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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance is not mutagenic in bacteria and in mammalian cells in vitro.


The substance is not clastogenic in vitro.


The studies were performed according to current OECD testing guidelines and under GLP.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
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:
26 Sep 2014
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Suitability of cells: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
liver S9 from rats induced with phenobarbital i.p. and β-naphthoflavone orally
Test concentrations with justification for top dose:
1st Experiment

4-hour exposure, 18-hour sampling time, without S9 mix
0; (6.25; 12.5; 25;) 50; 100; 200 μg/mL

4-hour exposure, 18-hour sampling time, with S9 mix
0; (6.25;) 12.5; 25; 50 (100; 200) μg/mL

2nd Experiment
18-hour exposure, 18-hour sampling time, without S9 mix
0; (6.25; 12.5; 25;) 50; 100; 200 μg/mL

4-hour exposure, 28-hour sampling time, with S9 mix
0; (6.25; 12.5; 25;) 50; 100; 200μg/mL

Concentrations in parenthesis were not scored.

The top dose was determined by precipitation and cytotoxicity as determined for V79 cells during the HPRT test.
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 - 30 hours
- Exposure duration: 4h or 18h
- Expression time (cells in growth medium): 10, 14 or 24 hours

- Fixation time (start of exposure up to fixation or harvest of cells): 28h


SPINDLE INHIBITOR (cytogenetic assays): colcemide
STAIN (for cytogenetic assays): 7.5% (v/v) Giemsa/Titrisol solution pH 7.2

NUMBER OF REPLICATIONS:2

NUMBER OF CELLS EVALUATED: 300 metaphases per dose group (150 per replicate)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Rationale for test conditions:
Concentrations were chosen based on range-finding studies.
Evaluation criteria:
The V79 in vitro cytogenetic assay is considered valid if the following criteria are met:
• The quality of the slides must allow the identification and evaluation of a sufficient number of analyzable metaphases.
• The numbers of cells with structural/numerical aberrations in the negative control has to be within the range of the historical negative control data.
• The positive control substances both with and without S9 mix have to induce a distinct increase of structural chromosome aberrations.

The test substance is considered as “positive” if the following criteria are met:
• A statistically significant, dose-related and reproducible increase in the number of cells
with structural chromosome aberrations (excl. gaps).
• The number of aberrant cells (excl. gaps) exceeds both the concurrent negative/vehicle
control value and the historical negative control data range.
A test substance generally is considered as “negative” if the following criteria are met:
• The number of cells with structural aberrations (excl. gaps) in the dose groups is not
statistically significant increased above the concurrent negative/vehicle control value and
is within the historical negative control data range.
Statistics:
The statistical evaluation of the data was carried out using the MUCHAN program system (BASF SE). The proportion of metaphases with structural aberrations was calculated for each group. A comparison of each dose group with the negative control group was carried out using Fisher's exact test for the hypothesis of equal proportions. This test was Bonferroni- Holm corrected versus the dose groups separately for each time and was performed one-sided. If the results of this test are statistically significant compared with the respective vehicle control, labels (* p ≤ 0.05, ** p ≤ 0.01) are printed in the tables.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
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: none
- Effects of osmolality: none
- Evaporation from medium: non volatile
- Water solubility: poorly soluble
- Precipitation: The highest applied stock solution of 270 mg/mL (Test group: 2700 μg/mL) was a homogeneous suspension in the most appropriate solvent DMSO. Test substance
suspensions in DMSO were obtained from 8.4 mg/mL (Test group: 84.4 μg/mL) onward. The test substance precipitation (macroscopically) in culture medium was observed at
84.4 μg/mL and above after 4 hours in the absence and presence of S9 mix.

RANGE-FINDING/SCREENING STUDIES: yes

HISTORICAL CONTROL DATA
- Negative (solvent/vehicle): negative control data range (0.0% - 4.7% aberrant metaphases, excl. gaps)
ADDITIONAL INFORMATION ON CYTOTOXICITY:
After 4 hours treatment in the absence and presence of S9 mix, no cytotoxicity was observed as indicated by a reduced relative cloning efficiency of about or below 20% relative survival upthe highest applied concentration of 2700 μg/mL.

