6-hydroxy-2-naphthoic acid

Genetic toxicity: in vitro

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:

key study

Study period:

1999-08-17 - 2000-02-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented GLP study performed in compliance with OECD 476

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

6-hydroxy-2-naphthoic acid

EC Number:

240-759-7

EC Name:

6-hydroxy-2-naphthoic acid

Cas Number:

16712-64-4

Molecular formula:

C11H8O3

IUPAC Name:

6-hydroxynaphthalene-2-carboxylic acid

Constituent 2

Reference substance name:

6-HNA

IUPAC Name:

6-HNA

Test material form:

other: Solid

Details on test material:

- Name of test material (as cited in study report): HNA
- Physical state: Solid.
- Analytical purity: 99.3%
- Impurities (identity and concentrations): Not specified.
- Composition of test material, percentage of components: 99.% test article, remainder not characterized.
- Isomers composition: Not determined.
- Purity test date: Not stated.
- Lot/batch No.: Lot No. 980808
- Expiration date of the lot/batch: 8 Aug 2000
- Stability under test conditions: Not indicated.
- Storage condition of test material: Room Temperature

Method

Target gene:

HPRT Locus

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

V79 cells supplied by Laboratory for Mutagenicity Testing, Technical University, D-64293 Darmstadt.
- Type and identity of media: MEM + 10% FCS
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

Mammalian S9

Test concentrations with justification for top dose:

Experiment 1:
- Without S9: 62.5, 125.0, 250.0, 500.0, 1000.0 and 1500.0 microg/mL
- With S9: 125.0, 250.0, 500.0, 1000.0, 1500.0 and 2000.0 microg/mL
Experiment 2:
- Without S9: 62.5, 125.0, 250.0, 500.0, 750.0 and 1000.0 microg/mL

Vehicle / solvent:

Vehicle selected was DMSO.
Vehicle was chosen based on test article solubility properties in vehicle and lack of toxicity to the V79 cells.

Controls

Untreated negative controls:

yes

Remarks:

no treatement; cells in culture medium

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

EMS used for "-S9"; DMBA used for "+S9"

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium.

DURATION
- Preincubation period: No pre-incubation for treatments.
- Exposure duration: 4 hours for Experiment No. 1; 24 hours for Experiment No. 2
- Expression time (cells in growth medium): 7 days.
- Selection time (if incubation with a selection agent): 8 days, post-expression time.
- Fixation time (start of exposure up to fixation or harvest of cells): 16 - 17 days.

SELECTION AGENT (mutation assays): 6-thioguanine (6-TG)
STAIN (for cytogenetic assays): 10% methylene blue in 0.01 KOH Solution.

NUMBER OF REPLICATIONS: One replicate each (with two flasks per condition) for each experiment (4 hr/-S9; 4 hr/+S9; 24 hr/-S9)

NUMBER OF CELLS EVALUATED: 1.5 x10^6 cells seeded for mutation rate experiment; 5.0 x10^2 cells seeded for toxicity experiment.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency.

Evaluation criteria:

Acceptability of the Assay:
The assay was considered acceptable if:
A. The number of mutant colonies per 10^6 cells found in the negative and/or solvent controls falls within the laboratory's historical control data range.
B. The positive control substances produce a significant increase in mutant colony frequencies.
C. The cloning efficiency (absolute value) of the negative and/or solvent controls must exceed 50%.

A test article is considered mutagenic if it induces either a concentration-dependent increase of the mutant frequency, or a reproducible and positive response at one of the test points. Additionally, the increase must be 3X above the spontaneous mutation frequency at least at one concentration. If the increase is not 3X, then if the dose-dependent increase is reproducible, the test article may still be considered to be mutagenic, dependent on a comparison to the spontaneous mutation frequency.

A test article not producing either a concentration-dependent increase in the mutant frequency or a reproducible positive response at any of the test points is considered non-mutagenic.

Statistics:

Since the distribution of mutant cells does not follow known statistical models, an adequate statistical method is not available.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity observed at highest doses.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation was observed only in the pre-test toxicity experiment, and only at the top dose.

