N-[2-[(2,6-dicyano-p-tolyl)azo]-5-(diethylamino)phenyl]methanesulphonamide

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:

weight of evidence

Study period:

08 Oct - 25 Nov 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1992

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

N-[2-[(2,6-dicyano-p-tolyl)azo]-5-(diethylamino)phenyl]methanesulphonamide

EC Number:

269-887-1

EC Name:

N-[2-[(2,6-dicyano-p-tolyl)azo]-5-(diethylamino)phenyl]methanesulphonamide

Cas Number:

68385-96-6

Molecular formula:

C20H22N6O2S

IUPAC Name:

N-[2-[(2,6-dicyano-p-tolyl)azo]-5-(diethylamino)phenyl]methanesulphonamide

Test material form:

solid: particulate/powder

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

The animals used were young adult male and virgin female mice, bred and supplied by F. Winkelmann, Borchen. They initially weighed 29-40 g, and were thus approximately 8 to 12 weeks of age.
The bread's state of health is regularly spot-checked for the major specific pathogene.
On the day of arrival, (Sept 27, 1991), the health of the animals was appraised before acclimatising them to the housing conditions for a period of at least one week. Only healthy animals without symptoms were used in the study.
They were kept in the group of a maximum of three to five mice, separated by sex and test group, in makrolon type I and II cages, using bedding of soft wood granules type S 8/15.
Husbandry was standardized, with twelve hours of electrical lighting daily (6.00 am to 6.00 pm), 21 - 23 °C room temperature, and 51 - 55 % mean relative humidity.
Setting for the animal room.22 ± 2 °C (rising with outside temperature above 24 °C).
At least 50 % humidity (rising with high absolute outside humidity), and air change at least ten times per hour.
Fixed-formula feed was Altromin 1324 standard Diet.
Water: drinking water quality.

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

suspension in 0.5 % aqueous Cremophor emulsion

Duration of treatment / exposure:

treatment: 24, 48 and 72 hrs
controls: 24 hours

Frequency of treatment:

once

Doses / concentrations

Dose / conc.:

10 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive control: Cyclophosphamide
- Route of administration: i.p.
- Doses / concentrations: 20 mg/kg bw

Examinations

Tissues and cell types examined:

bone marrow (from femur)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The selection of the test item was based on a pilot test, in which groups of five animals, including both males and females, were intraperitoneally administered 5000 mg/kg and 10000 mg/kg. No animals died. In addition the following symptoms were recorded, starting at 5000 mg/kg: apathy, roughened fur, staggering gait, spasm and reddish discoloured urine. Based on these results, 10000 mg/kg was chosen for the main test.

Evaluation criteria:

Assessment criteria:
A test was considered positive if, at any of the intervals, there was relevant and significant increase in the number of polychromatic erythrocytes showing micronucleated in comparison to the negative control.
A test was considered negative if there was no relevant or significant increase in rats of micronucleated polychromatic erythrocytes at any time.
In addition, a test was considered equivocal if there was an increase of micronucleated polychromatic erythrocytes above the range of attached historical negative controls, provided the increase was not significant. A test was also considered equivocal if there was a significant increase in that rate which, according to the laboratory's experience was within the range of negative controls. In either case, a second test had to be performed at the most sensitive interval.
Assay Acceptance criteria:
An assay was considered acceptable if the figures of negative and positive controls were within the expected range, in accordance with the laboratory's experience.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Apathy, roughened fur, staggering gait, spasm, reddish discoloured urine

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no mortality

Additional information on results:

Assessment:
Normally, cells with micronuclei (Howell-Jolly bodies) occur in polychromatic erythrocytes with an incidence of up 2.5/1000. The increase in the polychromatic erythrocytes, due for example, to chromosome breaks or spindle disorders, is the criterion for clastogenic effects in this test model.
The results with the test item gave no relevant indications of clastogenic effects after a single intraperitoneal treatment with 10000 mg/kg bw.
Furthermore the known mutagen and clastogen cyclophosphamide, had a clear clastogenic effect at an intraperitoneal dose of 20 mg/kg bw. The number of micronucleated polychromatic erythrocytes increase to a biological relevant degree.

Applicant's summary and conclusion

Conclusions:

In a GLP study according to OECD test guideline 474, no indication of a clastogenic effect of 10000 mg/kg bw in the micronucleus test on the mouse was observed. The substance is not considered to be a clastogen in mammalia.

Executive summary:

The micronucleus test was used in order to investigate the test item in male and female mice for a possible clastogenic effect on the chromosomes of bone-marrow erythroblasts. The known clastogen and cytostatic agent; i.e. cyclophosphamide, served as positive control.

The treated animals received a single intraperitoneal administration of either the test item or cyclophosphamide. The femoral marrow of groups treated with the test item was prepared 16, 24 and 48 hours after administration. All negative and positive control animals were sacrificed after 24 hours. The dose of the test item and the positive control, cyclophosphamide, were 10000 and 20 mg/kg body weight, respectively.

The animals treated with the test item, showed some symptoms of toxicity after administration. However all animals survived until the end of the test. There was an altered ratio between polychromatic and monochromatic erythrocytes.

No indications of a clastogenic effect of the test item were found after a single intraperitionael treatment with 10000 mg/kg bw. Furthermore the known mutagen and clastogen cyclophosphamide, the positive control, had a clear clastogenic effect at an intraperitoneal dose of 20 mg/kg bw as is shown by the biologically relevant increase in polychromatic erythrocytes with micronuclei. The ratio of polychromatic to monochromatic erythrocytes was not altered.