Table 1: Results

Genotoxicity Cytotoxicity**
Schedule Aberrant cells [%]  
Exp. Exposure/ mix Aberrant cells [%] Polyploid RPD Mitotic
preparation
period		 Test groups S9 mix precipitation incl.
gaps#		 excl.
gaps#		 rel.
[%]		 Polyploid cells
[%]		 RPD number
[%]		 Mitotic index [%]
1 4/18 hrs Vehicle control1 - - 9.0 4.3 0.7 1.3 100.0 100.0
6.25 µg/mL - - n.d. n.d. n.d. n.d. 100.9 n.d.
12.50 µg/mL - - n.d. n.d. n.d. n.d. 104.3 n.d.
25.00 µg/mL - - n.d. n.d. n.d. n.d. 103.3 n.d.
50.00 µg/mL - - 6.3 4.7 1.0 0.0 109.7 113.6
100.00 µg/mL - - 6.7 4.0 0.3 0.3 109.5 113.6
200.00 µg/mL - + 9.3 5.3 0.3 1.3 99.4 109.5
Positive control2 - - 12.3 9.0s 2.3 0.3 n.t. 92.8
2 18/18 hrs Vehicle control1 - - 12.7 3.7 0.7 0.0 100.0 100.0
6.25 µg/mL - - n.d. n.d. n.d. n.d. 99.2 n.d.
12.50 µg/mL - - n.d. n.d. n.d. n.d. 102.6 n.d.
25.00 µg/mL - - n.d. n.d. n.d. n.d. 100.4 n.d.
50.00 µg/mL - - 10.7 4.3 0.7 1.3 104.6 110.4
100.00 µg/mL - - 10.0 3.0 0.0 0.3 107.3 110.4
200.00 µg/mL - + 8.7 3.3 0.3 2.7 106.6 102.6
Positive control2 - - 46.3 34.7s 17.3 1.0 n.t. 78.3
1 4/18 hrs Vehicle control1 + - 2.7 0.3 0.0 1.0 100.0 100.0
6.25 µg/mL + - n.d. n.d. n.d. n.d. 107.5 n.d.
12.50 µg/mL + - 9.3 3.0s 1.3 1.7 117.1 106.9
25.00 µg/mL + - 10.0 3.3s 0.7 1.0 117.7 113.2
50.00 µg/mL + + 9.0 3.7s 0.7 0.7 123.8 128.6
100.00 µg/mL + + n.d. n.d. n.d. n.d. 113.6 n.d.
200.00 µg/mL + + n.d. n.d. n.d. n.d. 104.2 n.d.
Positive control2 + - 37.0 33.3s 16.0 0.0 n.t. 94.7
2 4/28 hrs Vehicle control1 + - 13.3 4.7 1.0 1.3 100.0 100.0
6.25 µg/mL + - n.d. n.d. n.d. n.d. 98.8 n.d.
12.50 µg/mL + - n.d. n.d. n.d. n.d. 102.9 n.d.
25.00 µg/mL + - n.d. n.d. n.d. n.d. 102.6 n.d.
50.00 µg/mL + - 10.3 5.3 0.7 1.7 102.4 90.1
100.00 µg/mL + - 9.0 2.7 0.3 0.0 93.2 90.1
200.00 µg/mL + + 12.7 5.7 1.0 0.7 95.7 68.6
Positive control2 + - 29.7 24.0s 9.7 0.0 n.t. 66.0

* Precipitation occured at the end of exposure period

** Relative values compared with the respective vehicle control

# Inclusive cells carrying exchanges

n.d. Not determined

n.t. Not tested

S Aberration frequency statistically significant higher than corresponding control values

