Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

negative, in vitro bacterial reverse mutation (with and without S-9 activation), OECD TG 471, 2007

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:
21-02-2007 to 16-03-2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected: August 2005 ; signature: November 2005
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine or tryptophan locus
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9
Test concentrations with justification for top dose:
Preliminary toxicity test (TA100 and WP2urvA): 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 1 (plate incorporation method): All strains: 0, 50, 150, 500, 1500, 5000 µg/plate
Experiment 2 (plate incorporation method): All strains: 0, 50, 150, 500, 1500, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in DMSO at the same concentration in solubility checks performed. DMSO was selected as the vehicle.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: Experiment 1. in medium; in agar (plate incorporation) ; Experiment 2. in medium; in agar (plate incorporation)

DURATION
- Exposure duration:
Experiment 1. All of the plates were incubated at 37 ± 3 ºC for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

Experiment 2. All of the plates were incubated at 37 ± 3 ºC for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).

A test item is considered non-mutagenic (negative) in the test system if the above criteria are not met.
In instances of data prohibiting definitive judgement about test item activity are reported as equivocal.
Statistics:
Statistical methods (Mahon, et al.); as recommended by the UKEMS Subcommittee on Guidelines for Mutagenicity Testing, Report - Part III (1989).
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
See table 1 and 2
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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, but tested up to precipitating concentrations
Remarks:
See table 1 and 2
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
See table 1 and 2
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
See table 1 and 2
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
See table 1 and 2
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The current Positive HCD dataset is presented in the full study report.
- Negative (solvent/vehicle) historical control data: The current background spontaneous revertant counts in concurrent untreated controls and/or or vehicle controls ; historic negative controls are presented in the full study report.
Remarks on result:
other: all strains/cell types tested

Table 1 : Test Results: Experiment 1: with and without metabolic activation and results of concurrent positive controls

S9-Mix

(-)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(DMSO)

96

101

86

(96)

4.5#

18

23

20

(20)

2.5

26

18

30

(25)

6.1

24

18

32

(25)

7.0

8

17

8

(11)

5.2

50 µg

93

85

81

(86)

6.1

17

22

21

(20)

2.6

21

19

23

(21)

2.0

20

13

23

(19)

5.1

9

10

14

(11)

2.6

150 µg

116

91

73

(93)

21.6

19

22

18

(20)

2.1

21

17

23

(20)

3.1

8

20

18

(15)

6.4

18

10

11

(13)

4.4

500 µg

85

76

82

(81)

4.6

24

19

18

(20)

3.2

21

17

23

(22)

5.5

19

25

20

(21)

3.2

8

11

7

(9)

2.1

1500 µg

81

91

82

(85)

5.5

15

20

9

(15)

5.5

24

20

21

(22)

2.1

23

18

23

(21)

2.9

7

2

13

(7)

6.0

5000 µg

81 P

54 P

85 P

(73)

16.9

18 P

14 P

14 P

(15)

2.3

21 P

18 P

16 P

(18)

2.5

28 P

24 P

23 P

(25)

2.6

14 P

14 P

10 P

(13)

2.3

Positive controls

S9-Mix

(-)

Name

Dose Level

No. of Revertants

ENNG

ENNG

ENNG

4NQO

9AA

3 µg

5 µg

2 µg

0.2 µg

80 µg

796

786

792

(791)

5.0

373

348

344

(355)

15.7

382

430

417

(410)

24.8

250

269

259

(259)

9.5

653

933

845

(810)

143.2

S9-Mix

(+)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(DMSO)

91

90

85

(89)

3.2#

8

16

10

(11)

3.8

27

29

23

(26)

3.1

25

28

30

(28)

2.5

14

7

8

(12)

2.3

50 µg

104

84

102

(97)

11.0

15

10

10

(12)

2.9

25

31

25

(27)

3.5

19

29

30

(26)

6.1

6

4

13

(8)

4.7

150 µg

96

108

96

(100)

6.9

10

11

10

(10)

0.6

25

23

24

(24)

1.0

21

15

26

(21)

5.5

13

10

9

(11)

2.1

500 µg

99

90

89

(93)

5.5

14

11

11

(12)

1.7

29

24

28

(27)

2.6

15

21

17

(18)

3.1

6

8

7

(7)

1.0

1500 µg

73

86

66

(75)

10.1

8

8

11

(9)

1.7

25

20

24

(23)

2.6

13

29

22

(21)

8.0

9

7

9

(8)

1.2

5000 µg

82 P

82 P

87 P

(84)

2.9

9 P

9 P

13 P

(10)

2.3

25 P

16 P

25 P

(22)

5.2

24 P

22 P

16 P

(21)

4.2

5 P

6 P

5 P

(5)

0.6

Positive controls

S9-Mix

(+)

Name

Dose Level

No. of Revertants

2AA

2AA

2AA

BP

2AA

1 µg

2 µg

10 µg

5 µg

2 µg

1585

1479

1661

(1575)

91.4

180

179

193

(184)

7.8

542

576

588

(569)

23.9

113

104

155

(124)

27.2

349

375

313

(346)

31.1

 

Table 2 : Test Results: Experiment 2: with and without metabolic activation and results of concurrent positive controls

S9-Mix

(-)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(DMSO)

127

110

138

(125)

14.1#

17

21

14

(17)

3.5

26

25

22

(24)

2.1

33

33

28

(21)

4.7

7

14

7

(9)

4.0

50 µg

115

105

124

(115)

