Sodium bis[3-[[1-(3-chlorophenyl)-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-4-yl]azo]-4-hydroxybenzenesulphonamidato(2-)]cobaltate(1-)

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

The in vivo test is required in case of in vitro positive findings.

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From the 11th of May to the 09 of August, 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Test conducted according to internationally accepted testing guidelines, in GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

In deviation to the protocol the body weight of two males exceeded the upper limit of 40 g (animal no. 4 = 40.5 g; animal no. 13 = 40.2 g) These deviations had no influence on the integrity and validity of the study.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Sodium bis[3-[[1-(3-chlorophenyl)-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-4-yl]azo]-4-hydroxybenzenesulphonamidato(2-)]cobaltate(1-)

EC Number:

277-554-7

EC Name:

Sodium bis[3-[[1-(3-chlorophenyl)-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-4-yl]azo]-4-hydroxybenzenesulphonamidato(2-)]cobaltate(1-)

Cas Number:

73612-40-5

Molecular formula:

C32H24Cl2CoN10NaO8S2

IUPAC Name:

sodium bis[3-[[1-(3-chlorophenyl)-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-4-yl]azo]-4-hydroxybenzenesulphonamidato(2-)]cobaltate(1-)

Test material form:

solid: particulate/powder

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Source: BRL, CH-4414 Füllinsdorf
Number of Animals: 72 (36 males/36 females)
Initial Age at Start of 8-12 weeks
Acclimatization: minimum 5 days
Initial Body Weight at Start of Treatment: males mean value 35.7 g (SD ± 2.9 g)
According to the suppliers assurance the animals were in healthy condition. The animals were under quarantine in the animal house of RCC - CCR 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.

ENVIRONMENTAL CONDITIONS
Housing
Cage Type: Makrolon Type I, with wire mesh top, (EHRET GmbH, D-79302 Emmendingen)
Bedding: granulated soft wood bedding
Feed: (ALTROMIN, D-32791 Lage/Lippe) pelleted standard diet, ad libitum
Water: tap water, ad libitum, (Gemeindewerke, D-64380 Roßdorf)
Environment: temperature 21 ±3°C
Humidity: relative humidity 30-70 %
Artficial light 6.00 a.m. - 6.00 p.m.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

PEG400

Details on exposure:

Approximately 18 hours before treatment the animals received no food but water ad libitum.
At the beginning of the treatment the animals (including the controls) were weighed and the individual volume to be administered was adjusted to the animals body weight. The animals received the test item, the vehicle or the positive control substance once. Twelve animals, six males and six females, were treated per dose group and sampling time.

Duration of treatment / exposure:

PRE-EXPERIMENT TOXICITY
1 h, 6h, 24h, 48 h after the administration

Frequency of treatment:

Single dose

Post exposure period:

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

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
200, 670, 2000 mg/Kg bw
Basis:
actual ingested
at 24 h

Doses / concentrations 2

Remarks:

Doses / Concentrations:
2000 mg/Kg bw
Basis:
actual ingested
at 48 h

No. of animals per sex per dose:

72 (36 males/36 females)

Control animals:

yes

Positive control(s):

CPA; Cyclophosphamide
deionised water
40 mg/kg b.w.
orally, once
10 ml/kg b.w.
The stability of CPA at room temperature is good. At 25°C only 3.5 % of its potency is lost after 24 hours.

Examinations

Tissues and cell types examined:

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-Griinwald (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.

Details of tissue and slide preparation:

Evaluation of the slides was performed using NIKON microscopes with lOOx oil immersion objectives. 2000 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 2000 the PCEs. The analysis was performed with coded slides.
Ten animals (5 males, 5 females) per test group were evaluated as described.

Evaluation criteria:

The study was considered valid as the following criteria are met:
- the vehicle controls are in the range of our historical control data (0.03 - 0.26 % PCEs with micronuclei)
- the positive controls show substantially increased values
- more than 80 % of the animals are évaluable

A test item 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 item 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.

.

Statistics:

The data generated are recorded in the laboratory protocol. The results are presented in tabular form, including experimental groups, vehicle and positive control. The micronucleated cells per 2000 and the ratio of polychromatic to normochromatic erythrocytes are presented for each animal.
This can be confirmed by means of the nonparametric Mann-Whitney test.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

at 2000 mg/Kg bw

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Toxic reactions: reduction of spontaneous activity, eyelid closure.

Applicant's summary and conclusion

Conclusions:

Non-mutagenic in the in vivo micronucleus assay

Executive summary:

This study was performed according to the in vivo OECD474, EU B.12, in order to investigate the potential of the substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test item was formulated in polyethylene glycol 400 (PEG 400). PEG 400 was used as vehicle control. The volume administered orally was 10 mL/kg b.w.. 24 h and 48 h after a single administration of the test item 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. 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei.

To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCEs per 2000 PCEs.

The following dose levels of the test item were investigated:

24 h preparation interval: 200, 670, and 2000 mg/kg b.w..

48 h preparation interval: 2000 mg/kg b.w.

The highest dose (2000 mg/kg; maximum guideline-recommended dose) was estimated by a pre-experiment to be suitable.

After treatment with the test item the number of NCEs was not substantially increased as compared to the mean value of NCEs of the vehicle control thus indicating that the substance had no cytotoxic effectiveness in the bone marrow.

In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used.

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

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

Therefore, the substance is considered to be non-mutagenic in this in vivo micronucleus assay.