in HAT medium (medium containing Hypoxanthine, Aminopterin and Thymidine).
Cleansed and for mycoplasma contamination screened stocks of cells were stored
in liquid nitrogen in the cell bank of LAUS GmbH to allow a continuous working stock of
cells, which guarantees similar parameters of the experiment and reproducible characteristics
of the cells.
The cells were thawed 6 - 9 d prior treatment and cultivated in DMEM complete culture
medium with 5 % HS in cell culture flasks at 37.0 ± 1.5 °C in a humidified atmosphere with
5.0 ± 0.5 % CO2. The normal cell cycle time as well as the detection of the spontaneous
mutant frequency of the used master cell stock is checked in each experiment. The data
are stored in the LAUS archive.

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9-Mix

Test concentrations with justification for top dose:

400, 200, 100, 50, 25, 12.5, 6.3 mg/ml
The test item was sufficiently soluble in DMSO
at the required concentration (400 mg/mL).

Vehicle / solvent:

DMSO
The test item was sufficiently soluble in DMSO at the required concentration (400 mg/mL).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium;
- Cell density at seeding (if applicable): 1 * 10^6 cells per 10 cm culture dish (for the determination of viability and mutagenicity) and 500 cells (for the determination of the cytotoxicity)
per 6 cm culture dish were seeded

DURATION
- Preincubation period: 24 h
- Exposure duration: 4 or 24 h
- Expression time (cells in growth medium): 7 days (for the determination of the cytotoxicity), 168 h (for the determination of viability and mutagenicity)
- Selection time (if incubation with a selection agent): 7 days
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays): 6-Thioguanin

SPINDLE INHIBITOR (cytogenetic assays): not applicable

STAIN (for cytogenetic assays): not applicable

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
0.1 % Löffler’s methylene blue solution in 0.01 % KOH solution

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: cloning efficiency;
- Any supplementary information relevant to cytotoxicity:

OTHER EXAMINATIONS:
none

Rationale for test conditions:

according to Guideline

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be
clearly positive if, in any of the experimental conditions examined:
 at least one of the test concentrations exhibits a statistically significant increase
compared with the concurrent negative control,
 the increase is concentration-related when evaluated with an appropriate trend test,
 any of the results are outside the distribution of the historical negative control data.
When all of these criteria are met, the test chemical is then considered able to induce gene
mutations in cultured mammalian cells in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly
negative if, in all experimental conditions examined:
 none of the test concentrations exhibits a statistically significant increase compared
with the concurrent negative control,
 there is no concentration-related increase when evaluated with an appropriate trend
test,
 all results are inside the distribution of the historical negative control data.
The test chemical is then considered unable to induce gene mutations in cultured mammalian
cells in this test system.
However, in a case by case evaluation this decision depends on the level of the corresponding
solvent control data. If there is by chance a low spontaneous mutation rate within
the laboratories historical control data range, a concentration-related increase of the mutations
within this range has to be discussed. The variability of the mutation rates of solvent
controls within all experiments of this study is also taken into consideration.
In cases when the response is neither clearly negative nor clearly positive as described
above, or in order to assist in establishing the biological relevance of a result, the data
should be evaluated by expert judgement and/or further investigations.

Statistics:

none applied

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

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

Additional information on results:

The gene mutation assay is considered acceptable if it meets the following criteria:
1. the mutant frequency found in the solvent controls falls within the laboratory historical
95% control limit.
2. the positive control substances must produce a significant increase (p < 0.05) in mutant
frequency and lies in the range of the laboratory historical 95% control limit.
3. two experimental conditions (+S9 and -S9) are tested unless one resulted in positive
results.
4. adequate number of cells (spontaneous MF is 5 - 20 per 106 cells) and concentrations
(minimum of 4) are analysable.
5. the criteria for the selection of top concentration are consistent with those described in
chapter 7.3.
All acceptability criteria were met. Therefore, the study is considered as valid.

Conclusions:

In conclusion, it can be stated that under the experimental conditions of this study
DENACOL EX-147 induced gene mutations at the HPRT locus in V79 cells in the absence
and presence of metabolic activation.
Therefore, the test item DENACOL EX-147 is considered to be “mutagenic under the conditions
of the HPRT assay”.

Executive summary:

This study was performed to investigate the potential of DENACOL EX-147 to induce mutations

at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese

Hamster cells (V79).

