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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

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.

Link to relevant study records
Reference
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
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
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
Target gene:
Thymidine kinase gene
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
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].
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]

 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

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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In vivo Geno toxicity of 1-phenylazo-2-naphthol (Sudan I) was examined by Matsumura et al (2015) using repeated-dose Bone marrow micronucleus (MN) assay in young adult male Crl: CD (SD) rats. They performed a dose range finding study. The animals were dosed once daily for 7 days with 75, 150, 300, and 600 mg/kg/day of Sudan I and were monitored for clinical signs and body weight changes. A decrease in body weight was observed in the 600 mg/kg/day group compared with the vehicle control group, but no lethality or decrease in motor activity was observed. Based on these observations, we determined the highest dose as 600 mg/kg/day and set the lower doses with the common ratio of two (300 and 150 mg/kg/day). BM cells were collected from the femurs. Immediately prior to microscopic observation, smear preparations were made with each sample and then stained with an AO solution and covered with a cover slip. The specimens were observed under a fluorescent microscope with B-excitation (Blue light excitation), and the number of micronucleated immature erythrocytes (MNIMEs) per 2000 immature erythrocytes (IMEs) was counted for each animal. There was statistically significant increase in the incidences of MNIMEs observed with the repeated administration of Sudan I. Therefore in vivo genetic toxicity of 1-phenylazo-2-naphthol (Sudan I) is positive.

Link to relevant study records
Reference
Endpoint:
genetic toxicity in vivo
Remarks:
Type of genotoxicity: genome mutation
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
Qualifier:
no guideline available
Principles of method if other than guideline:
Bone marrow MN micronucleus (MN) assay was performed to determine the mutagenic nature of Solvent yellow 14
GLP compliance:
not specified
Type of assay:
other: Bone marrow MN micronucleus (MN) assay
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: >95%
- Impurities (identity and concentrations): No data available
Species:
rat
Strain:
Crj: CD(SD)
Details on species / strain selection:
No data
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan Inc. (Yokohama,Japan)
- Age at study initiation: 6 weeks old
- Weight at study initiation: No data available
- Assigned to test groups randomly: No data available
- Fasting period before study: No data available
- Housing: The animals were housed in an air-conditioned room
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: No data available

ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data available
- Humidity (%): No data available
- Air changes (per hr): No data available
- Photoperiod (hrs dark / hrs light): 12 h light/dark cycle

IN-LIFE DATES: From: To: No data available
Route of administration:
oral: unspecified
Vehicle:
- Vehicle(s)/solvent(s) used: yes- methyl cellulose
- Justification for choice of solvent/vehicle: No data available
- Concentration of test material in vehicle: 0, 150, 300, and 600 mg/kg/day
- Amount of vehicle (if gavage or dermal): No data available
- Type and concentration of dispersant aid (if powder): No data available
- Lot/batch no. (if required): No data available
- Purity: No data available
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Sudan I was suspended in 0.5% (w/v) Methyl Cellulose Solution (Wako Pure Chemical Industries, Ltd., Osaka, Japan) each day before dosing to guve a dose range of 0, 150, 300 or 600 mg/Kg/day

DIET PREPARATION
- Rate of preparation of diet (frequency): No data available
- Mixing appropriate amounts with (Type of food): No data available
- Storage temperature of food: No data available
Duration of treatment / exposure:
14 days
Frequency of treatment:
Daily
Post exposure period:
No data available
Remarks:
Doses / Concentrations:
0, 150, 300, and 600 mg/kg/day
No. of animals per sex per dose:
Total: 20
0 mg/Kg/day: 5 male rats
150 mg/Kg/day: 5 male rats
300 mg/Kg/day: 5 male rats
600 mg/Kg/day: 5 male rats
Control animals:
yes, concurrent vehicle
Positive control(s):
No data available
Tissues and cell types examined:
Micronucleated Bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Dose range finding study was conducted, The animals were dosed once daily for 7 days with 75, 150, 300, and 600 mg/kg/day of Sudan I and were monitored for clinical signs and body weight changes. A decrease in body weight was observed in the 600 mg/kg/day group compared with the vehicle control group, but no lethality or decrease in motor activity was observed. Based on these observations, the highest dose was set as 600 mg/kg/day and set the lower doses with the common ratio of two (300 and 150 mg/kg/day).

