2-naphthol

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

no

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

2-naphthol

EC Number:

205-182-7

EC Name:

2-naphthol

Cas Number:

135-19-3

Molecular formula:

C10H8O

IUPAC Name:

2-naphthol

Test material form:

solid: flakes

Test animals

Species:

mouse

Strain:

B6C3F1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Japan SLC, Inc.
- Age at study initiation: 9 weeks of age
- Weight at study initiation: 24.5 to 27.7 g
- Assigned to test groups randomly: yes
- Housing: housed in a zyfone animal cage
- Diet: pellet diet (MF: Oriental Yeast Co., Ltd.; Lot No. 040310) ad libitum
- Water: tap water ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23.4 to 24.3°C
- Humidity (%): 54 to 67%
- Air changes (per hr): 18 times per hour
- Photoperiod (hrs dark / hrs light): 12-hour light/dark cycle

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: CMC
- Amount of vehicle: 0.1 mL per 10 g body weight
- Lot/batch no.: . PKG2104

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was suspended in 0.5 w/v% methylcellulose solution using mortar and pestle.

Duration of treatment / exposure:

Two days

Frequency of treatment:

Once a day

Post exposure period:

24 hours

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

62.5 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

125 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

250 mg/kg bw/day

No. of animals per sex per dose:

5 animals / dose

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive control: Mitomycin C
- Justification for choice of positive control(s): OECD TG 474
- Route of administration: intraperitoneal
- Doses / concentrations: 0.5 mg/kg

Examinations

Tissues and cell types examined:

Bone marrow erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
On the basis of the results of a dose-finding study, 250 mg/kg, which is near the dose inducing toxic signs, was selected as the highest dose, and three doses including 125 and 62.5 mg/kg (common ratio of 2) were selected. Since there was no evident sex difference in the incidences of toxic signs, only male mice were used for this study.

TREATMENT AND SAMPLING TIMES:
Mice were euthanized by inhalation of carbon dioxide at 24 hours after the final dosing (at 24 hours after dosing in case of the positive control).

DETAILS OF SLIDE PREPARATION:
Femur was removed, and the bone marrow cells were flushed out with small amount of calf serum (Invitrogen Corp.; Lot No. 1172087, inactivated at 56°C for 30 minutes) into a centrifuge tube. Excess serum was removed by centrifugation method and the bone marrow cells were smeared on 3 slides per animal. The smears were dried well and were then fixed using methanol. Two smears per animal were stained with 3% Giemsa solution (Merck KGaA; Lot No. OB279210) for 30 minutes. They were rinsed with 1/100 mol/L sodium phosphate buffer (Merck KGaA; pH 6.8; Lot No. TP601374) and purified water, and dried. Then, these slides were rinsed with 0.01% citric acid solution and purified water, and dried again.
The bone marrow smear slides were prepared from all animals.

METHOD OF ANALYSIS:
All samples were coded and then examined by the blind method. Two thousand polychromatic erythrocytes (PCE) per animal were analyzed using a microscope (×1000). The number of micronucleated polychromatic erythrocytes (MNPCE) was counted. To examine the effect of test substance on bone marrow cells, the number of PCE out of a total of 500 erythrocytes was also counted.

Evaluation criteria:

The results were evaluated to be positive when the statistically significant difference was detected in the each treatment group or in the positive control group as compared to the negative control group.

Statistics:

The Conditional Binomial test (Kastenbaum and Bowman method: upper-tailed significance level of 0.025) was performed to compare the frequency of MNPCE in the negative control group with that in each treatment group and positive control group.
The ratios of polychromatic erythrocytes to total erythrocytes were subjected to Dunnett’s multiple comparison test (two-tailed significance level of 0.05) for the analysis of significant differences between the negative control group and the groups treated with the test substance. The data obtained from the negative and positive control groups were subjected to Aspin-Welch t test (two-tailed significance level of 0.05).

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 62.5 to 1,000 mg/kg bw
- Clinical signs of toxicity in test animals: Mortality observed at 500 and 1,000 mg/kg bw in both male and female animals. Reduction of body weight was observed in both sexes at 250 mg/kg bw, as well as decreased locomotor activity and/or hypothermia.
- Rationale for exposure: Based on available information on the toxicity of the substance at the time of the study.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: The frequencies of MNPCE in the groups treated with the 2-Naphthol were 0.27, 0.22 and 0.29% in 62.5, 125 and 250 mg/kg groups, respectively.

Any other information on results incl. tables

Micronucleus assay

In the negative control group, 2 to 6 micronucleated cells (MNPCE) in 2000 polychromatic erythrocytes (PCE) per animal were noted indicating an incidence of 0.18%.  The ratio of PCE to the total number of erythrocytes was 57.6%.

The frequencies of MNPCE in the groups treated with the 2-Naphthol were 0.27, 0.22 and 0.29% in 62.5, 125 and 250 mg/kg groups, respectively.  No statistically significant increase was noted in any of the treatment groups as compared to the negative control group.  The ratios of PCE to the analyzed erythrocytes, an index of the effects of the test substance on the bone marrow cells, were 54.6, 54.8 and 48.7% in 62.5, 125 and 250 mg/kg groups, respectively.  No significant decrease was noted in any of the treatment groups as compared with that in the negative control group.

On the other hand, the incidence of MNPCE in the positive control group was markedly increased to 1.18% (15 to 32 MNPCEs in 2000 PCEs), and a statistically significant increase (p=0.025) was found as compared with the negative control group.  The ratio of PCE was 54.0%.

Body weight and clinical observations

Due to the treatment with 2-Naphthol, 1 out of 8 males died at 21 hours after the first dosing of 250 mg/kg.  In addition, toxic signs such as decrease in locomotor activity and so on were observed in the animals receiving 250 mg/kg from 1 hour after the first dosing, and 3 g reduction of mean body weight value was observed.

Applicant's summary and conclusion

Conclusions:

2-Naphthol did not induce micronucleated erythrocytes in mouse bone marrow cells, i.e. structural nor numerical chromosome aberrations, under the conditions employed in this study.

Executive summary:

To assess the potential of 2-Naphthol for inducing micronucleated erythrocytes, an in vivo micronucleus test was conducted on male mice using a method similar to the OECD Testing Guideline 474. The study was not GLP-compliant.

On the basis of the results of dose-finding study, 250 mg/kg, which is near the dose inducing toxic signs, was selected as high dose, and three doses including 125 and 62.5 mg/kg were selected. Since there was no evident sex difference in the incidences of toxic signs, only male mice were used for the present micronucleus assay.

The results revealed that the frequency of MNPCE in any of the 2-Naphthol-treated groups was comparable to that in the negative control group and no statistically significant increase was found. No statistically significant decrease in the ratio of PCE to the analyzed erythrocytes was observed in any of the test substance-treated groups.

It was concluded that 2-Naphthol did not induce micronucleated erythrocytes in mouse bone marrow cells, i.e. structural nor numerical chromosome aberrations, under the conditions employed in this study.