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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.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

The test item was not mutagenic to Salmonella typhimurium TA100, TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA, with or without an exogenous metabolic activation system (reference 7.6.1-1).

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
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:
SD Rat (7 week old, male) liver induced by phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
[1st trial]
-S9 mix: 0, 31.3, 62.5, 125, 250, 500 and 1000 μg/plate (TA1535, TA100 and TA1537)
0, 62.5, 125, 250, 500, 1000 and 2000 μg/plate (TA98 and WP2 uvrA)
+S9 mix: 0, 31.3, 62.5, 125, 250, 500 and 1000 μg/plate (TA1535 and TA1537)
0, 62.5, 125, 250, 500 and 1000 μg/plate (TA100, TA98 and WP2 uvrA)
[2nd trial]
-S9 mix: 0, 31.3, 62.5, 125, 250, 500 and 1000 μg/plate (all strains)
+S9 mix: 0, 31.3, 62.5, 125, 250, 500 and 1000 μg/plate (TA1535 and TA1537)
0, 62.5, 125, 250, 500 and 1000 μg/plate (TA100, TA98 and WP2 uvrA)
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent: The test substance is highly soluble in DMSO.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: -S9 mix: AF-2 (TA100, TA98 and WP2 uvrA), +S9 mix: 2-Aminoanthracene (all strains)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hrs

NUMBER OF PLATES: 3

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: other: growth inhibition
Evaluation criteria:
In any strain(s) tested under conditions with or without S9 mix, when the mean number of revertant colonies per plate increased twice more than that of the vehicle control and when the increase was shown to be dose-related and reproducible, the chemical was judged to be mutagenic.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
- Concentration range: 50 - 5000 μg/plate (common ratio: 3)
- Cytotoxic conc.: -S9 mix & +S9 mix: >=1500 µg/plate (TA100, TA98, WP2 uvrA);
>=500 μg/plate (TA1535,TA1537)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
[1st trial]
-S9 mix: 2000 μg/plate (WP2 uvrA); 1000 μg/plate (TA100, TA98, TA1537); 500 μg/plate (TA1535)
+S9 mix: 2000 μg/plate (WP2 uvrA, TA100, TA98); 1000 μg/plate (TA1537); 500 μg/plate (TA1535)
[2nd trial]
-S9 mix: 1000 μg/plate (WP2 uvrA, TA98); 500 μg/plate (TA100, TA1535, TA1537)
+S9 mix: 2000 μg/plate (WP2 uvrA, TA100); 1000 μg/plate (TA1535, TA98); 500 μg/plate (TA1537)

The test item did not induce mutation in the S. typhimurium and E. coli strains with and without S9 mix.

[1st trial]

-S9 mix: Negative (all test strains)

+S9 mix: Negative (all test strains)

 

Table 1. Mutagenicity of the test item** in reverse mutation test (I) on bacteria

With (+)

or without (-)

S9 mix

Test substance

dose

(μg/plate)

Number of revertants (Number of colonies/plate, Mean ± SD)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

S9 mix(-)

0

97, 114, 127

(113 ± 15.0)

10, 8, 13

(10 ± 2.5)

18, 22, 20

(20 ± 2.0)

26, 21, 18

(22 ± 4.0)

11, 8, 5

(8 ± 3.0)

31.3

106, 93, 122

(107 ± 14.5)

11, 11, 15

(12 ± 2.3)

ND

ND

10, 8, 6

(8 ± 2.0)

62.5

111, 121, 118

(117 ± 5.1)

13, 14, 8

(12 ± 3.2)

15, 25, 17

(19 ± 5.3)

23, 25, 26

(25 ± 1.5)

7, 9, 9

(8 ± 1.2)

125

102, 141, 123

(122 ± 19.5)

10, 11, 6

(9 ± 2.6)

13, 20, 24

(19 ± 5.6)

30, 13, 26

(23 ± 8.9)

9, 7, 9

(8 ± 1.2)