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): No data available

DETAILS OF SLIDE PREPARATION: smear preparations were made from the bone marroe cells and then stained with an AO solution and covered with a cover slip.

METHOD OF ANALYSIS: The specimens were observed under a fluorescent microscope with B-excitation (Blue light excitation), and the number of micronucleated immature erythrocytes (MNIMEs) per 2000 immature erythrocytes (IMEs) was counted for each animal.

OTHER: No data available
Evaluation criteria:
Micronucleated Bone marrow cells were observed
Statistics:
Differences in the incidences of MNHEPs between the test and the vehicle control groups were analyzed using the conditional binomial test reported by Kastenbaum and Bowman at the upper-tailed significance levels of 5% and 1%. The exception was that a Chi-squared test
was performed to analyze differences in the incidences of micronucleated immature erythrocytes (MNIMEs) between the test groups and the vehicle-treated control group.

The data were also statistically analyzed by a multiple comparison test according to the requirements of the organizing committee. Specifically, the homogeneity of the variance was examined using Bartlett’s test. When a homogeneous variance was demonstrated, one-way analysis of variance was applied. In cases of heterogeneous variance, the Kruskal–Wallis test was applied. When a statistically significant difference was demonstrated between groups, the difference was assessed by Dunnett’s test or a Dunnett-type multiple comparison test.
Sex:
male
Genotoxicity:
positive
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 75, 150, 300, and 600 mg/kg/day
- Solubility: No data available
- Clinical signs of toxicity in test animals: A decrease in body weight was observed in the 600 mg/Kg/bw dose level - Evidence of cytotoxicity in tissue analyzed: No data available
- Rationale for exposure: No data available
- Harvest times: No data available
- High dose with and without activation: No data available
- Other:No data available

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): No data available
- Induction of micronuclei (for Micronucleus assay): No data available
- Ratio of PCE/NCE (for Micronucleus assay): No data available
- Appropriateness of dose levels and route: No data available
- Statistical evaluation: No data available

Results of the Bone marrow MN assay with Sudan:

 

Sudan I (mg/kg/day)

No. of animals

MNIMEs (%) individual

data (mean±SD)

0

5

0.60, 0.65, 0.75, 0.25, 0.50 (0.55±0.19)    

150

5

0.75, 1.25, 1.75, 1.10, 1.25 (1.22±0.35*)

300

5

0.65, 1.30, 2.00, 3.25, 1.65 (1.77±0.96*)

600

5

1.80, 2.40, 3.25, 1.75, 2.70 (2.38±0.63*)

* p < 0.01, Chi-squared test.

Conclusions:
There was statistically significant increase in the incidences of MNIMEs observed with the repeated administration of 1-phenylazo-2-naphthol (Sudan I). Therefore in vivo genetic toxicity of 1-phenylazo-2-naphthol (Sudan I) is positive.
Executive summary:

The in vivo Geno toxicity of 1-phenylazo-2-naphthol (Sudan I) was examined using repeated-doseBone marrowmicronucleus (MN) assays in young adult maleCrl: CD (SD)rats.

They performed a dose range finding study. The animals were dosed once daily for 7 days with 75, 150, 300, and 600 mg/kg/day of Sudan I and were monitored for clinical signs and body weight changes. A decrease in body weight was observed in the 600 mg/kg/day group compared with the vehicle control group, but no lethality or decrease in motor activity was observed. Based on these observations, we determined the highest dose as 600 mg/kg/day and set the lower doses with the common ratio of two (300 and 150 mg/kg/day). BM cells were collected from the femurs. Immediately prior to microscopic observation, smear preparations were made with each sample and then stained with an AO solution and covered with a cover slip. The specimens were observed under a fluorescent microscope with B-excitation (Blue light excitation), and the number of micronucleated immature erythrocytes (MNIMEs) per 2000 immature erythrocytes (IMEs) was counted for each animal.

