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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:
September 28, 1992 - March 23, 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guidleline study with GLP certificate

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report date:
1993

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay

Test material

Constituent 1
Reference substance name:
2-benzyloxy-naphtalene
IUPAC Name:
2-benzyloxy-naphtalene
Constituent 2
Chemical structure
Reference substance name:
2-(phenylmethoxy)naphthalene
EC Number:
405-490-3
EC Name:
2-(phenylmethoxy)naphthalene
Cas Number:
613-62-7
Molecular formula:
C17H14O
IUPAC Name:
2-(benzyloxy)naphthalene
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): BNE
- Substance type: organic
- Physical state: solid
- Analytical purity: 99.8 %
- Lot/batch No.: 20979
- Expiration date of the lot/batch: August 31, 1994
- Stability under test conditions: Pure: stable for more than 1 year in corn oil (60 mg/ml) stable at least one week
- Storage condition of test material: at room temperature, light protected

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source:Charles River Wiga GmbH, Sandhofer Weg 7, D-8741 Sulzfeld 1
- Age at study initiation: 10 weeks
- Weight at study initiation: 27.6 - 41.1 g
- Assigned to test groups randomly: yes, under following basis: not specified

- Housing: single
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +-3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 / 12 hours

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil

- Concentration of test material in vehicle: 200, 670 and 2000 mg/kg bw
- Amount of vehicle (if gavage or dermal): 10 ml /kg bw
- Type and concentration of dispersant aid (if powder): non mentioned
Frequency of treatment:
once
No. of animals per sex per dose:
6
Control animals:
yes
Positive control(s):
Cyclophophamide, 30 mg/kg bw. in physiological saline

Examinations

Tissues and cell types examined:
only bone marrow is examined
Details of tissue and slide preparation:
The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a 5 ml syringe. The cell suspension was centrifuged at 1500 rpm for 10 minutes and the supernatant was discarded. A small drop of the unfixed, sesuspended cell pellet was spread on a slide. The smear was air dried and then stained with May-Grünwald/Giemsa. Cover slips were mounted with Eukitt. At least one slide was made from each bone marrow sample.
Evaluation criteria:
Evaluation of the slides was performed using Nikon microscopes with 100x oil immersion objectives. 1000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 1000 the PCEs. The analysis was perforemed with coded slides.

A test article is classified as mutagenic if it induces either a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes or a reproducible statistically significant positive response for at least one of the test points.
A test article producing neither a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant and reproducible positive response at any of the test points is considered non mutagenic in this system.
This can be confirmed by means of the nonparametric Mann-Whitney test.
However, both biological and statistical significance should be considere together.
Statistics:
Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.
Negative control
vs. Test group Significance p
200 mg/kg bw, 24 h - 0.12
670 mg/kg bw, 24 h - 0.16
2000mg/kg bw, 24 h not tested*
2000mg/kg bw, 48 h not tested*

* = mean micronucleus frequency was not above the mean corresponding negative control value

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw

- Clinical signs of toxicity in test animals: slight toxic reactions

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): see table
- Ratio of PCE/NCE (for Micronucleus assay): see table

Any other information on results incl. tables

 test group dose mg/kg bw  sampling time (h)  PCEs with micronuclei (%)  range  PCE/NCE 
 vehicle 24  0.03  0 - 2  1000 / 744 
 test article 200  24  0.08  0 - 2  1000 / 644 
 test article 670  24  0.08  0 - 3  1000 / 687 
 test article 2000  24  0.03  0 - 2  1000 / 663
cyclophosphamide  30  24  1.44  5 - 29  1000 / 569 
 vehicle 48  0.05  0 - 2  1000 / 560 
 test article 2000  48  0.03  0 - 1  1000 /  643

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
It can be stated that during the study described and 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.
Executive summary:

The test article 2-(phenylmethoxy)naphthalene was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

Tes test article was formulated in corn oil. This vehicle was used as negative control. The volume administered intraperitoneally was 10 ml/kg bw. 24 h and 48 h after a single application of the test article the bone marrow cells were collected for micronuclei analysis.

Ten animals (5 males/ 5 females) per test group were evaluated for the occurrence of micronuclei. 1ßßß polychromatic erythrocytes (PCE) per animal were scored fo rmicronuclei.

To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the sam esmaple and reported as the number of NCE per 1000 PCE.

The following dose levels of the test article were investigated:

24 h preparation interval: 200, 670, and 2000 mg/kg bw.

48 h preparation interval: 2000 mg/kg bw.

In a pre-experiment 2000 mg/kg bw of the test article were administered. The animals expressed slight toxic reaction. The mean number of normochromatic erythrocytes was not increased after treatment with the test article as compared to the mean values of NCEs of the corresponding negative controls, indicating that 2-(phenylmethoxy)naphthalene had no cytotoxic properties.

In comparison to the corresponding negative controls there was no significant enhancement in the frequency of the detected micronuclei at any preparation intervals and dose levels after application of the test articl. The mean values of micronuclei observed after treatment with 2-(phenylmethoxy)naphthalene were in the same range as compared to the negative control values.

30 mg/kg bw cycloposphamide administered intraperitoneally was used as positive control which showed a distinct increase of induced micronucleus frequency.

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