RANGE-FINDING/SCREENING STUDIES: Based on the pre-test toxicity experiment, the concentration ranges were selected to yield cytotoxic effects at the higher doses.

COMPARISON WITH HISTORICAL CONTROL DATA: All experimental results requiring comparison to historical values were within the limits of those historical values.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Pre-Test Cytotoxicity Experiment:

Concentration (microg/mL)	Duration of Treatment	S9	Cloning Efficiency (%; absolute)	Cloning Efficiency (%; relative)
Negative Control	4	-	62.2	100.0
Solvent Control	4	-	64.1	100.0
15.6	4	-	63.4	98.9
31.3	4	-	69.5	108.5
62.5	4	-	55.9	87.3
125.0	4	-	56.6	88.4
250.0	4	-	60.9	95.1
500.0	4	-	60.9	95.1
1000.0	4	-	23.2	36.2
2000.0	4	-	0.0	0.0
Negative Control	4	+	56.2	100.0
Solvent Control	4	+	58.7	100.0
15.6	4	+	53.8	91.6
31.3	4	+	55.6	94.8
62.5	4	+	56.3	96.0
125.0	4	+	58.5	99.7
250.0	4	+	54.5	92.7
500.0	4	+	57.4	97.8
1000.0	4	+	49.5	84.3
2000.0	4	-	0.0	0.0
Negative Control	24	-	76.4	100.0
Solvent Control	24	-	70.7	100.0
15.6	24	-	78.0	110.4
31.3	24	-	81.7	115.5
62.5	24	-	77.2	109.2
125.0	24	-	59.4	84.0
250.0	24	-	56.6	80.1
500.0	24	-	45.0	63.7
1000.0	24	-	4.1	5.7
2000.0	24	-	0.0	0.0

Main Experiment: Experiment 1, Culture 1

Concentration	S9	Cloning Efficiency (relative % survival)	Cloning Efficiency Factor	Mutant Colonies per 10^6 cells
Negative Control (untreated cells)	-	100.0	0.68	12.8
Solvent Control	-	100.0	0.68	9.6
Positive Control with EMS (300.0 microg/mL)	-	92.0	0.63	262.8
62.5 microg/mL	-	103.7	*culture not continued	*culture not continued
125.0 microg/mL	-	94.0	0.69	13.8
250.0 microg/mL	-	107.5	0.74	2.2
500.0 microg/mL	-	105.0	0.69	7.1
1000.0 microg/mL	-	85.2	0.69	2.6
1500.0 microg/mL	-	0.0	0.68	2.7
Negative Control (untreated cells)		100.0	0.82	2.9
Solvent Control	+	100.0	0.74	6.1
Positive Control (DMBA 2.5 microg/mL)	+	53.0	0.74	721.0
125.0 microg/mL	+	106.7	0.91	2.1
250.0 microg/mL	+	105.1	0.76	7.6
500.0 microg/mL	+	82.2	0.91	7.3
1000.0 microg/mL	+	90.9	0.74	7.4
1500.0 microg/mL	+	93.3	0.46	8.8
2000.0 microg/mL	+	13.4	0.41	4.4

Main Experiment: Experiment 1, Culture 2

Concentration	S9	Cloning Efficiency (relative % survival)	Cloning Efficiency Factor	Mutant Colonies per 10^6 cells
Negative Control (untreated cells)	-	100.0	0.50	7.8
Solvent Control	-	100.0	0.44	5.5
Positive Control with EMS (300.0 microg/mL)	-	105.0	0.67	267.0
62.5 microg/mL	-	103.8	*Culture not continued	*Culture not continued
125.0 microg/mL	-	108.8	0.62	5.7
250.0 microg/mL	-	97.2	0.40	9.2
500.0 microg/mL	-	96.2	0.52	8.2
1000.0 microg/mL	-	88.9	0.33	14.9
1500.0 microg/mL	-	0.0	0.45	4.9
Negative Control (untreated cells)	+	100.0	0.54	6.5
Solvent Control	+	100.0	0.54	2.7
Positive Control (DMBA 2.5 microg/mL)	+	45.4	0.33	1417.4
125.0 microg/mL	+	145.1	0.44	12.8
250.0 microg/mL	+	93.1	0.41	4.5
500.0 microg/mL	+	95.7	0.50	5.7
1000.0 microg/mL	+	90.6	0.44	22.7
1500.0 microg/mL	+	92.9	0.42	6.9
2000.0 microg/mL	+	1.1	0.45	3.6