The assay comprised two pre-tests and two independent experiments (experiment I and

II). The pre-tests were done to detect a potential cytotoxic effect of the test item. Based on

the results of those tests the concentrations for the main experiments were determined.

The first main experiment (experiment I) was performed with and without metabolic activation

(liver S9 mix from male rats, treated with Aroclor 1254) and a treatment period of 4 h.

The second experiment (experiment II) was performed with a treatment period of 24 hours

without metabolic activation.

The highest nominal concentration (experiment I +S9: 125 μg/mL; -S9: 31.3 μg/mL; experiment

II -S9: 15.6 μg/mL) applied was chosen with regard to the solubility of the test item in

organic solvents and aqueous media and the cytotoxicity.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in

mutant colonies and thus, showed enough sensitivity of the testing procedure and the activity

of the metabolic activation system.

The evaluated experimental points and the results are summarized in chapter 8, page 21.

No statistically significant dose-dependent effect was observed but a substantial and reproducible

increase in mutant colony numbers was observed in both experiments up to the

maximal concentration of the test item. Since this effect was observed in all evaluated

concentrations, the test item is considered as mutagenic despite the fact that one of the

criteria is not fulfilled

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16. August - 18. December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Name DENACOL EX-147
Batch no. CG272
Appearance yellow liquid
Composition Mono Constituent, [(2,4-Dibromophenoxy)methyl]oxirane
Purity ≥ 90% (GC)
Homogeneity homogeneous liquid
Expiry date 11. Jul. 2018
Storage Room Temperature (20 ± 5°C)

Target gene:

no single target gene

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Specification
Human whole blood treated with anti-coagulant (heparin).
BloodCollection
Blood samples were obtained from healthy donors who neither smoke nor receive medication.
Primary cultures of human peripheral lymphocytes are preferred for this type of study because
of their low and stable background rate of micronuclei. In addition, human cells are
generally the most relevant ones for risk assessment.

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9

Test concentrations with justification for top dose:

The solubility of the test item was determined in a non-GLP pre-test in cell culture medium
(DMEM) and dimethyl sulfoxide (DMSO). The test item was sufficiently soluble in DMSO at
the required concentration (400 mg/mL). Therefore DMSO was used as solvent for the test
item.
Preparation was the same for all experiments:
On the day of the exposure, a stock solution with an appropriate concentration (pre-test:
400 mg/mL, experiment I: 25 mg/mL, experiment II: 12.5 mg/mL) of the test item in DMSO
was prepared. DMSO was chosen as solvent, because this solvent has no effects on the
viability of cells, does not show genetic toxicity and because the test item was sufficiently
soluble in it. The stock solutions were used to prepare the geometric series of the concentrations
to be tested

Experimental Setup Experiment I
Nominal concentration of test
item solution (mg/mL)
25 12.5 6.25 3.13 1.56
Resulting nominal concentration
in the experiment (μg/mL)
125 62.5 31.3 15.6 7.8

Experiment I: Concentrations Selected for Scoring of Micronuclei
Test item concentrations (μg/mL)
With S9 mix 62.5 31.3 15.6
Without S9 mix 31.3 15.6 7.8

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Colchicine

Details on test system and experimental conditions:

Cell Cultivation, Treatment and Preparation
Cell Cultivation
The blood cultures were set up in defined time intervals within 24 h after collection in sterile
culture vessels, each containing 1 part of heparinised blood and 9 parts of complete
culture medium RPMI 1640 for cell proliferation. The cultures were incubated for 72 h at
37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2.
Cell Treatment
After the initial cell cultivation, duplicate cultures were prepared for each test group. After
centrifugation (10 min, 500 * g), the cells were resuspended in serum free RPMI 1640 and
solvent control, positive control or the single test item concentrations were added.
In the case of metabolic activation, 50 μL S9 mix per mL medium were used. The cell cultures
were incubated at 37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2 for 4 h
(exposure period).

