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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three in vitro tests with the test item were performed. All tests showed negative results. Therefore, the test item is not cosidered to be genotoxic.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 1987-06-29 to 1987-07-23
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:
(1983)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Remarks:
and TA 1538
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9 mix
Test concentrations with justification for top dose:
100, 333, 1000, 3330 and 5000 µg/plate
Vehicle / solvent:
- Vehicle/solvent used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: With metabolic activation: 2-aminoanthracene (all strains); Without metabolic activation: 4-nitro-o-phenylene-diamine (TA1538), daunomycine (TA 98)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

MUTATION ASSAY:
Five different doses of the test substance were tested in triplicate in each strain.
The test substance was tested both in the absence and presence of S9-mix in each strain.
Top agar in top agar tubes is melted and heated to 45 °C. The following solutions are successively added to 3 mL of top agar: 0.1 mL of a fresh bacterial culture (109 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test substance in dimethylsulphoxide (DMSO) of spectroscopic quality (Merck), and in the case of activation assays 0.5 mL of S9-mix. The ingredients are mixed on a Vortex and the contents of the top agar tube are poured onto a selective agar plate. After solidification of the top agar, the plates are turned and incubated in the dark at 37 °C for 48h. After this period revertant colonies (histidine independent) are counted automatically with an Artek model 880 colony counter or manually.

DETERMINATION OF CYTOTOXICITY
- Method: The percentage survival of the TA 100 culture is determined by comparing the number of colonies on the solvent control plate with those on the plates containing the test substance.
Evaluation criteria:
A test substance is considered negative (not mutagenic) in the Ames test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the Ames test if:
a) It induces at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is considered to be not significant. If the test substance shows in the first test only a positive response at one or two concentrations, the assay is repeated with doses just below and exceeding those showing positive effects in the first test.
b) The positive response should be reproducible in at least one independently repeated experiment.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
, but tested up to limit concentrations.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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
Remarks:
, but tested up to limit concentrations.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
, but tested up to limit concentrations.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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
Remarks:
, but tested up to limit concentrations.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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
Remarks:
, but tested up to limit concentrations.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Mutagenic Response of the Test Substance in the Ames Salmonella/Microsome Plate Test

Dose (µg/plate)

Mean number of revertant (His+) colonies/ 3 replicate plates (± S.D.)

with different strains of S. typhimurium

TA 1535

TA 1537

TA 1538

TA 98

TA 100

Without S9-mix

100

6 ± 4

7 ± 4

8 ± 2

27 ± 11

57 ± 9

333

9 ± 3

12 ± 3

8 ± 3

31 ± 6

70 ± 3

1000

8 ± 2

15; 8b)

8 ± 3

30 ± 5

54 ± 5

3330

8 ± 2

13 ± 3

11 ± 6

30 ± 9c)

57 ± 10

5000

7 ± 4

9 ± 4

8 ± 3

7 ± 7d)

56 ± 5

Solvent control

8 ± 6

12 ± 5

5 ± 3

23 ± 5

54 ± 12

Positive control

286 ± 27

1329 ± 79

544 ± 160

659; 175f)

890 ± 40

With S9-mix

100

8 ±

6 ± 3

13 ± 3

19 ± 2

54 ± 8

333

13 ±

3 ± 3

13 ± 3

22 ± 8

61 ± 4

1000

12 ±

3 ± 1

12 ± 3

20 ± 5

61 ± 6

3330

8 ±

4 ± 2

13 ± 4

19 ± 4

57 ± 6

5000

8 ±

4 ± 3c)

9 ± 5

23 ± 10

61 ± 12

Solvent control

11 ±

6 ± 0

15 ± 2

28 ± 3

91 ± 27

Positive control

132 ±

67 ± 7

744 ± 99

693 ± 28

1036 ± 123

a) 0.1 mL DMSO.

b) One plate infected with other bacteria.

c) Bacterial background lawn slightly reduced.

d) Bacterial background lawn moderately reduced.

e) Test substance precipitated in the top agar.

f) Inadvertently no bacteria plated on one plate; value of 175 is outside historical range.

