Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay. Under the experimental conditions described, the test item is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation. The test item showed no biologically relevant increase of mutants in the in vitro mammalian cell gene mutation test after treatment with the test item (with and without metabolic activation).

Link to relevant study records
Reference
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:
2013-08-29 to 2013-09-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
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)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
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:
Aroclor-induced rat liver S-9 mix
Test concentrations with justification for top dose:
5 mg/plate or 5 μL/plate were generally selected as maximum test dose.
1st experiment: 0; 33; 100; 333; 1 000; 2 500 and 5 000 μg/plate
2nd experiment: 0; 33; 100; 333; 1 000; 2 500 and 5 000 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
with metabolic activation: 2-aminoanthracene; without metabolic activation: N-methyl-N'-nitro-N-nitrosoguanidine, 4-nitro-o-phenylendiamine, 9-aminoacridine and 4-nitroquinoline-N-oxide
Details on test system and experimental conditions:
Standard Plate Test and Preincubation Test both with and without metabolic activation
METHOD OF APPLICATION: Standard plate test based on the method of Ames et al. + Preincubation test based on the method described by Yahagi et al. and Matsushima et al.

Conditions
- Preincubation period: 20 minutes using a shaker
- Exposure duration: 48 - 72 hours
- Temperatur: Incubation at 37 °C

Titer determination
- The titer was determined only in the experimental parts with S9 mix both for the negative controls (vehicle only) and for the two highest doses in all experiments.

DETERMINATION OF CYTOTOXICITY
Toxicity detected by a
- decrease in the number of revertants
- clearing or diminution of the background lawn (= reduced his- or trp- background growth)
- reduction in the titer was recorded for all test groups both with and without S9 mix in all experiments and indicated
in the tables.
Evaluation criteria:
The test chemical is considered positive in this assay if the following criteria are met:
- A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S-9 mix or after adding a metabolizing system. A test substance is generally considered nonmutagenic in this test if:
- The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

Standard plate test

Preincubation test

TA1535

TA1535

 

Dose (µg)

Without S9 Mix

With S9 Mix

Dose (µg)

Without S9 Mix

With S9 Mix

 

DMSO

11

11

DMSO

11

11

 

33

13

12

33

10

11

 

100

11

11

100

11

13

 

333

12

11

333

10

12

 

1000

12

11

1000

12

9

 

2500

11

10

2500

11

10

 

5000

12

11

5000

10

11

 

 

MNNG

1130

MNNG

1198

 

2-AA

362

2-AA

453

 

 

 

TA100

TA100

 

Dose (µg)

Without S9 Mix

With S9 Mix

Dose (µg)

Without S9 Mix

With S9 Mix

 

DMSO

48

55

DMSO

59

63

 

33

48

52

33

59

67

 

100

49

47

100

56

65

 

333

51

52

333

58

64

 

1000

50

50

1000

59

63

 

2500

52

48

2500

61

64

 

5000

50

50

5000

59

62

 

 

MNNG

1201

MNNG

1477

 

2-AA

1339

2-AA

1606

 

 

 

TA1537

TA1537

 

Dose (µg)

Without S9 Mix

With S9 Mix

Dose (µg)

Without S9 Mix

With S9 Mix

 

DMSO

5

7

DMSO

6

7

 

33

6

6

33

6

7

 

100

7

7

100

6

7

 

333

6

5

333

6

6

 

1000

6

6

1000

6

7

 

2500

6

7

2500

6

6

 

5000

5

8

5000

7

7

 

 

AAC

559

AAC

547

 

2-AA

234

2-AA

229

 

 

 

TA98

TA98

 

Dose (µg)

Without S9 Mix

With S9 Mix

Dose (µg)

Without S9 Mix

With S9 Mix

 

DMSO

18

22

DMSO

16

18

 

33

20

24

33

16

19

 

100

17

24

100

14

17

 

333

19

27

333

15

19

 

1000

16

28

1000

16

17

 

