6-hydroxy-2-naphthoic acid

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted under GLP (including certificate), according to OECD 474.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

Relative humidity during experiment ranged between 25 - 70%, which differed from protocol statement of "30 - 70%". Did not affect validity of the experiment.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)

Deviations:

yes

Remarks:

Relative humidity during experiment ranged between 25 - 70%, which differed from protocol statement of "30 - 70%". Did not affect validity of the experiment.

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

Relative humidity during experiment ranged between 25 - 70%, which differed from protocol statement of "30 - 70%". Did not affect validity of the experiment.

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus 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: 99.3% test article, remainder not characterized.
- 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.

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd.; Biotechnology and Animal Breeding Division
- Age at study initiation: 8 - 12 weeks.
- Weight at study initiation: Males - Mean value of 33.6 g (SD +/- 2.6 g); Females - Mean value of 28.0 g (SD +/- 1.9 g)
- Assigned to test groups randomly: Yes, method not stated.
- Fasting period before study: No food 18 hours prior to exposure, but received water ad libitum.
- Housing: Single; Makrolon Type 1 cage with wire mesh top, granulated soft wood bedding.
- Diet (e.g. ad libitum): Pelleted standard diet, ad libitum.
- Water (e.g. ad libitum): Tap water, ad libitum.
- Acclimation period: At least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 3 degrees C.
- Humidity (%): 25 - 70%
- Air changes (per hr): Not stated.
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: Vehicle chosen for its relative non-toxicity to the animals.
- Concentration of test material in vehicle: Varied to achieve standard dose volume of 10 mL/kg.
- Amount of vehicle (if gavage or dermal): 10 mL/kg by gavage.
- Lot/batch no. (if required): Lot No. 107H1699
- Purity: Not stated.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

On the day of the experiment, the test item was formulated in Corn Oil.

Duration of treatment / exposure:

Test article was administered as a single dose.

Frequency of treatment:

Single treatment.

Post exposure period:

A preliminary study on acute toxicity was performed with two animals per sex. The animals were treated orally, and examined for signs of acute toxicity at intervals of 1, 6, 24, and 48 hours after administration of the test article.

Based on the results of the preliminary study, post-exposure durations were set at 24 and 48 hours.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
24 Hour Post-Exposure Group: 150.0, 300.0 and 600.0 mg/kg b.w.
Basis:
actual ingested
Given by gavage.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
48 Hour Post-Exposure Group: 600.0 mg/kg b.w.
Basis:
actual ingested
Given by gavage.

No. of animals per sex per dose:

6 animals per dose, per sex.

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Justification for choice of positive control(s): Standard positive control used for mammalian chromosomal aberration studies.
- Route of administration: Oral gavage.
- Doses / concentrations: 40 mg/kg b.w., single dose.
- Preparation: Positive control prepared in deionised water on day of administration.

Examinations

Tissues and cell types examined:

Polychromatic Erythrocytes from bone marrow was evaluated.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Followed the recommendation to use the Maximum Tolerated Dose (MTD), or 2000 mg/kg as the upper limit for non-toxic test articles. A preliminary experiment for evaluating acute toxicity was performed with 2 animals per sex under conditions identical to the main study. The pre-experiment began exposure at 1000 mg/kg b.w., and stepped down 200 mg/kg b.w. until the MTD was determined. No deaths occurred at 600 mg/kg b.w., therefore this was selected as the MTD, and set as the high dose. The medium and low doses for the main study were sequential 2 fold reductions (300 mg/kg b.w. and 150 mg/kg b.w.).

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Animals were restricted from food for 18 hours pre-exposure, but were given water ad libitum. Animals were treated, and there were 2 post-treatment sacrifice intervals. All dose levels had a 24 hour-post exposure sacrifice group, and the high dose (600 mg/kg b.w.) had an additional 48 hour post-treatment sacrifice group.

DETAILS OF SLIDE PREPARATION: Details not given.

METHOD OF ANALYSIS: Slides were evaluated using a microscope with 100x oil immersion objectives. 2000 polychromatic erythrocytes (PCEs) were analyzed per animal for micronuclei.

