1-phenylazo-2-naphthol

Genetic toxicity: in vitro

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

Test material information

Constituent 1

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 results

Test results 1

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

Test results 2

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+/tk^–mouse 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.