250

111, 103, 102

(105 ± 4.9)

6, 9, 8

(8 ± 1.5)

18, 17, 21

(19 ± 2.1)

17, 22, 15

(18 ± 3.6)

8, 5, 10

(8 ± 2.5)

500

91, 93, 99

(94 ± 4.2)

7*, 11*, 6*

(8 ± 2.6)

20, 16, 26

(21 ± 5.0)

14, 13, 18

(15 ± 2.6)

4, 7, 6

(6 ± 1.5)

1000

97*, 100*, 100*

(99 ± 1.7)

10*, 11*, 11*

(11 ± 0.6)

16, 25, 18

(20 ± 4.7)

15*, 21*, 22*

(19 ± 3.8)

6*, 4*, 4*

(5 ± 1.2)

2000

12*, 24*, 16*

(17 ± 6.1)

25*, 17*, 14*

(19 ± 5.7)

S9 mix(+)

0

122, 108, 105

(112 ± 9.1)

10, 13, 18

(14 ± 4.0)

23, 22, 20

(22 ± 1.5)

43, 35, 33

(37 ± 5.3)

9, 7, 17

(11 ± 5.3)

31.3

ND

14, 16, 8

(13 ± 4.2)

ND

ND

13, 8, 13

(11 ± 2.9)

62.5

89, 123, 102

(105 ± 17.2)

8, 10, 7

(8 ± 1.5)

30, 24, 24

(26 ± 3.5)

29, 31, 39

(33 ± 5.3)

15, 11, 8

(11 ± 3.5)

125

91, 83, 105

(93 ± 11.1)

14, 15, 18

(16 ± 2.1)

22, 40, 39

(34 ± 10.1)

19, 30, 27

(25 ± 5.7)

14, 13, 11

(13 ± 1.5)

250

77, 98, 103

(93 ± 13.8)

10, 9, 6

(8 ± 2.1)

24, 28, 23

(25 ± 2.6)

31, 27, 30

(29 ± 2.1)

14, 9, 8

(10 ± 3.2)

500

89, 91, 91

(90 ± 1.2)

8*, 7*, 15*

(10 ± 4.4)

25, 23, 17

(22 ± 4.2)

33, 36, 23

(31 ± 6.8)

18, 11, 15

(15 ± 3.5)

1000

86, 74, 95

(85 ± 10.5)

7*, 15*, 11*

(11 ± 4.0)

33, 18, 23

(25 ± 7.6)

21, 21, 41

(28 ± 11.5)

18*, 14*, 15*

(16 ± 2.1)

2000

65*, 72*, 84*

(74 ± 9.6)

16*, 32*, 31*

(26 ± 9.0)

26*, 27*, 27*

(27 ± 0.6)

Positive control

S9 mix (-)

Chemical

AF2

SA

AF2

AF2

9AA

Dose (μg/plate)

0.01

0.5

0.01

0.1

80

Number of
colonies/plate

618, 627, 632

(626 ± 7.1)

268, 257, 282

(269 ± 12.5)

146, 138, 104

(129 ± 22.3)

727, 733, 772

(744 ± 24.4)

628, 786, 686

(700 ± 79.9)

Positive control

S9 mix (+)

Chemical

2AA

2AA

2AA

2AA

2AA

Dose (μg/plate)

1

2

10

0.5

2

Number of
colonies/plate

970, 981, 1008

(986 ± 19.6)

243, 223, 213

(226 ± 15.3)

1423, 1378, 1280

(1360 ± 73.1)

259, 263, 258

(260 ± 2.6)

184, 179, 194

(186 ± 7.6)

AF2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide, SA: Sodium azide, 9AA: 9-Aminoacridine, 2AA: 2-Aminoanthracene

*: Inhibition was observed against growth of the bacteria.

