Registration Dossier

Toxicological information

Genetic toxicity: in vivo

Currently viewing:

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
December 1989 to March 1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report date:
1990

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
EEC Directive 84/449, L 251, B 12, p. 137 - 139
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
6-chloro-2-[4-[ethyl(2-hydroxyethyl)amino]phenyl]-1-methylbenz[cd]indolium acetate
EC Number:
296-673-5
EC Name:
6-chloro-2-[4-[ethyl(2-hydroxyethyl)amino]phenyl]-1-methylbenz[cd]indolium acetate
Cas Number:
92952-73-3
Molecular formula:
C22H22ClN2O.C2H3O2
IUPAC Name:
6-chloro-2-{4-[ethyl(2-hydroxyethyl)amino]phenyl}-1-methylbenzo[cd]indolium acetate
Test material form:
solid: particulate/powder
Details on test material:
Basic Blue 145

Test animals

Species:
mouse
Strain:
NMRI
Details on species / strain selection:
Recommended by the guideline
Sex:
male/female
Details on test animals or test system and environmental conditions:
Source: BRL Tierfarm Flillinsdorf, CH-4414 Füllinsdorf/Basel, Switzerland
Number of Animals: 84 (42 males/42 females)
Initial Age at Start of Acclimatization: minimun 10 weeks
Acclimatization: minimum 5 days
Initial Body Weight at Start of Treatment: approx 30 g
According to the suppliers assurance the animals were in healthy condition. The animals were under quarantine in the animal house of C C R for a minimum of five days after their arrival. During this period the animals did not show any signs of illness or altered behaviour. The animals were distributed into the test groups at random and identified by cage number.

Husbandry
The animals were kept conventionally. The experiment was conducted under standard laboratory conditions.
Housing: single
Cage Type: Makrolon Type I, with wire mesh top (EBECO, D-4620 Castrop-Rauxel)
Bedding: granulated soft wood bedding (ALTROMIN, D-32791 Lage/Lippe)
Feed: pelleted standard diet, ad libitum (ALTROMIN 1324, D-32791 Lage/Lippe)
Water: tap water, ad libitum
Environment: temperature 21 ±3°C
relative humidity 30-70%
artificial light 6.00 a.m. - 6.00 p.m.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Carboxymethycellulose (1%)
- Justification for choice of solvent/vehicle: Widely accepted.

Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment, the test article was suspended in CMC (1%). The vehicle was chosen to its nontoxicity for the animals. All animals received a single standard dose volume of 20 ml/kg body weight orally.
Duration of treatment / exposure:
single injection
Frequency of treatment:
once
Post exposure period:
24, 48 and 72 hours
Doses / concentrations
Dose / conc.:
5 000 mg/kg bw (total dose)
No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide;
- Justification for choice of positive control(s): recommended by the guideline
- Route of administration: orally, once
- Doses / concentrations: 40 mg/kg b.w.
- Volume administered. 10 mL/kg body weight

Examinations

Tissues and cell types examined:
polychromatic erythrocytes (PCE)
Details of tissue and slide preparation:
Slide preparation:
The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a syringe. The cell suspension was centrifiiged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (MERCK, D-64293 Darmstadt)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-79110 Freiburg). At least one slide was made from each bone marrow sample.

Analysis of cells:
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 micronucleated 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 micronucleated polychromatic erythrocytes nor a statistically significant positive response at any of the test points is considered non-mutagenic in this system.
However, both biological and statistical significance should be considered together.
Statistics:
Statistical significance was evaluated by means of the 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
Additional information on results:
At preparation interval 24 hours one male and one female died.
At preparation interval 48 hours one male and one female died.
At preparation interval 72 hours one male died.

The mean number of normochromatic erythrocytes was increased after treatment with the test article at preparation interval 48 hours as compared to the mean value of NCEs of the corresponding negative control, indicating that the test substance had cytotoxic properties.
In comparison to the corresponding negative controls there was no enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. The mean values of micronuclei observed after treatment with the test substance were in the same range as compared to the negative control groups.
40 mg/kg b.w. cyclophosphamide administered per os was used as positive control which showed a distinct increase of induced micronucleus frequency.

Applicant's summary and conclusion

Conclusions:
The test substance did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse
Executive summary:

The test substance was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

The test article was suspended in carboxymethylcellulose-solution (1%). This suspending agent was used as negative control. The volume administered orally was 20 ml/kg bw. 24 h, 48 h and 72 h after a single application of the test article the bone marrow cells were collected for micronuclei analysis.

Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei.

To describe a cytotoxic effect due to the treatment with the test substance the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 1000 PCE.

The following dose level of the test article was investigated: 24 h, 48 h, and 72 h preparation interval: 5000 mg/kg bw.

In a pre-experiment this dose level was estimated to be the maximum attainable dose. The animals expressed slight toxic reactions. In the micronucleus assay three males and two females died after administration of the test article.

The mean number of normochromatic erythrocytes was increased after treatment with the test article at preparation interval 48 hours as compared to the mean value of NCEs of the corresponding negative control, indicating that the test substance had cytotoxic properties.

In comparison to the corresponding negative controls there was no enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. The mean values of micronuclei observed after treatment with the test substance were in the same range as compared to the negative control groups.

40 mg/kg bw cyclophosphamide administered per os was used as positive control which showed a distinct increase of induced micronucleus frequency.

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test substance did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.