1 DMSO 1% (v/v)

2 CPP 0.5 μg/mL

Conclusions:
The substance is not clastogenic in mammalian cells in vitro.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-2015
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:
28 Jul 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany
Type of assay:
mammalian cell gene mutation assay
Target gene:
hypoxanthine-guanine phosphoribosyl transferase (HGPRT)
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media:
- Culture medium: Ham's F12 medium containing stable glutamine and hypoxanthinesupplemented with 10% (v/v) fetal calf serum (FCS).
- Treatment medium (without S9 mix): Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with 10% (v/v) fetal calf serum.
- Treatment medium (with S9 mix): Ham's F12 medium containing stable glutamine and hypoxanthine.
- Pretreatment medium ("HAT" medium): Ham's F12 medium supplemented with: hypoxanthine (13.6 x 10-3 mg/mL), aminopterin (0.18 x 10-3 mg/mL), thymidine (3.88 x 10-3 mg/mL), 10% (v/v) fetal calf serum (FCS).
- Selection medium ("TG" medium): Hypoxanthine-free Ham's F12 medium supplemented with: 6-thioguanine (10 μg/mL), 1% (v/v) stable glutamine (200 mM), 10% (v/v) fetal calf serum (FCS).
- All media were supplemented with: 1% (v/v) penicillin/streptomycin and 1% (v/v) amphotericine B
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes; During the week prior to treatment, any spontaneous HPRT-deficient mutants were eliminated by pretreatment with "HAT" medium.
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and β-naphthoflavone induced rat liver S9 mix
Test concentrations with justification for top dose:
1st Experiment without S9 mix: 0; 4.7; 9.4; 18.8; 37.5; 75.0; 150.0 μg/mL; with S9 mix: 0; 4.7; 9.4; 18.8; 37.5; 75.0; 150.0 μg/mL
2nd Experiment: without S9 mix:0; (1.6; 3.1;) 6.3; 12.5; 25.0; 50.0; 100.0 μg/mL; with S9 mix: 0; (1.6; 3.1;) 6.3; 12.5; 25.0; 50.0; 100.0 μg/mL
Concentrations in parenthesis were not scored because the minimum number of concentrations were already reached.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, DMSO was selected as the vehicle which had been demonstrated to be suitable in the CHO/HPRT assay and for which historical data are available.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Remarks:
400 μg/mL EMS (with S9 mix), 1.25 μg/mL DMBA (without S9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h (with and without S9)
- Expression time (cells in growth medium): 7-9 days
- Selection time (if incubation with a selection agent): 6-7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 15 days

SELECTION AGENT (mutation assays): 6-thioguanine (10 μg/mL)
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: Duplicate cultures were used for all experimental groups.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
Acceptance criteria
The HPRT assay is considered valid if the following criteria are met:
• The absolute cloning efficiencies of the negative/vehicle controls should not be less than 50% (with and without S9 mix).
• The background mutant frequency in the negative/vehicle controls should be within our historical negative control data range of 0.00 – 16.43 mutants per 10E6 clonable cells
• The positive controls both with and without S9 mix have to induce distinctly increased mutant frequencies (historical positive control data).
• At least 4 dose levels should be tested ranging up to a toxic concentration or up to or beyond the limit of solubility under culture conditions. Freely soluble and apparently non-toxic substances are not tested at concentrations higher than 5 mg/mL or 10 mM.

Assessment criteria
A finding is assessed as positive if the following criteria are met:
• Increase in the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and our historical negative control data range.
• Evidence of the reproducibility of any increase in mutant frequencies.
• A statistically significant increase in mutant frequencies and the evidence of a doseresponse relationship.
Isolated increases of mutant frequencies above our historical negative control range (i.e. 15 mutants per 10E6 clonable cells) or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.
The test substance is considered non-mutagenic according to the following criteria:
• The corrected mutation frequency (MFcorr.) in the dose groups is not statistically significantly increased above the concurrent negative control and is within our historical negative control data range.
Statistics:
An appropriate statistical trend test was performed to assess a dose-related increase of mutant frequencies. The number of mutant colonies obtained for the test substance treated groups was compared with that of the respective vehicle control groups. A trend is judged as statistically significant whenever the p-value (probability value) is below 0.10 and the slope is greater than 0. However, both, biological and statistical significance will be considered together.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS: none

RANGE-FINDING/SCREENING STUDIES:The pretest was performed following the method described for the main experiment. The cloning efficiency 1 (survival) was determined as a toxicity indicator for dose selection and various parameters were checked for all, or at least some, selected doses.
A slight pH shift was observed at the highest required concentration prior to testing. Therefore, the test substance formulation were prediluted 1:10 with culture medium without FCS and the pH was adjusted to a physiological value prior to application, using small amounts of 32% (w/v) HCl.
The pH value was not influenced by the addition of the test substance preparation to the culture medium at the concentrations measured.
The highest applied concentration of 2.7 mg/mL (Test group: 2700 µg/mL) was a homogeneous suspension in the most appropriate solvent DMSO. Test substance suspensions in DMSO were obtained from 84.4 mg/mL onward. Besides, test substance precipitation in culture medium was observed at 84.4 µg/mL and above after 4 hours in the absence and presence of S9 mix
After 4 hours treatment in the absence and presence of S9 mix, no cytotoxicity was observed as indicated by a reduced relative cloning efficiency of about or below 20% relative survival up the highest required concentration of 2700 µg/mL



COMPARISON WITH HISTORICAL CONTROL DATA: yes


ADDITIONAL INFORMATION ON CYTOTOXICITY: The substance was not cytotoxic at non-precipiating concentrations.

Table: Summary of results

 

Exp.