**: Purity was above 98.0 % and water was contained below 0.2 % as impurity.

ND: Not done

[2nd trial]

-S9 mix: Negative (all test strains)

+S9 mix: Negative (all test strains)

 

Table 2. Mutagenicity of the test item** in reverse mutation test (II) on bacteria

With (+) or without (-)

S9 mix

Test substance

dose

(μg/plate)

Number of revertants (Number of colonies/plate, Mean ± SD)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

S9 mix(-)

0

88, 114, 113

(105 ± 14.7)

18, 15, 16

(16 ± 1.5)

20, 15, 19

(18 ± 2.6)

22, 22, 14

(19 ± 4.6)

11, 14, 9

(11 ± 2.5)

31.3

103, 110, 105

(106 ± 3.6)

14, 11, 14

(13 ± 1.7)

17, 17, 19

(18 ± 1.2)

30, 22, 20

(24 ± 5.3)

7, 4, 10

(7 ± 3.0)

62.5

95, 100, 100

(98 ± 2.9)

17, 14, 8

(13 ± 4.6)

20, 21, 34

(25 ± 7.8)

20, 17, 18

(18 ± 1.5)

5, 11, 10

(9 ± 3.2)

125

93, 93, 108

(98 ± 8.7)

14, 13, 13

(13 ± 0.6)

22, 32, 18

(24 ± 7.2)

21, 14, 20

(18 ± 3.8)

9, 14, 8

(10 ± 3.2)

250

106, 82, 114

(101 ± 16.7)

13, 7, 14

(11 ± 3.8)

21, 19, 22

(21 ± 1.5)

17, 22, 21

(20 ± 2.6)

10, 7, 11

(9 ± 2.1)

500

80*, 68*, 78*

(75 ± 6.4)

15*, 5*, 5*

(8 ± 5.8)

22, 14, 16

(17 ± 4.2)

16, 23, 29

(23 ± 6.5)

8*, 10*, 7*

(8 ± 1.5)

1000

3*, 78*, 79*

(53 ± 43.6)

4*, 2*, 4*

(3 ± 1.2)

25*, 28*, 19*

(24 ± 4.6)

18*, 16*, 19*

(18 ± 1.5)

7*, 7*, 9*

(8 ± 1.2)

2000

S9 mix(+)

0

112, 121, 120

(118 ± 4.9)

8, 12, 12

(11 ± 2.3)

33, 28, 32

(31 ± 2.6)

44, 36, 29

(36 ± 7.5)

19, 10, 19

(16 ± 5.2)

31.3

ND

13, 13, 18

(15 ± 2.9)

ND

ND

10, 17, 12

(13 ± 3.6)

62.5

138, 129, 124

(130 ± 7.1)

17, 14, 6

(12 ± 5.7)

35, 33, 29

(32 ± 3.1)

45, 32, 42

(40 ± 6.8)

17, 26, 16

(20 ± 5.5)

125

133, 103, 118

(118 ± 15.0)

17, 18, 17

(17 ± 0.6)

28, 26, 31

(28 ± 2.5)

27, 38, 44

(36 ± 8.6)

14, 22, 20

(19 ± 4.2)

250

124, 95, 104

(108 ± 14.8)

9, 14, 13

(12 ± 2.6)

23, 29, 21

(24 ± 4.2)

43, 38, 25

(35 ± 9.3)

11, 10, 13

(11 ± 1.5)

500

81, 96, 107

(95 ± 13.1)

12, 12, 11

(12 ± 0.6)

24, 23, 26

(24 ± 1.5)

36, 24, 33

(31 ± 6.2)

13*, 11*, 9*

(11 ± 2.0)

1000

75, 96, 104

(92 ± 15.0)

13*, 6*, 6*

(8 ± 4.0)

24, 23, 19

(22 ± 2.6)

25*, 27*, 23*

(25 ± 2.0)

7*, 10*, 11*

(9 ± 2.1)

2000

59*, 72*, 84*

(72 ± 12.5)

18*, 20*, 22*

(20 ± 2.0)

20*, 23*, 22*

(22 ± 1.5)

Positive control

S9 mix (-)

