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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

OECD guideline, GLP-compliant in vitro study to assess gene mutations in bacteria was available. All 4 strains of S. Typhimurium (TA 98, TA 100, TA 1535, TA 1537) and E. Coli WP2 uvr A were negative for genotoxicity with and without metabolic activation.


 


OECD guideline, GLP-compliant in vitro study to assess cytogenicity in mammalian cells was available. The substance increased the number of polyploid cells and cells with chromosomal structural abnormality with and without metabolic activation. Under the conditions of the test, the substance was positive for genotoxicity.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 Jan - 09 Mar 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
other: The guideline of the Act on the Evaluation of Chemical Substances and Regulation of Their Manufacture, etc. (Kanpogyo No. 700, Yakuhatsu No. 1039, 61 Kikyoku No. 1014, 1986)
Deviations:
not specified
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
not specified
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon; trp operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254.
Test concentrations with justification for top dose:
First experiment: 50, 100, 500, 1000, 2000 and 5000 µg/plate with and without metabolic activation
Second experiment: 15.6, 31.3, 62.5, 125, 250, 500 and 1000 µg/plate with and without metabolic activation
Vehicle / solvent:
- Solvent used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
furylfuramide
other: 2-Aminoanthracene: +S9: 2 or 20 µg/plate for TA1535 and WP2uvrA
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

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

NUMBER OF REPLICATIONS: Duplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: Reduction of bacterial growth
Evaluation criteria:
From these results, the mutagenicity of the test substance was evaluated as negative:
- The number of colonies with reverse mutation of test bacterial strains induced by the test substance was within the range of naturally induced reverse mutation found in the bacterial strains of each test.
- The test substance showed antibacterial activity against the test bacterial strains.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment I: ≥ 500 µg/plate with and without metabolic activation; Experiment II: ≥ 500 µg/plate without metabolic activation and ≥ 250 µg/plate with metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment I: ≥ 500 µg/plate with and without metabolic activation; Experiment II: ≥ 250 µg/plate with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment I: ≥ 500 µg/plate with and without metabolic activation; Experiment II: ≥ 250 µg/plate without metabolic activation and ≥ 500 µg/plate with metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment I: ≥ 500 µg/plate with and without metabolic activation; Experiment II: ≥ 250 µg/plate without metabolic activation and ≥ 500 µg/plate with metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment I: ≥ 500 µg/plate with and without metabolic activation; Experiment II: ≥ 500 µg/plate without metabolic activation and at 1000 µg/plate with metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Table 1: Results of Experiment I

Presence of metabolic activation system

Concentration of test substance (μg/plate)

Number of reverse mutation (colonies/plate)

Base substitution

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

- S9

Solvent control

159

174

(167)

9

11

(10)

24

17

(21)

20

26

(23)

10

10

(10)

50

187

149

(168)

11

16

(14)

29

21

(25)

23

16

(20)

9

9

(9)

100

152

146

(149)

17

11

(14)

28

18

(23)

20

12

(16)

7

4

(6)

500

0*

0*

10

15

(13)*

1

0

(1)*

0*

1000

0*

0*

0*

0*

0*

2000

0*

0*

0*

0*

0*

5000

0*C

0*C

0*C

0*C

0*C

+ S9

Solvent control

163

212

(188)

16

15

(16)

26

24

(25)

33

37

(35)

13

5

(9)

50

209

192

(201)

18

13

(16)

37

28

(33)

37

48

(43)

9

8

(9)

100

178

166

(172)

17

26

(22)

21

15

(18)

41

55

(48)

7

6

(7)

500

79

65

(72)*

9

7

(8)*

16

19

(18)*

32

9

(21)*

0*

1000

7

12

(10)*

3

1

(2)*

5

6

(6)*

0*

0*

2000

0*

0*

0*

0*

0*

5000

0*C

0*C

0*C

0*C

0*C

Positive control

- S9

Name

AF-2

SA

ENNG

AF-2

9-AA

Concentration (μg/plate)

0.01

0.5

2

0.1

80

Colonies/plate

606

543

(575)

294

284

(289)

669

653

(661)

344

354

(349)

423

565

(497)