Conclusions:
Based on the results of this study, the test substance can be considered as non mutagenic in the Ames Salmonella/microsome assay.
Executive summary:

The test item was tested in the Ames Salmonella/microsome test up to 5000 µg/plate. The test substance induced no dose-related increase in the numbers of revertant (His+) colonies in each of the five tester strains (TA1535; TA1537; TA1538; TA98 and TA100). These results were confirmed in an independently repeated experiment. The test substance can, therefore, be considered as nonmutagenic in this test system.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
(July 1997)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
(June 2000)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
No target gene
Species / strain / cell type:
lymphocytes: primary culture
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9 mix
Test concentrations with justification for top dose:
The test item was tested as supplied (15 µL of the supplied item into 5.5 mL of the culture medium in order to reach a final concentration of active material of 2077.75 µg/mL corresponding to 7.25 mM) or diluted in the vehicle. All the dose-levels and concentrations were expressed as active item, taking into account the peroxid content of 85.6% and the density of 890 kg/m³. The preparations were made immediately before use.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Details on test system and experimental conditions:
METHODS:
The test item was tested in two independent experiments, both with and without a liver metabolising system (S9 mix), obtained from rats previously treated with Aroclor 1254.
No preliminary cytotoxicity test was performed. The highest dose-level for treatment in the first experiment was selected on the basis of pH, osmolality and solubility. For selection of the dose-levels for the second experiment, toxicity indicated by the reduction of mitotic index (MI) in the first experiment, if any, was also taken into account.
For each culture, heparinised whole blood was added to culture medium containing a mitogen (phytohaemagglutinin) and incubated at 37 °C, for 48 hours.
In the first experiment, lymphocyte cultures were exposed to the test or control items, with or without S9 mix, for 3 hours then rinsed. Cells were harvested 20 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles.
As this first experiment was negative, a second experiment was performed as follows:
- without S9 mix, cells were exposed continuously until harvest to the test or control items, with S9 mix, cells were exposed to the test or control items for 3 hours and then rinsed.
- Cells were harvested 20 hours and 44 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles and 24 hours later, respectively.
One and a half hour before harvest, each culture was treated with a colcemid solution (10 µg/mL) to block cells at the metaphase-stage of mitosis. After hypotonic treatment (KCl 0.075 M), the cells were fixed in a methanol/acetic acid mixture (3/1; v/v), spread on glass slides and stained with Giemsa. All the slides were coded for scoring. All the dose-levels and concentrations were expressed as active item, taking into account the peroxid content of 85.6 %.
Evaluation criteria:
A reproducible and statistically significant increase in the frequency of cells with structural chromosome aberrations for at least one of the dose-levels and one of the two harvest times was considered as a positive result. Reference to historical data or other considerations of biological relevance, was also taken into account in the evaluation of the findings.
Statistics:
For each test and for each harvest time, the frequency of cells with structural chromosome aberrations (excluding gaps) in treated cultures was compared to that of the vehicle control cultures. If necessary, the comparison was performed using the x² test, in which p = 0.05 was used as the lowest level of significance.
Key result
Species / strain:
lymphocytes: primary culture
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
After the 3-hour treatment, a slight to marked decrease in the mitotic index was noted at dose-levels > 1.81 mM
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
lymphocytes: primary culture
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At the 20-hour harvest time, a 32-75% decrease in the mitotic index was induced at dose-levels >0.91 mM.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
All the dose-levels and concentrations were expressed as active item, taking into account the peroxid content of 85.6 %.

In the culture medium, the dose-level of 7.25 mM (corresponding to 2077.75 µg/mL) showed a moderate emulsion. At this dose-level, the pH was approximately 7.1 (7.1 for the vehicle control) and the osmolality equal to 299 mOsm/kg H2O (298 for the vehicle control).
With a treatment volume of 15 µL/5.5 mL culture medium, the dose-levels were as follows: 0.06, 0.11, 0.23, 0.45, 0.91, 1.81, 3.63 and 7.25 mM, for the first experiment with and without S9 mix, 0.23, 0.45, 0.91, 1.81, 3.63 and 7.25 mM, for the second experiment with and without S9 mix.
A slight to moderate emulsion was observed at the end of the treatment period, generally at dose-levels > 0.91 mM.

