Tetra(sodium/potassium)-7-[(E)-{2-acetamido-4-[(E)-(4-{[4-chlor-6-({2-[(4-fluor-6-{[4-(vinylsulfonyl)phenyl]amino}-1,3,5-triazin-2-yl)amino]propyl}amino)-1,3,5-triazin-2-yl]amino}-5-sulfonato-1-naphthyl)diazenyl]-5-methoxyphenyl}diazenyl]-1,3,6-naphthalintrisulfonate

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:

Study initiation date - 23 October 2006; Experiment start date - 01 November 2006; Experiment completion date - 19 December 2006; Study completion date - 20 March 2007.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Commission Directive 2000/32/EC, Annex 4C, dated May 19, 2000.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Test material form:

other: solid

Details on test material:

Colour: Black
Molecular Weight: 1272.7 g/mol as free acid
Purity: Content of organic part (Na-salt): approx. 80 %; Oligomers: 13 %; Main component: approx. 53 %
Solubility in Water: Approx > 100 g/L at room temperature
Stability in Solvent: Stable for 7 days in water, saline, polyethylene glycol, and CMC at room temperature
Storage: At room temperature in the desiccator

Specific details on test material used for the study:

Identity: FAT 40827/A
Batch: T2 5572 BOP 01/06
Purity: determined in this study
Appearance: black sticky powder
Expiration date: 28.02.2011
Storage: at room temperature

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

Strain: NMRI
Source Harlan Winkelmann GmbH D-33178 Borchen
Number of Animals: 72 (36 males/36 females)
Initial Age at Start of Acclimatisation: 6 - 9 weeks
Acclimatization: minimum 5 days
Initial Body Weight at Start of Treatment: males mean value 31.1 g (SD ± 1.5 g) females mean value 31.2 g (SD ± 1.6 g);
The animals were kept conventionally. The experiment was conducted under standard laboratory conditions.
Housing: single
Cage Type: Makrolon Type I, with wire mesh top
Bedding: granulated soft wood bedding
Feed: pelleted standard diet, ad libitum
Water: tap water, ad libitum
Environment: temperature 22 ± 3 °C
relative humidity 30 - 73 %
artificial light 6.00 a.m. - 6.00 p.m.

Administration / exposure

Route of administration:

oral: unspecified

Vehicle:

deionised water

Details on exposure:

On the day of the experiment, the test item was formulated in deionised water and the animals (including the controls) were weighed and the individual volume to be administered was adjusted to the animal’s body weight (All animals received a single standard volume of 10 mL/kg body weight orally). The animals received the test item, the vehicle or the positive control substance once.

Duration of treatment / exposure:

24 and 48 hours

Frequency of treatment:

once

Post exposure period:

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Remarks:

24h interval

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

24h interval

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

Remarks:

24 and 48h interval

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

CPA, Cyclophosphamide, dissolved in deionised water, dosing: 40 mg/kg b.w., route and frequency of administration: orally, once, volume administered: 10 mL/kg bw

Examinations

Tissues and cell types examined:

The marrow of the femora;
Two types of erythrocytes were observed in the bone marrow smears: normochromatic (mature red blood cells about to pass into the blood stream) and polychromatic (immature red blood cells).

Details of tissue and slide preparation:

The animals were sacrificed using CO2 followed by bleeding. The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal 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 re-suspended 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.

Evaluation criteria:

A test item is 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 item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.

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

Remarks:

no cytotoxic properties in the bone marrow

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The urine of the treated animals was stained red to orange indicating the systemic distribution of the test article and hence confirming its bioavailability.

Any other information on results incl. tables

Micronuclei in polychromatic erythrocytes (PCE) and relationship PCE/ total erythrocytes scoring 24 hours after treatment

Test group	Dose mg/kg b.w.	Micronucleated cells per 2000 PCEs per animal	Percent cells with micronuclei	PCE per 2000 erythrocytes
Vehicle (a. deion.)	0	2.5	0.125	1110
Test item	500	3.5	0.175	1108
	1000	2.0	0.100	1100
	2000	2.7	0.135	1114
Positive control(CPA)	40	43.2	2.160	971

 

Micronuclei in polychromatic erythrocytes (PCE) and relationship PCE/ total erythrocytes scoring 48 hours after treatment

Test group	Dose mg/kg b.w.	Micronucleated cells per 2000 PCEs per animal	Percent cells with micronuclei	PCE per 2000 erythrocytes
Test item	2000	1.6	0.080	1211

Applicant's summary and conclusion

Conclusions:

During the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the
micronucleus test in the bone marrow cells of the mouse.

Executive summary:

A GLP-compliant study was performed according to OECD test guideline 474 to investigate the potential of the test item to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test item was formulated in deionised water, which was also used as vehicle control. The volume administered orally was 10 mL/kg bw. 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. At least 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 was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes. The following dose levels of the test item were investigated:

24 h preparation interval: 500, 1000, and 2000 mg/kg bw,

48 h preparation interval: 2000 mg/kg bw.

The highest dose (2000 mg/kg; maximum guideline-recommended dose) was estimated by pre-experiments to be suitable. After treatment with the test item 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 item did not exert any cytotoxic effects in the bone marrow. However, the urine of the treated animals was stained red to orange indicating the systemic distribution of the test item and hence confirming its bioavailability. In comparison to the corresponding vehicle controls 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 item and with any dose level used. 40 mg/kg bw cyclophosphamide administered orally was used as positive control which showed a substantial increase of induced micronucleus frequency. In conclusion, the test item was considered to be non-mutagenic in this micronucleus assay.