Hexasodium 2,2'-[vinylenebis[(3-sulphonato-4,1-phenylene)imino[6-[(2-cyanoethyl)[2-(2-hydroxyethoxy)ethyl]amino]-1,3,5-triazine-4,2-diyl]imino]]bis[benzene-1,4-disulphonate]

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Remarks:

source of read across

Adequacy of study:

key study

Study period:

From the 02nd February to the 23th March, 1989

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:

1989

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted on 26th May, 1983

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

mammalian germ cell cytogenetic assay

Test material

Test material information

Constituent 1

Reference substance name:

Similar Substance 01

IUPAC Name:

Similar Substance 01

Test animals

Species:

mouse

Strain:

Swiss

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga GmbH, Sulzfeld, FRG
- Age at study initiation: ca. 8 weeks
- Weight at study initiation: males 27 - 32 g; females 20 - 25 g
- Fasting period before study: withheld the night before dosing until 4 hours after administration.
- Diet: standard laboratory animal diet (RMH-B); certificate of analysis performed.
- Water: tap water; certificate of analysis performed.
- Accomodation: in groups of 5 per sex in polycarbonate cages.
- Acclimation: at least 6 days under laboratory conditions.
- Identification: by unique cage number and a mark on the tail.

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 3 °C
- Humidity: 40 - 70 %
- Air changes: 7.5 ACH
- Photoperiod: 12 hours dark

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle: Milli-RO water.
- Concentration of test material in vehicle: 4000 mg/kg b.w., single dose.
- Amount of vehicle: 10 ml/kg b.w.
- Sampling time: 24, 48 and 72 hours.

Details on exposure:

Single oral dose of 4000 mg/kg b.w.

Duration of treatment / exposure:

Bone marrow was sampled at 24, 48 and 72 hours after dosing for both treated and control animal groups. Groups A and B were dosed with 4000 and 2000 mg/kg b.w. substance respectively, and did not show any signs of reaction to the treatment.
Concentration higher than 4000 could not be assessed due to aggregation of test article in suspension.

Doses / concentrations

Dose / conc.:

4 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 males and 5 females.

Control animals:

yes, concurrent vehicle

Positive control(s):

Yes, cyclophosphamide.
- Route of administration: oral gavage.
- Dose: single dose at 50 mg/kg b.w. dissolved in 0.9 % NaCl in Milli-RO water.
- Sampling time: 48 hours after treatment.

Examinations

Tissues and cell types examined:

Bone marrow and erythrocytes.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION
A pilot study was performed on 3 males and 3 females per each dose group (five).

DETAILS OF SLIDE PREPARATION
Both femurs were flushed with 2 ml of foetal calf serum. The cell suspension was collected and centrifuged at 1000 rmp for 5 minutes. A drop of the cell suspension was placed on a slide which was previously cleaned (24 hours immersed in a 1:1 mixture of 96 % ethanol/ether). The preparations were then air-dried and thereafter fixed for 5 min in 100 % methanol and air-dried overnight. Two slides were prepared per animal.
- Staining: stained for 3 minutes in undiluted May-Grunwald solution followed by 2 minutes in May Grunwald solution diluted 1:1 with Sorensen buffer pH 6.8. Thereafter slides were rinsed and stained for 25 minutes in 5 % (v/v) Giemsa solution in Sorensen buffer pH 6.8. The preparations were rinsed for 1 minute in running tap-water and blotted dry between filter paper.

METHOD OF ANALYSIS
Slides were scored at a magnification of 1000 x. The number of micronuclei was counted in 1000 polychromatic erythrocytes. The ratio polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Averages and standard deviations were calculated.

Evaluation criteria:

Test substance is considered negative if:
- None of the tested concentrations or sampling times showed a statistically significant (p < 0.05) increase in the incidence of micronuclei neither in the combined data for both sexes nor in the data for male or female groups alone.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: no.
- Statistical evaluation: incidence of micronuclei in the control animals was found to be in the range of historical data of the performing laboratory. Positive control showed a statistical significant increase in the number of micronuclei.

Any other information on results incl. tables

Mean number of micronuclei

Sex	Group	Treatment	Dose [mg/kg b.w.]	Sampling time [h]	Micronuclei no. per 1000 polychromatic erythrocytes		Ratio polychromatic / normochromatic erythrocytes
					Mean	SD	Mean	SD
M	A	Vehicle	-	24	0.0	0.0	1.50	0.27
	B	Vehicle	-	48	0.0	0.0	1.27	0.44
	C	Vehicle	-	72	0.0	0.0	1.31	0.17
	D	Test item	4000	24	0.0	0.0	1.46	0.89
	E	Test item	4000	48	0.6	0.9	1.06	0.16
	F	Test item	4000	72	0.0	0.0	1.64	0.45
	G	Positive control	50	48	8.6	2.1	0.44	0.12
F	A	Vehicle	-	24	0.0	0.0	2.23	0.86
	B	Vehicle	-	48	0.4	0.9	1.94	1.45
	C	Vehicle	-	72	0.0	0.0	1.38	0.40
	D	Test item	4000	24	0.2	0.4	1.33	0.77
	E	Test item	4000	48	0.2	0.4	1.39	0.41
	F	Test item	4000	72	0.0	0.0	1.26	0.21
	G	Positive control	50	48	8.0	0.7	0.40	0.11

Applicant's summary and conclusion

Conclusions:

The test substance can be considered as not mutagenic in the Mouse Micronucleus Test, under the experimental conditions of the test.

Executive summary:

The substance was tested in the Micronucleus Test in mice, according to the method and procedures outlined into the OECD guideline 474. Three groups, each comprising 5 males and 5 females, received a single oral dose of 4000 mg/kg body weight. Bone marrow was sampled at 24, 48 and 72 hours after dosing. Corresponding vehicle treated groups served as negative controls. Bone marrow from a positive control group, treated with a single oral dose of cyclophosphamide (CP) at 50 mg/kg body weight, was harvested at 48 hours after dosing only. The test substance was found to respond negatively in the Micronucleus Test, whereas the positive control substance (CP) produced a statistically significant increase in the incidence of micronuclei in polychromatic erythrocytes.

Conclusion

The test substance can be considered as not mutagenic in the Mouse Micronucleus Test, under the experimental conditions of the test.