9.5

17

13

21

(17)

4.0

29

13

29

(24)

9.2

32

26

24

(24)

7.6

8

5

6

(6)

1.5

150 µg

98

104

99

(100)

3.2

9

24

15

(16)

7.5

10

24

22

(19)

7.6

37

24

28

(17)

5.7

7

5

6

(6)

1.0

500 µg

129

136

158

(141)

15.1

10

14

15

(13)

2.6

18

13

18

(16)

2.9

16

28

24

(19)

5.5

7

8

4

(6)

2.1

1500 µg

95

78

75

(83)

10.8

10

18

15

(14)

4.0

23

28

8

(20)

10.4

26

29

27

(20)

5.6

7

3

8

(6)

2.6

5000 µg

79 P

80 P

74 P

(78)

3.2

16 P

13 P

19 P

(16)

3.0

19 P

18 P

18 P

(18)

0.6

17 P

26 P

27 P

(21)

4.2

2 P

5 P

9 P

(5)

3.5

Positive controls

S9-Mix

(-)

Name

Dose Level

No. of Revertants

ENNG

ENNG

ENNG

4NQO

9AA

3 µg

5 µg

2 µg

0.2 µg

80 µg

416

447

471

(445)

27.6

267

266

367

(300)

58.0

235

357

331

(308)

64.3

425

330

306

(364)

62.9

1338

1402

1382

(1374)

32.7

S9-Mix

(+)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(DMSO)

65

74

94

(78)

14.8#

10

9

10

(10)

0.6

21

22

23

(22)

1.0

29

34

22

(28)

6.0

9

10

8

(9)

1.0

50 µg

69

64

81

(71)

8.7

9

10

9

(9)

0.6

16

16

23

(18)

4.0

28

29

32

(30)

2.1

2

8

5

(5)

3.0

150 µg

78

97

76

(84)

11.6

8

8

10

(9)

1.2

23

29

24

(25)

3.2

25

25

22

(24)

1.7

8

6

5

(6)

1.5

500 µg

73

71

77

(74)

3.1

8

9

9

(9)

0.6

24

17

24

(22)

4.0

14

14

15

(14)

0.6

6

3

3

(4)

1.7

1500 µg

66

73

76

(72)

5.1

8

9

8

(8)

0.6

17

28

21

(22)

5.6

17

19

20

(19)

1.5

2

2

4

(3)

1.2

5000 µg

61 P

70 P

73 P

(68)

6.2

8 P

13 P

9 P

(10)

2.6

21 P

23 P

19 P

(21)

2.0

14 P

24 P

20 P

(19)

5.0

7 P

8 P

11 P

(9)

2.1

Positive controls

S9-Mix

(+)

Name

Dose Level

No. of Revertants

2AA

2AA

2AA

BP

2AA

1 µg

2 µg

10 µg

5 µg

2 µg

1879

2004

1965

(1949)

64.0

328

296

340

(321)

22.7

527

503

541

(524)

19.2

371

643

903

(639)

266.0

424

518

310

(417)

104.2

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO: 4-Nitroquinoline-1-oxide

9AA: 9-Aminoacridine

BP: Benzo(a)pyrene

2AA: 2-Aminoanthracene

N/T: Not tested at this dose level

S: partial absence of bacterial background lawn

P : precipitate

#: Standard deviation

Conclusions:
Interpretation of results:
Negative
Under the conditions of this study the test item was considered to be non-mutagenic in the presence and absence of S9 activation.
Executive summary:

The study was performed to the requirements of OECD Guideline 471, EU Method B.13/14, US EPA OCSPP 870.5100 and Japanese guidelines for bacterial mutagenicity testing under GLP, to evaluate the potential mutagenicity of the test item in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in both the presence and absence of S-9 mix. The test strains were treated with the test item the Ames plate incorporation method at up to five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined based on the results of a preliminary toxicity assay. The dose levels were 50 to 5000 µg/plate for all Salmonella strains: TA98, TA100, TA1535, TA1537 and for E.coli strain WP2uvrA-. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test item formulations. The vehicle (DMSO) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate or based on absence of cytotoxicity (reduction in growth of the bacterial background law) in all tester strains. A item item (oily in appearance) precipitate was observed on the plates at 5000 μg/plate. This did not prevent scoring of the revertant colonies. There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9 mix). It was concluded that, under the conditions of this assay, the test item gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in the presence and absence of S-9 mix.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Key study: OECD TG 471, 2007 : The study was performed to the requirements of OECD Guideline 471, EU Method B.13/14, US EPA OCSPP 870.5100 and Japanese guidelines for bacterial mutagenicity testing under GLP, to evaluate the potential mutagenicity of the test item in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in both the presence and absence of S-9 mix. The test strains were treated with the test item the Ames plate incorporation method at up to five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined based on the results of a preliminary toxicity assay. The dose levels were 50 to 5000 µg/plate for all Salmonella strains: TA98, TA100, TA1535, TA1537 and for E.coli strain WP2uvrA-. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test item formulations. The vehicle (DMSO) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate or based on absence of cytotoxicity (reduction in growth of the bacterial background law) in all tester strains. A item item (oily in appearance) precipitate was observed on the plates at 5000 μg/plate. This did not prevent scoring of the revertant colonies. There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9 mix). It was concluded that, under the conditions of this assay, the test item gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in the presence and absence of S-9 mix.

Justification for classification or non-classification

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for mutagenicity