After the exposure time of 4 h, the cells were spun down by gentle centrifugation for 5 min
(500 * g). The supernatant was discarded, the cells were re-suspended in 5 mL Saline G
and centrifuged again. The washing procedure was repeated once as described.
After washing, the cells were re-suspended in complete culture medium RPMI 1640, cytochalasin
B (final concentration 5 μg/mL) was added and the cells were incubated at
37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2 for 19 h until preparation.
In experiment II without metabolic activation, 72 h after seeding, the blood cultures were
centrifuged (10 min, 500 * g). The cell pellet was re-suspended in complete culture medium
RPMI 1640, cytoB (final concentration 5 μg/mL) and solvent control, positive control or
the test item concentrations were added. The exposure duration was 23.5 h.
7.2.3 Harvesting Procedure
Each cell culture was harvested and processed separately. The cells were spun down by
gentle centrifugation (10 min, 500 * g). The supernatant was discarded and the cells were
resuspended in 12 ml hypotonic KCl solution. The cell suspension was allowed to stand for
15 min (exp. II: 10 min) at room temperature (20 ± 5°C). After removal of the hypotonic
solution by centrifugation (10 min, 500 * g), the cell pellet was fixed with a mixture of
methanol and glacial acetic acid (3:1). After fixation at 2 – 8 °C for minimum 30 min, the
cell suspension was spun down by gentle centrifugation (10 min, 500 * g), the supernatant
was discarded and the cell pellet was re-suspended in fixative again. The washing procedures
were repeated until the cell pellet was white.
7.2.4 Preparation of Slides
The slides were prepared by dropping the cell suspension onto a clean microscope slide.
The cells were then stained with a 10% solution of Giemsa. All slides were independently
coded before microscopic analysis.
7.2.5 Determination of the Cytokinesis-Block Proliferation Index
In all replicates, the cytokinesis-block proliferation index (using at least 500 cells per culture)
was determined in order to assess the cytotoxicity of the test item. From these determinations,
the test item concentrations which were evaluated for scoring of micronuclei
were defined.
7.2.6 Determination of Binucleated Cells with Micronuclei
At least 1000 binucleated cells per culture were scored for micronuclei. Only cells with sufficiently
distinguishable cytoplasmic boundaries and clearly visible cytoplasm were included
in the analysis.

Rationale for test conditions:

according to Guideline

Evaluation criteria:

The CBPI was calculated using the following equation: CBPI = (MONC*1+BINC*2+MUNC*3)/n
n= total number of cells
MONC Mononucleated cells
BINC Binucleated cells
MUNC Multinucleate cells

Cytotoxicity was calculated as reduction in CBPI compared to the CBPI of the concurrent
solvent control.

The genotoxicity assay is considered acceptable if it meets the following criteria:
 All experimental conditions are tested (short exposure with and without metabolic activation,
extended exposure without metabolic activation) unless a positive result is
achieved in any experiment.
 In each experiment, an adequate number of cells is analysable both in the controls and
in at least 3 test item concentrations.
 The micronucleus induction of the solvent and positive controls is compatible with the
historical laboratory control data or the literature data.
 The positive control shows a statistically significant increase of binucleated cells with
micronuclei compared with the concurrent solvent control.
 The criteria for cell proliferation and for the selection of concentrations are fulfilled.

Statistics:

The number of binucleated cells with micronuclei in each treatment group was compared
with the solvent control. Statistical significance was tested using Fisher’s exact test at the
five per cent level (p<0.05) was used.
For positive controls with high values of binucleated cells with micronuclei, the chi-squaretest was used.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In experiment I without metabolic activation, a strong cytotoxic effect was observed at the
two highest chosen concentrations (125 μg/mL and 62.5 μg/mL) of the test item, therefore
no micronuclei ratio was determined. None of the lower test item concentrations induced a
critical cytotoxic effect.
In experiment I with metabolic activation, the highest chosen concentration (125 μg/mL)
also showed complete cytotoxicity. Therefore, in this concentration no micronuclei ratio
was determined. None of the lower test item concentrations induced a critical cytotoxic
effect.
In the approach without metabolic activation, a concentration-dependent effect (not statistically
significant) was observed. In addition, the two highest test item concentrations induced
a statistically significant increase in micronuclei whereby the value of the highest
test item concentration (% MBNC: 1.05) was also outside the range of the historical data of
the solvent control but within the historical range of other solvents used in the test facility.
Also in the approach with metabolic activation, a dose-dependent effect was detected.
Again, the two highest test item concentrations induced a statistically significant increase
in micronuclei but both values remained within the historical control data of the solvent
control.
In conclusion, no clearly positive result was obtained. Therefore a 2nd experiment (experiment
II without metabolic activation, extended exposure) was performed.