Exposure period

[h]

Test groups [µg/mL]

S9

mix

 

Prec.*

Genotoxicity**

MFcorr.[per 106cells]

Cytotoxicity***

CE1

[%]

CE2

[%]

1

 

 

 

 

 

 

 

 

 

 

 

 

2

4

 

 

 

 

 

 

 

 

 

 

 

 

4

Vehicle cont1 4.7

9.4

18.8

37.5

75.0

150.0

Positive control2

 

Vehicle cont1

1.6

3.1

6.3

12.5

25.0

50.0

100.0

Positive control2

-

n.d.

3.87

100.0

100.0

-

-

1.54

98.9

114.1

-

-

2.54

110.2

110.9

-

+

1.41

108.0

99.6

-

+

2.35

85.2

104.9

-

+

4.29

88.9

98.6

-

+

8.86

92.9

95.4

-

n.d.

359.80s

41.5

71.8

 

-

 

n.d.

 

1.64

 

100.0

 

100.0

-

-

n.c

95.6

n.c

-

-

n.c

95.6

n.c

-

-

2.31

99.4

88.5

-

-

5.43

85.3

113.1

-

+

1.46

89.5

168.0

-

+

3.76

87.3

87.3

-

+

2.85

80.6

115.2

-

n.d.

291.38s

69.0

71.3

*       Macroscopically visible precipitation in culture medium at the end of exposure period

**      Mutant frequency MFcorr.: mutant colonies per 106cells corrected with the CE2value

***    Cloning efficiency related to the respective vehicle control

s         Mutant frequency statistically significant higher than corresponding controlvalues

n.c.        Culture was not continued since a minimum of only four analysable concentrations is required

n.d.        Not determined

1         DMSO 1%(v/v)               2     EMS 400µg/mL

Conclusions:
Under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April - July 2021
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:
26 June 2020
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesamt für Umwelt, Rheinland Pfalz, Deutschland
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
solid, beige
purity > 99%
Target gene:
his, trp
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : liver of 5 adult male Wistar rats induced by a combination of phenobarbital and ß-naphthoflavone
- method of preparation of S9 mix: 24 hours after the last administration, the rats were sacrificed, and the livers were prepared. The livers were weighed, washed and homogenized in KCl solution. After centrifugation of the homogenate, portions of the supernatant (S9 fraction) were stored at -70°C to -80°C. The S9 mix was prepared freshly prior to each experiment. For this purpose, a sufficient amount of S9 fraction was thawed at room temperature and 1 part of S9 fraction is mixed with 9 parts of S9 supplement (cofactors). This mixture of both components (S9 mix) was kept on ice until used.
- concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL S9 mix
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): To demonstrate the efficacy of the S9 mix in this assay, the S9 batch was characterized with benzo(a)pyrene.
Test concentrations with justification for top dose:
0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone

- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, Acetone was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
other: 2-aminoanthracene (2.5 μg/plate, 60 μg/plate; dissolved in DMSO; with S9-mix); N-methyl-N'-nitro-N-nitrosoguanidine (5 μg/plate; dissolved in DMSO; without S9-mix); 4-nitro-o-phenylenediamine (10 μg/plate; dissolved in DMSO; without S9-mix)
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration : triplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): Fresh cultures of bacteria were grown up to late exponential or early stationary phase of growth (approximately 10^9 cells per mL). These cultures grown overnight were kept in iced water from the beginning of the experiment until the end in order to prevent further growth.
- Test substance added: Standard plate test, Preincubation Test

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: about 20 minutes
- Exposure duration/duration of treatment: 48 – 72 hours

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

Evaluation criteria:
Generally, the experiment was considered valid if the following criteria were met:
• The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
• The sterility controls revealed no indication of bacterial contamination.
• The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies compatible with the range of the historical positive control data or above.
• Fresh bacterial culture containing approximately 10^9 cells per mL were used.

The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: toxicity and tested up to limit concentation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: toxicity and tested up to limit concentation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: toxicity and tested up to limit concentation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: toxicity and tested up to limit concentation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: toxicity and tested up to limit concentation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: no data
- Data on osmolality: no data
- Water solubility: insoluble
- Precipitation and time of the determination: Precipitation of the test substance was observed depending on the test conditions at and above 33 µg/plate. (See tables)

RANGE-FINDING/SCREENING STUDIES (if applicable):
1st Experiment
Strains: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA
Doses: 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
Type of test: Standard plate test with and without S9 mix
Number of plates: 3 test plates per dose or per control

STUDY RESULTS
- Concurrent vehicle negative and positive control data : see Tables

