REAKTIV-GELB F-68 072 FW

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:

19 April 1993 to 19 May 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

lithium sodium 2-amino-4-{[4-(cyanoazanidyl)-6-[(3-sulfonatophenyl)amino]-1,3,5-triazin-2-yl]amino}-5-(2-{4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl)benzene-1-sulfonate

EC Number:

413-090-5

Cas Number:

189574-45-6

Molecular formula:

Hill formula: C24H18LixN10NayO12S4

IUPAC Name:

lithium sodium 2-amino-4-{[4-(cyanoazanidyl)-6-[(3-sulfonatophenyl)amino]-1,3,5-triazin-2-yl]amino}-5-(2-{4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl)benzene-1-sulfonate

Test material form:

solid: particulate/powder

Details on test material:

See below

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species: NMRI mouse
Strain: Hoe: NMRKf (SPF71)
Origin: HOECHST AG, Kastengrund, SPF breeding colony
Initial age at test: 7 weeks
Number of animals: 70 (35 males / 35 females)
Bodyweight at start of study: males: x= 28.5 g (23 - 32 g) females: x= 23.7 g (21 - 27 g)
Acclimatization: at least 5 days
Food / water: rat/mice diet Altromin 1324 (Altromin-GmbH,Lage/Lippe), ad libitum
tap water in plastic bottles, ad libitum
Housing: in fully air-conditioned rooms in Macrolon cages (Type 3), on softwood granulate in groups of 5 animals

Room temperature: 20 ± 3 ⁰C
Relative humidity: approx. 30%
Lighting time: 12 hours daily
Animal identification: fur-marking with KMnO4 and cage numbering

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

The test compound dilutions were freshly prepared at the day of administration. 5000 mg Reaktiv-Gelb F-68 072 Pi were weight in a mortar, ground well, suspended with starch mucilage, washed out in a 25 ml flask and topped up to the calibration mark. A suspension was formed.

Details on exposure:

The test compound was suspended in starch mucilage and dosed once orally at 2000 mg per kg bodyweight to male and female mice

Duration of treatment / exposure:

24, 48 and 72 hours

Frequency of treatment:

Single dose

Post exposure period:

None

Doses / concentrations

Remarks:

Doses / Concentrations:
2000 mg/kg body weight
Basis:
actual ingested

No. of animals per sex per dose:

Group Dose (mg/kg bwt.) Conc. (%) (w/v) Volume (ml/kg bwt.) Number of animals and sex Killing-time (hours p.a.)
1 0 0 10 5 males/5 females 24
3 2000 20.0 10 5 males/5 females 24
5* 50 0.5 10 5 males/5 females 24
1 0 0 10 5 males/5 females 48
3 2000 20.0 10 5 males/5 females 48
1 0 0 10 5 males/5 females 72
3 2000 20.0 10 5 males/5 females 72
* : Endoxan® (positive control)
Hours p.a. : hours after administration

Control animals:

yes

Positive control(s):

POSITIVE CONTROL: Cyclophosphamide-Endoxan®

For the Endoxan® stock solution, 5 ml distilled water were added to 100 mg Endaxan®in an injection phial and shaken to form a clear solution. The solutions for administration were prepared from this stock solution. For this purpose, 2 ml of the 2 % stock solution were mixed with 6 ml distilled water.

Examinations

Tissues and cell types examined:

polychromatic erythrocytes

Details of tissue and slide preparation:

Extraction of the bone marrow
In conformity with the test procedure the animals were killed by carbon dioxide asphyxiation 24, 48 or 72 hours after application. For each animal, about 3 ml foetal bovine serum was poured into a centrifuge tube. Both femora were removed and the bones freed of muscle tissue. The proximal ends of the femora were opened and the bone marrow flushed into the centrifuge tube. A suspension was formed. The mixture was then centrifuged for 5 minutes at approx. 1000 rpm and almost all the supernatant discarded. One drop of the thoroughly mixed sediment was smeared on a cleaned slide, identified by project code and animal number and air-dried for about 12 hours.

