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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)

Data source

Reference
Reference Type:
publication
Title:
Mutagenic activity of 27 dyes and related chemicals in the Salmonella/microsome and mouse lymphoma TK +/- assays
Author:
T.P. Cameron, T.J. Hughes, P.E. Kirby, V.A. Fung and V.C. Dunkel
Year:
1987
Bibliographic source:
Mutation Research, 189 (1987) 223-261

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: as per mentioned below
Principles of method if other than guideline:
Evaluate mutagenicity of test substance by the mouse lymphoma TK assay.
GLP compliance:
no
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): C.I. Acid blue 74
- Molecular formula (if other than submission substance):
C16-H10-N2-O8-S2.2Na
C16-H8-N2-Na2-O8-S2
C16-H8-N2-O8-S2.2Na
- Molecular weight (if other than submission substance): 466.3572 g/mol
- Substance type: Organic
- Physical state: Solid
Purity: 85%

Method

Target gene:
mouse lymphoma assay
Species / strain
Species / strain:
mouse lymphoma L5178Y cells
Details on mammalian cell lines (if applicable):
Type and identity of media: The cells were grown in Fischer's medium for leukemic cells of mice (Gibco, Grand Island, NY) supplemented with 10% horse serum (Gibco, Grand Island, NY), antibiotics (50 U
penicillin/mi and 50 /µg streptomycin/ml), and 0.02% Pluronic F-68 (BASF Wyandotte Corp., Wyandotte, MI). All serum lots were prescreened for their ability to support optimal growth.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: : No data
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Test concentrations with justification for top dose:
Without S9: 971, 971, 1229, 1229, 1486, 1486, 1743, 1743, 2000 and 2000 µg/ml
With S9: 92, 206, 332, 439, 439, 556 and 556 µg/ml
Vehicle:
Vehicle
- Vehicle(s)/solvent(s) used: Yes
- Justification for choice of solvent/vehicle: No data available
Controls
Negative controls:
not specified
Solvent controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: Without metabolic activation: Ethyl methanesulfonate at 0.5 µl/ml with metabolic activation: 3-methylcholanthrene at 5.0 or 10.0 µg/ml.
Details on test system and conditions:
Details on test system and conditions
METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: No data available
- Exposure duration: 4 hr
- Expression time (cells in growth medium): 48 hr
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): No data available

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

NUMBER OF REPLICATIONS: No data available

NUMBER OF CELLS EVALUATED: No data available

DETERMINATION OF CYTOTOXICITY
- Method: No data available

OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other: No data available
Evaluation criteria:
Mutant frequencies were expressed as mutants per 10⁴ surviving cells. In general, a response was considered positive if there was a dose-related increase in the mutant frequency above the spontaneous control frequency, with a 2-fold increase at more than 1 dose and relative total growth greater than 10%.
Statistics:
No data available

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: Aroclor-1254-induced male Fischer 344 rats
Additional information on results:
No data available

Any other information on results incl. tables

Concentration µl/ml

S9

Relative suspension growth (%)

Relative cloning efficiency (%)

Relative total growth (%)

Average no. colonies TFT/viable

Mutant frequency (per 10⁴)

0.0

-

 

 

 

 

x0.61

971

-

100

113

112

82/230

0.71

971

-

103

108

111

71/221

0.64

1229

-

45

95

43

59/194

0.61

1229

-

102

84

86

66/172

0.7

1486

-

102

91

93

62/185

0.67

1486

-

100

96

95

49/195

0.50

1743

-

101

88

88

47/179

0.53

1743

-

93

92

86

47/187

0.50

2000

-

96

86

83

44/175

0.50

2000

-

108

94

101

55/192

0.57

Positive control

-

47

29

13

306/58

10.55

Solventᵃ

-

 

 

 

 

x0.56

 

 

 

 

Concentration µl/ml

S9

Relative suspension growth (%)

Relative cloning efficiency (%)

Relative total growth (%)

Average no. colonies TFT/viable

Mutant frequency (per 10⁴)

0.0

+

 

 

 

 

X0.53

92

+

51

45

23

131/106

2.47

206

+

37

44

16

131/102

2.57

332

+

37

49

18

137/114

2.40

439

+

34

40

14

130/94

2.77

439

+

31

46

14

124/108

2.30

556

+

26

50

13

134/116

2.31

556

+

25

40

10

125/93

2.69

Positive control

+

65

56

36

191/96

3.98

Solventb

+

 

 

 

 

X0.61

 

a: Solvent control for test chemical.

b: Solvent for positive control.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative Negative (with and without)

The substance is considered to be not mutagenic in L5178Y TK +/- mouse lymphoma cells with and without metabolic activation.
Executive summary:

Test chemical tested for mutagenicity in mouse lymphoma TK⁺′⁻ assay. toxicity of test chemical was determined both with and without S9 prepared from Aroclor-1254-induced male Fischer 344 rats. L5178Y TK +/- mouse lymphoma cells was exposed to Without S9: 971, 971, 1229, 1229, 1486, 1486, 1743, 1743, 2000 and 2000 µg/ml and positive control is Ethyl methanesulfonate at 0.5 µl/ml. With S9: 92, 206, 332, 439, 439, 556 and 556 µg/ml concentration of test chemical and positive control is 3-methylcholanthrene at 5.0 or 10.0 µg/ml. There was no dose-related increase in the mutant frequency above the spontaneous control frequency, no 2-fold increase at more than 1 dose and not relative total growth greater than 10% at any exposed concentration.

Hence, the substance is considered to be not mutagenic inL5178Y TK +/- mouse lymphoma cells with and without metabolic activation.