Ames test:
- Signs of toxicity :A bacteriotoxic effect was observed depending on the strain and test conditions at and above 333 µg/plate.
- Individual plate counts : see Tables
- Mean number of revertant colonies per plate and standard deviation : see Tables

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
see Tab. 5 and 6

Tab. 1: Assay conditions: Standard plate test ( Experiment: 1st_SPT), without S9

Strain          Test group      Dose (µg/plate)

Mean revertants

per plate

 Standard deviation Factor           Individual revertant colony
counts
                   
TA 1535 Acetone - 10.0 1.0 - 9, 11, 10
Test item 33 7.3 3.5 0.7 11, 7, 4
100 9.3 3.5 0.9 9 P, 6 P, 13 P
333 8.0 3.5 0.8 10 P, 10 P, 4 P
1000 8.7 0.6 0.9 9 P, 8 P, 9 P
2500 5.7 4.7 0.6 11 P, 4 P, 2 P
5000 4.0 1.0 0.4 4 P, 5 P, 3 P
  MNNG 5.0 5819.7 423.9 582.0 5342, 5966, 6151
TA 100 Acetone - 131.0 13.0 - 138, 139, 116
Test item 33 133.0 12.8 1.0 136, 119, 144
100 134.3 4.6 1.0 137 P, 129 P, 137 P
333 125.3 27.0 1.0 98 P, 126 P, 152 P
1000 113.3 11.7 0.9 100 P, 122 P, 118 P
2500 112.7 19.0 0.9 112 P, 94 P, 132 P
5000 90.0 17.4 0.7 98 P, 102 P, 70 P
  MNNG 5.0 4769.7 329.0 36.4 4670, 4502, 5137
             
TA 1537 Acetone - 12.7 4.2 - 14, 16, 8
Test item 33 16.0 3.6 1.3 20, 15, 13
100 12.7 4.7 1.0 18 P, 11 P, 9 P
333 7.3 2.1 0.6 5 P, 8 P, 9 P
1000 7.7 1.5 0.6 6 P, 8 P, 9 P
2500 6.0 2.0 0.5 8 P, 4 P, 6 P
5000 6.0 1.0 0.5 7 P, 6 P, 5 P
  AAC 100 1556.0 337.2 122.8 1668, 1177, 1823
             
TA 98 Acetone - 23.7 1.2 - 25, 23, 23
Test item 33 24.7 2.9 1.0 28, 23, 23
100 20.0 1.0 0.8 19 P, 21 P, 20 P
333 20.7 3.2 0.9 23 P, 17 P, 22 P
1000 20.7 3.1 0.9 18 P, 20 P, 24 P
2500 5.7 1.5 0.2 4 P, 6 P, 7 P
5000 5.7 0.6 0.2 6 P, 5 P, 6 P
  NOPD 10 637.3 36.9 26.9 678, 606, 628
E. coli Acetone - 39.0 11.4 - 47, 44, 26
Test item 33 35.7 3.2 0.9 37, 32, 38
100 34.3 2.5 0.9 37 P, 34 P, 32 P
333 40.7 5.0 1.0 36 P, 46 P, 40 P
1000 30.0 10.5 0.8 40 P, 19 P, 31 P
2500 22.7 2.5 0.6 20 P, 25 P, 23 P
5000 21.0 2.0 0.5 21 P, 19 P, 23 P
  4-NQO 5 872.0 53.8 22.4 822, 865, 929
P Precipitation

 

Tab. 2: Assay conditions: Standard plate test ( Experiment: 1st_SPT), with S9

Strain          Test group      Dose (µg/plate)

Mean revertants

per plate

Standard

deviation

 Factor           Individual revertant colony
counts
  
TA 1535 Acetone - 14.0 3.6 - 11, 18, 13
Test item 33 12.0 6.0 0.9 18, 6, 12
100 12.3 3.2 0.9 16 P, 10 P, 11 P
333 9.3 1.2 0.7 8 P, 10 P, 10 P
1000 6.0 1.0 0.4 5 P, 7 P, 6 P
2500 4.0 2.0 0.3 6 P, 2 P, 4 P
5000 9.3 5.5 0.7 13 P, 12 P, 3 P
  2-AA 2.5 237.3 1.5 17.0 239, 237, 236
TA 100 Acetone - 132.0 13.0 - 147, 124, 125
Test item 33 127.0 15.0 1.0 142, 127, 112
100 115.3 8.3 0.9 118 P, 122 P, 106 P
333 117.3 11.4 0.9 114 P, 130 P, 108 P
1000 112.0 7.2 0.8 120 P, 110 P, 106 P
2500 121.3 4.6 0.9 124 P, 116 P, 124 P
5000 59.0 10.1 0.4 70 P, 50 P, 57 P
  2-AA 2.5 2341.7 215.7 17.7 2552, 2121, 2352
             
