Black HM 2482

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:

From March 11th to April 1st, 1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1985

Report date:

1985

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted May 26, 1983

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EEE Directive 84/449

GLP compliance:

not specified

Remarks:

Study procedures were periodically inspected and this report was audited by the Quality Assurance Unit.

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

400-920-6

EC Name:

-

Cas Number:

89857-06-7

Molecular formula:

C50 H53 N11 O14 S

IUPAC Name:

5'-[2-(7-{2-[4-(2-{1'-[3-(dimethylazaniumyl)propyl]-6'-hydroxy-4'-methyl-2'-oxo-1',2'-dihydro-1λ⁵-[1,3'-bipyridin]-1-ylium-5'-yl}diazen-1-yl)phenyl]diazen-1-yl}-8-hydroxy-6-sulfonatonaphthalen-2-yl)diazen-1-yl]-6'-hydroxy-3,4'-dimethyl-2'-oxo-1',2'-dihydro-1λ⁵-[1,3'-bipyridin]-1-ylium bis(2-hydroxypropanoate)

Test animals

Species:

mouse

Strain:

NMRI

Remarks:

KFM (outbred, SPF-quality)

Details on species / strain selection:

Mice are recommended for micronucleus assays as the international standard.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Kleintierfarm Madoerin AG, 4414 Fuellinsdorf / Swiitzerland
- Age at study initiation: 7 and 8 weeks
- Weight at study initiation: males: 28 -37 g; females: 24-36 g
- Fasting period before study: not specified
- Housing: groups of 6 in makrolon type 3, with wire mesh top and granulated softwood bedding
- Diet: pelleted standard Kliba 343, mouse diet, ad libitum
- Water: tap water ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 55 ± 10 %
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12 hours Iight/dark per day

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

2% carboxymethylcellulose sodium salt

Frequency of treatment:

Single oral application

Doses / concentrations

Dose / conc.:

5 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

18

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclohexylamine
- Route of administration: oral
- Doses / concentrations: 50 mg/kg bw/day dissolved in 0.9% saline solution

Examinations

Tissues and cell types examined:

bone marrow, polychromatic erythrocytes (PCE), normochromatic erythrocytes (NCE)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The maximum tolerated dose was based on acute oral toxicity data.
A preliminary acute oral LD50 (Iimit test; 2 doses, 3 males and 3 females per dose) performed with the same mouse species as was used in this study showed the following results after 14 days observation:
1000 mg/ks bw: 0 mortality in 6 animals
5000 mglkg bw: 0 mortality in 6 animals
The 5000 mg/kg bw dose uwas used in this study as the maximum tolerated dose.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
All animals were given a single application of 20 ml/kg bw by gavage.
24, 48 and 72 hours after treatment, six mice per sex and group were sacrificed and bone marrow was removed from the femora for examination.

DETAILS OF SLIDE PREPARATION:
The femora were removed from each mouse and freed of adherent tissue. The proximal end of the femur was cut with scissors; the distal end was left intact. The needle of a plastic syringe containing 0.2 ml calf serurn was inserted into the proximal part of the marrow canaI.
The bone marrow cells were dispersed in 1.5 ml of calf serurn as a homogeneous suspension. The tube containing the bone marrow ceIIs of both femora was centrifuged at 1000 r.p.m. for 5 mlnutes.
The supernatant was removed, Ieaving a thin Iayer of serum. The cells of the sediment were suspended by aspiration in a siliconized pasteur pipette. A small drop of the marrow serum suspension was smeared on the sIide, which was identified by project code and the animal number, and allowed to dry overnight. Two slides per animal were prepared. The following day, the smears were stained using the panoptic stain method developed by Pappenheim (3).

METHOD OF ANALYSIS:
The slides were coded before microscopic analysis. If macroscopic evaluation revealed technical imperfections, the first slide was replaced by the second sIide prepared. From each animal, one thousand polychromatic erythrocytes (PCE) were scored under the microscope (magnification 1000x), for the incidence of micronuclei. Additional information could be obtained by scoring normochromatic enythrocytes for micronuclei.
The calculated ratio of polychromatic to normochromatic erythrocytes (PCE/NCE), based on 1000 enythrocytes scored per slide, measured the toxic efficacy of the test article.

Evaluation criteria:

The frequencies of micronuclei of the treated male and female groups were compared with those of the negative control groups at each sampling time. A regression model assuming a Poisson
distribution was applied. Estimation and testing were performed by maximum likelihood method (4). If a test article produced no statistically significant and reproducible positive response at any one of the test points, it was considered to be non-mutagenic in this system.

Statistics:

The data were statistically analyzed by means of a regression model assuming a Poisson distribution. Estimation and testing were performed by maximum likelihood method (4).

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Test article induced no chromosome mutations by chromosome-breaking activity or by damage to the mitotic apparatus.

Executive summary:

This in vivo experiment was performed to assess the potential mutagenic activity induced by test substance through damage to the chromosomes of to the mitotic apparatus according to Schmid (1) with the modifications of Salomone et al. (2).
The experirnent was performed with three groups, each containing 18 males and 18 females, for a total of 108 mice.
The negative control groups received the test article vehicle, i.e. 2% carboxymethylcellulose sodium salt in distilled water. The test groups received 5000 mg/kg bw as the maximurn tolerated dose of the test article. The positive control groups received 50 mg/kg bw cyclophosphamlde dissolved in 0.9 % saline solution.
All animals were given a single application of 20 ml/kg bw by gavage.
24, 48 and 72 hours after treatment, six mice per sex and group were sacrificed and bone marrow was removed from the femora for examination. The first five animals were used for evaluation. One thousand polychromatic erythrocytes per animal were scored for micronuclei. The ratio of polychromatic to normochromatic erythrocytes was used to assess the toxicity of the test article by counting a total of 1000 erythrocytes.
All animals treated with the test article showed light sedation on the first day of the test. No toxic effect of the test article was observed.
After single application of the test article at 5000 mg/kg bw by gavage, no significant test article-related
increase of micronucleated polychromatic erythrocytes was observed in either male and female treated groups, when compared with corresponding negative control groups These results were found at the three examination times, 24, 48 and 72 hours post-application, respectively. The positive control groups, which received cyclophosphamide, exhibited a significant and clear increase in the number of micronucrlated polychromatic erythrocytes and thus validated the test.
In conclusion, it can be stated that under the extrerimental conditions reported, the test article induced no chromosome mutations by chromosome-breaking activity or by damage to the mitotic apparatus.
Therefore, test article is not considered to be mutagenic in this in vivo mouse micronucleus assay.