reaction mass of Benzenesulfonic acid, 2,2'-(1,2-ethenediyl)bis[5-[[4-[bis(2- hydroxyethyl)amino]-6-(phenylamino)-1,3,5-triazin-2-yl]amino]-, disodium salt and Benzenesulfonic acid, 5-[[4-[bis(2-hydroxyethyl)amino]-6-(phenylamino)-1,3,5-triazin-2-yl]amino]-2-[2-[4-[[4-[(2-hydroxyethyl)amino]-6-(phenylamino)-1,3,5-triazin-2-yl]amino]-2-sulfophenyl]ethenyl]-, disodium salt

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:

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

Adequacy of study:

key study

Study period:

Form February 22th to March 08th, 2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted according to internationally accepted testing guidelines and performed in compliance with Good Laboratory Practice. Justification for read across approach is given in the endpoint summary and in the read across justification report attached to the Section 13 of this dossier.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted July 21, l997

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH.
- Age at study initiation: 8 - 10 weeks.
- Weight at study initiation: males mean value 34.0 g (SD ± 2.5 g); females mean value 28.7 g (SD ± 2.1 g).
- Assigned to test groups randomly: yes.
- Housing: single in cages Makrolon Type I, with wire mesh top (EHRET GmbH, D-79302 Emmendingen). Bedding was made by granulated soft wood bedding (Harlan Winkelmann GmbH, D-33178 Borchen).
- Diet: pelleted standard diet, ad Iibitum (Harlan Winkelmann GmbH, D-33178 Borchen).
- Water: tap water, ad Iibitum (Gemeindewerke, D-64380 Rossdorf).
- Acclimation period: minimum 5 dasy.
- Quarantine: according to the suppliers assurance the animals were in healthy condition. The animals were under quarantine in the animal house of the testing laboratory for a minimum of five days after their arrival. During this period the animals did not show any signs of illness or altered behaviour.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C.
- Humidity: 26 - 70 %.
- Photoperiod: artificial light 6.00 a.m. - 6.00 p.m.

Administration / exposure

Route of administration:

oral: unspecified

Vehicle:

- Vehicle used: deionised water.
- Route of administration: oral.
- Frequency of administration: once.
- Volume administered: 10 ml/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
The test item was formulated in deionised water, which was also 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.

TREATMENT
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. The animals of all dose groups were examined for acute toxic symptoms at intervals of around 1 h, 2 - 4 h, 6 h and 24 h after administration ofthe test item.

Frequency of treatment:

Single treatment

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
500, 1000 and 2000 mg/kg bw
Basis:
nominal conc.
24 hrs preparation

Doses / concentrations 2

Remarks:

Doses / Concentrations:
2000 mg/kg bw
Basis:
nominal conc.
48 hrs preparation

No. of animals per sex per dose:

six males and six females per group

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (CPA)
- Justification for choice of positive control: the stability of CPA at room temperature is sufficient. At 25 °C only 3.5 % of its potency is lost after 24 hours.
- Route of administration: oral.
- Frequency of administration: once.
- Volume administreted: 10 ml/kg bw
- Dissolved in: deionised water.

Examinations

Tissues and cell types examined:

Micronucleus in bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION
It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or 2000 mg/kg as the upper limit for non-toxic test items.
The maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on survival within 48 hours.
The volume to be administered should be compatible with physiological space available.
Three adequate spaced dose levels spaced by a factor of 2 were applied at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 48 h after treatment.

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

DETAILS OF SLIDE PREPARATION
The animals were sacrificed using CO2 following by bleeding. The femora were removed, the epiphyses were cutoff 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 (KINDLER, D-79110 Freiburg). At least one slide was made from each bone marrow sample.

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 normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides. Ten animals (5 males, 5 females) per test group were evaluated as described. The remaining G"` animal of each sex in the respective test group test group is usually evaluated in case an animal dies in its test group spontaneously.

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

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

PRE-EXPERIMENT FOR TOXICITY
All the animals treated with 2000 mg/kg b.w. showed ruffled fur between 2 and 6 hours post-treatment. At 24 hours no signs of toxicity were observed. On the basis of the preliminary test outcomes, 2000 mg/kg b.w. were estimated to be suitable.

TOXIC SYMPTOMS IN THE MAIN EXPERIMENT
Animals treated with 2000 mg/kg bw showed ruffled fur during the first 6 hours; these effects were not more observed at 24 hours.
Ruffled fur was also recorded in animals treated with 1000 mg/kg bw, at 2 - 4 hours. At 6 hours this effect was not observed.
The animals treated with 500 mg/kg bw and the vehicle control (deionised water) animals did not show any toxic reactions.

Any other information on results incl. tables

SUMMARY OF MICRONUCLEUS TEST RESULTS

Test group	Dose mg/kg bw	sampling time (h)	PCEs with micronuclei (%)	Range	PCE per 2000 erythocytes
Vehicle	0	24	0.125	0 - 6	1154
Test item	500	24	0.115	0 - 4	1166
Test item	1000	24	0.090	1 - 4	1157
Test item	2000	24	0.095	0 - 5	1216
Positive control	40	24	3.030	42 - 92	988
Test item	2000	48	0.075	0 - 3	1081

Biometry

Vehicle control versus test group	Significance	p
500 mg test item/kg bw; 24 h	n.t.	-
1000 mg test item/kg bw; 24 h	n.t.	-
2000 mg test item/kg bw; 24 h	n.t.	-
40 mg CPA/kg bw; 24 h	+	< 0.0001
2000 mg test item/kg bw; 48 h	n.t.	-

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

HISTORICAL CONTROLS - 1999 - 2004

	Vehicle controls			Positive control (CPA)
	Males	Females	Total	Males	Females	Total
Mean*± SD	0.078± 0.04	0.058± 0.033	0.069± 0.028	0.078± 0.07	1.368± 0.497	1.632± 0.468
Range**	0.01- 0.23	0.0- 0.19	0.01- 0.15	0.70- 3.46	0.49- 3.55	0.77- 3.48
N. of experiments	229	217	230	228	217	229

*: mean value (percent micrcnucleated cells)

**: range of the mean group values (percent micronucleated cells)

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
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.

Executive summary:

This study was performed 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 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. 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 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 PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that the substance did not exert any cytotoxic effects in the bone marrow.

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 b.w. cyclophosphamide administered orally was used as positive control which showed a substantial increase of induced micronucleus frequency.

Conclusion

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 micronucleus assay.