Registration Dossier

Diss Factsheets

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:
1992-03-17 to 1993-01-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP 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

Constituent 1
Chemical structure
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-[[(4-methoxyphenyl)methylhydrazono]methyl]-1,3,3-trimethyl-3H-indolium methyl sulphate
Details on test material:
- Name of test material (as cited in study report): C. I. Basic Yellow 28

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Winkelmann, Borchen, Germany
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 28-43 g
- Assigned to test groups randomly: yes
- Housing: males: single; females: 3/cage (Macrolon type I cages)
- Diet (ad libitum): ad libitum
- Water (ad libitum): ad libitum
- Acclimation period: at least one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.5 to 23 °C
- Humidity (%): 39 to 43%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
physiological saline
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item 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:
N.A.
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 bw

Examinations

Tissues and cell types examined:
Femoral bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The selection of the test item 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 bw of the test item. 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. Based on these results, 5 mg/kg test item was chosen as 1 MTD for this test.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Single administration
Sampling time was:
Test substance: 16, 24, and 48 hours after administration
Negative control: 24 hours after administration
Positive Control: 24 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. Moreover, the ratio of polychromatic to normochromatic erythrocytes was determined (number of normochromatic erythrocytes per 1000 polychromatic ones). Additionally, the number of normochromatic erythrocytes showing micronuclei was also established.
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 test item 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:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF DEFINITIVE STUDY
Clinical signs:
After a single intraperitoneal administration of 5 mg/kg test item, 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 test item. No symptoms were recorded for the control groups. No animals died in these groups.

Microscopic evaluation:
Concerning the assessment of the clastogenic potential of the test item there were no relevant variation in results between males and females.Therefore, they were evaluated jointly.
The ratio of polychromatic to normochromatic erythrocytes was altered by the treatment with the test item, being 1000: 811 (1s=208) in the negative control, 1000: 1770 (1s=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 the test item gave no relevant induction of micronucleated polychromatic erythrocytes after a single intraperitoneal treatment with 5 mg/kg. Similarly, the number of micronucleated normochromatic erythrocytes did not increase relevantly in any of the treatment groups.
The positive control caused a clear increase in the number of micronucleated polychromatic erythrocytes.

Any other information on results incl. tables

Table 1: Summary of results of micronucleus test with the test item
experimental groups Number of evaluated poly-chromatic erythrocytes (PCE) Number of normo-chromatic erythrocytes per 1000 PCE micronucleated cells per 1000
normo-chromatic erythrocytes poly-chromatic erythrocytes
Negative control 10000 811 +/- 208 1.2 +/- 1.6 1.5 +/- 1.1
Test item_16 hours 10000 1770* +/- 662 0.8 +/- 0.7 1.9 +/- 1.4
Test item_24 hours 10000 1620 +/- 745 1.1 +/- 0.5 1.9 +/- 1.4
Test item_48 hours  10000 1210 +/- 892 0.9 +/- 1.0 1.9 +/- 1.4
Positive control 10000 557 +/- 231 0.4 +/- 1.3 12.6* +/- 6.9

Concentration: test item: 5 mg/kg bw; positive control: 20 mg/kg bw

* p<0.01 (tested by non-parametric Wilcoxon ranking test

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The test item did not induce structural and/or numerical chromosomal damage in the immature erythrocytes of the mouse. Therefore, the test item is considered to be non-mutagenic with respect to clatogenicity and aneugenicity in the mammalian erythrocyte micronucleus test.
Executive summary:

In a NMRI mouse bone marrow micronucleus assay (OECD 474), 5 animals/sex/dose were treated intraperitoneal with the test item (98.5%) at doses of 0 and 5 mg/kg bw. Bone marrow cells were harvested at 16, 24 and 48 hours post-treatment. The vehicle was physiological saline. There were signs of toxicity during the study such as apathy, roughened fur, staggering gait, spasm, difficulty in breathing and slitted eyes. One animal died during the test period. The test item was tested at an adequate dose based on the results from a dose range finding study. The positive control induced the appropriate response. There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 474 for in vivo cytogenicity.

 

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