Reaction mass of trisodium [m[7-[[4'-[[6-benzamido-1-hydroxy-3-sulpho-2-naphthyl]azo]-3,3'-dihydroxy[1,1'-biphenyl]-4-yl]azo]-8-hydroxynaphthalene-1,6-disulphonato(7-)]]dicuprate(3-) and trisodium [m[4-[[4'-[[6-benzamido-1-hydroxy-3-sulpho-2-naphthyl]azo]-3,3'-dihydroxy[1,1'-biphenyl]-4-yl]azo]-3-hydroxynaphthalene-2,7-disulphonato(7-)]]dicuprate(3-)

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Remarks:

source of read across study record

Adequacy of study:

key study

Study period:

From October 22th to November 27th, 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

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)

Version / remarks:

adopted May 26, 1983

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Similar Substance 02

IUPAC Name:

Similar Substance 02

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.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL Tierfarm Füllinsdorf, Basel, Switzerland.
- Number of animals: 84 (42 males and 42 females).
- Age at start of acclimatization: minimum 10 weeks.
- Weight at study initiation: approximately 30 g.
- Health conditions: according to the suppliers assurance, the animals were in healthy condition. The animals were under quarantine in the animal house of testing laboratory for a minimum of five days after their arrival. During this period the animals did not show any signs of illness or altered behaviour.
- Assigned to test groups randomly: the animals were distributed into the test groups at random and identified by cage number.
- Fasting period before study: approximately 18 hours before treatment with the test article the animals received no food but water ad libitum.
- Housing: sinle; Makrolon Type I cages, with wire mesh top. Granulated soft wood bedding.
- Diet: pelleted standard diet.
- Water: tap water, ad libitum.
- Acclimation period: minimum 5 days.

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle: aqua dest.
- Justification for choice of vehicle: the vehicle was chosen to its nontoxicity for the animals.

Details on exposure:

DOSE VOLUME
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 volume administered was 20 ml/kg bw.

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

Frequency of treatment:

The animals received the test article once.

Post exposure period:

Sampling of the bone marrow was collected at 24, 48 and 72 hours.

Doses / concentrations

Dose / conc.:

400 mg/kg bw/day

No. of animals per sex per dose:

Six males and six females were assigned to each test group.

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive control: cyclophosphamide.
- Vehicle: physiological saline.
- Route of administration: orally, singly.
- Doses / concentrations: 30 mg/kg bw.

Examinations

Tissues and cell types examined:

Bone marrow.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: a preliminary study (4 pre-experiments) on acute toxicity was performed with the same strain and under identical conditions as in the mutagenicity study.

TREATMENT AND SAMPLING TIMES: sampling of the bone marrow was collected at 24, 48 and 72 hours.

DETAILS OF 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 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-Grfinwald. Cover slips were mounted with EUKITT. 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 immersion 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. 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 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

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The mean number of normochromatic erythrocytes was not substantially after treatment with the test article as compared to the mean values of NCEs of the corresponding negative controls indicating that test item had no cytotoxic properties.
In comparison to the corresponding negative controls there was no significant enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article.

POSITIVE CONTROL
Cyclophosphamide administration showed a distinct increase of induced micronuleus frequency.

PRE-EXPERIMENT FOR TOXICITY
First pre-experiment: in the pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 5000 mg/kg b.w. test item dissolved in aqua dest.. The volume administered was 20 ml/kg b.w.
The treated animals expressed toxic reactions: reduction of spontaneous activity, abdominal position, eyelid closure, apathy. All animals died within 6 hours after treatment.

Second pre-experiment: in the pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 2000 mg/kg b.w. test item dissolved in aqua dest.. The volume administered was 20 ml/kg b.w.
All treated animals expressed toxic reactions: reduction of spontaneous reaction one male and two females; abdominal position two females; eyelid closure two females; apathy one male and two females; lethalities one male and two females.

Third pre-experiment: in the pre-experiment 4 animals (2 males, 2 females) per dose group received orally a single dose of 400, or 500 mg/kg b.w. test item, respectively, dissolved in aqua dest. The volume administered was 10 ml/kg b.w.
400 mg/kg b.w.: reduction of spontaneous reaction two males and two females; eyelid closure one male and one female.
500 mg/kg b.w.: reduction of spontaneous reaction two males and two females; eyelid closure in one male and one female; apathy one male and one female; lethalities one male.

Fourth pre-experiment: in the pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 300 mg/kg b.w. test item dissolved in aqua dest.. The volume administered was 10 ml/kg b.w.
The animals expressed no toxic reactions.
On the basis of these results 400 mg/kg b.w. test item were estimated to be the maximum tolerated dose.

Any other information on results incl. tables

Summary of results

Test group	Dose mg/kg bw	Sampling time (h)	PCEs with micronuclei	Range	PCE/NCE
Solvent	0	24	0.06 %	0 - 3	1000 / 880
Test item	400	24	0.06 %	0 - 3	1000 / 935
Cyclophosphamide	30	24	1.44 %	7 - 30	1000 / 899
Solvent	0	48	0.06 %	0 - 2	1000 / 917
Test item	400	48	0.09 %	0 - 3	1000 / 973
Solvent	0	72	0.07 %	0 - 3	1000 / 765
Test item	400	72	0.06 %	0 - 4	1000 / 911

Applicant's summary and conclusion

Conclusions:

The test item is considered to be non-mutagenic in the micronucleus assay.

Executive summary:

The study was performed to investigate the potential of test item to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test article was dissolved in aqua dest.; the 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. 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, 400 mg/kg b.w.

In pre-experiments this dose level was estimated to be the maximum tolerated dose.

After treatment with the test article the ratio between PCEs and NCEs was not substantially affected when 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 micronuclei at any preparation interval.

An appropriate reference mutagen was used as positive control which showed a distinct increase of induced micronucleus frequency.

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