Reaction mass of Cobaltate(3-), bis[2-[[[3-[2-[1-[[(2-chlorophenyl)amino]carbonyl]-2-(oxo-κO)propyl]diazenyl-κN1]-4-(hydroxy-κO)phenyl]sulfonyl]amino]benzoato(3-)]-, sodium (1:3) and tetrasodium bis[2-[[[3-[[1-[(2-chloroanilino)carbonyl]-2-oxopropyl]azo]-4-hydroxyphenyl]sulphonyl]amino]benzoato(3-)]cobaltate(4-)

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 23 September, 2002 to 17 January, 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of Trisodium bis[2-({[3-({1-[(2-chlorophenyl)amino]-3-(hydroxy-kO)-1-oxobut-2-en-2-yl}diazenyl-kN1)-4-(hydroxy-kO)phenyl]sulfonyl}amino)benzoato(3-)]cobaltate(3-) and Trisodium [2-({[3-({1-[(2-chlorophenyl)amino]-3-(hydroxy-kO)-1-oxobut-2-en-2-yl}diazenyl-kN1)-4-(hydroxy-kO)phenyl]sulfonyl}amino)benzoato(3-)][2-({[3-({1-[(2-chlorophenyl)amino]-3-(hydroxy-kO)-1-oxobut-2-en-2-yl}diazenyl-kN2)-4-(hydroxy-kO)phenyl]sulfonyl}amino)benzoato(3-)]cobaltate(3-)

EC Number:

941-792-6

Cas Number:

not given

Molecular formula:

C46H32Cl2CoN8O14S2.3Na

IUPAC Name:

Reaction mass of Trisodium bis[2-({[3-({1-[(2-chlorophenyl)amino]-3-(hydroxy-kO)-1-oxobut-2-en-2-yl}diazenyl-kN1)-4-(hydroxy-kO)phenyl]sulfonyl}amino)benzoato(3-)]cobaltate(3-) and Trisodium [2-({[3-({1-[(2-chlorophenyl)amino]-3-(hydroxy-kO)-1-oxobut-2-en-2-yl}diazenyl-kN1)-4-(hydroxy-kO)phenyl]sulfonyl}amino)benzoato(3-)][2-({[3-({1-[(2-chlorophenyl)amino]-3-(hydroxy-kO)-1-oxobut-2-en-2-yl}diazenyl-kN2)-4-(hydroxy-kO)phenyl]sulfonyl}amino)benzoato(3-)]cobaltate(3-)

Test material form:

solid

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd., Biotechnology and Animal Breeding Division; CH-4414 Füllinsdorf
- Age at study initiation: 8 -10 weeks
- Weight at study initiation: Males mean value 37.9 g (SD ±3.1 g); Females mean value 26.5 g (SD ±3.1 g)
- Assigned to test groups randomly: Yes
- Housing: Single
- Diet (e.g. ad libitum): Pelleted standard diet, ad libitum (ALTROMIN 1324, D-32791 Lage/Lippe)
- Water (e.g. ad libitum): Tap water, ad libitum, (Gemeindewerke, D-64380 Roßdorf)
- Acclimation period: 5 d

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30-72 %
- Artificial light: 6.00 a.m-6.00 p.m

IN-LIFE DATES: From: 07 October, 2002 to 02 November, 2002

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Deionised water
- Justification for choice of solvent/vehicle: The vehicle was chosen to its relative non-toxicity for the animals.
- Volume administered: 10 mL/kg bw

Details on exposure:

On the day of the experiment, the test substance was formulated in deionised water. The vehicle was chosen for its relative non-toxicity for the animals. All animals received a single standard volume of 10 mL/kg bw orally.

Duration of treatment / exposure:

Single administration

Frequency of treatment:

Once

Post exposure period:

no data

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

437.5 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

875 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 750 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5/sex/group

Control animals:

yes, concurrent vehicle

Positive control(s):

Positive control: Cyclophosphamide
Dissolved in: Deionized water
Dosing: 40 mg/kg bw
Route and frequency of administration: Orally, once
Volume Administered: 10 mL/kg bw
Solution prepared on day of administration. The stability of positive control at room temperature is sufficient. At 25 °C only 3.5 % of its potency is lost after 24 h.

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 centrifuged 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-Grünwald/Giemsa. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The highest dose (1750 mg/kg bw) was estimated by a pre-experiment to be suitable.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): 24 h and 48 h after a single administration of the test substance the bone marrow cells were collected for micronuclei analysis.

METHOD OF ANALYSIS: Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and total erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides.