Main Experiment: Experiment 2, Culture 1

Concentration	S9	Cloning Efficiency (relative % survival)	Cloning Efficiency Factor	Mutant Colonies per 10^6 cells
Negative Control	-	100.0	0.69	5.8
Solvent Control	-	100.0	0.85	9.8
Positive Control with EMS (300.0 microg/mL)	-	76.9	0.49	1064.7
62.5 microg/mL	-	100.0	*Culture not continued	*Culture not continued
125.0 microg/mL	-	88.6	0.76	4.9
250.0 microg/mL	-	81.3	0.71	3.0
500.0 microg/mL	-	59.7	0.67	10.6
750.0 microg/mL	-	27.0	0.63	11.2
1000.0 microg/mL		25.4	0.63	4.6

Main Experiment: Experiment 2, Culture 2

Concentration	S9	Cloning Efficiency (relative % survival)	Cloning Efficiency Factor	Mutant Colonies per 10^6 cells
Negative Control	-	100.0	0.86	6.6
Solvent Control	-	100.0	0.92	5.0
Positive Control with EMS (300.0 microg/mL)	-	75.6	0.62	707.5
62.5 microg/mL	-	100.4	*Culture not continued	*Culture not continued
125.0 microg/mL	-	85.6	0.81	14.1
250.0 microg/mL	-	78.3	0.67	3.3
500.0 microg/mL	-	51.9	0.63	5.1
750.0 microg/mL	-	28.3	0.76	8.0
1000.0 microg/mL		21.5	0.73	9.7

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, it can be stated that during the described mutagenicity test, and under the experimental conditions reported, the test article did not induce gene mutations at the HPRT locus in V79 cells.

Therefore, 6-hydroxy-2-naphthoic acid is considered to be non-mutagenic in this assay.

Executive summary:

The study was performed to investigate the potential of the test article to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments. The cells were exposed to the test article for 4 hours in the first experiment with and without metabolic activation (S9 mix). In the second experiment, cells were exposed to test article for 24 hours, and solely in the absence of metabolic activation.

In the main experiments, the following concentrations were evaluated:

Experiment 1

- without S9 mix: 125.0, 250.0, 500.0, 1000.0 and 1500.0 microg/mL

- with S9 mix: 125.0, 250.0, 500.0, 750.0 and 1000.0 microg/mL

Experiment 2

- without S9 mix: 125.0, 250.0, 500.0, and 1000.0 microg/mL

Visible precipitation of the test item only occurred in the pre-experiment at the highest concentration of test article (2000.0 microg/mL). Toxic effects occurred in the pre-experiment at 1000.0 microg/mL and above without metabolic activation (4h and 24h treatments), and at 2000.0 microg/mL with metabolic activation. Based on the results of the pre-test on toxicity, the concentrations were selected to yield concentration-related toxic effects. The highest concentration produced a low-level of survival, and the survival at the lowest concentration was relatively close to the negative control.

The concentration range of the test article was limited by toxicity. In the first experiment, strong toxic effects occurred at 1500.0 microg/mL in the absence of metabolic activation and 2000.0 microg/mL in the presence of metabolic activation. In the second experiment, relevant toxic effects were observed at 500.0 microg/mL and above.

No relevant and reproducible increases in mutant frequency were observed at any concentration, neither in the absence or presence of metabolic activiation. Appropriate reference mutagens were used as positive controls, and showed a distinct increase in mutant frequencies.

In conclusion, it can be stated that during the described mutagenicity test, and under the experimental conditions reported, the test article did not induce gene mutations at the HPRT locus in V79 cells.

Therefore, 6-hydroxy-2-naphthoic acid is considered to be non-mutagenic in this assay.