Staining procedure
5 minutes in methanol
5 minutes in May-Grunwalds solution
brief rinsing twice in distilled water
10 minutes staining in 1 part Giemsa solution to 6 parts buffer solution, pH 7.2 (Heise)
rinsing in distilled water
drying
coating with Entellan

Evaluation
1000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded, not the number of individual micronuclei. As a control measure 1000 mature erythrocytes were also counted and examined for micronuclei. In addition, the ratio of polychromatic to normochromatic erythrocytes was determined. All bone marrow smears for evaluation are coded to ensure that the group which they belonged to remains unknown to the investigator. The number of polychromatic erythrocytes with micronuclei occurring in the 1000 polychromatic erythrocytes counted, and the number of normocytes with micro-nuclei occurring in the 1000 normocytes counted, were evaluated statistically.

Evaluation criteria:

Criteria for a positive response
Both biological and statistical significances are considered together in evaluation.
A substance is considered as positive if there is a significant increase in the number of micronucleated polychromatic erythrocytes for at least one of the time points. A test substance producing no significant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.

Statistics:

A Wilcoxon-Test (one-sided) [7,8] is evaluated to check the validity of the study. This is done by comparing the number of polychromatic erythrocytes with micronuclei in the positive control with the negative control.

A Wilcoxon-Test (one-sided) [7,8] is calculated for each measurement group (24h, 48h, 72h) and for polychromatic and normochromatic erythrocytes. These tests are performed sequentially with a multiple level of significance of 5% [5]. Tests on lower dose groups are only performed if the higher dose group is significantly different from the control [6].

The presupposition to make any of the fol1owing comparisons is a difference between the positive control and the negative control (24h). This is tested with a Wilcoxon-Test (two-sided) [7,8] with 5%-level of significance. Wilcoxon-Tests (two-sided) are performed sequentially for the ratio of polychromatic erythrocytes for each measurement group (24h, 48h, 72h) at a multiple level of significance of 5% [5]. Lower dose groups are only tested 1f the higher dose group is significantly different from the control [6]. Actual data were also compared with historical controls.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Mice were treated with 2000 mg Reaktiv-Gelb F-68 072 FW per kg bodyweight to study the induction of micronuclei in bone marrow cells.

All animals survived after application of Reaktiv-Gelb F-68 072 FW. Only orange coloured faeces were observed within 3 hours after application.

The dissection of the animals revealed no test substance related findings.

The bone marrow smears were examined for the occurance of micronuclei in red blood cells.

The incidence of micronucleated polychromatic and normochromatic erythrocytes in the dose groups of Reaktiv-Gelb F-68 072 FW was within the normal range of the negative control groups. No statistically significant increase of micronucleated polychromatic erythrocytes has been observed. The number of normochromatic erythrocytes with micronuclei did not differ from the values of the simultaneous control animals for each of the three killing times investigated. The ratio of polychromatic erythrocytes to normocytes remained essentially unaffected by the test compound.

Cyclophosphamide (Endoxan®) induced a marked and statistically significant increase of the number of polychromatic erythrocytes with micronuclei in both males and females indicating the sensitivity of the test system.

Summarizing it can be stated that administration of Reaktiv-Gelb F-68 072 FW did not lead to a substantial increase of micronucleated polychromatic erythrocytes and is not mutagenic in the micronucleus test.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
The results indicate that, under the conditions of the present study, Reaktiv-Gelb F-68 072 FW is not mutagenic in the micronucleus test.

Executive summary:

This test was performed according to EU test guidance 84/449/EEC and OECD test guideline 474. No unforeseen circumstances were observed, which may have affected the quality and integrity of this study. The study was conducted in compliance with the principles of Good Laboratory Practice (GLP).

 

Reaktiv-Gelb F-68 072 FWwas tested inthemicronucleus test. The test compound was suspended in starch mucilage and dosed once orally at 2000 mg per kg bodyweight to male and female mice, upon the results of the previously conducted dose range finding assay. According to the test procedure the animals were killed 24, 48 or 72 hours after administration.

 

Endoxan®was used as positive control substance and was administered orally at a dose of 50 mg per kg bodyweight.

 

The number of polychromatic and normochromatic erythrocytes containing micro-nuclei was not increased. The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained unaffected by the treatment with Reaktiv-GelbF-68 072 FW and was statistically not different from the control values.

 

Endoxan®induced in both males and females a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the system. The ratio of polychromatic erythrocytes to normocytes was not changed to a significant extent.

 

The results indicate that, under the conditions of the present study, Reaktiv-Gelb F-68 072 FW is not mutagenic in the micronucleus test.