3-hydroxy-2-naphthoic acid

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 10 to September 14, 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Only 50 metaphases were examined per animal. Apart from that, study conducted according to OECD 475 test guideline.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

yes

Remarks:

only 50 metaphases were examined per animal.

GLP compliance:

yes

Type of assay:

chromosome aberration assay

Test material

Test material information

Constituent 1

Reference substance name:

3-hydroxy-2-naphthoic acid

EC Number:

202-180-8

EC Name:

3-hydroxy-2-naphthoic acid

Cas Number:

92-70-6

Molecular formula:

C11H8O3

IUPAC Name:

3-hydroxynaphthalene-2-carboxylic acid

Details on test material:

- Name of test material (as cited in study report): Beta-Oxynaphthoesäure (BONS)

Test animals

Species:

hamster, Chinese

Strain:

other: Han:Chin

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Zentralinstitut für Versuchstiere, Hannover
- Age at study initiation: 10 - 13 weeks
- Weight at study initiation: males mean = 31.8 g (27 - 37 g), females: mean = 26.0 g (21 - 31 g)
- Housing: in fully air-conditioned rooms in Makrolon cages (Type 2) on soft wood granulate, one animal per cage
- Diet (e.g. ad libitum): Altromin 7010 hamster diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20+/-3
- Humidity (%): approx. 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

other: oral, suspended in starch mucilage

Vehicle:

starch mucilage

Details on exposure:

The test substance was administered orally to the test animals in a dose of 2000 mg/kg bodyweight. Starch mucilage was administered in the same way to the negative control groups. Simultaneously to negative control s and dose groups EndoxanR was used as positive control substance and was administered orally at a dose of 50 mg per kg bodyweight. Two hours before killing by carbon dioxide asphyxation, each of the hamsters received an intraperitoneal injection of 3.3 mg demecolcin (ColcemidR) per kg bodyweight.

Frequency of treatment:

single oral dose

Post exposure period:

12, 24 or 48 hours

Doses / concentrations

Remarks:

Doses / Concentrations:
2000 mg/kg bodyweight
Basis:
actual ingested

No. of animals per sex per dose:

negative control (killing time 12h p.a.): 5 male , 5 female
2000 mg/kg (killing time 12h p.a.): 5 male, 5 female

negative control (killing time 24h p.a.): 5 male , 5 female
2000 mg/kg (killing time 24h p.a.): 5 male, 5 female
positive control (killing time 24h p.a.): 5 male, 5 female

negative control (killing time 48h p.a.): 5 male , 5 female
2000 mg/kg (killing time 48h p.a.): 5 male, 5 female

Control animals:

yes, concurrent vehicle

Positive control(s):

Endoxan at 50 mg/kg bodyweight orally administered. 5 males and 5 females each killed 24 hours post administration.

Examinations

Tissues and cell types examined:

Femoral bone marrow cells. 2-4 slides were prepared from each animal and 50 metaphases per animal were evaluated for numerical and structural chromosome aberrations.

Details of tissue and slide preparation:

After sacrifice, bone marrow was obtained by flushing with Hanks solution (approx. 2 ml/both femora) at approx. 37°C. Hypotonic treatment with approx. 0.075 M potassium chloride solution at approx. 37°C. The resulting suspension was incubated for 10 minutes at approx. 37°C and then approx. 1.5 ml fixative (methanol:glacial acetic acid 3 + 1) were added and the suspension aerated. A series of centrifugation and covering/withdrawal of fixative followed. After storage for at least 12 hours (overnight) in a refrigerator at approx. 4°C, re-centrifugation and formation of a fresh sususpension. Finally the cells were spread on slides and stained as follows:

- staining for 10 minutes in approximately 2 % orcein solution
- rinsing 3 times in distilled water
- rinsing twice in acetone
- brief rinsing in acetone/xylene
- 2 minutes in acetone/xylene
- 5 minutes in xylene
- minimum 10 minutes in xylene
- embedding in EntellanR or EukittR.

