disodium 5-{4-chloro-6-[N-ethyl-3-(vinylsulfonyl)anilino]-1,3,5-triazin-2-ylamino}-4-hydroxy-3-[(4-vinylsulfonyl)phenylazo]naphthalene-2,7-disulfonate; reaction mass of: trisodium 5-{4-chloro-6-[N-ethyl-(3-(2-sulfonatooxy)ethylsulfonyl)anilino]-1,3,5-triazin-2-ylamino}-4-hydroxy-3-[4-(vinylsulfonyl)phenylazo]naphthalene-2,7-disulfonate; tetrasodium 5-{4-chloro-6-[N-ethyl-3-(2-(sulfonatooxy)ethylsulfonyl)anilino]-1,3,5-triazin-2-ylamino}-3-[4-(2-(sulfonatooxy)ethylsulfonyl)phenylazo]-4-hydroxynaphthalene-2,7-disulfonate; trisodium 5-{4-chloro-6-[N-ethyl-3-(vinylsulfonyl)anilino]-1,3,5-triazin-2-ylamino}-4-hydroxy-3-[4-(2-(sulfonatooxy)ethylsulfonyl)phenylazo]naphthalene-2,7-disulfonate

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:

Date of Study Plan: October 29, 2002 - Date of Final Report: February 07, 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

9th addendum to the OECD Guideline for Testing of Chemicals, Section 4, No. 474, adopted July 21, 1997, "Mammalian Erythrocyte Micronucleus Test".

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: micronucleus assay in bone marrow cells of the mouse

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

444-050-5

EC Name:

-

IUPAC Name:

dodecasodium 5-({4-chloro-6-[ethyl({3-[2-(sulfooxy)ethanesulfonyl]phenyl})amino]-1,3,5-triazin-2-yl}amino)-3-[(E)-2-[4-(ethenesulfonyl)phenyl]diazen-1-yl]-4-hydroxynaphthalene-2,7-disulfonate 5-({4-chloro-6-[ethyl({3-[2-(sulfooxy)ethanesulfonyl]phenyl})amino]-1,3,5-triazin-2-yl}amino)-4-hydroxy-3-[(E)-2-{4-[2-(sulfooxy)ethanesulfonyl]phenyl}diazen-1-yl]naphthalene-2,7-disulfonate 5-[(4-chloro-6-{[3-(ethenesulfonyl)phenyl](ethyl)amino}-1,3,5-triazin-2-yl)amino]-3-[(E)-2-[4-(ethenesulfonyl)phenyl]diazen-1-yl]-4-hydroxynaphthalene-2,7-disulfonate 5-[(4-chloro-6-{[3-(ethenesulfonyl)phenyl](ethyl)amino}-1,3,5-triazin-2-yl)amino]-4-hydroxy-3-[(E)-2-{4-[2-(sulfooxy)ethanesulfonyl]phenyl}diazen-1-yl]naphthalene-2,7-disulfonate

Test material form:

solid

Details on test material:

Identity: Red Rwa 4565
Appearance: Solid, red powder
Storage: at room temperature at about 20°C

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 (at start of the treatment): males mean value 32.7 g (SD ± 2.7 g); females mean value 26.4 g (SD ± 1.8 g)
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing: single. Cage type: Makrolon Type 1, with wire mesh top (EHRET GmbH, D-79302 Emmendingen). Bedding: granulated soft wood bedding (ALTROMIN, D-32791 Lage/Lippe)
- 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 Rof1dorf)
- Acclimation period: minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 25-82 *
- Photoperiod: artificial light 6.00 a.m. - 6.00 p.m.

* ln the present study, the relative humidity under which the experiment was conducted ranged between 25 - 82 % and not between 30 -70 % as described in the study plan. This deviation, however, does not affect the validity of the experiment.

Administration / exposure

Route of administration:

oral: unspecified

Vehicle:

- Vehicle used: deionised water
- Justification for choice of solvent/vehicle: the vehicle was chosen to its relative non-toxicity for the animals.
- Amount of vehicle: 10 ml/kg b.w.

Details on exposure:

Pre-experiment for toxicity
A preliminary study on acute toxicity was performed with two animals per sex under identical conditions as in the mutagenicity study concerning: starvation period, animal strain; vehicle; route, frequency, and volume of administration.
The animals were treated orally with the test item and examined for acute toxic symptoms at intervals of around 1 h, 2-4 h, 6 h, 24 h, 30 h, and 48 h after administration of the test item.

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
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 positíve control substance once. Twelve animals, six males and six females, were treated per dose group and sampling time. The animals of the highest dose group were examined for acute toxic symptoms at intervals of around t h, 24h, 6 h and 24 h after administration of the test item.

Duration of treatment / exposure:

acute exposure

Frequency of treatment:

1 treatment per animal

Post exposure period:

24 and 48 h

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

24h preparation interval

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

24h preparation interval

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

24h and 48h preparation intervals
maximum guideline-recommended dose and estimated by a pre-experiment to be suitable

No. of animals per sex per dose:

10 animals (5 males, 5 females) per test group were evaluated for the occurence of micronuclei.

Control animals:

yes, concurrent vehicle

Positive control(s):

CPA; Cyclophosphamide
- 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
- Doses / concentrations: 40 mg/kg b.w.

Examinations

Tissues and cell types examined:

Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively (to cover the intervals in which maximum frequencies of micronuclei occur).

Details of tissue and slide preparation:

Preparation of the Animais:
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 (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.

Analysis of Cells:
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.
Ten animals (5 males, 5 females) per test group were evaluated as described.

Data Recording
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 PCEs and the ratio of polychromatic to total erythrocytes are presented for each animal.