+ S9

Name

BP

2-AA

2-AA

BP

BP

Concentration (μg/plate)

5

2

20

5

5

Colonies/plate

1105

1155

(1130)

169

210

(190)

999

1062

(1031)

413

421

(417)

174

214

(194)

 ( ): The mean colony number is given in parentheses

*: Bacterial growth suppression was observed

C: Precipitation (crystals) was observed

AF-2: 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (furfurylamide)

SA: Sodium azide

ENNG: N-ethyl-N-nitroso-N’-nitroguanidine (ENNG)

9-AA: 9-Aminoacridine

BP: Benzo[a]pyrene

2-AA: 2-Aminoanthracene

Table 2: Results of Experiment II

Presence of metabolic activation system

Concentration of test substance (μg/plate)

Number of reverse mutation (colonies/plate)

Base substitution

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

- S9

Solvent control

143

176

(160)

13

9

(11)

23

13

(18)

33

23

(28)

8

7

(8)

15.6

161

146

(154)

10

14

(12)

22

18

(20)

23

13

(18)

6

3

(5)

31.3

138

167

(153)

15

17

(16)

11

21

(16)

22

12

(17)

5

10

(8)

62.5

175

148

(162)

21

13

(17)

14

12

(13)

24

25

(25)

11

5

(8)

125

148

162

(155)

16

11

(14)

9

18

(14)

16

21

(19)

5

4

(5)

250

38

10

(24)*

11

13

(12)

16

13

(15)

12

9

(11)*

0*

500

0*

2

2

(2)*

4

5

(5)*

0*

0*

1000

 

 

0*

 

 

+ S9

Solvent control

185

139

(162)

19

15

(17)

25

18

(22)

48

46

(47)

7

11

(9)

15.6

190

157

(174)

11

13

(12)

17

16

(22)

45

60

(53)

9

6

(8)

31.3

164

162

(163)

16

16

(16)

16

17

(17)

49

45

(47)

13

20

(17)

62.5

176

171

(174)

17

20

(19)

17

28

(23)

62

54

(58)

9

15

(12)

125

176

195

(186)

20

16

(18)

22

20

(21)

57

54

(56)

5

13

(9)

250

165

131

(148)

0*

20

21

(21)

34

37

(36)

10

14

(12)*

500

69

85

(77)*

0*

10

17

(14)

32

31

(32)*

0*

1000

 

 

7

2

(5)*

 

 

Positive control

- S9

Name

AF-2

SA

ENNG

AF-2

9-AA

Concentration (μg/plate)

0.01

0.5

2

0.1

80

Colonies/plate

516

597

(557)

204

188

(196)

239

296

(268)

320

393

(357)

780

432

(606)

+ S9

Name

BP

2-AA

2-AA

BP

BP

Concentration (μg/plate)

5

2

20

5

5

Colonies/plate

1357

1371

(1364)

318

354

(336)

1286

1229

(1258)

367

447

(407)

215

210

(213)

( ): The mean colony number is given in parentheses

*: Bacterial growth suppression was observed

C: Precipitation (crystals) was observed

AF-2: 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (furfurylamide)

SA: Sodium azide

ENNG: N-ethyl-N-nitroso-N’-nitroguanidine (ENNG)

9-AA: 9-Aminoacridine

BP: Benzo[a]pyrene

2-AA: 2-Aminoanthracene

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 Jan - 31 Mar 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted Jul 2016
Deviations:
not specified
GLP compliance:
yes
Type of assay:
other: in vitro mammalian chromosome aberration
Target gene:
Not applicable
Species / strain / cell type:
other: CHL/IU
Remarks:
(Chinese hamster)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Dainippon Pharmaceutical Co., Ltd.
- Cell doubling time: Approximately 15 h
- Number of passages: 22
- Methods for maintenance in cell culture: Cells were cultured at 37 °C and 5% CO2.
- Modal number of chromosomes: 25

MEDIA USED
- Type and identity of media including CO2 concentration: Eagle`s MEM (Nissui Pharmaceutical Co., Ltd.) supplemented with 10% fetal calf serum (Gibco Lab., Lot No. 32P3481)
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
Test concentrations with justification for top dose:
Experiment I:
24 h treatment: 4, 8 and 16 µg/mL without metabolic activation
48 h treatment: 4, 8 and 16 µg/mL without metabolic activation