2-4 slides were prepared from each animal.

Evaluation criteria:

The test substance is considered to be mutagenic if it induces a statistically significantly increased aberration rate (excluding gaps) as compared with the negative controls for at least one of the time points.

The test substance producing no significant increase of the aberration rate is classified as non mutagenic.

Statistics:

Statistics was not performed, as all aberration rates were within the range of the negative control values.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Remarks:

12, 24 and 48 h post single oral dose of 2000 mg/kg bw.

Toxicity:

no effects

Remarks:

No clinical signs of toxicity, no sign of cytotoxicity in the bone marrow cells (no reduction of the mitotic index) and no macroscopic pathology findings

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

The test substance did not induce a significant increase in the number of chromosomal aberrations. A marked increase in the number of chromosomal aberrations was found in the groups treated with the positive control substance.

The percentages of metaphases with aberrations (excluding gaps) were as follows:

at 12 hours post administration: negative control - 0.0%, test substance - 0.0%

at 24 hours post administration: negative control - 0.2%, test substance - 0.0%, positive control - 13.0%

at 48 hours post administration: negative control - 0.0%, test substance - 0.2%.

The percentages of metaphases with aberrations (including gaps) were as follows:

at 12 hours post administration: negative control - 0.4%, test substance - 1.6%

at 24 hours post administration: negative control - 0.8%, test substance - 0.8%, positive control - 14.0%

at 48 hours post administration: negative control - 0.4%, test substance: 1.8%.

No clinical signs of toxicity were observed. There was no sign of cytotoxicity in the bone marrow cells (no reduction of the mitotic index). At necropsy, no pathological changes were found.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
In this study there was no indication of clastogenic activity in test substance treated animals, as the percentages of aberrations (including and excluding gaps) found in bone marrow cells 12, 24 or 48 hours post single oral dosing at the maximum recommended dose of 2000 mg/kg bw did not distinguish test substance treated animals from concurrent vehicle controls. In addition, signs of toxicity (clinical signs, macroscopic pathology findings, and cytogenicity expressed as reduced mitotic index) were not evident in the present study.

In the present study only 50 metaphases per animal were examined and there was no indication that the target tissue was reached by the test substance. However, in acute toxicity studies and repeated dose toxicity studies clear treatment related effects were evident, demonstrating that the test substance or its metabolites reach the general circulation and cause systemic toxicity after oral application. Therefore, there is no evidence, that the test substance will not reach the target tissue.

Executive summary:

3-Hydroxy-2-naphthoic acid was tested for mutagenicity in an in vivo chromosome aberration assay in general equivalent to OECD Guideline 475. A group of 15 male and 15 female Chinese hamsters was treated by single oral administration with 3-hydroxy-2-naphthoic acid at the maximum recommended dose of 2000 mg/kg bw. For administration, the test substance was suspended in starch mucilage. The animals, 5 males and 5 females each, were killed at 12, 24 and 48 hours after dosing, approximately two hours after intraperitoneal injection of demecolcin (ColcemidR) for metaphase arrestment. In addition, vehicle controls (hamsters receiving the vehicle starch mucilage alone) and positive controls (dosed with Endoxan at 50 mg/kg bw) were included in the study. Bone marrow cells were collected for chromosome aberration analysis from vehicle control and test animals at 12, 24 and 48 hours post administration and from positive control animals 24 hours post administration. Signs of toxicity (clinical signs, macroscopic pathology findings, and cytogenicity expressed as reduced mitotic index) and clastogenic activity were not evident in the test substance treated group.

In the present study only 50 metaphases per animal were examined and there was no definite indication in this study that the target tissue was reached by the test substance. However, in further studies for acute toxicity or repeated dose toxicity clear treatment related effects were evident, demonstrating that the test substance or its metabolites reach the general circulation and cause systemic toxicity after oral application. Therefore, there is adequate evidence, that the test substance will reach the target tissue.