TA 1537 Acetone - 12.3 2.1 - 10, 14, 13
Test item 33 13.0 2.6 1.1 10, 14, 15
100 8.7 2.5 0.7 9 P, 11 P, 6 P
333 9.3 1.5 0.8 11 P, 8 P, 9 P
1000 6.3 2.1 0.5 8 P, 4 P, 7 P
2500 8.0 0.0 0.6 8 P, 8 P, 8 P
5000 4.0 1.0 0.3 3 P, 5 P, 4 P
  2-AA 2.5 148.7 9.1 12.1 157, 150, 139
             
TA 98 Acetone - 32.7 3.8 - 37, 31, 30
Test item 33 24.7 3.8 0.8 23, 29, 22
100 21.3 3.2 0.7 20 P, 25 P, 19 P
333 19.7 2.3 0.6 17 P, 21 P, 21 P
1000 20.3 2.1 0.6 21 P, 18 P, 22 P
2500 9.7 3.1 0.3 7 P, 9 P, 13 P
5000 12.0 3.6 0.4 9 P, 11 P, 16 P
  2-AA 2.5 1602.7 557.6 49.1 1910, 1939, 959
E. coli Acetone - 39.7 9.5 - 49, 40, 30
Test item 33 39.3 10.1 1.0 38, 30, 50
100 34.0 7.0 0.9 37 P, 26 P, 39 P
333 36.7 3.1 0.9 36 P, 40 P, 34 P
1000 32.7 1.5 0.8 31 P, 34 P, 33 P
2500 20.0 2.6 0.5 22 P, 17 P, 21 P
5000 13.7 1.5 0.3 12 P, 14 P, 15 P
  2-AA 60 152.3 4.2 3.8 157, 149, 151
P Precipitation

 

 

 

Table 3: Second experiment
Preincubation test,Without metabolic activation
Strain          Test group      Dose (µg/plate) Mean revertants per plate Standard deviation   Factor    Individual revertant colony
counts
   
TA 1535 Acetone - 13.3 2.3 - 12, 12, 16
Test item 33 13.0 4.0 1.0 13 P, 9 P, 17 P
100 15.7 5.5 1.2 16 P, 10 P, 21 P
333 11.3 0.6 0.9 11 P, 12 P, 11 P
1000 11.0 3.0 0.8 11 P, 14 P, 8 P
2500 7.7 1.5 0.6 6 P, 9 P, 8 P
5000 6.7 2.1 0.5 6 P, 5 P, 9 P
  MNNG 5.0 3331.0 71.8 249.8 3387, 3356, 3250
TA 100 Acetone - 129.3 2.9 - 131, 131, 126
Test item 33 123.3 6.5 1.0 117 P, 130 P, 123 P
100 123.0 12.8 1.0 109 P, 126 P, 134 P
333 104.0 8.7 0.8 98 P, 100 P, 114 P
1000 94.0 2.0 0.7 94 P, 92 P, 96 P
2500 46.0 8.7 0.4 40 P, 56 P, 42 P
5000 29.7 6.0 0.2 24 P, 29 P, 36 P
  MNNG 5.0 2974.7 298.5 23.0 3046, 3231, 2647
TA 1537 Acetone - 9.0 3.5 - 7, 7, 13
Test item 33 8.7 5.0 1.0 4 P, 8 P, 14 P
100 6.3 3.1 0.7 7 P, 3 P, 9 P
333 6.0 1.7 0.7 5 P, 8 P, 5 P
1000 6.0 0.0 0.7 6 P, 6 P, 6 P
2500 4.3 2.9 0.5 1 P, 6 P, 6 P
5000 3.0 1.0 0.3 3 P, 4 P, 2 P
  AAC 100 909.0 31.3 101.0 945, 888, 894
TA 98 Acetone - 24.0 1.0 - 24, 25, 23
Test item 33 17.7 7.5 0.7 22 P, 22 P, 9 P
100 18.7 5.7 0.8 25 P, 14 P, 17 P
333 21.0 1.0 0.9 22 P, 20 P, 21 P
1000 16.3 2.5 0.7 16 P, 14 P, 19 P
2500 9.7 2.5 0.4 7 P, 10 P, 12 P
5000 2.7 1.5 0.1 1 P, 4 P, 3 P
  NOPD 10 632.3 21.6 26.3 617, 657, 623
E. coli Acetone - 42.0 16.5 - 34, 31, 61
Test item 33 41.7 3.8 1.0 39 P, 46 P, 40 P
100 37.0 3.6 0.9 36 P, 34 P, 41 P
333 31.7 2.3 0.8 33 P, 29 P, 33 P
1000 23.0 1.7 0.5 24 P, 21 P, 24 P
2500 14.3 2.1 0.3 15 P, 16 P, 12 P
5000 7.7 1.5 0.2 6 P, 8 P, 9 P
  4-NQO 5 605.0 11.0 14.4 616, 605, 594
P Precipitation

