Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames Test: negative (non guideline study in Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537; OECD 471 study in E. coli WP2 uvrA);

gene mutation (HPRT): negative (OECD 476);
chromosomal aberration: negative (OECD 473)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 Feb 2014 - 21 Feb 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
yes
Remarks:
only E. coli strain tested
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
trp operon for the E. coli strain
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 liver mix prepared from Wistar rats treated with 80 mg/kg bw phenobarbital i.p. and β-naphthoflavone orally, each on three consecutive days.
Test concentrations with justification for top dose:
First experiment (standard plate test, with and without metabolic activation, 3 plates/dose or control): 0, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Second experiment (preincubation test with and without metabolic activation, 3 plates/dose or control): 0, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: due to the limited solubility of the test substance in water, DMSO was used as vehicle.
Untreated negative controls:
yes
Remarks:
sterility control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
with S9-mix
Positive control substance:
other: 2-aminoanthracene (2-AA)
Remarks:
60 µg/plate in DMSO
Positive controls:
yes
Remarks:
without S9-mix
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
5 µg/plate in DMSO
Details on test system and experimental conditions:
STANDARD PLATE TEST (SPT)
According to Ames et al., Mut Res 31: 347-364 (1975) and Maron & Ames, Mut Res 113: 173-215 (1983)

In the standard plate test, tubes were filled with 2mL portions of soft agar and kept in a water bath at 42 to 45°C. This soft agar consisted of 100 mL agar and 10 mL amino acid solution.
Then following components are added:
0.1 mL test solution or vehicle
0.1 mL fresh bacterial culture
0.5 mL S9 -mix or phosphate buffer
After mixing samples were poured onto Merckoplate® (minimal glucose agar plates) plate and incubated for 48 - 72 hrs in the dark at 37°C.

PREINCUBATION TEST (PIT)
According to Yahagi et al. Mut Res 48: 121-129 (1977) and Matsushima et al., In: Norpoth, K.H. and R.C. Garner, Short-Term Test Systems for Detecting Carcinogens, Springer Verlag Berlin, Heidelberg, New York (1980)

For the preincubation test 0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL of either S9 mix or phosphate buffer were incubated at 37°C for 20 minutes. After addition of 2 mL soft agar, samples were poured onto agar plates and incubated again at 37°C for 48 to 72 hrs.

Evaluation criteria:
Acceptance 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 the tester strain.
• The sterility controls revealed no indication of bacterial contamination (see Appendix 3).
• The positive control substances both with and without S9 mix induced a distinct increase in
the number of revertant colonies within the range of the historical positive control data or
above.
• Fresh bacterial culture containing approximately 109 cells per mL were used.

Assessment criteria
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 of the spontaneous mutation rate in at least in the 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 the tester strains were within the historical negative control
data range under all experimental conditions in at least two experiments carried out
independently of each other.
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
no increase in number of revertants was observed
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
from about 2500 µg/plate onward
Vehicle controls validity:
valid
Untreated negative controls validity:
other: sterility control, yes
Positive controls validity:
valid
Additional information on results:
A biologically relevant increase in the number of 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.
No bacteriotoxic effect was observed in the standard plate test.
In the preincubation assay bacteriotoxicity (reduced trp- background growth, slight decrease in the number of trp+ revertants) was occasionally observed depending on the test conditions from about 2 500 μg/plate onward.
Conclusions:
Under the experimental conditions of this study, the test substance is not mutagenic in the Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.
Executive summary:

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of Escherichia coli WP2 uvrA, in a reverse mutation assay. The test concentrations were 0, 33, 100, 333, 1000, 2500, and 5000 µg/plate for the standard plate test with and without S9 mix, and for the preincubation test with and without S9 mix, respectively. Negative (sterility and solvent) and positive controls were considered.

Precipitation of the test substance was found from about 2 500 μg/plate onward with and without S9 mix. A weak bacteriotoxic effect was occasionally observed depending on the test conditions from about 2 500 μg/plate

onward.

