Reaction mass of Cuprate(4-), [4,5-dihydro-4-[[8-hydroxy-7-[[2-hydroxy-5-methoxy-4-[[2-(sulfooxy)Vinyl]phenyl]azo]-6-sulfo-2-naphthalenyl]azo]-5-oxo-1-(4-sulfophenyl)-1H-pyrazole-3-carboxylato(6-)], trisodium salt and Cuprate(4-), [4,5-dihydro-4-[[8-hydroxy-7-[[2-hydroxy-5-methoxy-4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]azo]-6-sulfo-2-naphthalenyl]azo]-5-oxo-1-(4-sulfophenyl)-1H-pyrazole-3-carboxylato(6-)]-, sodium and Cuprate(4-), [4,5-dihydro-4-[[8-hydroxy-7-[[2-hydroxy-5-methoxy-4-[2-(sulfooxy)Ethanol]phenyl]azo]-6-sulfo-2-naphthalenyl]azo]-5-oxo-1-(4-sulfophenyl)-1H-pyrazole-3-carboxylato(6-)], trisodium salt

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

From August 10 to 13, 1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Complete read across justification is attached in section 13. Source study has reliability 2.

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)

Version / remarks:

1983

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

RA Substance 01

IUPAC Name:

RA Substance 01

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: HOECHST AG, Kastengrund, SPF breeding colony (own breed)
- Strain: Hoe: NMRKf (SPF71)
- Age at study initiation: 7 weeks
- Weight at study initiation (mean): males: 29.3 g; females: 22.5 g
- Assigned to test groups randomly: yes, under following basis: computer assisted randomization plan
- Fasting period before study: no data
- Housing: in fully air-conditioned rooms in Macrolon cages (type 3), on softwood granulate in groups of 5 animals
- Diet: Altromin 1324, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 17 - 23 °C
- Humidity: 30 - 70 %
- Photoperiod: 12 / 12 dark / light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Deionised water.

Details on exposure:

The test compound dilutions were freshly prepared at the day of administration. 12500 mg of test substance were weighed in a beaker, mixed with deionised water, washed out in a 25 ml flask and topped up to the calibration mark. A suspension was formed.

As for the positive control, 5 ml distilled water were added to 100 mg of positive control in an injection phial and shaken to form a clear solution. The solutions for administration were prepared from this stock solution. For this purpose, 2 ml of the 2 % stock solution were mixed with 6 ml distilled water.

Duration of treatment / exposure:

24, 48 and 72 hours.

Frequency of treatment:

Once.

Post exposure period:

None.

Doses / concentrations

Dose / conc.:

5 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 males and 5 females per dose group and killing time-point.

Control animals:

yes

Positive control(s):

- Substance name: cyclophosphamide.
- Justification for choice of positive control: proven cytostatic agent and known clastogen with bifunctional alkylation action.
- Route of administration: oral gavage.
- Dose: 50 mg/kg bw.

Examinations

Tissues and cell types examined:

Chromosomes of bone-marrow erythroblasts.

Details of tissue and slide preparation:

Rationale for dose selection
Oral administration of 5000 mg test substance kg bw did not lead to a partial lethality in male and female mice. Consequently, the maximum applicable dose was selected as dose level for the main study.

Extraction of the bone marrow
In conformity with the test procedure the animals were killed by carbon dioxide asphyxiation 24, 48 or 72 hours after application. For each animal, about 3 ml foetal bovine serum was poured into a centrifuge tube. Both femora were removed and the bones freed of muscle tissue. The proximal ends of the femora were opened and the bone marrow flushed into the centrifuge tube. A suspension was formed. The mixture was then centrifuged for 5 minutes at ca. 1000 rpm and almost all the supernatant discarded. One drop of the thoroughly mixed sediment was smeared on a cleaned slide, identified by project code and animal number and air-dried for about 24 hours.

