(2,2'-(3,3'-dioxidobiphenyl-4,4'-diyldiazo)bis(6-(4-(3-(diethylamino)propylamino)-6-(3-(diethylammonio)propylamino)-1,3,5-triazin-2-ylamino)-3-sulfonato-1-naphtholato))dicopper(II) acetate lactate

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:

From October 22, 1990 to November 27, 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

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

May 26, 1983

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

(2,2'-(3,3'-dioxidobiphenyl-4,4'-diyldiazo)bis(6-(4-(3-(diethylamino)propylamino)-6-(3-(diethylammonio)propylamino)-1,3,5-triazin-2-ylamino)-3-sulfonato-1-naphtholato))dicopper(II) acetate lactate

EC Number:

407-240-9

EC Name:

(2,2'-(3,3'-dioxidobiphenyl-4,4'-diyldiazo)bis(6-(4-(3-(diethylamino)propylamino)-6-(3-(diethylammonio)propylamino)-1,3,5-triazin-2-ylamino)-3-sulfonato-1-naphtholato))dicopper(II) acetate lactate

Cas Number:

159604-94-1

Molecular formula:

C66H88Cu2N20O10S2.C3H5O3.C2H3O2

IUPAC Name:

7,7'-bis[4-(3-diethylaminopropylamino)-6-(3-diethylammoniopropylamino)-1,3,5-triazin-2-ylamino]-{μ-4,4'-dihydroxy-1:2k2O4:O4'-3,3'-[3,3'-dihydroxy-1:2k2O3:O3'-biphenyl-bisazo-1:2(N3,N4-η:N3',N4'-η)]dinaphthalene-2-sulphonato(6-)}dicuprate(2-), mixed (1:1) acetic/lactic acid salts

Test material form:

solid

Test animals

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

The mouse is an animal which has been used for many years as suitable experimental animal in cytogenetic investigations. There are many data available from such investigations which may be helpful in the interpretation of results from the micronucleus test. In addition, the mouse is an experimental animal in many physiological, pharmacological and toxicological studies. Data from such experiments also may be useful for the design and the performance of the micronucleus test
Strain: NMRI
Source: BRL Tierfarm Füllinsdorf
CH— 4414 Füllinsdorf/Basel, Switzerland
Number of Animals: 84 (42 males/42 females)
Initial age at start of acclimatization: minimum 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.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL Tierfarm Füllinsdorf
CH— 4414 Füllinsdorf/Basel, Switzerland
- Age at study initiation: minimum 10 weeks
- Weight at study initiation: approx. 30 g
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing: single
- Water: ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C ± 3°C
- Humidity (%): 30 - 70%
- Photoperiod: artificial light 6.00 a.m. - 6.00 p.m.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: The vehicle was chosen to its nontoxicity for the animals

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: On the day of the experiment, the test article was dissolved in aqua dest. 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:

24, 48, 72 hours

Frequency of treatment:

Singly

Doses / concentrations

Dose / conc.:

5 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12 animals (6m/6f) per 5000 mg/kg bw

Control animals:

yes

Positive control(s):

cyclophosphamide;
- Route of administration: orally
- Doses / concentrations: 30 mg/kg b. w.

Examinations

Tissues and cell types examined:

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 same sample and expressed in normochromatic erythrocytes per 1000 the PCEs. The analysis was performed with coded slides.

Details of tissue and slide preparation:

TREATMENT AND SAMPLING TIMES: sampling of the bone marrow was done 24, 48 and 72 hours after treatment. 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 1500 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—Grünwald (MERCK, Darmstadt / Giemsa ) (Gurr, BDH Limited Poole, Great Britain) Cover slips were mounted with EUKITT (KINDLER, D—7800 Freiburg). At least one slide was made from each bone marrow sample.


METHOD OF ANALYSIS: Evaluation of the slides was performed using NIKON microscopes with 100x oil irnmersion objectives. 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 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 (if available) 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:

A test article is classified as mutagenic if it induces a statistically significant increase in the number of micronucleated polychromatic erythrocytes at for at least one of the test points.
A test article producing no statistically significant increase in the number of micronucleated polychromatic erythrocytes at anyone of the test points is considered non-mutagenic in this system.
This can be confirrned 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.
Negative control versusTest group Significance

5000 mg/kg b. w. ; 24 h -
5000 mg/kg b.w. ; 48 h -
5000 mg/kg b. w. ; 72 h -
- not significant;
+ significant

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative

Executive summary:

This study was performed to investigate the potential of the test item to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse

The test article was dissolved in aqua des t. This solvent 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, with the exception of the test article groups at preparation intervals 24 and 48 hours in which only nine animals could be investigated because of lethalities. 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) 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 b. w..

In a pre-experiment this dose level was estimated to be the maximum attainable dose. The animals expressed slight toxic reactions .

However, in the larger sample of the mutagenicity study 7 out of 36 treated animals died.

After treatment with the test article the ratio between PCEs and NCEs was not substantially affected as compared to the corresponding negative controls thus indicating no cytotoxic effects.

In comparison to the corresponding negative controls there was no significant enhancement in the frequency of the detected micronuc lei at any preparation interval after application of the test article .

An appropriate reference mutagen 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 article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse .

Therefore, the test item is considered to be non-mutagenic in this micronucleus assay.