EXPERIMENTS WITHOUT S9 MIX:

Cytotoxicity:
After the 3-hour treatment, up to 58 % decrease in the mitotic index was noted at dose-levels > 1.81 mM.
After the 20-hour treatment, no decrease in the mitotic index was noted.
After the 44-hour treatment, a 31 % decrease in the mitotic index was noted at 7.25 mM.

Chromosomal aberration analysis:
The dose-levels selected for metaphase analysis were as follows:
- 1.81, 3.63, and 7.25 mM, for the 3-hour and the 20-hour treatments, the latter inducing 58 % decrease in the mitotic index following the 3-hour treatment,
- 7.25 mM, for the 44-hour treatment (highest dose-level used for treatment and inducing 31 % decrease in the mitotic index).
No significant increase in the frequency of cells with structural chromosomal aberrations was noted after 3, 20 as well as 44 hours treatments.

EXPERIMENTS WITH S9 MIX:

Cytotoxicity:
At the 20-hour harvest time, a 32 - 75 % decrease in the mitotic index was induced at dose-levels >0.91 mM.
At the 44-hour harvest time, a 43 % decrease in the mitotic index was noted at 7.25 mM.

Chromosomal aberration analysis:
The dose-levels selected for metaphase analysis were as follows:
- 1.81, 3.63 and 7.25 mM, for the 20-hour harvest time in the first experiment, the latter being the highest dose used for treatment and inducing 57 % decrease in the mitotic index, 0.23, 0.45 and 0.91 mM, for the 20-hour harvest time in the second experiment, the latter inducing 48 % decrease in the mitotic index,
- 7.25 mM, for the 44-hour harvest time, this dose-level being the highest dose used for treatment and inducing 43 % decrease in the mitotic index.
No significant increase in the frequency of cells with structural chromosomal aberrations was noted in both experiments and at both harvest times.
The frequencies of cells with structural chromosome aberrations of the vehicle and positive controls were as specified in acceptance criteria. The study was therefore considered valid.
Remarks on result:
other: all strains/cell types tested

Acceptance criteria

This study was considered valid since the following criteria were met:

- the frequency of cells with structural chromosome aberrations in the vehicle controls was consistent with our historical data,

- the frequency of cells with structural chromosome aberrations in the positive controls was significantly higher than that of the controls and consistent with our historical data.

Conclusions:
Under the experimental conditions, the test item (batch No. 11572 426109-002, peroxid content: 85.6 %) did not induce chromosome aberrations in cultured human lymphocytes.
Executive summary:

The test substance (peroxid content: 85.6 %) was tested in cultured human lymphocytes according to OECD guideline 473 and EU method B.10. The test item was tested in two independent experiments, both with and without a liver metabolising system (S9 mix), obtained from rats preciously treated with Aroclor 1254. Under the experimental conditions, the test item did not induce chromosome aberrations in cultured human lymphocytes.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2015-04-13 to 2015-07-06
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)
Version / remarks:
(July 1997)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
(May 2008)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
(August 1998)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: Mammalian cell gene mutation assay
Target gene:
The objective of this study was to determine whether the test item or its metabolites can induce forward mutation at the hypoxanthine-guanine phosphoribosyl transferase enzyme locus (hprt) in cultured Chinese hamster ovary cells.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Ham's F12 medium
- Properly maintained: yes
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Experiment 1, 5-hour treatment period without S9 mix:
20, 25, 30, 35, 40, 42.5, 45 and 47.5 μg/mL
Experiment 1, 5-hour treatment period with S9 mix:
50, 60, 70, 80, 90 and 100 μg/mL
Experiment 2, 20-hour treatment period without S9 mix:
20, 25, 27.5, 30, 32.5, 35 and 37.5 μg/mL
Experiment 2, 5-hour treatment period with S9 mix:
50, 60, 70, 80, 90 and 100 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Suitable solvent. This vehicle is comparativle with the survival of the CHO cells and the S9 activity and was chosen based on the results of the preliminary Solubility Test.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
no
Remarks:
With and without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without S9 Mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
With S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 hours
- Exposure duration: 5 and 20 hours
- Expression time: Aliquots of 5x10^5 cells were taken on days 1, 3, and 5 or 6
- Selection time: day 8

