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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
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:
Responses of the L5178Y Mouse Lymphoma Cell Forward Mutation Assay. V: 27 Coded Chemicals
Author:
Douglas B. McGregor, Alison G. Brown, Susan Howgate, Douglas McBride, Colin Riach, and William J. Caspary
Year:
1991
Bibliographic source:
Environmental and Molecular Mutagenesis 17:196 - 219 (1991)

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
The study was performed to determine the mutagenic potential of 1-(phenyldiazenyl)-2-naphthol in the L5178Y tk+/tk¯ mouse lymphoma cells
GLP compliance:
not specified
Type of assay:
other: Mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
1-phenylazo-2-naphthol
EC Number:
212-668-2
EC Name:
1-phenylazo-2-naphthol
Cas Number:
842-07-9
Molecular formula:
C16H12N2O
IUPAC Name:
1-[(E)-2-phenyldiazen-1-yl]naphthalen-2-ol
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): Solvent yellow 14
- Molecular formula (if other than submission substance): C16H12N2O
- Molecular weight (if other than submission substance): 248.28 g/mole
- InChl (if other than submission substance):1S/C16H12N2O/c19-15-11-10-12-6-4-5-9-14(12)16(15)18-17-13-7-2-1-3-8-13/h1-11,19H/b18-17+
- Substance type: Organic
- Physical state: Solid
- Analytical purity: No data
- Impurities (identity and concentrations): No data
Specific details on test material used for the study:
- Name of test material: Solvent yellow 14
- Molecular formula: C16H12N2O
- Molecular weight: 248.284 g/mol
- Substance type: Organic
- Physical state: Solid
- Purity: No data available
- Impurities (identity and concentrations): No data available

Method

Target gene:
Thymidine kinase gene
Species / strain
Species / strain / cell type:
mammalian cell line, other: L5178Y tk+/tk- 3.7.2 C mouse lymphoma cells
Details on mammalian cell type (if applicable):
- Type and identity of media: The tkt/tk- -3.7.2C heterozygote of L5178Y mouse lymphoma cells were maintained in Fischer’s medium at 37°C on gyratory tables. Fischer’s medium (designated F0) was supplemented with 2 mM L-glutamine, sodium pyruvate, 110 µg/ml, 0.05% pluronic F68, antibiotics, and 10% heat-inactivated donor horse serum (v/v) (designated Flop)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes, Laboratory cultures were confirmed as free from mycoplasma by cultivation or Hoechst staining techniques
- Periodically checked for karyotype stability: No data available
- Periodically "cleansed" against high spontaneous background: No data available
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
S9 mix, Post-mitochondria1 supernatant fractions of liver homogenates (S9) were preparedfrom 200 g, male, Fischer 344 rats
Test concentrations with justification for top dose:
0, 1, 2, 4, 8, 16 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ACET
- Justification for choice of solvent/vehicle: No data available
Controls
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
ethylmethanesulphonate
methylmethanesulfonate
Details on test system and experimental conditions:
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 6 X 10 6 cells

DURATION
- Preincubation period: No data
- Exposure duration: 4 hrs
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11-14 dats
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): TFT

SPINDLE INHIBITOR (cytogenetic assays): No data

STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: Duplicate

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: No data

NUMBER OF CELLS EVALUATED: Three aliquots (each containing 10 6 cells)

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): No data

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Toxicity was expressed as either a reduction of cell population growth in suspension during the expression period or a reduction in cloning efficiency. A measure of the overall toxicity was the relative total growth (RTG)
- Any supplementary information relevant to cytotoxicity: Mutant fraction was also calculated

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No data

- OTHER: Mutant selection was done by seeding 1000000 cells by mixing with cloning medium at 37°C to give final concentrations of 0.35% Noble agar and 3 pg TFT/ml, then poured into 90
mm Petri plates. The agar was solidified at 4°C for 5-10 min, then the plates were incubated for 11-14 days in 5% C02:95% air at 37°C. Colonies were counted using an Artek 880 Automated
Colony Counter, with the colony size discriminator control in the “off” position. A check on the instrument indicated that colonies of about 8.1 mm in diameter were counted.
Rationale for test conditions:
No data available
Evaluation criteria:
Mutant colonies were counted and mutant fraction was determined. Primary judgments were made at the level of individual experiments, but judgment on the mutagenic potential of a chemical was made on a basis of consensus of all valid experimental results.
Positive (+)
A test was considered positive when, out of three trials, a positive

Negative (-)
A test was considered negative when, out of three trials, a positive response or a positive dose was not reproducible.