A biologically relevant increase in the number of 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.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable well documented publication which meets basic scientific principles.
Principles of method if other than guideline:
The test substance was evaluated for mutagenicity in the Salmonella/microsome preincubation assay using a standard protocol approved by the National Toxicology Program. Doses of 0, 100, 333, 1000, 1666, 3333, 6666 and 10000 µg/plate were tested in four Salmonella typhimurium strains (TA98, TA100, TA1535, and TA1537) in the presence and absence of Aroclor-induced rat or hamster liver S9.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
induced male Sprague Dawley rat liver S9, induced male Syrian hamster liver S9
Test concentrations with justification for top dose:
0, 100, 333, 1000, 1666, 3333, 6666, 10000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: Absence of S9: TA98, 2-nitrofluorene, TA100 and TA1535, sodium azide, TA1537, 9-aminoacridine. With S9: All strains, 2-aminoanthracene.
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
from 3333 µg/plate onwards
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Strain: TA1535

Dose

No Activation
(Negative)

No Activation
(Negative)

10% HLI
(Equivocal)

10% HLI
(Negative)

10% HLI
(Negative)

10% RLI
(Equivocal)

10% RLI
(Negative)

10% RLI
(Negative)

Protocol

Preincubation

Preincubation

Preincubation

Preincubation

Preincubation

Preincubation

Preincubation

Preincubation

ug/Plate

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

0     

25

1.2

15

1.7

10

0.9

9

1.3

16

0.9

12

1.5

10

1.2

12

1.7

100     

26

2.3

11

1.5

 

 

 

 

 

 

 

 

 

 

 

 

333     

28

5.2

12

2.5

11

0.7

6

0.9

11

1.2

12

2.3

10

1.2

5

0.9

1000     

24

0.7

10

2.1

11

2.1

8

2.6

9

1.5

13

0.3

10

2.1

6

1.2

1666     

17

0.6

9

1.8

 

 

7

1.2

10

2.5

 

 

11

0.6

8

2.1

3333     

8s

2.8

1s

0.9

35

5.1

10

2.3

10

1.5

31

3.2

11

2.3

9

1.5

6666     

 

 

 

 

24

3.5

1s

0.7

12

1.9

31

1.2

0s

0

10

1.8

10000     

 

 

 

 

5s

5.3

 

 

 

 

0s

0

 

 

 

 

Positive Control

359

15

180

2.1

483

4.5

114

7.3

312

11.7

287

35.3

137

0.3

129

17.6

 

Strain: TA100

Dose

No Activation
(Negative)

No Activation
(Negative)

10% HLI
(Equivocal)

10% HLI
(Negative)

10% HLI
(Negative)

10% RLI
(Equivocal)

10% RLI
(Negative)

10% RLI
(Equivocal)

Protocol

Preincubation

Preincubation

Preincubation

Preincubation

Preincubation

Preincubation

Preincubation

Preincubation

ug/Plate

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

0     

116

4.3

96

2.6

137

3

96

3.2

128

18.1

141

7.3

108

2.6

128

6.7

100     

117

7.7

95

10.5

 

 

 

 

 

 

 

 

 

 

 

 

333     

136

5.8

104

10.3

139

13.9

106

3.3

129

12

141

13.4

109

8.1

136

4.2

1000     

137

13

101

1.5

138

18

112

2.3

126

7.9

150

18.9

133

10.1

143

4.9

1666     

136

7.2

101

13.7

 

 

105

7.1

143

7.8

 

 

133

10.1

152

7.8

3333     

28

1.2

46s

32.5

177

3.2

113

7.2

127

7.6

183

13.6

135

8.7

196

5.5

6666     

 

 

 

 

190

4.3

83s

8.1

137

12.9

185

14.6

43s

43

187

2

10000     

 

 

 

 

120s

5.8

 

 

 

 

132s

10.2

 

 

 

 