SELECTION AGENT (mutation assays): 6-thioguanine (6-TG)

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
The test item would have been considered to be mutagenic in this assay if all the following criteria were met:
- The assay is valid.
- The mutant frequency at one or more doses is significantly greater than that of the relevant control.
- Increase of the mutant frequency is reproducible.
- There is a clear dose-response relationship.
The test item would have been considered to have shown no mutagenic activity if no increases were observed which met the criteria listed above.
Statistics:
Statistical analysis was done with SPSS PC+ software for the following data:
- Mutant frequency between the negative (solvent) and the test item or positive control item treated groups.
The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences.
Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. In case of a none-normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there was a positive result, the inter-group comparisons were performed using the Mann-Whitney U-test.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Precipitation: For all test item concentrations examined, no precipitation in the medium was noted.

RANGE-FINDING/SCREENING STUDIES:
Treatment concentrations for the mutation assay were selected on the basis of the result of a Pre-test on cell toxicity. During the cytotoxicity assay, 1-3 day old cultures (more than 50 % confluent) were trypsinised and cell suspensions were prepared in Ham's F12-10 medium. Cells were seeded into 90 mm petri dishes (tissue culture quality: TC sterile) at 10^6 cells each and incubated in culture medium. After 24 hours the cells were treated with the suitable concentrations of the test item in DMSO in absence (10 concentrations) or in presence (8 concentrations) of S9 mix (50 μL/mL) and incubated at 37 °C for 5 hours. After the treatment cells were washed and incubated in fresh Ham's F12-10 medium for 19 hours. Additional groups of cells were treated for 20 hours without metabolic activation (9 concentrations). 24 hours after the beginning of treatment, the cultures were washed with medium, covered with trypsin-EDTA solution and then counted. The cell concentration was adjusted to 40 cells/mL with Ham's F12-10 medium. For each dose, 5 mL was plated in parallel into 3 sterile dishes (diameter is approx. 60 mm). The dishes were incubated at 37 °C in a humidified atmosphere of 5 % CO2 in air for 7 days for colony growing. Colonies were then fixed with methanol, stained with Giemsa and the colonies were counted. Survivals were assessed by comparing the colony forming ability of the treated groups to the negative (solvent) control. Precipitation of the test item in the final culture medium was examined visually at beginning and end of the treatments.
In addition, pH and osmolality were considered for dose level selection. Results of the Pre-test on cell toxicity were used for dose selection of the test item used in the Main Mutation Assays (Experiments 1 and 2). In Experiment 1 eight concentrations were selected for the treatment without and six concentrations with metabolic activation system, respectively. In Experiment 2 seven concentrations were selected for the treatment without and six concentrations with metabolic activation system, respectively.

COMPARISON WITH HISTORICAL CONTROL DATA:
The mutant frequency in the negative (solvent) control cultures is within the range (min-max) of historical laboratory control data. The positive control chemicals induce a statistically significant and biologically relevant increase in mutant frequency.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The following concentrations were tested but were very toxic and there were not enough cells to start the phenotypic expression period after the treatment:

Experiment 1, 5-hour treatment period without S9 mix: 45 and 47.5 µg/mL
Experiment 1, 5-hour treatment period with S9 mix: 100 µg/mL
Experiment 2, 20-hour treatment period without S9 mix: 37.5 µg/mL
Experiment 2, 5-hour treatment period with S9 mix: 100 µg/mL
Remarks on result:
other: strain/cell type: Sub-line (KI) of Chinese hamster ovary cell line CHO

The summarized results of the HPRT test are attached as PDF under "Attached background material".

