2-[[(4-methoxyphenyl)methylhydrazono]methyl]-1,3,3-trimethyl-3H-indolium acetate

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:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

March to June 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

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)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

2-[[(4-methoxyphenyl)methylhydrazono]methyl]-1,3,3-trimethyl-3H-indolium methyl sulphate

EC Number:

258-946-7

EC Name:

2-[[(4-methoxyphenyl)methylhydrazono]methyl]-1,3,3-trimethyl-3H-indolium methyl sulphate

Cas Number:

54060-92-3

Molecular formula:

C20H24N3O.CH3O4S C21H27N3O5S

IUPAC Name:

2-{[2-(4-methoxyphenyl)-2-methylhydrazin-1-ylidene]methyl}-1,3,3-trimethyl-3H-indol-1-ium methyl sulfate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material: C I Basic Yellow 28 (Astrazon Goldgelb GLN)

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Winkelman, Borchen, Germany
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 28-43 g
- Assigned to test groups randomly: yes
- Fasting period before study: -
- Housing: males: single; females: 3/cage
- Diet (ad libitum): Altromin 1324
- Water (ad libitum): tap
- Acclimation period: at least one week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.5 to 23
- Humidity (%): 39 to 43
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 24. March To: 25. March 1992

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

physiological saline

Details on exposure:

C I Basic Yellow 28 was dissolved in physiological saline solution using sonication for 15 minutes and injected intraperitoneally at a volume of 10 mL/kg body weight

Duration of treatment / exposure:

negative control 24 h
test substance 16, 24, and 48 h
positive control 24 h

Frequency of treatment:

single

Post exposure period:

see duration of exposure

Doses / concentrations

Remarks:

Doses / Concentrations:
5 mg/kg body weight
Basis:
nominal conc.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: intraperitoneal
- Doses / concentrations: 20 mg/kg

Examinations

Tissues and cell types examined:

femoral bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The selection of the C. I. Basic Yellow 28 (Astrazon Goldgelb GLN) dose was based on a pilot test, in which groups of five animals, including both males and females, were intraperitoneally administered 1 mg/kg, 5 mg/kg, 7.5 mg/kg, 10 mg/kg, 50 mg/kg and 100 mg/kg C. I. Basic Yellow 28 (Astrazon Goldgelb GLN). The following symptoms were recorded for up to 48 hours, starting at 5 mg/kg: apathy, roughened fur, staggering gait, sternal recumbency, spasm and difficulty in breathing. In addition, 3 of 5 animals died in the 7.5 mg/kg group and all animals died in the higher dose groups.


TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
single administration
sampling 16, 24, and 48 hours after administration

DETAILS OF SLIDE PREPARATION:
Bone marrow was flushed into a tube containing fetal bovine serum and centrifuged (5 min, 1000 rpm)
Air dried smears were automatically stained with an Ames HemaTek Slide Stainer and then destained with methanol, rinsed with deionized water and dried. After drying, the slides were covered with xylene and a cover glass.


METHOD OF ANALYSIS:
1000 polychromatic erythrocytes per animal were scored for incidence of micronuclei. The number of cells with micronuclei was recorded, not the number of individual micronuclei.
the ratio of polychromatic to normochromatic erythrocytes was determined

Evaluation criteria:

A test was considered positive if, at any of the intervals, there was a relevant and significant increase in the number of polychromatic erythrocytes showing micronuclei in comparison to the negative control.
A test was considered negative if there was no relevant or significant increase in the rate of micronucleated polychromatic erythrocytes at any time. A test was also considered negative if there was a significant increase in that rate which, according to the laboratory‘s experience was within the range of negative controls.
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 and the result of the negative control was not closely related to the data of the respective treatment group. In this case, a second test had to be performed at the most sensitive interval.