Evaluation criteria:

Acceptance criteria:
The study was considered valid as the following criteria are met:
- the negative controls are in the range of our historical control data (0.01 - 0.15 %; mean = 0.066 x 0.032 PCEs with micronuclei).
- the positive controls are in the range of our historical control data (0.91 - 2.975 %imean = 1.644 + 0.446 PCEs with micronuclei).
- at least 80 % of animals are evaluable

Evaluation of Results:
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 (8)) will be used as an aid in evaluating the results. However, the primary point of consideratrion 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.
Ref.: Krauth, J. (1971). Locally most powerful tied rank test in a Wilcoxon situation Annals of Mathematical Statistics, 42, 1949 - 1956

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg b.w. (maximum guideline-recommended dose)
- Clinical signs of toxicity in test animals: no

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): significant with positive control (substantial increase); not significant with test item (no substantial decrease)
- PCE per 2000 erythrocytes (for Micronucleus assay): 1090 for vehicle; 1050 for test item (500 mg/kg b.w.; 24h sampling); 1094 for test item (1000 mg/kg b.w.; 24h sampling); 1152for test item (1000 mg/kg b.w.; 24h sampling); 1040 for Positive control (40 mg/kg b.w.; 24h sampling); 1022 for test item (2000 mg/kg b.w.; 48h sampling)
- Appropriateness of dose levels and route: yes
- Statistical evaluation: Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.
Not significant with P = 0.0518 for test item at 2000 mg/kg bw; 24h
Not significant with P = 0.5000 for test item at 2000 mg/kg bw; 48h
Significant with P<0.0001 for positive control at 40 mg/kg bw; 24h

Any other information on results incl. tables

PRE-EXPERIMENT FOR TOXICITY

ln a pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 2000 mg/kg b.w. RED RWA 4565 formulated in deionised water. The volume administered was 10 ml/kg b.w..

The animals treated with 2000 mg/kg b.w. expressed toxic reactions as shown in the table:

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

On the basis of these data 2000 mg/kg b.w. were estimated to be suitable.

TOXICITY SYMPTOMS IN THE MAIN EXPERIMENT

ln the main experiment for the highest dose group 24 animals (12 males, 12 females) received orally a single dose of 2000 mg /kg b.w. RED RWA 4565 formulated in deionised water. The volume administered was 10 ml/kg b.w.. The animals treated with 2000 mg /kg b.w. expressed toxic reactions as shown in the table:

	Hours post-treatment male/female
Toxic reactions	1h	2-4h	6h	24h
Reduction of spontaneous activity	8/11	5/6	5/5	1/0
Abdominal position	3/5	0/0	0/0	0/0
Eyelid closure	5/5	0/1	0/0	0/0
Ruffled fur	12/12	12/10	12/9	1/0
Urine colour	-	-	-	red

ln the main experiment for the mid dose group 12 animals (6 males, 6 females) received orally a single dose of 1000 mg /kg b.w. RED RWA 4565 formulated in deionised water.

The volume administered was 10 ml/kg b.w.. The animals treated with 1000 mg /kg b.w. expressed toxic reactions as shown in the table:

	Hours post-treatment male/female
Toxic reactions	1h	2-4h	6h	24h
Reduction of spontaneous activity	2/2	2/3	2/3	0/0
Abdominal position	0/1	0/0	0/0	0/0
Eyelid closure	0/1	0/1	0/1	0/0
Ruffled fur	3/4	4/4	2/4	0/0

ln the main experiment for the mid dose group 12 animals (6 males, 6 females) received orally a single dose of 500 mg /kg b.w. RED RWA 4565 formulated in deionised water.

The volume administered was 10 ml/kg b.w.. The animals treated with 500 mg /kg b.w. expressed toxic reactions as shown in the table:

	Hours post-treatment male/female
Toxic reactions	1h	2-4h	6h	24h
Reduction of spontaneous activity	1/1	1/2	1/2	1/1
Eyelid closure	0/0	0/0	0/0	1/0
Ruffled fur	1/1	1/1	1/2	1/1
Urine colour	-	-	-	Red/pink

SUMMARY OF MICRONUCLEUS TEST RESULTS

Test group	dose mg/kg b.w.	sampling time (h)	PCEs with micronuclei (%)	range	PCE per 2000 erythocytes
Vehicle	0	24	0.025	0-2	1090
Test item	500	24	0.045	0-4	1050
Test item	1000	24	0.015	0-1	1094
Test item	2000	24	0.065	0-3	1152
Positive control	40	24	1.315	10-51	1040
Test item	2000	48	0.030	0-3	1022

 

BIOMETRY

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

Vehicle control versus test group	Significance	p
500 mg RED RWA 4565/kg b.w.;24 h	-	0.3214
1000 mg RED RWA4565/kg b.w.;24h	n.t.	-
2000 mg RED RWA 4565/kg b.w.;24 h	-	0.0518
40 mg CPA/kg b.w.;24 h	+	< 0.0001
2000 mg RED RWA 4565/kg b.w.; 48 h	-	0.5000

n.t. = not tested as the mean micronucleus frequency was not above the vehicle contraol value

- = not significant

+ = significant

Applicant's summary and conclusion

Conclusions:

ln 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, RED RWA 4565 is considered to be non-mutagenic in this micronucleus assay.

Executive summary:

This study was performed to investigate the potential of RED RWA 4565 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 total 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:

- 24h 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 RED RWA 4565 d¡d not exert any cytotoxic effects in the bone marrow. However, the urine of the treated animals had taken over the colour of the test item indicating its systemic distribution and thus its bioavailability.

ln 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.