Evaluation criteria:

A test substance classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group. Statistical methods (nonparametric Mann-Whitney test) will be used as an aid in evaluating the results. However, the primary point of consideration is the biological relevance of the results. A test substance that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system

Statistics:

no data

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

The mean number of polychromatic erythrocytes was not decreased after treatment with the test substance as compared to the mean value of PCEs of the vehicle control indicating that the test substance had no cytotoxic properties in the bone marrow. However, the urine of the treated animals was brown indicating the systemic distribution of the test substance and thus its bioavailibility. 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 and dose level after administration of the test substance. The mean values of micronuclei observed after treatment with the test substance were below or near to the value of the vehicle control group. 40 mg/kg bw cyclophosphamide administered orally was used as positive control which showed a statistically significant increase of induced micronucleus frequency.

Any other information on results incl. tables

Pre-experiment for toxicity:

In the first pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 2000 mg/kg bw formulated in deionised water. The volume administered was 10 mL/kg bw. The animals treated with 2000 mg/kg bw expressed toxic reactions.

Toxic reactions	Hours post-treatment
	1 h	2-4 h	6 h	24 h	30 h	48 h
Reduction of spontaneous activity	2/2	2/2	2/2	1/-	1/-	1/-
Abdominal position	2/2	1/2	1/2	0/-	0/-	0/-
Eyelid closure	2/2	2/2	2/2	0/-	0/-	0/-
Ruffled fur	2/2	2/2	2/2	1/-	1/-	1/-
Apathy	2/2	1/1	1/2	0/-	0/-	0/-
Urine colour	-	brown	-	brown	-	-
Death	0/0	0/0	0/0	1/2	0/0	0/0

 

In the second pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 1500 mg/kg bw formulated in deionised water. The volume administered was 10 mL/kg bw. The animals treated with 1500 mg/kg bw expressed toxic reactions.

Toxic reactions	Hours post-treatment
	1 h	2-4 h	6 h	24 h	30 h	48 h
Reduction of spontaneous activity	2/2	2/2	2/2	2/2	2/2	2/2
Abdominal position	1/1	0/0	0/0	0/0	0/0	0/0
Eyelid closure	1/2	1/2	2/2	0/0	0/0	0/0
Ruffled fur	2/2	2/2	2/2	2/2	2/2	2/2
Apathy	1/1	0/0	1/1	0/0	0/0	0/0

 

In the third pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 1750 mg/kg bw formulated in deionised water. The volume administered was 10 mL/kg bw. The animals treated with 1750 mg/kg bw also expressed toxic reactions.

Toxic reactions	Hours post-treatment
	1 h	2-4 h	6 h	24 h	30 h	48 h
Reduction of spontaneous activity	2/2	2/2	2/2	2/2	2/2	1/1
Abdominal position	1/2	1/2	2/1	0/0	0/0	0/0
Eyelid closure	1/2	1/2	2/2	0/0	1/1	1/1
Ruffled fur	2/2	2/2	2/2	1/1	1/1	1/1
Apathy	1/2	2/2	2/1	0/0	1/1	1/1

On the basis of these data 1,750 mg/kg bw were estimated to be suitable.

Toxic symptoms in the main experiment: In the main experiment for the highest dose group 24 animals (12 males, 12 females) received orally a single dose of 1750 mg /kg bw formulated in deionized water. The volume administered was 10 mL/kg bw. The animals treated with 2000 mg /kg bw expressed toxic reactions.

Toxic reactions	Hours post-treatment
	1 h	2-4 h	6 h	24 h
Reduction of spontaneous activity	12/10	12/11	12/12	3/6
Abdominal position	2/0	2/0	0/3	0/1
Eyelid closure	10/10	10/9	9/10	1/1
Ruffled fur	12/12	12/12	12/12	11/11
Brown urine colour	-/-	+/+	-/-	-/-
Death	0/0	0/0	0/0	1/1*

*1 female died after the 24 h observation interval

Applicant's summary and conclusion

Conclusions:

The test substance did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

Executive summary:

The study was performed to investigate the potential of the test substance (at ca. 70-80 % purity) to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse according to OECD Guideline 474 in compliance with GLP. The substance was formulated in deionised water, which was also used as vehicle control. 24 h and 48 h after a single administration, bone marrow cells were collected for micronuclei analysis. The highest dose was chosen based on a preliminary experiment. The following dose levels were investigated:

- 24 h preparation interval: 437.5, 875 and 1750 mg/kg bw

- 48 h preparation interval: 1750 mg/kg bw

 

In the main experiment, 3 animals died after treatment at this dose. After treatment with the test substance, the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control, thus indicating that the test substance did not exert cytotoxic effects in the bone marrow. However, the urine of the treated animals was brown, suggesting systemic distribution of the substance and thus bioavailability. Further, there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test substance and with any dose level used. Hence, it can be concluded that the test substance did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.