Reaction mass ofDisodium [1-{[2-(hydroxy-kO)-3,5-dinitrophenyl]diazenyl-kN1}naphthalen-2-olato(2-)-kO][3-(hydroxy-kO)-4-{[2-(hydroxy-kO)naphthalen-1-yl]diazenyl-kN1}-7-nitronaphthalene-1-sulfonato(3-)]chromate(2-) andDisodium [1-{[2-(hydroxy-kO)-3,5-dinitrophenyl]diazenyl-kN2}naphthalen-2-olato(2-)-kO][3-(hydroxy-kO)-4-{[2-(hydroxy-kO)naphthalen-1-yl]diazenyl-kN1}-7-nitronaphthalene-1-sulfonato(3-)]chromate(2-)

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 - 07 April, 1998; Experimental start date- 05 May 1998; End of Experiment - 23 June, 1998; Study Completion Date - 30 June 1998.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

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:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Disodium [1-[(2-hydroxy-3,5-dinitrophenyl)azo]-2-naphtholato(2-)][3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]-7-nitronaphthalene-1-sulphonato(3-)]chromate(2-)

EC Number:

274-487-5

EC Name:

Disodium [1-[(2-hydroxy-3,5-dinitrophenyl)azo]-2-naphtholato(2-)][3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]-7-nitronaphthalene-1-sulphonato(3-)]chromate(2-)

Cas Number:

70236-55-4

Molecular formula:

C36H18CrN7O13S.2Na

IUPAC Name:

disodium [1-[(2-hydroxy-3,5-dinitrophenyl)azo]-2-naphtholato(2-)][3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]-7-nitronaphthalene-1-sulphonato(3-)]chromate(2-)

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

None

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Identification of the test material as used in the study report: FAT 20037/C
- Source and batch No.of test material: 9300001
- Expiration date of the batch: December 1998
- Purity: approximately 80%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability of the test substance in the solvent/vehicle: at least 24 hours in water, saline, PEG 400, and CMC.

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL, CH-4414 Füllinsdorf
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 36.5 g (mean weight for males); 28.8 g (mean weight for females)
- Assigned to test groups randomly: yes
- Fasting period before study: 18 hours
- Housing: single in Makrolon Type I, with wire mesh top (EHRET GmbH, D-79302 Emmendingen)
- Diet: pelleted standard diet, ad libitum (ALTROMIN 1324, D-32791 Lage/Lippe)
- Water (e.g. ad libitum): tap water, ad libitum, (Gemeindewerke, D-64380 Roßdorf)
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 3 °C
- Humidity: 30-88 %
- Photoperiod: Artificial light 6.00 a.m. - 6.00 p.m.

IN-LIFE DATES: From: 05 May, 1998 To: 23 June, 1998

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Deionised water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: On the day of the experiment, the test article was formulated in deionised water. The vehicle was chosen to its non-toxicity for the animals. All animals received a single standard volume of 10 mL/kg body weight orally.

Duration of treatment / exposure:

once orally (gavage)

Frequency of treatment:

Single dose

Post exposure period:

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control group

Doses / concentrations 2

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

24 hour interval only

Doses / concentrations 3

Dose / conc.:

670 mg/kg bw/day (nominal)

Remarks:

24 hour interval only

Doses / concentrations 4

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

24 h and 48 h each.

No. of animals per sex per dose:

Six males and six females were assigned to each test group.

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive control used: Cyclophosphamide
- Route of administration: orally once
- Doses: 40 mg/kg bw

Examinations

Tissues and cell types examined:

Bone marrow and polychromatic erythrocytes (PCE)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: In a pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 2000 mg/kg b.w. FAT 20037/C formulated in deionised water. The volume administered was 10 mL/kg bw. The dose was well tolerated, hence it was chosen to be the highest dose. Based on the above information, three adequate spaced dose levels extending over a single log range 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: 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 article, the vehicle or the positive control substance once orally (gavage). Twelve animals, six males and six females, were treated per dose group and sampling time. Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively.

DETAILS OF SLIDE PREPARATION: 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.