There was statistically significant increase in the incidences of MNIMEs observed with the repeated administration of Sudan I. Therefore in vivo genetic toxicity of 1-phenylazo-2-naphthol (Sudan I) is positive.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Additional information

Gene mutation in vitro:

Various peer reviewed publications were reviewed to determine the mutagenic nature of 1-phenylazo-2-naphthol (C. I. Solvent Yellow 14). The summary is as mentioned below:

 

1-phenylazo-2-naphthol (C. I. Solvent Yellow 14) (CAS no 842 -07 -9) was tested by for its mutagenic potential in the L5178Y tk+/tk–mouse lymphoma cell forward mutation assay at concentration of 0, 1, 2, 4, 8, 16 µg/ml (McGregor et al, 1991). 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 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. 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.

 

In vitro Sister Chromatid Exchange Test was performed by Ivett et al (1989) for 1-phenylazo-2-naphthol (C. I. Solvent Yellow 14) (CAS no 842 -07 -9) in Chinese Hamster Ovary Cells both with and without metabolic activation. In the SCE trials without metabolic activation, cells were exposed to the test chemical at concentration 0.0000, 2.4700, 8.2200, 24.6500 (trial 1) and 0.000, 40.2000, 55.3000, 70.500 (trial 2) for approximately 25 hr.; for the trials (concentration 0.000, 24.6500, 82.2000, 246.500) with metabolic activation the exposure was for 2 hr. For both testing conditions, 10 µM bromodeoxyuridine (BrdUrd) was added 2 hr after dosing. The cells were continuously exposed to BrdUrd up until the time of harvest with 0.1 µg/mlColcemid present for the last 2-2.5 hr of incubation. Under standard conditions the total incubation time was 27.5-28 hr. Dose-related increases of SCE were observed in both trials without activation; all positive responses occurred at doses that induced cell cycle delay. The SCE trial with activation was judged positive, both at doses that induced delay and at doses that did not and hence 1-phenylazo-2-naphthol (C. I. Solvent Yellow 14) is likely to classify as a gene mutant in vitro.

 

In the same study by Ivett et al (1989), In vitro mammalian chromosome aberration tests was performed in Chinese Hamster Ovary Cells with 1-phenylazo-2-naphthol (C. I. Solvent Yellow 14) (CAS no 842 -07 -9) both with and without metabolic activation. In this assay without activation, cells were exposed to the test chemical for 8 hr. The test chemical was washed off, and the cells were treated with 0.1 µg/ml Colcemid for 2-2.5 hr. With metabolic activation, the cells were exposed to the test chemical plus the metabolic activation mixture for 2 hr., washed, incubated for 8 hr., and then treated with Colcemid for 2-2.5 hr.The mitotic cells were treated with 0.075 M KCl and fixed in 3:1 methanol: glacial acetic acid. In order to evaluate cell cycle kinetics, slides from the highest doses were stained with Hoechst 33258 (0.5 µg/ml) and examined by fluorescence microscopy. Fifty cells were scored per dose in the initial trial, and, generally 25 were scored in the repeat trials. The slides were observed for chromosomal aberrations like chromatid breaks, chromosome breaks, triradials. However, these data were combined as the percent of cells with simple (deletions), complex (exchanges), and total (simple, complex and other) aberrations. 1-phenylazo-2-naphthol (C. I. Solvent Yellow 14) did not induce chromosomal aberrations with or without activation when tested at toxic doses.

 

L5178Y TK +/- mouse lymphoma assay was performed using 1-phenylazo-2-naphthol (C. I. Solvent Yellow 14) at different concentrations both with and without S9 metabolic activation system. Cells in duplicate cultures were exposed to the test chemical, positive control, and solvent control for 4 h at 37 ° C; washed twice with growth medium; and maintained at 37 °C for 48 h in log phase growth to allow recovery and mutant expression. The cultures were adjusted to 0.3 × 106cells/ml at 24-h intervals. They were then cloned in soft agar medium containing Fischer's medium, 20% horse serum, 2 mM sodium pyruvate, 0.02% Pluronic F-68 and 0.35% Noble agar. Resistance to trifluorothymidine (TFT) was determined by adding 3 µg/ml TFT to one set of plates. The 100 X stock solution of TFT in saline was stored at -70°C and thawed immediately before use. Plates were incubated at 37°C in 5% CO 2 in air for 12 days, and then counted with an automatic colony counter. Mutant frequencies were expressed as mutants per 104surviving 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%. 1-phenylazo-2-naphthol (C. I. Solvent Yellow 14) induced mutation in the L5178Y TK +/- cell line and hence is positive (with and without S9 mix) in L5178Y TK +/- mouse lymphoma assay.

