4-[(1-amino-9,10-dihydro-4-hydroxy-9,10-dioxo-2-anthryl)oxy]-N-(3-ethoxypropyl)benzenesulphonamide

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 November 1996 to 04 March 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Refer chapter 13 for detailed read-across justification

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

Lower limit of Body weight range of one animals; relative humidity lower than 30 % sometimes, Due to technical reasons 20 mg/kg bw instead of 40 mg/kg bw cyclophosphamide applied as positive control.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

N-(4-amino-9,10-dihydro-3-methoxy-9,10-dioxo-1-anthryl)benzenesulphonamide

EC Number:

274-040-4

EC Name:

N-(4-amino-9,10-dihydro-3-methoxy-9,10-dioxo-1-anthryl)benzenesulphonamide

Cas Number:

69563-51-5

Molecular formula:

C21H16N2O5S

IUPAC Name:

N-(4-amino-3-methoxy-9,10-dioxo-9,10-dihydroanthracen-1-yl)benzenesulfonamide

Test material form:

other: solid

Details on test material:

None

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 9300102
- Expiration date of the lot/batch: 31 July 1998

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability of the test substance in the solvent/vehicle: 24 h in PEG 400

Test animals

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

Historical experience as suitable experimental animal in cytogenetic investigations.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL, CH-4414 Fullinsdorf
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: males mean value 30.8 g (SD ± 2.9 g); females mean value 22.5 g (SD ± 1.5 g)
- Assigned to test groups randomly: The animals were distributed into the test groups at random and identified by cage number.
- Housing: Makrolon Type I, with wire mesh top (EHRET GmbH, D-79302 Emmendingen) granulated soft wood bedding
- Diet: pelleted standard diet, ad libitum (ALTROMIN 1324, D-32791 Lage/Lippe)
- Water: tap water, ad libitum, (Gemeindewerke, D-64380 Rofidorf)
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 5
- Humidity (%): 20-70
- Photoperiod (hrs dark / hrs light): 6.00 am. - 6.00 p.m.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: polyethylene glycol 400 (PEG 400)
- Justification for choice of solvent/vehicle: The vehicle was chosen to its relative non-toxicity for the animals.

Details on exposure:

Approximately 18 hours before treatment the animals received no food but water ad libitum. At the beginning of the treatment the animals (including the controls) were weighed and the individual volume to be administered was adjusted to the animals body weight. The animals received the test article, the vehicle or the positive control substance once. Twelve animals, six males and six females, were treated per dose group and sampling time. Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively.

Duration of treatment / exposure:

24 and 48 h

Frequency of treatment:

once

Post exposure period:

Not available

Doses / concentrations

Dose / conc.:

2 000 mg/kg bw/day

Remarks:

24 h preparation interval: 200, 670 and 2000 mg/kg b.w.;
48 h preparation interval: 2000 mg/kg b.w.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

CPA; Cyclophosphamide
- Justification for choice of positive control(s):The stability of CPA at room temperature is good. At 25 °C only 3.5 % of its potency is lost after 24 h.
- Route of administration: orally, once
- Doses / concentrations: 20 mg/kg b.w.

Examinations

Tissues and cell types examined:

micronucleated polychromatic erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or 2000 mg/kg as the upper limit for non-toxic test articles. The maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on survival within 48 hours. The volume to be administered should be compatible with physiological space available. Three adequate spaced dose levels extending over a single log range were applied at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 48 h after treatment.

METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 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 performed with coded slides. Five animals per sex and group were evaluated as described. The remaining 6th animal of each test group was evaluated in case an animal had died in its test group.

Evaluation criteria:

A test article is classified as mutagenic if it induces either a dose-related increase in the number of micro nucleated polychromatic erythrocytes or a statistically significant positive response for at least one of the test points. A test article producing neither a dose-related increase in the number of micro-nucleated polychromatic erythrocytes nor a statistically significant 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 considered together.

Statistics:

nonparametric Mann-Whitney test

Results and discussion

Test results

Key result

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:

FAT 36034/D is considered to be non-clastogenic in the in-vivo micronucleus assay.

Executive summary:

A GLP-compliant OECD guideline 474 study was performed to investigate the potential of FAT 36034/D to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The following dose levels of the test article were investigated: 24 h preparation interval: 200, 670 and 2000 mg/kg b.w., 48 h preparation interval: 2000 mg/kg b.w. The highest guideline-recommended dose (2000 mg/kg) was estimated by a pre-experiment to be suitable. The animals expressed slight toxic reactions. After treatment with the test article the number of NCEs was not increased as compared to the corresponding negative controls thus indicating that FAT 36034/D had no cytotoxic effectiveness in the bone marrow. In comparison to the corresponding vehicle controls, there was no substantial enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test article and with any dose level used. 20 mg/kg b.w. cyclophosphamide administered was used as positive control which showed a statistically significant increase of induced micronucleus frequency. In conclusion, 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 with bone marrow cells of the mouse. Therefore, FAT 36034/D is considered to be non-clastogenic in this micronucleus assay.