Questionable (?)
A test was considered questionable when, out of three trials, neither a positive nor a negative response was reproduced.
Statistics:
The statistical analysis was based upon the mathematical model proposed for this system [Lee and Caspary, 19831 and consisted of a dose trend test [Barlow et al., 1972, p. 2151 and a variance analysis of pair-wise comparisons of each dose against the vehicle control. Significant differences from concurrent vehicle control values at the 5% level after variance analysis are
indicated by underlines of the average mutant fractions at the appropriate concentrations. For further discussion of the data evaluation procedure readers should refer to McGregor et al. [1988b].

Results and discussion

Test resultsopen allclose all
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
No data availableTEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: Yes, precipitation was noted at and above 12.5 µg/ml without S9 metabolic activation system
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: Ten-fold differences in test compound concentrations were used in the toxicity test, the highest being 5 mg/ml unless a much lower concentration was indicated by the poor solubility of a compound. This test was followed by at least two experiments in the absence of S9 mix. If no clear positive response was observed, then two experiments were performed in the presence of S9 mix. Test compound concentrations were primarily two-fold dilutions from the highest testable concentration, as estimated from the toxicity test.

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]

Any other information on results incl. tables

 Results with activation(S9):

Conc. µg/ml

Trial 1

Trial 2

Trial 3

 

CE

RTG

MC

Avg. MF

CE

RTG

MC

Avg. MF

CE

RTG

MC

Avg. MF

ACET

82

115

86

 

96

117

86

 

92

103

145

 

0

84

57

58

98

92

94

115

114

113

53

90

90

97

90

88

105

103

73

108

33

101

97

95

103

96

97

135

132

154

49

1

72

63

91

87

113

120

58

67

65

82

83

73

89

41

105

94

87

77

155

175

56

2

60

57

72

73

152

102

72

84

64

73

52

109

107

49

96

84

70

71

163

176

63

4

51

56

37

36

134

118

79

47

63

27

28

133

144

85

83

86

39

42

134

199

66

8

38

24

18

11

172

143

175

39

38

13

9

86

124

91

72

68

19

16

151

178

79

16

62

52

32

28

137

101

69

24

33

6

9

45

85

75

54

47

9

6

148

142

96

MCA

2.5

18

15

5

4

134

177

418

13

14

3

2

193

262

566

58

54

28

21

570

603

349

CE=cloning efficiency (%); RTG=relative total growth; MC= mutant colony count; MF= mutant fraction (mutant colonies per 1000000 clonable cells)

PRECIPITATION AT 16. UG/ML

Results without activation (S9):

Conc. µg/ml

Trial 1

Conc. µg/ml

Trial 2

 

CE

RTG

MC

Avg. MF

 

CE

RTG

MC

Avg. MF

Acet

75

101

75

33

ACET

90

106

120

45

0

95 96 113

98

101 100

111

165

126

39 58 37

0

78 76 68

102 107 85

98 114 115

48

3.125

138 r

121 r

97 87

201 147

48 40

1

70 67

84 83

108 113

52

56

6.25

108 86

118 93

170 99

52 39

2

77 88

87 100

109 109

47

41

12.5

70 79

54 59

104 116

50 49

4

86 106

67 92

141 139

55

44

25

37 48

6

6

1157

1330

1042

927

8

73 88

89

80

82 141

38

54

50

Lethal

Lethal

 

 

 

16

74

71

34

26

133

114

60

53

EMS

250µg/mL

82

66

124

80

223

156

90

79

 

73

74

69

73

399

427

182

193

MMS

15µg/mL

37

45

25

25

146

182

131

136

50

52

41

32

31

261

218

167

176

Precipitation at 12.5 and 16 µg/ml

Applicant's summary and conclusion

Conclusions:
1-phenylazo-2-naphthol shows failed to induce mutation in L5178Y tk+/tk– mouse lymphoma cell without S9 metabolic activation and did induce mutation with of S9 metabolic activation system.
Executive summary:

1-phenylazo-2-naphthol (C. I. Solvent Yellow 14) was tested for its mutagenic potential in the L5178Y tk+/tkmouse lymphoma cell forward mutation assay at concentration of 0, 1, 2, 4, 8, 16 µg/ml. Two trials were conducted in the absence of S9 mix.

In the first trial, no mutagenicity was observed up to a concentration of 12.5 µg/ml, where precipitation occurred. In the second trial, no mutagenicity was observed when the concentration of solvent yellow 14 was restricted to 16 µg/ml, giving an RTG of about 30%.

In three trials performed in the presence of S9 mix, statistically significant dose-related mutagenic responses were obtained at doses below those at which precipitation occurred. The LOED was 8 µg/ml, and it was noted that significant responses occurred only when the RTG was below 30% and associated with decreases in cloning efficiency.

1-phenylazo-2-naphthol failed to induce mutation in L5178Y tk+/tk– mouse lymphoma cell without S9 metabolic activation and did induce mutation with of S9 metabolic activation system.

Since mutagenic response is observed for the chemical with metabolic activation system, hence, 1-phenylazo-2-naphthol is likely to classify as a gene mutant in vitro.

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