Chemical

AF2

SA

AF2

AF2

9AA

Dose (μg/plate)

0.01

0.5

0.01

0.1

80

Number of colonies/plate

506, 526, 513

(515 ± 10.1)

207, 224, 235

(222 ± 14.1)

100, 116, 124

(113 ± 12.2)

756, 631, 787

(725 ± 82.6)

890, 726, 734

(783 ± 92.5)

Positive control

S9 mix (+)

Chemical

2AA

2AA

2AA

2AA

2AA

Dose (μg/plate)

1

2

10

0.5

2

Number of colonies/plate

1164, 1206, 1145

(1172 ± 31.2)

254, 288, 268

(270 ± 17.1)

913, 869, 854

(879 ± 30.7)

372, 303, 455

(377 ± 76.1)

163, 180, 169

(171 ± 8.6)

AF2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide, SA: Sodium azide, 9AA: 9-Aminoacridine, 2AA: 2-Aminoanthracene

*: Inhibition was observed against growth of the bacteria.

**: Purity was above 98.0 % and water was contained below 0.2 % as impurity.

ND: Not done

Conclusions:
The test item was not mutagenic to Salmonella typhimurium TA100, TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA, with or without an exogenous metabolic activation system.
Executive summary:

The test item was examined for its mutagenic activity in two series of a bacterial reverse mutation assay (OECD 471) employing Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA as indicator organisms.

The plate incorporation test with and without addition of liver S9 mix from SD Rat (7 week old, male) liver, induced by phenobarbital and 5,6-benzoflavone, was used. In this study, two experimental series were performed. Treatments of all tester strains were performed using the test item solved in DMSO in the absence and in the presence of S9 mix, using final concentrations between 31.3 and 2000 µg/plate, plus vehicle and positive controls.

The strain specific positive control test materials, namely AF-2, sodium azide and 9-aminoacridine in the absence of S9 mix, yielded the expected mutant frequencies. The genotype of the tester strains used was thus confirmed. 2-Aminoanthracene which requires metabolic activation, was strongly mutagenic. This indicates that the exogenous metabolizing system used in the present investigation (S9 mix) was functioning. Thus, the study was considered valid.

No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. During the experiments there was no evidence of toxicity to the bacteria caused by the test material exposure.

Under the conditions described, there were no relevant increases in revertant numbers after treatment with the test item observed in both, the absence and presence of S9 mix.

In conclusion, the test material was considered to be non-mutagenic under the described experimental conditions.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Bacterial Reverse Mutation Test

The test item was examined for its mutagenic activity in two series of a bacterial reverse mutation assay (OECD 471) employing Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA as indicator organisms (reference 7.6.1-1).

The plate incorporation test with and without addition of liver S9 mix from SD Rat (7 week old, male) liver, induced by phenobarbital and 5,6-benzoflavone, was used. In this study, two experimental series were performed. Treatments of all tester strains were performed using the test item solved in DMSO in the absence and in the presence of S9 mix, using final concentrations between 31.3 and 2000 µg/plate, plus vehicle and positive controls.

The strain specific positive control test materials, namely AF-2, sodium azide and 9-aminoacridine in the absence of S9 mix, yielded the expected mutant frequencies. The genotype of the tester strains used was thus confirmed. 2-Aminoanthracene which requires metabolic activation, was strongly mutagenic. This indicates that the exogenous metabolizing system used in the present investigation (S9 mix) was functioning. Thus, the study was considered valid.

No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. During the experiments there was no evidence of toxicity to the bacteria caused by the test material exposure.

Under the conditions described, there were no relevant increases in revertant numbers after treatment with the test item observed in both, the absence and presence of S9 mix.

In conclusion, the test material was considered to be non-mutagenic under the described experimental conditions.

Justification for classification or non-classification

Classification, Labeling, 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. The results indicate that the substance is non-mutagenic. Based on available data on genetic toxicity, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the twelfth time in Regulation (EC) No 2019/521.