METHOD OF ANALYSIS: Evaluation of the slides was performed using NIKON microscopes with 100 x 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 the PCEs. The analysis was performed with coded slides.
Five animals per sex and group were evaluated.

Evaluation criteria:

Acceptance Criteria:
The study was considered valid as the following criteria are met:
- the vehicle controls are in the range of our historical control data (0.03 - 0.26 % PCEs with micronuclei).
- the positive controls show substantially increased values
- more than 80 % of animals are évaluable

Evaluation of Results:
- A test article is classified as mutagenic if it induces either a dose-related increase in the number of micronucleated polychromatic erythrocytes or a statistically significant positive response for at least one of the test points.
- A test article producing neither a dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant positive response at any of the test points is considered non-mutagenic in this system.
- This can be confirmed by means of the nonparametric Mann-Whitney test.
- However, both biological and statistical significance should be considered together.

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

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose: 2000 mg/kg bw
- Solubility: soulble in water
- Clinical signs of toxicity in test animals: reduction of spontaneous activity was the only sign observed at 1 and 6 hours post treatment
- High dose with and without activation: 2000 mg/kg bw

Any other information on results incl. tables

Summary of Micronucleus Test Results:

Test Group	Dose mg/kg b.w	sampling time (h)	PCEs with micronuclei (%)	Range	PCE/NCE
Vehicle	0	24	0.090	0-5	2000/1508
Test Article	200	24	0.060	0-3	2000/1533
Test Article	670	24	0.120	0-5	2000/1488
Test Article	2000	24	0.110	0-5	2000/1571
Cyclophosphamide	40	24	1.290	11-37	2000/2108
Test Article	2000	48	0.045	0-2	2000/2252

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
200 mg FAT 20037/C/kg bw; 24 h	n.t.	-
670 mg FAT 20037/C/kg bw; 24 h	-	0.2257
2000 mg FAT 20037/C/kg bw; 24 h	-	0.3341
40 mg CPA/kg bw; 24h	+	< 0.0001
2000 mg FAT 20037/C/kg bw; 48 h	n.t.	-

+ = significant; - not significant n.t. = not tested, as the mean micronucleus frequency was not above the vehicle control value

The mean number of normochromatic erythrocytes was increased after treatment with the test article as compared to the mean value of NCEs of the vehicle controls at preparation interval 48 hours, indicating that FAT 20037/C had cytotoxic properties in the bone marrow.

In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test article. The mean values of micronuclei observed after treatment with FAT 20037/C were near to the range of the vehicle control group.

40 mg/kg b.w. cyclophosphamide administered per os was used as positive control which showed a statistically significant increase of induced micronucleus frequency.

Applicant's summary and conclusion

Conclusions:

FAT 20037/C was considered to be devoid of clastogenic potential in this micronucleus assay.

Executive summary:

A study was performed to investigate the potential of FAT 20037/C to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse according to OECD Guideline 474 and EU Method B.12, in compliance with GLP. The test article was formulated in deionised water. Deionised water was used as vehicle control. The volume administered orally (gavage) was 10 mL/kg bw. 24 h and 48 h after a single administration of the test article the bone marrow cells were collected for micronuclei analysis. 12 animals (6 males, 6 females) per test group were evaluated for the occurrence of micronuclei. At least 2000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 2000 PCE. The following dose levels of the test article were investigated: 24 h preparation interval: 200, 670, and 2000 mg/kg bw. 48 h preparation interval: 2000 mg/kg bw. The highest guideline-recommended dose (2000 mg/kg bw) was estimated by a pre-experiment to be suitable. The animals expressed slight toxic reactions. After treatment with the test article the mean number of NCEs at preparation interval 48 hours was increased as compared to the vehicle controls thus indicating that FAT 20037/C had cytotoxic effectiveness. In comparison to the corresponding vehicle controls there was no statistically significant or biological relevant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test article and with any dose level used. 40 mg/kg bw cyclophosphamide administered per os was used as positive control which showed a statistically significant increase of induced micronucleus frequency. Based on the findings of the study, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, FAT 20037/C was considered to be devoid of clastogenic potential in this micronucleus assay.