3,10-diamino-6,13-dichloro-2-((6-(((4-(1,1-dimethylethyl)phenyl)sulfonyl)amino)-2-naphthalenyl)sulfonyl)-4,11-triphenodioxazinedisulfonic acid, lithium potassium sodium salt

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:

2002 April, 26 to 2002 August, 09

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:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted July 21, 1997

GLP compliance:

yes

Type of assay:

other: in vivo mammalian somatic cell / structural and numerical chromosome aberration

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

440-770-9

EC Name:

-

Cas Number:

371921-63-0

Molecular formula:

C38 H29 Cl2 N5 O12 S4 .x K .x Li .x Na

IUPAC Name:

3,10-diamino-2-{[6-(4-tert-butylbenzenesulfonamido)naphthalen-2-yl]sulfonyl}-6,13-dichloro-5,12-dioxa-7,14-diazapentacene-4,11-disulfonic acid lithium hydride potassium hydride sodium hydride

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 35.0 g (SD ± 3.1 g); females mean value 26.9 g (SD ± 2.3 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 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 4
- Humidity (%): 40-50
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: unspecified

Vehicle:

- Vehicle used: corn oil
- Justification for choice of solvent/vehicle:
- 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 animais per sex under identical conditions as in the mutagenicity study concerning: starvation period, animal strain; vehicle; route, frequency, and volume of administration.
The animais 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
lt 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 animais received no food but water ad libitum. At the beginning of the treatment the animais (including the contrais) were weighed and the individual volume to be administered was adjusted to the animais body weight. The animais received the test item, the vehicle or the positive contrai substance once. Twelve animais, six males and six females, were treated per dose group and sampling time. The animais of the highest dose group were examined for acute toxic symptoms at intervals of around 1 h, 2-4h, 6 h and 24 h after administration of the test item.

Duration of treatment / exposure:

Acute exposure

Frequency of treatment:

1 treatment/animal

Post exposure period:

24 and 48h

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

No. of animals per sex per dose:

10 (5 males and 5 females)

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
- Doses / concentrations: 10 ml/kg b.w. & 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.

Details of tissue and slide preparation:

Preparation of the Animais:
The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were eut 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). Caver slips were mounted with EUKITI (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 normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 2000 PCEs. The analysis was performed with coded slides.
Ten animais (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 contrai. The micro­ nucleated cells per 2000 PCEs and the ratio of polychromatic to normochromatic erythrocytes are presented for each animal.

Evaluation criteria:

Acceptance criteria
The study was considered valid as the following criteria are met:
a) The negative contrais are in the range of our historical contrai data (0.02 - 0.15 %; mean = 0.086 ± 0.032 PCEs with micronuclei).
b) The positive contrais are in the range of our historical contrai data (1.0 - 2.71 %; mean = 1.678 ± 0.479 PCEs with micronuclei).
c) At least 80 % of animais 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.
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 methods (nonparametric Mann-Whitney test; Krauth J, 1971) will be used as an aid in evaluating the results. However, the primary point of consideratrion is the biological relevance of the results.

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:

yes

Remarks:

toxic reactions to mouse at dose of 2000 mg/kg b.w. (but no cytotoxic to bone marrow)

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: yes

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): significant with positive control; not significant with test item
- Ratio of PCE/NCE (for Micronucleus assay): 2000/1590 for vehicle; 2000/1646 for test item (500 mg/kg b.w.; 24h sampling); 2000/1809 for test item (1000 mg/kg b.w.; 24h sampling); 2000/1809 for test item (1000 mg/kg b.w.; 24h sampling); 2000/1786 for Positive control (40 mg/kg b.w.; 24h sampling); 2000/1873 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 = 0.1494 for test item at 2000 mg/kg bw; 24h
Not significant with P = 0.4133 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 forToxicity

ln a pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 2000 mg/kg b.w. BLUE GS 5664.80 formulated in corn oil. The volume administered was 10 ml/kg b.w.

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

 

toxicreactions	hours post-treatment male / female
	1 h	2-4 h	6h	24 h	30 h	48 h
reduction of spontaneous activity	2/2	2/2	1/1	0/0	0/0	0/0
eyelid closure	1/1	1/1	0/0	0/0	0/0	0/0
ruffled fur	2/2	2/2	1/1	0/0	0/0	0/0

 

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

 

Toxic symptoms in the Main Experiment

ln the main experiment for the highest dose group 24 animais (12 males, 12 females) received orally a single dose of 2000 mg /kg b.w. BLUE GS 5664.80 formulated in corn oil. The volume administered was 10 ml/kg b.w.

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

 

toxicreactions	hours post-treatment male / female
	1 h	2-4 h	6h	24 h
reduction of spontaneous activity	12/12	12/12	12/12	12/12
abdominal position	8/8	8/7	9/8	4/4
eyelid closure	8/8	8/8	8/7	3/3
ruffled fur	8/7	8/6	7/4	4/1
apathy	8/9	8/7	5/3	3/2

 

Summary of Micronucleus Test Results

 

 

test group	dose mg/kg b.w.	sampling time (h)	PCEs with micronuclei (%)	range	PCE/	NCE
vehicle	0	24	0.055	0-3	2000/	1590
test item	500	24	0.050	0-2	2000/	1646
test item	1000	24	0.045	0-4	2000/	1809
test item	2000	24	0.090	0-4	2000/	1676
cycle-phosphamide	40	24	1.235	9-54	2000/	1786
test item	2000	48	0.065	0-3	2000/	1873

 

Statistical significance

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 BLUE GS 5664.80/kg b.w.; 24 h	n.t.	-
1000 mg BLUE GS 5664.80/kg b.w.; 24 h	n.t.	-
2000 mg BLUE GS 5664.80/kg b.w.; 24 h	-	0.1494
40 mg CPA/kg b.w.; 24 h	+	<0.0001
2000 mg BLUE GS 5664.80/kg b.w.; 48 h	-	0.4133

n.t. = not tested as the mean micronucleus frequency was not above the vehicle contrai 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, BLUE GS 5664.80 is considered to be non-mutagenic in this micronucleus assay.

Executive summary:

This study was performed to investigate the potential of BLUE GS 5664.80 to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse in a test following OECD 474 and GLP.

The test item was formulated in corn oil, 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 animais (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 (NCE) was determined in the same sample and reported as the number of NCEs per 2000 PCEs.

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

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 NCEs was not substantially increased as compared to the mean value of NCEs of the vehicle contrai thus indicating that BLUE GS 5664.80 did not exert any cytotoxic effects in the bone marrow.

ln comparison to the corresponding vehicle contrais 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.

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, BLUE GS 5664.80 is considered to be non-mutagenic in this micronucleus assay.