Conclusions:
The test substance tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency over the background (negative solvent control) in this in vitro test in Chinese hamster ovary cells, when tested up to cytotoxic concentrations.
Thus, test item was not mutagenic under the conditions of this study.
Executive summary:

The test item was tested in a Mammalian Gene Mutation Test in CHO cells according to OECD guideline 476. The test item was dissolved in DMSO and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (without and with metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver). Two independent main experiments (both run in duplicate) were performed at the concentrations and treatment intervals given below:

Experiment 1, 5-hour treatment period without S9 mix:

20, 25, 30, 35, 40, 42.5, 45* and 47.5* μg/mL

Experiment 1, 5-hour treatment period with S9 mix:

50, 60, 70, 80, 90 and 100* μg/mL

Experiment 2, 20-hour treatment period without S9 mix:

20, 25, 27.5, 30, 32.5, 35 and 37.5* μg/mL

Experiment 2, 5-hour treatment period with S9 mix:

50, 60, 70, 80, 90 and 100* μg/mL

*: These concentrations were tested but were very toxic and there were not enough cells to start the phenotypic expression period after the treatment.

In Experiment 1, there were no biologically or statistically significant increases in mutation frequency at any concentration tested, either in the absence or in the presence of metabolic activation, when tested up to cytotoxic concentrations. There were no biologically significant differences between treatment and control groups and no dose-response relationships were noted.

In Experiment 2, the mutant frequency of the cells did not show biologically or statistically significant alterations compared to the concurrent control, when the test item was tested without S9 mix over a prolonged treatment period (20 hours) up to cytotoxic concentrations. Furthermore, a five-hour treatment in the presence of S9 mix did not cause significant increases in mutant frequency even when cytotoxicity occurred.

As in Experiment 1, in Experiment 2 no statistical differences between treatment and solvent control groups and no dose-response relationships were noted.

The sensitivity of the tests and the efficacy of the S9 mix were demonstrated by large increases in mutation frequency in the positive control cultures with Ethyl methanesulfonate (0.4 or 1.0 μL/mL) and 7,12-Dimethyl benz[a]anthracene (20 μg/mL).

The test item tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency over the background (negative solvent control) in this in vitro test in Chinese hamster ovary cells, when tested up to cytotoxic concentrations.

Thus, the test item was not mutagenic under the conditions of this study.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames Test:
The test item was tested in the Ames gene mutation assay according to OECD guideline 471. Strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 of S. typhimurium were exposed to the test substance at concentrations of 100 to 5000 µg/plate with and without metabolic activation (Aroclor induced tar liver S-9 mix). The test substance induced no dose-related increase in the numbers of revertant (His+) colonies in each of the five tester strains. These results were confirmed in an independently repeated experiment. The test substance can, therefore, be considered as nonmutagenic in this test system.


 


Chromosome aberration test:


The test item (peroxid content: 85.6 %) was tested in cultured human lymphocytes according to OECD guideline 473 and EU method B.10. The test item was tested in two independent experiments, both with and without a liver metabolising system (S9 mix), obtained from rats preciously treated with Aroclor 1254. Under the experimental conditions, the test item did not induce chromosome aberrations in cultured human lymphocytes.


 


Mammalian Cell Gene Mutation Test: HPRT Assay


The test item was tested in a Mammalian Gene Mutation Test in CHO cells according to OECD guideline 476. The test item was dissolved in DMSO and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (without and with metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver). Two independent main experiments (both run in duplicate) were performed at the concentrations and treatment intervals given below:


 


Experiment 1, 5-hour treatment period without S9 mix:


20, 25, 30, 35, 40, 42.5, 45* and 47.5* μg/mL


 


Experiment 1, 5-hour treatment period with S9 mix:


50, 60, 70, 80, 90 and 100* μg/mL


 


Experiment 2, 20-hour treatment period without S9 mix:


20, 25, 27.5, 30, 32.5, 35 and 37.5* μg/mL


 


Experiment 2, 5-hour treatment period with S9 mix:


50, 60, 70, 80, 90 and 100* μg/mL


 


*: These concentrations were tested but were very toxic and there were not enough cells to start the phenotypic expression period after the treatment.


 


The test item tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency over the background (negative solvent control) in this in vitro test in Chinese hamster ovary cells, when tested up to cytotoxic concentrations.


Thus, the test item was not mutagenic under the conditions of this study.

Justification for classification or non-classification

Based on the available results the test substance was not classified and labelled for genotoxicity according to Regulation (EC) No 1272/2008 (CLP), as amended fo the fifteenth time in Regulation (EU) 2020/1182.