Table 4: Second experiment
Preincubation test, With metabolic activation
Strain          Test group      Dose (µg/plate) Mean revertants per plate Standard deviation  Factor

 Individual revertant colony counts
             
TA 1535 Acetone - 11.7 5.5 - 17, 12, 6
Test item 33 15.0 4.6 1.3 16 P, 19 P, 10 P
100 13.0 6.9 1.1 17 P, 5 P, 17 P
333 9.3 1.5 0.8 9 P, 11 P, 8 P
1000 11.7 1.5 1.0 13 P, 10 P, 12 P
2500 8.0 1.0 0.7 7 P, 8 P, 9 P
5000 4.3 1.5 0.4 3 P, 4 P, 6 P
  2-AA 2.5 199.7 7.6 17.1 205, 203, 191
TA 100 Acetone - 132.0 3.6 - 135, 133, 128
Test item 33 130.0 10.8 1.0 118 P, 133 P, 139 P
100 132.7 4.7 1.0 129 P, 131 P, 138 P
333 110.0 14.0 0.8 104 P, 126 P, 100 P
1000 106.7 17.9 0.8 116 P, 86 P, 118 P
2500 74.7 9.5 0.6 82 P, 64 P, 78 P
5000 55.7 4.7 0.4 61 P, 54 P, 52 P
  2-AA 2.5 1389.7 138.1 10.5 1426, 1237, 1506
TA 1537 Acetone - 8.7 3.8 - 7, 6, 13
Test item 33 7.0 2.0 0.8 5 P, 9 P, 7 P
100 6.3 2.3 0.7 9 P, 5 P, 5 P
333 7.3 0.6 0.8 7 P, 8 P, 7 P
1000 7.0 1.0 0.8 7 P, 6 P, 8 P
2500 2.3 0.6 0.3 2 P, 2 P, 3 P
5000 1.3 0.6 0.2 2 P, 1 P, 1 P
  2-AA 2.5 109.0 17.5 12.6 126, 110, 91
TA 98 Acetone - 25.0 2.0 - 25, 23, 27
Test item 33 22.0 5.6 0.9 23 P, 16 P, 27 P
100 21.7 1.5 0.9 22 P, 20 P, 23 P
333 23.7 3.8 0.9 22 P, 21 P, 28 P
1000 23.3 4.2 0.9 28 P, 22 P, 20 P
2500 15.3 3.5 0.6 15 P, 19 P, 12 P
5000 10.0 4.0 0.4 6 P, 14 P, 10 P
  2-AA 2.5 1038.0 21.6 41.5 1020, 1062, 1032
E. coli Acetone - 40.3 8.1 - 45, 31, 45
Test item 33 40.0 1.0 1.0 39 P, 40 P, 41 P
100 34.0 5.0 0.8 39 P, 34 P, 29 P
333 29.7 7.4 0.7 24 P, 27 P, 38 P
1000 29.3 2.5 0.7 32 P, 29 P, 27 P
2500 15.7 5.9 0.4 9 P, 18 P, 20 P
5000 16.0 1.7 0.4 17 P, 14 P, 17 P
  2-AA 60 142.0 35.6 3.5 108, 139, 179
P Precipitation

Tab. 5: Historical Negative Controls

S9 Mix Vehicle No. of
Plates		 No. of
Values		 Min Max Mean SD
               
TA 1535 Without (Alle) 288 107 6 20 13 2.8
With (Alle) 297 107 7 21 13 2.7
               
TA 100 Without (Alle) 288 107 73 157 112 13.5
With (Alle) 300 107 78 145 113 14.0
               
