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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
January 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from study report

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: * OECD No. 471 (July 21, 1997) * EEC Directive 92/69, B14 and B13 (December 1992)
Principles of method if other than guideline:
Standard plate test and Preincubation test was performed to determine the mutagenic nature of the test chemical
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of substance:ferrate(4-), hexakis(cyano-C)-, Et 2-[6-(ethylamino)-3-(ethylimino)-2,7-dimethyl-3H-xanthen-9-yl]benzoate copper(2+) salts
- Appearance : red powder
- Common name: Pigment red 169
- Substance type: UVCBs-organometallic

Method

Target gene:
Histidine for Salmonella strains and Tryptophan for E. coli strain
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
No data
Additional strain / cell type characteristics:
other: histidine auxotrophs (his-)
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
No data
Additional strain / cell type characteristics:
other: tryptophan auxotrophy (trp-)
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S-9 mix
Test concentrations with justification for top dose:
0, 20, 100, 500, 2,500 or 5,000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, DMSO was selected as the vehicle, which had been
demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Additional plates are treated with soft agar, S-9 mix, buffer, vehiele or the test substance but without the addition of tester strains
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Remarks:
with S9
Positive control substance:
other: 2-aminoanthracene (2-AA)
Remarks:
strains: TA 1535, TA 100, TA 1537, TA 98: 2.5 µg/plate, dissolved in DMSO; Strain: Escherichia coli WP2 uvrA: 60 µg/plate, dissolved in DMSO
Untreated negative controls:
yes
Remarks:
Additional plates are treated with soft agar, S-9 mix, buffer, vehicle or the test substance but without the addition of tester strains
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Remarks:
without S9
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosuguanidine (MNNG)
Remarks:
Strains: TA 1535, TA 100: 5 µg/plate, dissolved in DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylendiamine (NOPD)
Remarks:
Strain: TA 98: 10 µg/plate, dissolved in DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Strain: TA 1537: 100 µg/plate, dissolved in DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Strain: E. coli WP2 uvrA: 10 µg/plate, dissolved in DMSO
Details on test system and experimental conditions:
Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (Aroclor-induced rat liver S-9 mix)

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk
- Cell density at seeding (if applicable):

DURATION
- Preincubation period: For PIT: 20 mins
- Exposure duration: 48 - 72 hours
- Expression time (cells in growth medium): 48 - 72 hours
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays):

SPINDLE INHIBITOR (cytogenetic assays):

STAIN (for cytogenetic assays):

NUMBER OF REPLICATIONS: Triplicate

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:

NUMBER OF CELLS EVALUATED:

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:
- Any supplementary information relevant to cytotoxicity:

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable):

- OTHER:
Rationale for test conditions:
No data
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 nuinber 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 adcling 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.
Statistics:
No data

Results and discussion

Test resultsopen allclose all
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:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: Precipitation of the test substance was found from about 500 µg/plate onward.
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: No data

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: No data

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: No data
- Indication whether binucleate or mononucleate where appropriate: No data

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No data
- Negative (solvent/vehicle) historical control data: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable: No data
Remarks on result:
other: No mutagenic potential

Any other information on results incl. tables

Standard plate assay

Table: Mutagenic potential

Strain TA1535 Without S9

 

Dose

Rev

M

SD

FAC

DMSO

16

20

3

1.0

21

22

20 µg

20

20

1

1.0

19

20

100 µg

21

20

1

1.0

19

19

500 µg

18P

19

1

1.0

20P

19P

2500 µg

5P

5

1

0.3

4P

6P

5000 µg

5P

3

2

0.2

3P

1P

MNNG

5.0µg

1004

1002

24

50.9

977

1024

2-AA

2.5 µg

 

 

 

 


Strain TA1535 With S9

Dose

Rev

M

SD

FAC

TITRE

DMSO

18

19

2

1.0

29

18

22

21

27

20 µg

19

18

1

1.0

 

18

18

100 µg

15

14

1

0.7

 

13

14

500 µg

17P

17

2

0.9

 

19P

19P

2500 µg

7P

6

2

0.3

15

8P

14

4P

20

 

5000 µg

0P

 

 

 

0

0P

0

0P

0

2-AA

176

171

14

9.0

 

155

 

181

 

 

 

Strain TA100 Without S9

 

Dose

Rev

M

SD

FAC

DMSO

124

115

8

1.0

108

113

20 µg

104

101

8

0.9

107

94

100 µg

98

102

7

0.9

110

99

500 µg

55P

60

5

0.5

61P

65P

2500 µg

11P

8

3

0.1

5P

8P

5000 µg

0P

 