Statistics:

The C. I. Basic Yellow 28 group(s) with the highest mean and the positive control were checked by Wilcoxon’s non parametric rank sum test with respect to the number of polychromatic erythrocytes having micronuclei and the number of normochromatic erythrocytes A variation was considered statistically significant if its error probability was below 5.% and the treatment group figure was higher than that of the negative control.
The rate of normochromatic erythrocytes containing micronuclei was examined if the micronuclear rate for polychromatic erythrocytes was already relevantly increased. In this case, the group with the highest mean was compared with the negative control using the one-sided chi²-test. A variation was considered statistically significant if the error probability was below 5.% and the treatment group figure was higher than that of the negative control.
In addition, standard deviations (1s ranges) were calculated for all the means.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY
Clinical signs: After single intraperitoneal administration of 5 mg/kg C.I. Basic Yellow 28 (Astrazon Goldgelb GLN), treated animals showed the following compound-related symptoms until sacrifice: apathy, roughened fur, staggering gait, spasm, difficulty in breathing and slitted eyes. Their feeding behavior was normal. One of 40 treated animals died during the test period, due to the acute toxicity of 5 mg/kg C.I. Basic Yellow 28 (Astrazon Goldgelb GLN). No symptoms were recorded for the control groups. No animals died in these groups.

Microscopic evaluation: Concerning the assessment of the clastogenic potential of C.I. Basic Yellow 28 (Astrazon Goldgelb GLN) there were no relevant variations in results between males and females. Therefore, they were evaluated jointly.
The ratio of polychromatic to normochromatic erythrocytes was altered by the treatment with C.I. Basic Yellow 28 (Astrazon Goldgelb GLN), being 1000: 811 (1s=208) in the negative control, 1000: 1770 (ls=652) in the 16 hours group, 1000: 1620 (1s=745) in the 24 hours group and 1000: 1210 (1s=892) in the 48 hours group.
The results with C.I. Basic Yellow 28 (Astrqgon Goldgelb GLN) gave no relevant indications of clastogenic effects after a single intraperitoneal treatment with 5 mg/kg. The number of micronucleated normochromatic erythrocytes did not increase relevantly in any of the groups.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
No indications of a clastogenic effect of C I Basic Yellow 28 (Astrazon Goldgelb GLN) were found after a single intraperitoneal treatment with 5 mg/kg. The ratio of polychromatic to normochromatic erythrocytes was not altered.

Executive summary:

The micronucleus test was employed to investigate C. I. Basic Yellow 28 methyl sulfate (Astrazon Goldgelb GLN) in male and female mice for a possible clastogenic effect on the chromosomes of bone marrow erythroblasts. The known clastogen and cytostatic agent, cyclophosphamide, served as positive control.

The treated animals received a single intraperitoneal administration of either C. I. Basic Yellow 28 (Astrazon Goldgelb GLN) or cyclophosphamide. The femoral marrow of groups treated with C. I. Basic Yellow 28 (Astrazon Goldgelb GLN) was prepared 16, 24 and 48 hours after administration. All negative and positive control animals were sacrificed after 24 hours. The doses of C. I. Basic Yellow 28 (Astrazon Goldgelb GLN) and the positive control, cyclophosphamide, were 5 and 20 mg/kg body weight, respectively.

The animals treated with C. I. Basic Yellow 28 (Astrazon Goldgelb GLN) showed symptoms of toxicity after administration. One of forty animals died before the end of the test due to the acute intraperitoneal toxicity of 5 mg/kg C. I. Basic Yellow 28 (Astrazon Goldgelb GLN).

There was an altered ratio between polychromatic and normochromatic erythrocytes.

No indications of a clastogenic effect of C I Basic Yellow 28 (Astrazon Goldgelb GLN) were found after a single intraperitoneal treatment with 5 mg/kg.

Cyclophosphamide, the positive control, had a clear clastogenic effect as is shown by the biologically relevant increase in polychromatic erythrocytes with micronuclei.

The ratio of polychromatic to normochromatic erythrocytes was not altered.