OTHER:

Evaluation criteria:

The study was considered valid if the following criteria were met:
- the vehicle controls were in the range of the laboratory's historical control data (0.03 - 0.15% PCEs with micronuclei).
- The positive controls showed substantially increased values.
- More than 80% of the animals were evaluable.

The test article was considered as mutagenic if it induced either a dose-related increase in the number of micronucleated PCEs, or a statistically significant positive response for at least one of the test points. The test article was considered non-mutagenic if neither of these criteria were met.

Statistics:

Non-parametric Mann-Whitney test, with consideration of biological significance.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Toxicity observed below 2000 mg/kg b.w. limit dose; MTD established through pre-experiment acute toxicity study.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: Started at 1000 mg/kg b.w., stepped down 200 mg/kg b.w. until no deaths observed. MTD determined to be 600 mg/kg b.w., medium and low dose set at sequential 2-fold reductions (300 mg/kg b.w. and 150 mg/kg b.w.).
- Solubility: Not stated, but not applicable, as this was an oral gavage study.
- Clinical signs of toxicity in test animals: For pre-experiment toxicity testing, "reduction in spontaneous activity", "eyelid closure", "apathy", "abdominal position", "convulsions" and "death" were monitored.
- Rationale for exposure: Not stated.
- Harvest times: Not applicable. Preliminary study was to determine MTD only.
- High dose with and without activation: In vivo mammalian exposure, so no metabolic activation required

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay):
- Induction of micronuclei (for Micronucleus assay): See Table Below.
- Ratio of PCE/NCE (for Micronucleus assay): See Table Below.
- Evidence of cytotoxicity in tissue analyzed: Ratio of PCE to NCE given as sign of bone marrow toxicity.
- Appropriateness of dose levels and route: Dose levels followed guidance to use MTD or 2000 mg/kg b.w. as limit dose.
- Statistical evaluation: Statistical results given in table.

Any other information on results incl. tables

Summary Table of Results:

Dose	Post-Exposure Sampling Time (hours)	PCEs with Micronuclei (%)	PCE/NCE Ratio	Statistical Significance (compared to Vehicle Control)
Vehicle	24	0.090	2000/1616	Not Applicable
150 mg/kg b.w Test Article	24	0.050	2000/1574	Not tested (mean frequency below Vehicle Control)
300 mg/kg b.w Test Article	24	0.055	2000/1603	Not tested (mean frequency below Vehicle Control)
600 mg/kg b.w Test Article	24	0.095	2000/1910	No significance (p = 0.4322)
600 mg/kg b.w Test Article	48	0.100	2000/1805	No significance (p = 0.3933)
Positive Control (40 mg/kg b.w. cyclophosphamide)	24	2.315	2000/1672	Significant (p < 0.0001)

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
In conclusion, it can be stated that during the study described, under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

Therefore, HNA is considered to be non-mutagenic in this micronucleus assay.

Executive summary:

This study was performed to investigate the potential of HNA to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

The test article was formulated in corn oil. Corn oil was used as vehicle control. The volume administered orally was 10 ml/kg b.w. At 24 and 48 hours after a single administration, the bone marrow cells were collected for micronuclei analysis. Ten animals (5M/5F) per test group were evaluated for the occurrence of micronuclei. 2000 PCEs per animal were scored for micronuclei. Cytotoxicity was determined by evaluating the ratio of PCEs to normochromatic erythrocytes (NCEs).

The following dose levels were evaluated:

- 24 hour preparation interval: Vehicle Control, 150, 300 and 600 mg/kg b.w.

- 48 hour preparation interval: 600 mg/kg b.w.

The high dose was estimated by a pre-experiment, and was considered to be the Maximum Tolerated Dose (MTD).

After treatment with the highest test article dose, the number of NCEs remained higher than the mean value of NCEs in the Vehicle Control. This was stated to indicate the bioavailability of the test article.

In comparison to the corresponding vehicle controls, there was no relevant enhancement in the frequency of the detected micronuclei at any dose level, at either preparation interval. The positive control (40 mg/kg b.w. cyclophosphamide) showed a statistically significant increase in micronucleus frequency.