Staining procedure
- 5 minutes in methanol
- 5 minutes in May-Grünwalds solution
- brief rinsing twice in distilled water, 10 minutes staining in 1 part Giemsa solution to 6 parts buffer solution, pH 7.2 (Weise)
- rinsing in distilled water
- drying
- coating with Entellan

Evaluation criteria:

1000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded, not the number of individual micronuclei.
As a control measure, 1000 mature erythrocytes were also counted and examined for micronuclei. In addition, the ratio of polychromatic to normochromatic erythrocytes was determined.
All bone marrow smears for evaluation are coded to ensure that the group which they belonged to remains unknown to the investigator.
Statistical evaluation of the number of polychromatic erythrocytes with micronuclei occurring in 1000 polychromatic erythrocytes and the number of normocytes with micronuclei occurring in 1000 normocytes counted was done.

Both biological and statistical significance are considered together in evaluation.
A substance is considered as positive if there is a significant increase in the number of micronucleated polychromatic erythrocytes for at least one of the time points.
A substance producing no significant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.

Statistics:

A Wilcoxon-Test (one-sided) was evaluated to check the validity of the study. This is done by comparing the number of polychromatic erythrocytes with micronuclei in the positive control with the negative control.
A Wilcoxon-Test (one-sided) was calculated for each measurement group (24h, 48h, 72h) and for polychromatic and normochromatic erythrocytes. These tests were performed sequentially with a multiple level of significance of 5%. Tests on lower dose groups are only performed if the higher dose group is significantly different from the control.

The presupposition to make any of the following comparisons is a difference between the positive control and the negative control (24 h). This is tested with a Wilcoxon-Test (two-sided) with 5 %-level of significance.
Wilcoxon-Tests (two-sided) are performed sequentially for the ratio of polychromatic erythrocytes for each measurement group (24 h, 48 h, 72 h) at a multiple level of significance of 5 %.
Lower dose groups are only tested if the higher dose group is significantly different from the control. Actual data were also compared with historical controls.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

All animals survived after application of 5000 mg/kg bw. The following signs of toxicity were observed: faeces blue coloured and diarrhea. 24 h after application all animals were free of clinical signs of toxicity.
The dissection of the animals revealed no test substance related findings.
The bone marrow smears were examined for the occurrence of micronuclei in red blood cells.
The incidence of micronucleated polychromatic and normochromatic erythrocytes in the dose groups of test substance was within the normal range of negative control groups. No statistically significant increase of micronucleated polychromatic erythrocytes was seen.
The number of normochromatic erythrocytes with micronuclei did not differ from the values of the simultaneous control animals for each of the three killing times investigated.
The ratio of polychromatic erythrocytes to normocytes remained essentially unaffected by the test compound.

Positive control substance, i.e. cyclophosphamide, induced a marked and statistically significant increase of the number of polychromatic erythrocytes with micronuclei in both males and females, thus showing the sensitivity of the test system.

Applicant's summary and conclusion

Conclusions:

Administration of test substance at a limit dose of 5000 mg/kg bw did not lead to a substantial increase of micronucleated polychromatic erythrocytes and is not mutagenic in the mouse micronucleus test.

Executive summary:

Method

Micronucleus test was carried out according to OECD guideline 474 (1983).

Test substance was suspended in deionised water and administered orally as a single dose of 5000 mg/kg bw to male and female mice, based on results obtained in a previous dose range finding assay. According to the test procedure the animals were killed 24, 48 or 72 hours after administration.

Cyclophosphamide was used as positive control and was administered orally at a dose of 50 mg/kg bw.

Results

All animals survived after application of 5000 mg/kg bw. Signs of toxicity were noted, in terms of blue faeces and diarrhea. However, these signs were no more evident 24 hours after application. The dissection of the animals revealed no test substance related findings.

The number of polychromatic and normochromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained unaffected by the treatment with test substance and was statistically not different from the control values.

Positive control induced in both males and females a marked statistically significant increase in the number of polychromatic cells with micronuclei, thus proving the sensitivity of the system. The ratio of polychromatic erythrocytes to normocytes was not changed to a significant extent.

The results indicate that, under the conditions of the present study, test substance is not mutagenic in the micronucleus test.