TA 1537 Without (Alle) 288 107 4 16 10 2.1
With (Alle) 294 107 5 14 10 2.0
               
TA 98 Without (Alle) 282 107 13 31 22 3.2
With (Alle) 294 107 15 53 26 5.4
               
E. Coli Without (Alle) 285 107 18 38 27 4.3
With (Alle) 288 107 19 39 27 3.9

Tab. 6: Historrical Positive Control

S9 Mix Positive
control		 No. of
Plates		 No. of
Values		 Min Max Mean SD
               
TA 1535 Without MNNG 200 73 406 8303 4032 1720.0
With 2-AA 207 73 99 433 235 100.3
               
TA 100 Without MNNG 201 73 611 6430 2853 1237.9
With 2-AA 204 73 534 4037 2080 965.0
               
TA 1537 Without AAC 204 73 342 2381 1008 364.6
With 2-AA 210 73 46 301 161 66.7
               
TA 98 Without NOPD 198 73 391 1711 693 204.0
With 2-AA 201 73 337 3025 1676 754.9
               
E. Coli Without 4-NQO 195 73 344 2265 987 423.9
With 2-AA 198 72 98 276 206 36.2

 

Conclusions:
A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

HPRT-Test (OECD 476, GLP)


The substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro.Two independent experiments werec arried out,both with and without the addition of liver S9 mix from phenobarbital- andb-naphthoflavone induced rats (exogenous metabolic activation). According to an initial range-finding cytotoxicity test for the determination of the experimental doses, the following concentrations were tested. Test groups printed in bold type were evaluated for gene mutations


1st Experiment without S9 mix


0; 4.7; 9.4; 18.8; 37.5; 75.0; 150.0µg/mL


with S9 mix


0; 4.7; 9.4; 18.8; 37.5; 75.0; 150.0µg/mL


2nd Experiment without S9 mix


0; 1.6; 3.1;6.3; 12.5; 25.0; 50.0; 100.0µg/mL


with S9 mix


0; 1.6; 3.1;6.3; 12.5; 25.0; 50.0; 100.0µg/mL


Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6 -8 days and then selected in 6 -thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted.


The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, ethyl methanesulfonate (EMS) and 7,12-dimethylbenz[a]- anthracene (DMBA), led to the expected increase in the frequencies of forward mutations. In this study, in the absence and the presence of metabolic activation, no cytotoxicity was observed up to the highest applied concentration evaluated for gene mutations. Based on the results of the present study, the test substance did not cause any relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other.


 


Chromosome aberration in vitro (OECD 473, GLP)


The test substance material was assessed for its potential to induce structural chromosome aberrations (clastogenic activity) and/or changes in the number of chromosomes (aneugenic activity) in V79 cells in vitro. Two independent experiments were carried out, both with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation). Based on the observations and the toxicity data of a previously performed pretest for a HPRT study the following concentrations were evaluated for cytogenetic damage.


 


1st Experiment


4-hour exposure, 18-hour sampling time, without S9 mix 0; 50; 100; 200 μg/mL


4-hour exposure, 18-hour sampling time, with S9 mix 0; 12.5; 25; 50 μg/mL


2nd Experiment


18-hour exposure, 18-hour sampling time, without S9 mix 0; 50; 100; 200 μg/mL


4-hour exposure, 28-hour sampling time, with S9 mix 0; 50; 100; 200 μg/mL


A sample of 300 metaphases for each test group was analyzed for chromosomal aberrations.


The vehicle controls gave frequencies of aberrations within the 95% control limit of our historical negative control data range for V79 cells. Both positive control substances, ethyl methanesulfonate (EMS) and cyclophosphamide (CPP), led to the expected increase in the number of cells containing structural chromosome aberrations. Thus, all validity criteria for this study were fulfilled. In this study, no cytotoxicity indicated by reduced relative population doubling (RPD) or mitotic rates was observed up to the highest applied test substance concentration. The highest scored test substance concentrations showed clear precipitates in culture medium at the end of exposure period. On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of structurally aberrant metaphases incl. and excl. gaps at both sampling times either without S9 mix or after adding a metabolizing system. No biologically relevant increase in the frequency of cells containing numerical chromosome aberrations was demonstrated either.


 


Ames Test (OECD 471, GLP)


The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay ( strains TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA). Tested concentrations were 33 μg - 5 000 μg/plate (SPT and PIT). Both experiments were performed with and without metabolic activation (liver S9 mix from induced rats). Precipitation of the test substance was found in the Standard plate test from 33 μg/plate onward. A bacteriotoxic effect was observed depending on the strain and test conditions from about 333 μg/plate onward. A biologically relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

No indication of genotoxicity was observed in the Ames test (OECD 471, GLP), the HPRT Test (OECD 476, GLP) and the in-vitro chromosome aberration assay (OECD 473, GLP). As a result the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008.