In experiment II, no cytotoxic effect was observed.
A statistically significant increase in the number of binucleated cells containing micronuclei
was observed at the evaluated concentrations 62.5μg/mL and 31.3 μg/mL. The micronucleus
frequency at the highest evaluated concentration was outside the range of the historical
control data of DMSO but still within the historical range of other solvents used in the
test facility. As in experiment I, no statistically significant dose-dependent effect was observed
but also in experiment II it is clearly visible that the effect of the formation of micronuclei
is reduced at the lower test item concentrations.

Conclusions:

In conclusion, under the experimental conditions reported, DENACOL EX-147 is possibly
able to induce the formation of micronuclei in human lymphocytes in vitro. But since not all
criteria for a clearly positive result are fulfilled, the result of the micronucleus assay with
the test item DENACOL EX-147 is considered as “inconclusive” under the conditions of the
test.

Executive summary:

This study was performed to assess the genotoxic potential of DENACOL EX-147 to induce

formation of micronuclei in human lymphocytes cultured in vitro in the absence and

the presence of an exogenous metabolic activation system (liver S9 mix from male rats,

treated with Aroclor 1254).

The test item was dissolved in DMSO to prepare a stock solution (pre-test: 400 mg/mL,

experiment I: 25 mg/mL, experiment II: 12.5 mg/mL), that was used to prepare a geometric

series of dilutions.

Human peripheral blood lymphocytes, on whole blood culture, were stimulated to divide by

addition of phytohaemagglutinin and exposed to solvent control, test item and positive control

item.

After the culture harvest time, the cells were harvested and slides were prepared. Then,

the proportion of cells containing micronuclei was determined.

A pre-experiment and two further independent experiments were performed. In each experiment,

all cell cultures were set up in duplicates. In order to assess the toxicity of the

test item to cultivated human lymphocytes, the cytokinesis-block proliferation index (CBPI)

was calculated for all cultures treated with solvent control, positive control and test item

(except in pre-experiment). On the basis of the data of the cytokinesis-block proliferation

index, 3 concentrations were selected for micronuclei scoring in experiment I and II.

In the pre-test, cytotoxicity was observed in all tested test item concentrations in both approaches.

For that reason, in experiment I the highest test item concentration corresponds

to the lowest concentration in the pre-test (125 μg/mL). In this experiment cytotoxic effects

were detected at the concentrations 125 μg/mL and 62.5 μg/mL in the approach without

metabolic activation and only at the concentration 125 μg/mL in the approach with metabolic

activation.

In experiment II, no cytotoxic effects were detected up to the maximum tested concentration

of 62.5 μg/mL.

In experiment I, a statistically significant increase in the number of binucleated cells containing

micronuclei was observed at the concentrations 31.3 μg/mL and 15.6 μg/mL in the

approach without metabolic activation. In this approach, the micronucleus frequency at the

highest evaluated concentration was outside the range of the historical control data for the

concurrent solvent control DMSO but within the historical range of other solvents used in

the test facility. A statistically significant dose-dependent effect was not detected but the

number of micronuclei was reduced at the lower tested concentrations.

In the approach with metabolic activation again, a statistically significant increase in the

number of binucleated cells containing micronuclei was observed at the concentrations

62.5 μg/mL and 31.3 μg/mL. In this approach, the micronucleus frequency at the highest

evaluated concentration remained within the 95.5 % control range of the historical data. A

statistically significant dose-dependent effect was not detected, but again, the number of

micronuclei was reduced at the lower tested concentrations.

In experiment II, a statistically significant increase in the number of binucleated cells containing

micronuclei was observed at the evaluated concentrations 62.5 μg/mL and

31.3 μg/mL. The micronucleus frequency at the highest evaluated concentration was also

outside the range of the historical control data for the solvent control DMSO but within the

historical range of other solvent controls used in the test facility. As in experiment I, no statistically

significant dose-dependent effect was observed but also in experiment II it is

clearly visible that the effect on the formation of micronuclei is reduced at the lower test

item concentrations.

All positive control compounds caused large, statistically significant increases in the proportion

of binucleate cells with micronuclei, demonstrating the sensitivity of the test system.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Mode of Action Analysis / Human Relevance Framework

based on the reactive epoxy-group, reaction with DNA is a possible mode of action

Justification for classification or non-classification

The available information is conclusive and sufficient for classification as Mutagen, category 2.