 

Based on the key study and its supporting data summarized, 1-phenylazo-2-naphthol (C. I. Solvent Yellow 14) is likely to classify as a gene mutant in vitro.

 

Gene mutation in vivo:

Various peer reviewed publications were reviewed to determine the mutagenic nature of 1-phenylazo-2-naphthol (C. I. Solvent Yellow 14). The summary is as mentioned below:

 

In vivo Geno toxicity of 1-phenylazo-2-naphthol (Sudan I) was examined by Matsumura et al (2015) using repeated-dose Bone marrow micronucleus (MN) assay in young adult male Crl: CD (SD) rats. They performed a dose range finding study. The animals were dosed once daily for 7 days with 75, 150, 300, and 600 mg/kg/day of Sudan I and were monitored for clinical signs and body weight changes. A decrease in body weight was observed in the 600 mg/kg/day group compared with the vehicle control group, but no lethality or decrease in motor activity was observed. Based on these observations, we determined the highest dose as 600 mg/kg/day and set the lower doses with the common ratio of two (300 and 150 mg/kg/day). BM cells were collected from the femurs. Immediately prior to microscopic observation, smear preparations were made with each sample and then stained with an AO solution and covered with a cover slip. The specimens were observed under a fluorescent microscope with B-excitation (Blue light excitation), and the number of micronucleated immature erythrocytes (MNIMEs) per 2000 immature erythrocytes (IMEs) was counted for each animal. There was statistically significant increase in the incidences of MNIMEs observed with the repeated administration of Sudan I. Therefore in vivo genetic toxicity of 1-phenylazo-2-naphthol (Sudan I) is positive.

 

The genotoxic effect of food colorant 1-phenylazo-2-naphthol (Sudan I) (CAS no 842 -07 -9) was investigated by Sadek et al (2010) using chromosomal aberration test. Three doses of sudan I 0.325, 0.65 and 1.3 mg/kg.b.wt. which represent (Accepted Daily Intake ADI, double of the ADI, and four times of the ADI) were investigated. The doses were dissolved in distilled water and orally administrated and tested for the induction of chromosomal aberrations in bone marrow cells and primary- spermatocytes. Cyclophosphamide was used as positive control. The results showed statistically significant increase in chromosomal aberrations in bone marrow cells and spermatocytes. Sudan I is a potent inducer of chromosomal aberrations in bone-marrow cells and spermatocytes with a dose-related relationship. The present results indicate that 1-phenylazo-2-naphthol (Sudan I) is genotoxic in the test examined.

 

Bone marrow micronucleus assay was performed by Elliott et al (1997) to determine the mutagenic nature of 1-phenylazo-2-naphthol (C. I. Solvent Yellow 14) in vivo. 6-12 week old C57BL/6JfBL10/Alpk male mice were used for the assay. An initial dose ranging study was conducted to assess the toxicity of CI Solvent Yellow 14. For the main studies, groups of five animals were given a single oral dose of test compound at 5000 mg/Kg or control substance and killed after 24 or 48 h by exposure to halothane or a rising concentration of CO2 followed by cervical dislocation. Bone marrow smears were prepared and stained with polychrome Methylene Blue and eosin (mouse) or haematoxylin and eosin (rat). All slides were coded before scoring. Initially 1000, but eventually up to 6000 polychromatic erythrocytes (PCEs) per animal were examined for micronuclei, and 1000 erythrocytes per animal to determine the percentage of polychromatic erythrocytes. Following an assessment of 1000 PCEs, small increases in MPEs were seen at both 24 and 48 h following a dose of 5000 mg/kg, with the increase at the 48 h sampling time being statistically significant compared with controls. Following an assessment of 6000 PCEs, the increase in MPEs remained at the 48 h sampling time, and retained statistical significance, whilst the 24 h sampling time MPE values for control and test were essentially the same. 1-phenylazo-2-naphthol (C. I. Solvent Yellow 14) induced Polychromatic mononucleated erythrocytes and hence is likely to be mutagenic in vivo.