Positive Control

397

20.2

221

20.9

1516

77.3

446

28.7

1112

18.2

500

27.7

215

10.6

572

32.9

 

TA 98

Dose

No Activation
(Negative)

No Activation
(Negative)

10% HLI
(Negative)

10% HLI
(Negative)

10% RLI
(Negative)

10% RLI
(Negative)

Protocol

Preincubation

Preincubation

Preincubation

Preincubation

Preincubation

Preincubation

ug/Plate

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

0     

14

2

14

1.5

40

2.5

20

1.9

35

3.3

27

0.6

100     

14

1.5

15

2.5

 

 

 

 

 

 

 

 

333     

12

0.9

13

2

31

0.6

22

3.3

29

1.3

17

1.3

1000     

12

1.2

11

0.7

37

8.4

23

3.1

37

2.3

16

1.5

1666     

11

0.9

14

2

 

 

22

2.8

 

 

16

2.1

3333     

0s

0

0s

0

21

3.6

11

2.6

31

6.2

18

1.9

6666     

 

 

 

 

21

6.6

0s

0

23

2.6

0s

0

10000     

 

 

 

 

0s

0

 

 

15s

7.8

 

 

Positive Control

373

15.6

430

4.6

1033

95.2

338

5.2

398

8.4

118

7

 

Strain: TA1537

Dose

No Activation
(Negative)

No Activation
(Negative)

10% HLI
(Negative)

10% HLI
(Negative)

10% RLI
(Negative)

10% RLI
(Negative)

Protocol

Preincubation

Preincubation

Preincubation

Preincubation

Preincubation

Preincubation

ug/Plate

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

0     

7

0.6

5

0.9

4

0.7

4

0.7

6

1.2

3

0.9

100     

7

1.7

3

0.3

 

 

 

 

 

 

 

 

333     

5

0.9

4

0.6

6

0.6

5

1.3

8

2.3

6

1.2

1000     

4

0.3

4

0.9

9

2.9

5

1.3

8

2.3

4

0.6

1666     

6

1.5

3

0.3

 

 

5

1.3

 

 

3

0.6

3333     

0s

0

0s

0

6

0.6

5

1

6

1.2

5

1.5

6666     

 

 

 

 

7

1.3

2s

0.9

7

0.9

0s

0

10000     

 

 

 

 

7s

1.2

 

 

3s

3

 

 

Positive Control

78

3.8

86

4.9

473

16.9

135

17.5

174

7.8

105

5

Abbreviations:
RLI = induced male Sprague Dawley rat liver S9
HLI = induced male Syrian hamster liver S9
s = Slight Toxicity; p = Precipitate; x = Slight Toxicity and Precipitate; T = Toxic; c = Contamination

Under the conditions tested the test substance was non mutagenic in the bacterial AMES-Test. The positive control gave the expected values.
Conclusions:
Under the experimental conditions of this study, the test substance was not mutagenic in the Salmonella/microsome preincubation assay in the absence and the presence of metabolic activation.
Executive summary:

The test substance was evaluated for mutagenicity in the Salmonella/microsome preincubation assay using a standard protocol approved by the National Toxicology Program. Doses of 0, 100, 333, 1000, 1666, 3333, 6666, 10000 µg/plate were tested in four Salmonella typhimurium strains (TA98, TAl00, TAl535 and TAl537) in the presence and absence of Aroclor-induced rat or hamster liver S9. These tests were negative and the highest ineffective dose level tested in all four Salmonella tester strains under all treatment conditions was 3333 µg/plate. Cytotoxicity was observed from this concentration onwards.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
HPRT-locus
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix
Test concentrations with justification for top dose:
46, 93, 185, 371, 741 µg/ml
Vehicle / solvent:
water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with metabolic activation
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Conclusions:
In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells and is therefore considered to be non-mutagenic in this HPRT assay.
Executive summary:

The study was performed to investigate the potential of the test substance to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The highest applied concentration (741 μg/mL) was equal to a molar concentration of approximately 10 mM.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test item and the activity of the metabolic activation system.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix
Test concentrations with justification for top dose:
1st Experiment
4-hour exposure, 18-hour sampling time, without S9 mix
0; 46.3; 92.5; 185; 370; 740 μg/mL
4-hour exposure, 18-hour sampling time, with S9 mix
0; 46.3; 92.5; 185; 370; 740 μg/mL

2nd Experiment
18-hour exposure, 18-hour sampling time, without S9 mix
0; 92.5; 185; 370; 555; 740 μg/mL
18-hour exposure, 28-hour sampling time, without S9 mix
0; 185.0; 370; 555; 740 μg/mL
4-hour exposure, 28-hour sampling time, with S9 mix
0; 46.3; 92.5; 185; 370; 740 μg/mL
Vehicle / solvent:
culture medium (Minimal Essential Medium: MEM)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Species / strain:
Chinese hamster lung fibroblasts (V79)
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
Conclusions:
Under the experimental conditions described, the test substance is considered not to have a chromosome-damaging (clastogenic) effect under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.
Executive summary:

The test substance was assessed for its potential to induce structural chromosomal aberrations (clastogenic activity) and/or changes in the number of chromosomes (aneugenic activity) in V79 cells in vitro both in the absence and the presence of a metabolizing system. According to an initial range-finding cytotoxicity test for the determination of the experimental doses, the test substance did not exhibit any pronounced toxicity up to the highest recommended dose of 740 μg/mL (approx. 10 mM). Thus, the test substance was tested up to and including this dose.

The negative controls gave frequencies of aberrations within the range expected for the V79 cell line. Both of the positive control substances, EMS and cyclophosphamide, led to the expected increase in the number of cells containing structural chromosomal aberrations.

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 at both sampling times either without S9 mix or after adding a metabolizing system in two experiments performed independently of each other.

No relevant increase in the frequency of cells containing numerical chromosome aberrations was demonstrated either.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In vitro studies:

The test substance was evaluated for mutagenicity in the Salmonella/microsome preincubation assay using a standard protocol approved by the National Toxicology Program. Doses of 0, 100, 333, 1000, 1666, 3333, 6666, 10000 µg/plate were tested in four Salmonella typhimurium strains (TA98, TAl00, TAl535 and TAl537) in the presence and absence of Aroclor-induced rat or hamster liver S9. These tests were negative and the highest ineffective dose level tested in all four Salmonella tester strains under all treatment conditions was 3333 µg/plate.

In a follow up study a fifth strain was tested in the Ames test according to OECD 471. The test substance was tested for mutagenicity in the reverse mutation assay both in the standard plate test and in the preincubation test with and without the addition of a metabolizing system (S9 mix) obtained from rat liver using the strain Escherichia coli WP2 uvrA. The test substance was not mutagenic in the Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation either.

The potential of the test substance to induce gene mutations was investigated at the HPRT-locus in V79 cells of the chinese hamster in an OECD 476 guideline study. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The highest applied concentration (741 μg/mL) was equal to a molar concentration of approximately 10 mM.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test item and the activity of the metabolic activation system.

The test substance did not induce gene mutations at the HPRT locus in V79 cells neither with nor without metabolic activation.

The test substance was assessed for its potential to induce structural chromosomal aberrations in V79 cells in vitro both in the absence and the presence of a metabolizing system up to a dose of 740 µg/ml (OECD 473). The test substance did not cause any biologically relevant increase in the number of structurally aberrant metaphases at both sampling times either without S9 mix or after adding a metabolizing system in two experiments performed independently of each other. No relevant increase in the frequency of cells containing numerical chromosome aberrations was demonstrated either. Thus, the test substance is considered not to have chromosome-damaging effects under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.


Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for genotoxicity under Regulation (EC) No. 1272/2008, as amended for the 13th time in Regulation (EU) 2018/1480.