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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from peer reviewed publication

Data source

Reference
Reference Type:
publication
Title:
Salmonella Mutagenicity Tests: II. Results From The Testing Of 270 Chemicals
Author:
Mortelmans,K, Haworth,S, Lawlor,T, Speck,W, Tainer,B And Zeiger,E
Year:
1986
Bibliographic source:
Environ. Mutagen. 8(Suppl. 7):1 -119, 1986

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
Gene mutation toxicity study was performed for Direct Blue 25 to evaluate its mutagenic nature
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material: Direct Blue 25
- IUPAC name: Tetrasodium 3,3'-[(3,3'-dimethyl[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[4,5-dihydroxynaphthalene-2,7-disulphonate]
- Molecular formula: C34H26N4O16S4.4Na
- Molecular weight: 962.7838 g/mol
- Substance type: Organic
Specific details on test material used for the study:
- Name of test material: Direct Blue 25
- IUPAC name: Tetrasodium 3,3'-[(3,3'-dimethyl[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[4,5-dihydroxynaphthalene-2,7-disulphonate]
- Molecular formula: C34H26N4O16S4.4Na
- Molecular weight: 962.7838 g/mol
- Substance type: Organic
- Physical state: No data
- Purity: No data
- Impurities (identity and concentrations): No data

Method

Target gene:
Histidine
Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell lines (if applicable):
Not applicable
Additional strain characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
Male Sprague-Dawley rats and male Syrian hamsters were routinely used for the S9 preparation of the liver fractions
Test concentrations with justification for top dose:
0, 100.0, 333.0, 1000.0, 3333.0, 10000.0 µg/plate
Vehicle:
- Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: The test chemical was soluble in water
Controls
Negative controls:
not specified
Solvent controls:
yes
Remarks:
Water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 4-nitro-o-phenylenediamine (TA98; -S9), 2-aminoanthracene (all strains, +S9)
Details on test system and conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 mins
- Exposure duration: 48 hr
- Expression time (cells in growth medium): 48 hr
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: At least five dose levels of the chemicals were tested, with three plates per dose level.

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: No data
Rationale for test conditions:
No data
Evaluation criteria:
1) mutagenic response: a dose-related, reproducible increase in the number of revertants over background, even if the increase was less than twofold;
2) nomutagenic response: when no increase in the number of revertants was elicited by the chemical;
3) questionable response: when there was an absence of a clear-cut dose-related increase in revertants; when the dose-related increases in the number of revertants were not reproducible;
or when the response was of insufficient magnitude to support a determination of mutagenicity
Statistics:
Mean and Standard error of mean

Results and discussion

Test results
Species / strain:
other: TA100, TA1535, TA1537, TA98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
For strain 1537, at 10000 µg/plate
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes
Remarks on result:
other: No mutagenc potential
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: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: The chemical was initially tested with strain TA100 in the presence and the absence of the metabolic activation systems, over a wide dose range with an upper limit of 10 mg/plate, or less when solubility problems were encountered. Toxicity was evidenced by one or more of the following phenomena: appearance of his+ pinpoint colonies, reduced numbers of revertant colonies per plate, or thinning or absence of the bacterial lawn. Nontoxic chemicals were tested in the initial experiment up to the 10 mg/plate dose level, or to a level determined by their solubility. Toxic chemicals were tested up to a high dose which exhibited some degree of toxicity.

COMPARISON WITH HISTORICAL CONTROL DATA: No data

Any other information on results incl. tables

Table: Mutation data for the test chemical Direct Blue 25

Dose (µg/plate)

TA100

NA

10% HLI

10% RLI

Mean

SEM

Mean

SEM

Mean

SEM

0

101

2.4

160

9.6

156

10.2

100

105

4.4

162

1.7

166

6.3

333

128

2.6

163

10.7

187

14.2

1000

101

18.1

173

13.9

136

6.8

3333

72

5.9

142

2.9

125

10.1

10000

82

3.8

99

6.1

87

3.0

Positive control

782

27.7

1136

43.5

1146

58.1

 

Dose (µg/plate)

TA1535

NA

10% HLI

10% RLI

Mean

SEM

Mean

SEM

Mean

SEM

0

20

0.9

13

1.9

15

1.2

100

24

2.7

16

2.1

17

1.0

333

17

0.3

12

0.9

15

2.6

1000

15

2.0

13

0.3

17

2.1

3333

16

2.9

10

1.7

19

2.3

10000

17

2.7

8

1.2

10

2.0

Positive control

1037

70.4

447

84.8

319

24.9

 

Dose (µg/plate)

TA1537

NA

10% HLI

10% RLI

Mean

SEM

Mean

SEM

Mean

SEM

0

8

1.2

13

2.3

13

3.3

100

10

3.2

15

1.2

17

4.3

333

11

1.8

11

0.0

16

2.0

1000

9

0.9

11

0.3

7

1.2

3333

6

0.6

7

1.8

7

1.5

10000

T

 

T

 

T

 

Positive control

165

11.8

293

8.5

307

9.0

 

Dose (µg/plate)

TA98

NA

10% HLI

10% RLI

Mean

SEM

Mean

SEM

Mean

SEM

0

18

1.0

37

5.0

31

3.3

100

20

5.0

45

3.7

28

3.0

333

23

3.1

41

5.5

33

1.5

1000

27

2.7

37

1.0

19

3.7

3333

15

2.6

42

3.5

21

1.5

10000

15

0.3

20

4.5

12

1.2

Positive control

465

36.1

1035

27.4

1026

47.0

 

T: Complete clearing of background lawn

Applicant's summary and conclusion

Conclusions:
Direct Blue 25 did not induce mutation in the Salmonella typhimurium strain TA100, TA1535, TA1537, TA98 both in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.c v
Executive summary:

Gene mutation toxicity study was performed for direct blue 25 to evaluate its mutagenic nature. The study was performed as per the preincubation protocol using Salmonella typhimurium strain TA100, TA1535, TA1537, TA98 both in the presence and absence of S9 metabolic activation system at doses of 0, 100.0, 333.0, 1000.0, 3333.0 or 10000.0 µg/plate. Water was used as the vehicle. The plates were incubated for 48 hrs after 20 mins preincubation before the evaluation of the revertant colonies could be made. Direct blue 25 did not induce mutation in the Salmonella typhimurium strain TA100, TA1535, TA1537, TA98 both in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.