 

In the same study performed by Matsumura et al (2015), in vivo genotoxicity of 1-phenylazo-2-naphthol (Sudan I) was examined using repeated-dose liver micronucleus (MN) assays in young adult rats. Sudan I was administered orally to rats for 14-days, and twenty-four hours after the 14-day administration, the rats were euthanized under anesthesia. The MN frequency in the liver, stomach, colon, and bone marrow were analyzed. Based on the results of the dose range finding study, the dose range selected was 0, 150, 300, and 600 mg/kg/day. The livers were excised, and a portion of the left lateral lobe was sliced into several pieces. These strips were rinsed with Hanks’ balanced salt solution and treated with HBSS containing 100 units/mL of collagenase in a flask shaken for 1 h at 37 ◦C. The resulting material was repeatedly pipetted to break apart cell clumps and then forced through a cell strainer. The obtained cell suspension was then fixed with 10% neutral buffered formalin. Immediately before observation, the cell suspension was stained with acridine orange (AO) and 4,6-diamidino-2-phenylindole dihydrochloride (DAPI), and specimens were prepared. The specimens were microscopically observed with U-excitation (Ultraviolet ray excitation), and the number of micronucleated hepatocytes (MNHEPs) per 2000 parenchymal hepatocytes (HEPs), including mono-, bi-, and multi-nucleated cells, was counted for each animal. There was however no statistically significant increase in the incidences of MNHEPs with the administration of Sudan I. Therefore in vivo genetic toxicity 1-phenylazo-2-naphthol (Sudan I) is negative. The possible explanation for a lack of liver MN induction might be possible under the hypothesis that 1-phenylazo-2-naphthol (Sudan I) and its metabolites were readily eliminated from the liver by phase II detoxification enzymes and hence gave a negative result.

 

Same study was also performed to determine the in vivo Geno toxicity of 1-phenylazo-2-naphthol (Sudan I)using repeated-dose Gastrointestinal tract micronucleus (MN) assays in young adult male Crl: CD (SD)rats. The stomach was dissected and the forestomach was removed, leaving the gastric cardia. After the contents of the stomach were rinsed out with HBSS, a glass rod was inserted into the gastric cardia, and the glandular stomach was everted on a ball at the end of the glass rod. The everted tissue was incubated in a tube containing a solution composed of 1mM ethylenediamine-tetraacetic acid disodium salt (EDTA) and 2mM dithiothreitol in HBSS at 35 ◦C for 1 h. After incubation, the tissue was vortexed for several minutes to isolate the epithelial cells. The colons were dissected, rinsed with HBSS, and everted on a glass rod. These colons were incubated in a solution of 1mM EDTA in HBSS at 35 ◦C for 30 min. The tissue was vortexed every 15 min to isolate the epithelial cells. The collected cells were strained through nylon mesh to remove cell clumps, and fixed in 10% neutral-buffered formalin. Immediately before observation, the cells were stained withAOand DAPI. The stained cells were analyzed under a fluorescence microscope with U excitation. Two thousand intact cells were scored per animal to determine the frequency of micronucleated gastrointestinal tract cells (MNGTCs). There were no statistically significant increases in the incidences of micro nucleated epithelial cells with the repeated administration of 1-phenylazo-2-naphthol (Sudan I). Therefore in vivo genetic toxicity of 1-phenylazo-2-naphthol (Sudan I) is negative. Azo compounds like 1-phenylazo-2-naphthol (Sudan I) may be less likely to induce DNA strand breaks in rat colon due to differences in their gut micro floral metabolism. This might be the possible reason for negative result in gastrointestinal tract MN assay.

 

Based on the data summarized, the test chemical 1-phenylazo-2-naphthol (C. I. Solvent Yellow 14) is likely to be a mutagen in vivo.

Justification for classification or non-classification

Based on the key study and its supporting data summarized, 1-phenylazo-2-naphthol (C. I. Solvent Yellow 14) is likely to classify as a gene mutant in vitro and in vivo.

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