2500

15

21

2500

15

19

 

5000

20

22

5000

15

17

 

 

NOPD

510

NOPD

864

 

2-AA

1239

2-AA

1153

 

 

 

E.coli

E.coli

 

Dose (µg)

Without S9 Mix

With S9 Mix

Dose (µg)

Without S9 Mix

With S9 Mix

 

DMSO

74

63

DMSO

34

32

 

33

66

65

33

34

29

 

100

65

66

100

35

32

 

333

64

70

333

34

33

 

1000

80

68

1000

34

31

 

2500

65

67

2500

36

30

 

5000

69

66

5000

35

29

 

 

4-NQO

824

4-NQO

1278

 

2-AA

554

2-AA

434

 

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

Additional information

Additional information from genetic toxicity in vitro:

Ames-Test

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 according to OECD guideline 471, EU method B.13/14 and EPA OPPTS 870.5100. The dose range tested was 33 µg/plate - 5000 µg/plate (Standard plate test and preincubation test) with and without metabolic activation. 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. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

In vitro Micronucleus Test

The substance was assessed for its potential to induce micronuclei in V79 cells in vitro (clastogenic or aneugenic activity). Two independent experiments were carried out, both with and without the addition of liver S9 mix from 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. The test groups printed in bold type were evaluated.

1st Experiment

4 hours exposure; 24 hours harvest time; without S9 mix:

0; 187.50; 375.00; 750.00; 1500.00 μg/mL

4 hours exposure, 24 hours harvest time, with S9 mix:

0; 187.50; 375.00; 750.00; 1500.00 μg/mL

2nd Experiment

24 hours exposure, 24 hours harvest time, without S9 mix

0; 187.50; 375.00; 750.00; 1500.00 μg/mL

4 hours exposure, 44 hours harvest time, with S9 mix

0; 187.50; 375.00; 750.00; 1500.00 μg/mL

A sample of at least 1000 cells for each culture were analyzed for micronuclei, i.e. 2000 cells for each test group. The negative controls gave frequencies of micronucleated cells within our historical negative control data range for V79 cells. Both positive control substances, EMS and cyclophosphamide, led to the expected increase in the number of cells containing micronuclei. In this study, no clear cytotoxicity indicated by reduced cell counts or proliferation index (CBPI) was observed up to the highest required test substance concentration. On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system. Thus, under the experimental conditions described, the test item is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.

In vitro Mammalian Cell Gene Mutation Test (HPRT Locus)

The test item was assessed for its potential to induce mutations at the HPRT locus using V79 cells of the Chinese Hamster. The selection of the concentrations was based on data from the pre-experiment. Experiment I and II with and without metabolic activation were performed as a 4 h short-term exposure assay. The test item was investigated in duplicate cultures at the following concentrations:

Experiment I

with and without metabolic activation:

0.5, 1.0, 2.5, 5.0, 7.5, 10.0 mM (1460 μg/mL)

Experiment II

with and without metabolic activation:

0.75, 2.0, 4.0, 6.0, 8.0, 10.0 mM (1460 μg/mL)

No precipitation of the test item was noted in the experiments. Biologically relevant growth inhibition (reduction of relative growth below 70%) was observed in experiment I and II without metabolic activation. In experiment I without metabolic activation the relative growth was 44. 1 % ( culture A) and 47.1% (culture B) for the highest concentration (10.0 mM) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 10.0 mM with a relative growth of 87.0% (culture A) and 80.9% (culture B). In experiment II without metabolic activation the relative growth was 69.9% (culture A) and 64.3% ( culture B) for the highest concentration (10.0 mM) evaluated. The highest concentration evaluated with metabolic activation was 10.0 mM with a relative growth of72.2% (culture A) and 70.1% (culture B). In both experiments no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). No dose-response relationship was observed. DMBA and EMS were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified under Regulation (EC) No 1272/2008, as amended for the sixth time in Regulation No 605/2014.