Experiment II
6 h treatment: 12.5, 25 and 50 µg/mL with and without metabolic activation
Vehicle / solvent:
- Solvent used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding: 4 x 10E03 cells/mL

DURATION
- Exposure duration: 6, 24 and 48 h
- Expression time (cells in growth medium): 6 h treatment: 18 h
- Fixation time (start of exposure up to fixation or harvest of cells): 6 h treatment: 26 h; 24 h treatment: 26 h; 48 h treatment: 50 h

SPINDLE INHIBITOR: colcemid 0.1 µg/mL medium

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: Parallel cultures were treated at each concentration.100 metaphases per culture were scored, in total 200 cells per concentration.

DETERMINATION OF CYTOTOXICITY
- Method: cell proliferation rate (%)

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
Key result
Species / strain:
other: CHL/IU
Remarks:
Chinese hamster
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
starting at 15 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
other: CHL/IU
Remarks:
Chinese hamster
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
under cytotoxic conditions
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
starting at 50 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
In a range-finding study, the cytotoxic properties of the test substance were investigated. The cells were treated for 48 h at concentrations of 5, 7.5, 10, 15, 25 and 30 µg/mL without metabolic activation and for 6 h at concentrations of 30, 40, 50, 60, 70, 80 and 90 µg/mL with metabolic activation, respectively. The concentrations evaluated in the main experiments were based on the results of the pre-experiment.

Table 1: Results of Experiment 1 (24-h and 48-h treatment without metabolic activation)

Treatment Processing time (h) Treatment level (µg/mL) Number of observed cells Number of polyploid cells Number and frequency (%) of the cells with chromosomal structural abnormality*
(%) Evaluation** Gap Chromatid type Chromosome type Total Evaluation**
g ctb cte csb cse without gap with gap
Negative control (DMSO) 24 0 100 0 n.a. 0 0 0 0 0 0 0 n.a.
100 0 0 0 0 0 0 0 0
200 0 0 0 0 0 0 0 0
48 0 100 1 n.a. 0 0 0 0 0 0 0 n.a.
100 0 2 1 0 0 0 1 3
200 1 (0.5) 2 (1.0) 1 (0.5) 0 0 0 1 (0.5) 3 (1.5)
Test substance 24 4 100 0 - 0 0 0 0 0 0 0 -
100 0 1 0 0 0 0 0 1
200 0 1 (0.5) 0 0 0 0 0 1 (0.5)
8 100 1 - 0 1 0 1 0 2 2 +
100 0 0 2 0 0 0 2 2
200 1 (0.5) 0 3 (1.5) 0 1 (0.5) 0 4 (2.0) 4 (2.0)
16 100 7 ++ 1 1 0 1 0 2 3 -
100 10 0 0 0 0 0 0 0
200 17 (8.5) 1 (0.5) 1 (0.5) 0 1 (0.5) 0 2 (1.0) 3 (1.5)
48 4 100 0 - 0 0 0 1 0 1 1 -
100 0 1 0 0 0 0 0 1
200 0 1 (0.5) 0 0 1 (0.5) 0 1 (0.5) 2 (1)
8 100 5 + 0 2 1 0 0 3 3 -
100 3 1 0 0 1 0 1 2
200 8 (4.0) 1 (0.5) 2 (1.0) 1 (0.5) 1 (0.5) 0 4 (2.0) 5 (2.5)
16 100 72 ++ 0 2 2 0 0 2 2 +
100 70 0 1 5 0 0 5 5
200 142 (71.0) 0 3 (1.5) 7 (3.5) 0 0 7 (3.5) 7 (3.5)
Positive control MMC 24 0.05 100 0 - 3 11 11 0 0 20 23 ++
100 0 5 3 19 1 0 22 26
200 0 8 (4.0) 17 (7.0) 30 (15.0) 1 (0.5) 0 42 (21.0) 49 (24.5)
48 0.05 100 0 - 6 9 8 1 0 18 23 ++
100 0 6 3 15 1 1 20 25
200 0 12 (6.0) 12 (6.0) 23 (11.5) 2 (1.0) 1 (0.5) 38 (19.0) 48 (24.0)

