Reaction mass of 6-Chloro-2-{4[ethyl(2-hydroxyethyl)amino]phenyl}-1-methylbenzo[cd]indolium hydroxide and 6-chloro-2-[4-[ethyl(2-hydroxyethyl)amino]phenyl]-1-methylbenz[cd]indolium acetate

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:

Test start date: 02 January, 1990; Test end date: 12 February, 1990; Study completion date: 19 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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Environmental Protection Agency, Code of Federal Regulations, Title 40, Subpart F-Genetic Toxicity, Revision July 1, 1986 "In vivo mammalian bone marrow cytogenetics tests: Micronucleus assay."

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 6-chloro-2-[4-[ethyl(2-hydroxyethyl)amino]phenyl]-1-methylbenz[cd]indolium acetate and 6-bromo-2-[4-[ethyl(2-hydroxyethyl)amino]phenyl]-1-methylbenz[cd]indolium acetate and 6-chloro-2-[4-[ethyl(2-hydroxyethyl)amino]phenyl]-1-methylbenz[cd]indolium hydroxide

IUPAC Name:

Reaction mass of 6-chloro-2-[4-[ethyl(2-hydroxyethyl)amino]phenyl]-1-methylbenz[cd]indolium acetate and 6-bromo-2-[4-[ethyl(2-hydroxyethyl)amino]phenyl]-1-methylbenz[cd]indolium acetate and 6-chloro-2-[4-[ethyl(2-hydroxyethyl)amino]phenyl]-1-methylbenz[cd]indolium hydroxide

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Identification of the test material as used in the study report: FAT 31'064/F
- Source and batch No.of test material: EN 158496.82/ HEW 133/6
- Expiration date of the batch: November 1994
- Aggregate State at RT: Solid
- Colour: Dark blue

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability of the test substance in the solvent/vehicle: Pure: stable for five years; In vehicle: stable in water, ethanol, acetone, DMSO, DMF for 48 hours

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL Tierfarm Flillinsdorf, CH- 4414 Villinsdorf/Basel, Switzerland
- Age at study initiation: 10 weeks
- Weight at study initiation: approximately 30 g

- Housing: single in Makrolon Type I, with wire mesh top (EBECO, D-4620 Castrop-Rauxel, F.R.G.)
- Diet: pelleted standard diet, ad libitum (ALTROMIN, D-4937 Lage/Lippe, F.R.G.)
- Water: tap water, ad libitum, (SUdhessische Gas- and Wasser AG, D-6100 Darmstadt)
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 3°C
- Photoperiod: artificial light 6.00 a.m. - 6.00 p.m.

IN-LIFE DATES: From: January 02, 1990 to February 12, 1990

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Carboxymethycellulose (1 %)

Details on exposure:

Approximately 18 hours before treatment with the test article the animals received no food but water ad libitum. At the beginning of the treatment the animals were weighed and the individual volume to be administered was adjusted to the animal's body weight. The animals received the test article once. Twelve animals, six males and six females, were treated per dose group. Sampling of the bone marrow was done 24, 48 and 72 hours after treatment.

Duration of treatment / exposure:

Once orally (gavage)

Frequency of treatment:

Single dose

Post exposure period:

Sampling of the bone marrow was done 24, 48 and 72 hours after treatment.

Doses / concentrations

Dose / conc.:

5 000 mg/kg bw (total dose)

No. of animals per sex per dose:

Six males and six females were assigned to each test group. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei.

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive control used: Cyclophosphamide
- Route of administration: Orally once
- Dissolved in: Physiological saline
- Doses: 40 mg/kg bw
- Volume administered: 10 ml/kg b.w.

Examinations

Tissues and cell types examined:

Bone marrow and polychromatic erythrocytes (PCE)

Details of tissue and slide preparation:

Preparation of the Animals:
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 5 ml syringe. The cell suspension was centrifuged at 1,500 rpm for 5 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-Grunwald/Giemsa. Cover slips were mounted with EUKITT (KINDLER, D-7800 Freiburg F.R.G.). 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.. 1,000 polychromatic erythro¬cytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochro-matic erythrocytes was determined in same sample and expressed in hormochromatic erythrocytes per 1000 PCEs. The analysis was per¬formed with coded slides. Five animals per sex and group were evaluated as described. The remaining animal of each test group was evaluated in case an animal had died in its test group spontaneously or due to gavage error.

Evaluation criteria:

EVALUATION OF RESULTS
A test article is classified as mutagenic if it induces either a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes or a reproducible statistically significant positive response for at least one of the test points.
A test article producing neither a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant and reproducible positive response at anyone 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:

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

PRE-EXPERIMENT FOR TOXICITY
In a pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 5000 mg/kg b.w., FAT 31064/F suspended' in carboxymethylceilulose (1 %).
The volume administered was 20 ml/kg b.w.
All treated animals expressed slight toxic reactions: reduction of spontaneous activity.

Higher dosing was not attainable:
a) Appropriate suspensions (homogeneity, viscosity) could be obtained only up to 250 mg/ml.
b) Application volumes higher than 20 ml/kg b.w. were not justifiable for the rodents used.

LETHALITIES IN THE MICRONUCLEUS ASSAY
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.

Any other information on results incl. tables

Summary of Micronucleus Test Results:

Test Group	Dose mg/kg b.w	sampling time (h)	PCEs with micronuclei (%)	Range	PCE/NCE
Vehicle	0	24	0.092	0 -3	1000/827
Test Article	5000	24	0.092	0-3	1000/925
cyclo-phosphamide	40	24	6.692	3-13	1000/964
Vehicle	0	48	0.072	0 -2	1000/714
Test article	5000	48	0.062	0 -2	1000/1358
Vehicle	0	72	0.09	0 -3	1000/780
Test article	5000	72	0.092	0 -3	1000/821

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 FAT 31'064/F were in the same range as compared to the negative control groups.

40 mg/kg b.w. cyclophosphamide (positive control) which showed a distinct increase of induced micronuleus frequency.

Applicant's summary and conclusion

Conclusions:

FAT 31064/F was considered to be devoid of clastogenic potential in this micronucleus assay.

Executive summary:

The test article FAT 31064/F 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 b.w. 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 article the ratio between polychromatic and normochromatic erythrocytes (NCE)wasdetermined 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 b.w. 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 FAT 31064/F 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 FAT 31064/F were in the same rangeascompared to thenegativecontrol groups. 40 mg/kg b.w. cyclophosphamide administered per os was used as positive control which showed a distinct increase of induced micronuleus 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 in the bone marrow cells of the mouse.