Sodium 6-amino-5-[[2-[(cyclohexylmethylamino)sulphonyl]phenyl]azo]-4-hydroxynaphthalene-2-sulphonate

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:

experimental study

Adequacy of study:

key study

Study period:

1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

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

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 6-amino-5[2-(2-chlorophenoxy)-5-chlorophenylazo]-4- hydroxy- naphthalene-2-sulphonate

Cas Number:

103241-64-1

Molecular formula:

Hill formula: C22 H14 Cl2 N3 Na O5 S

IUPAC Name:

Sodium 6-amino-5[2-(2-chlorophenoxy)-5-chlorophenylazo]-4- hydroxy- naphthalene-2-sulphonate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Specific details on test material used for the study:

Name: FAT 20'070/C
Batch No.: UO-Vers. 1310/87
Aggregate State at RT: solid
Colour: dark brown
Stability: Pure: stable for 12 months. In vehicle: stable in DMSO and DMF for > 8 hours
Storage: room temperature / light protected
Expiration date: March 1992

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

Housing: Single
Cage Type: Makrolon Type I, with wire mesh top (EBECO, D-4620 Castrop-Rauxel, F.R.G.)
Bedding :granulated soft wood bedding (ALTROMIN, D-4937 Lage/Lippe, F.R.G.)
Feed: pelleted standard diet (ALTROMIN, D-4937 Lage/Lippe, F.R.G.)
Water : tap water, ad libitum (Südhessische Gas- und Wasser AG, D-6100 Darmstadt)
Environment : temperature 21 +/- 3 °C; relative humidity not regulated; artificial light 6.00 a.m. - 6.00 p.m.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

The vehicle of the test article was used as negative control.
Name: Dimethylsulfoxide (DMSO)
Supplier: MERCK, D-6100 Darmstadt, F.R.G,

Details on exposure:

Approximately 18 hours before treatment with the test article the animals received no food but water ad libitum. At the beginning of the treatment the animals were weighed and the individual volume to be administered was adjusted to the animal's body weight. The animals received the test article once. Twelve animals, six males and six females, were treated per dose group. Sampling of the bone marrow was done 24, 48 and 72 hours after treatment.

Frequency of treatment:

Once

Post exposure period:

24, 48 and 72 hours

Doses / concentrations

Remarks:

Doses / Concentrations:
3000 mg/kg bw.
Basis:
nominal conc.

No. of animals per sex per dose:

5 males / 5 females

Control animals:

yes, concurrent vehicle

Positive control(s):

Name: CPA; Cyclophosphamide
Supplier: SERVA, D-6900 Heidelberg, F.R.G.
Catalogue no.: 17681
Dissolved in: physiological saline
Dosing: 30 mg/kg b.w.
Route and Frequency of Administration: orally, singly
Volume Administered: 10 ml/kg b.w.

Examinations

Tissues and cell types examined:

Polychromatic erythrocytes (PCE) in the bone marrow

Details of tissue and slide preparation:

Preparation of the Animals :
The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with 2.0 ml fetal calf serum, using a 5 ml syringe, into 1 ml fetal calf serum. The cell suspension was centrifuged at 1,000 rpm for 5 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/Giemsa. Cover slips were mounted with EUKITT (KINDLER,D-7800 Freiburg F.R.G.). At least one slide was made from eachbone marrow sample.

Analysis of Cells:
Evaluation of the slides was performed using NIKON microscopes with lOOx oil immersion objectives. 1,000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in same sample and expressed in normochromatic erythrocytes per 1000 PCEs. The analysis was performed with coded slides. Five animals per sex and group were evaluated as described. The remaining animal of each test group was evaluated in case an animal had died in its test group spontaneously or due to gavage error.

Evaluation criteria:

The frequencies of micronucleated PCEs in the groups treated with the test article are compared with their corresponding negative controls. This is achieved by means of the nonparametric Mann- Whitney test (6) .
Positive responses are those in which an increase of micronucleated PCEs can be confirmed statistically significant at the five per cent level (p < 0.05).
However, both biological and statistical significance should be considered together in the evaluation.

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:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

Test Group	Dose (mg/kg bw)	Time	PCEs with micronuclei	Range	PCE/NCE
Solvent	0	24	0.12%	0-3	1000/600
Solvent	0	48	0.05%	0-2	1000/508
Solvent	0	72	0.10%	0-2	1000/547
CPA	30	24	1.07%	4-20	1000/693
Test Article	3000	24	0.11%	0-3	1000/522
Test Article	3000	48	0.10%	0-2	1000/551
Test Article	3000	72	0.10%	0-3	1000/399

Applicant's summary and conclusion

Conclusions:

FAT 20'070/C is considered to be not clastogenic in this micronucleus assay.

Executive summary:

This study was performed to investigate the potential of FAT 20'070/C to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. This study was performed according to OECD test guideline 474. The test article was dissolved in DMSO which served as a negative control. The volume administered orally was 5 ml/kg bw. 24 h, 48 h and 72 h after a single application of the test article 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. 1000 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 1000 PCE. The following dose level of the test article was investigated: 24 h, 48 h, and 72 h preparation interval: 3000 mg/kg bw. In a pre-experiment this dose level was estimated to be the maximum attainable dose. The animals expressed toxic reactions. After treatment with the test article the ratio between PCEs and NCEs was not affected as compared to the corresponding negative controls thus indicating that FAT 20'070/C had no cytotoxic properties.

In comparison with the corresponding negative controls, there was no substantial enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. An appropriate reference mutagen was used as positive control which showed a distinct increase of induced micronucleus frequency.

Hence, 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 20'070/C is considered to be not clastogenic in this micronucleus assay.