*ctb: chromatid break, cte: chromatid exchange, csb: chromosome break, cse: chromosome exchange

** χ2 test was used to evaluate significance; + (p<0.05), ++ (p<0.001)

MMC: Mitomycin C

Table 2: Results of Experiment II (6-h treatment with and without metabolic activation)

Treatment S9 mix Treatment level (µg/mL) Number of observed cells Number of polyploid cells Number and frequency (%) of the cells with chromosomal structural abnormality*
(%) Evaluation** Gap Chromatid type Chromosome type Total Evaluation**
g ctb cte csb cse without gap with gap
Negative control (DMSO) - 0 100 1 n.a. 0 0 0 0 0 0 0 n.a.
100 0 0 0 0 0 0 0 0
200 1 (0.5) 0 0 0 0 0 0 0
+ 0 100 1 n.a. 0 0 0 0 0 0 0 n.a.
100 2 0 0 0 0 0 0 0
200 3 (1.5) 0 0 0 0 0 0 0
Test substance - 12.5 100 1 - 0 0 1 0 0 1 1 -
100 0 1 0 0 0 0 0 0
200 1 (0.5) 1 (0.5) 0 1 (0.5) 0 0 1 (0.5) 1 (0.5)
25 100 1 - 0 0 0 0 0 0 0 -
100 1 1 0 1 0 0 1 2
200 2 (1.0) 1 (0.5) 0 1 (0.5) 0 0 1 (0.5) 2 (1.0)
50 100 Because of the strong cytotoxicity, samples were not prepared.
100
200
+ 12.5 100 0 - 0 0 0 0 0 0 0 -
100 0 1 0 0 1 0 1 2
200 0 1 (0.5) 0 0 1 (0.5) 0 1 (0.5) 2 (1)
25 100 0 - 0 0 0 0 0 0 0 -
100 0 2 0 0 0 0 0 2
200 0 2 (1.0) 0 0 0 0 0 2 (1.0)
50 100 0 - 2 2 1 0 0 3 4 ++
100 0 1 2 2 2 0 5 5
200 0 3 (1.5) 4 (2.0) 3 (1.5) 2 (1.0) 0 8 (4.0) 9 (4.5)

Positive control

B(a)P

- 10 100 0 - 1 0 0 0 0 0 1 -
100 2 0 0 1 1 0 2 2
200 2 (1.0) 0 1 (0.5) 1 (0.5) 0 2 (1.) 3 (1.5)
+ 10 100 0 - 7 9 19 1 0 32 35 ++
100 0 5 6 12 0 0 19 22
200 0 12 (6.0) 15 (7.5) 31 (15.5) 1 (0.5) 0 51 (25.5) 57 (28.5)

*ctb: chromatid break, cte: chromatid exchange, csb: chromosome break, cse: chromosome exchange

** χ2 test was used to evaluate significance; + (p<0.05), ++ (p<0.001)

B(a)p: Benzo(a)pyrene

Table 3: Cell viability

  Concentration (µg/mL) Cell proliferation rate (%)
Without metabolic activation (48 h treatment) 0 (solvent) 100
5 85
7.5 74
10 56
15 44
20 39
25 4.5
30 0
With metabolic activation (6 h treatment) 0 (solvent) 100
30 79
40 67
50 35
60 18
70 6.2
80 0
90 0
Conclusions:
Under the conditions of the conducted study the test substance increased the number of polyploid cells and cells with chromosomal structural abnormality after a treatment period of 24 and 48 h in the absence of metabolic activation, respectively. In the presence of a metabolic activation system the number of cells with chromosomal structural abnormality were increased after 6 h exposure with the test substance.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available (further information necessary)

Additional information

Justification for classification or non-classification

There is not enough data available to conclude on the classification and labelling under CLP Regulation (EC) 1272/2008 for this endpoint. An in vivo cytogenicity study has been proposed to provide more data to be used in the assessment of classification and labelling in accordance with the CLP Regulation (EC 1272/2008, as amended).