 

 

0P

0P

MNNG

5.0µg

1312

1319

49

11.5

1273

1371

2-AA

2.5 µg

 

 

 

 

 

Strain TA100 With S9

Dose

Rev

M

SD

FAC

TITRE

DMSO

112

111

9

1.0

22

102

21

120

29

20 µg

110

110

2

1.0

 

108

111

100 µg

104

114

8

1.0

 

118

119

500 µg

57P

52

17

0.5

 

66P

33P

2500 µg

5P

5

3

0.0

1

8P

2

2P

5

5000 µg

0P

 

 

 

0

0P

0

0P

0

2-AA

2.5 µg

1135

1274

180

11.4

 

1477

1210

 

Strain TA1537 Without S9

 

Dose

Rev

M

SD

FAC

DMSO

9

10

1

1.0

10

10

20

9

9

1

1.0

9

10

100

9

10

1

1.0

10

11

500

5P

5

2

0.6

7P

4P

2500

1P

1

0

0.1

1P

1P

5000

0P

 

 

 

0P

0P

AAC

100 µg

621

649

27

67.2

674

653

2-AA

2.5 µg

 

 

 

 

 

Strain TA1537 With S9

Dose

Rev

M

SD

FAC

TITRE

DMSO

11

11

1

1.0

38

11

29

10

28

20 µg

9

9

2

0.9

 

8

11

100 µg

7

8

1

0.8

 

9

9

500 µg

8P

8

0

0.8

 

8P

8P

2500 µg

1P

1

1

0.1

2

1P

5

2P

1

5000 µg

0P

 

 

 

0

0P

0

0P

0

AAC

100 µg

 

 

 

 

 

 

2-AA

2.5 µg

173

185

13

17.5

 

198

185

 

Strain TA 98 Without S9

 

Dose

Rev

M

SD

FAC

DMSO

20

27

7

1.0

29

33

20 µg

24

21

3

0.8

19

21

100 µg

21

23

2

0.9

24

25

500 µg

21P

20

1

0.7

19P

19P

2500 µg

2P

3

1

0.1

3P

4P

5000 µg

0P

 

 

 

0P

0P

NOPD

10 µg

937

945

25

34.6

925

973

2-AA

2.5 µg

 

 

 

 

 

Strain TA 98 With S9

Dose

Rev

M

SD

FAC

TITRE

DMSO

42

38

3

1.0

17

36

24

36

24

20 µg

35

33

2

0.9

 

31

32

100 µg

34

33

2

0.9

 

31

34

500 µg

20P

22

2

0.6

 

24P

23P

2500 µg

11P

8

3

0.2

2

8P

2

6P

2

5000 µg

0P

 

 

 

0

0P

0

0P

0

NOPD

10 µg

 

 

 

 

 

2-AA

2.5 µg

936

943

13

24.8

 

925

958

 

 

StrainE.coli WP2uvrA Without S9

 

Dose

Rev

M

SD

FAC

DMSO

36

36

1

1.0

37

35

20 µg

31

32

4

0.9

37

29

100 µg

34

32

3

0.9

33

29

500 µg

29P

32

3

0.9

31P

35P

2500 µg

25P

28

3

0.8

30P

30P

5000 µg

15P

18

3

0.5

17P

21P

ENNG

10 µg

999

1009

18

28.0

998

1029

2-AA

60 µg

 

 

 

 

 

Strain E.coli WP2 uvrAWith S9

Dose

Rev

M

SD

FAC

TITRE

DMSO

39

40

2

1.0

40

42

45

39

39

20 µg

35

32

3

0.8

 

31

29

100 µg

37

35

3

0.9

 

37

31

500 µg

31P

31

1

0.8

 

31P

30P

2500 µg

20P

24

4

0.6

8

26P

6

27P

7

5000 µg

12P

12

1

0.3

0

12P

0

13P

0

ENNG

10 µg

 

 

 

 

 

2-AA

60 µg

326

314

40

7.9

 

347

269

Applicant's summary and conclusion

Conclusions:
According to the results of the present study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay with and without 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 several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay. The study was performed using Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA. The test chemical was dissolved in DMSO and used at dose level of 0, 20, 100, 500, 2,500 or 5,000 µg/plate. Concurrent solvent and positive control chemicals were also included in the study. According to the results of the present study, the test substance pigment red 169 is not mutagenic in the Salmonella typhimurium /Escherichia coli reverse mutation assay with and without metabolic activation.