Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
10 April 2002 to 30 April 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to EU, OECD & US EPA test guidance in compliance with GLP. The read across substance, Reaktiv Gelb BZK5934 is considered to be chemically equivalent and analogous.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Reaktiv Gelb BZK 5934
- Chemical name: 7-{4-[4-Chloro-6-(3-ethenesulfonylphenylamino)-[ 1,3,5] triazin-2-ylamino]-2-ureido-phenylazo}-naphthalene-1,3,6-trisulfonic acid sodium salt.
- Molecular formula (if other than submission substance): C28H29ClN9Na3O12S4
- Molecular weight (if other than submission substance): 906.2
- Smiles notation (if other than submission substance): C1=C(S(=O)(=O)O[Na])C2C=C(C(=CC=2C=C1S(=O)(=O)O[Na])S(=O)(=O)O[Na])N=NC(C1)=C(C=C(C=1)NC(=NC(Cl)=N1)N=C1NC(C=C(C1)S(=O)(=O)C=C)=CC=1)NC(N)=O
- InChl (if other than submission substance): InChI=1S/C28H22ClN9O12S4.3Na/c1-2-51(40,41)17-5-3-4-15(10-17)31-27-34-25(29)35-28(36-27)32-16-6-7-20(21(11-16)33-26(30)39)37-38-22-13-19-14(9-24(22)54(48,49)50)8-18(52(42,43)44)12-23(19)53(45,46)47;;;/h2-13H,1H2,(H3,30,33,39)(H,42,43,44)(H,45,46,47)(H,48,49,50)(H2,31,32,34,35,36);;;/q;3*+1/p-3/b38-37+;;;
- Structural formula attached as image file (if other than submission substance): see "Illustrations"
- Physical state: Orange powder
- Analytical purity: 61.7%
- Purity test date: 04-Dec-2002
- Lot/batch No.: BZK 5934
- Expiration date of the lot/batch: Sep-2007
- Stability under test conditions: Stability and homogeneity in the vehicle: is acceptable for 28 days in deionised water (HPLC method)
- Storage condition of test material: at approximately 20 deg C in a fume cupboard

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Species of animals: rat
Strain of animals: Hsd:Sprague Dawley
Origin (supplier) of animals: Harlan Winkelmann GmbH Gartenstrasse 27, 33178 Borchen
Animal identification: fur marking with KMn04 and cage numbering
Body weight at start of study: male animals
mean = 189.8 g (= 100 %)
min = 178 g (-6.2%)
max = 202 g (+6.4%)
n = 15
female animals
mean = 152.7 g (= 100 %)
min = 148 g (-3.1%)
max = 162 g (+6.1%)
n = 15
Age at start of study: male animals approximately 6 weeks, female animals approximately 6 weeks
Randomisation procedure: Randomisation schemes 2003.0198 and 2003.0199
Animal maintenance: five animals per cage in transparent macrolon cages (type IV) on soft wood granulate in an air-conditioned room (room number 011)
Room temperature: 22 °C ± 3 °C (except short lasting deviations due to disturbances of air condition)
Relative humidity: 50 % ± 20 % (except short lasting deviations due to disturbances of air condition)
Lighting times: 12 hours light / dark cycle
Acclimatisation: 5 days under study conditions
Food: rat/mice diet ssniff® R/M-H (V 1534), ad libitum ssniff® GmbH, Postbox 2039, 59480 Soest
Water:tap water in plastic bottles, ad libitum

Administration / exposure

Route of administration:
oral: unspecified
Vehicle:
- Vehicle(s)/solvent(s) used:Deionised water
- Justification for choice of solvent/vehicle: Historical use within the laboratory.
- Concentration of test material in vehicle: 200 mg/ml
Details on exposure:
Frequency of administrations: two doses separated by an interval of 24 hours (except positive control with only one dose).
Duration of treatment / exposure:
24 hours
Frequency of treatment:
Two doses
Post exposure period:
No post exposure period
Doses / concentrations
Remarks:
Doses / Concentrations:
2000 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
Group Dose Concentration Volume No. of animals Animal No. Killing time
(mg/kg bwt.) (mg/ml) (ml/kg bwt.) and sex (hours p.a.)
1 0 0 10 5 males 1 – 5 24
5 females 6 -10
2 2000 200 10 5 males 11 – 15 24
5 females 16 – 20
3* 40 4.0 10 5 males 21 – 25 24
5 females 26 – 30

* = positive control: Endoxan® (batch no. 9M642A) containing cyclophosphamide, dissolved in distilled water
Hours p.a. = hours after administration
Control animals:
yes, concurrent vehicle
Positive control(s):
Name or number of compound (I.N.N. orU.S.A.N.): cyclophosphamide
Synonyms: Endoxan®
Chemical name: C7H15CI2N2O2P - H20
CAS-Register number: 6055-19-2
Supplier of reference compound: ASTA Medica AG WeismiillerstraBe 45 D-60314 Frankfurt Germany
Batch number: 2K645B
Certificate of analysis: certified by the supplier, Quality Control, Dr. Hofmann dated 10-Dec-2002

Examinations

Tissues and cell types examined:
chromosomal damage (clastogenicity) in a rat bone marrow
Details of tissue and slide preparation:
PREPARATION AND STAINING

Extraction of the bone marrow
Animals were killed by carbon dioxide asphyxiation 24 hours after second dosing. One femoral bone was removed and the bone freed of muscle tissue. The proximal end of the femoral bone was opened, the bone marrow flushed into a centrifuge tube containing about 3 ml of fetal bovine serum and a suspension was prepared. The mixture was then centrifuged for 5 minutes at approx. 1200 rpm, after which almost all the supernatant was discarded. One drop of the thoroughly mixed sediment was smeared onto a cleaned slide, identified by project code and animal number and air-dried for about 12 hours.

Subsequently the slides were stained as follows:
Staining procedure
5 minutes in methanol
5 minutes in May-Grunwald's solution
brief rinsing twice in distilled water
10 minutes staining in 1 part Giemsa solution to 6 parts buffer solution, pH 7.2 (Weise)
rinsing in distilled water drying
coating with Entellan®
Evaluation criteria:
SCORING AND EVALUATION OF DATA

Scoring
2000 polychromatic erythrocytes were counted for each animal. The number of cells with micro-nuclei was recorded, not the number of individual micronuclei. In addition, the ratio of polychro-matic erythrocytes to 200 normochromatic erythrocytes was determined. Main parameter for the statistical analysis, i.e. validity assessment of the study and mutagenicity of the test substance, was the proportion of polychromatic erythrocytes with micronuclei out of the 2000 counted erythrocytes. All bone marrow smears for evaluation were coded to ensure that the group from which they were taken remained unknown to the investigator.

Criteria for assay validity
An one-sided Wilcoxon-Test was performed to check the validity of the study. The study was considered as valid if the proportion of polychromatic erythrocytes with micronuclei in the positive control was significantly higher than in the negative control (p<0.05).

Data analysis
Assuming the study is valid based on a monotone-dose-relationship, one-sided Wilcoxon tests were performed initially comparing control values with those of the highest dose group. A significance level of 5% is adopted for all tests.

CRITERIA FOR A POSITIVE RESPONSE
Both biological and statistical significances were considered together for evaluation purposes.
A substance is considered as positive if there is a significant or dose-related increase in the number of micronucleated polychromatic erythrocytes compared with the concurrent negative control group. A test substance producing no significant or dose-related increase in the number of micronucleated polychromatic erythrocytes is considered as non-clastogenic in this system.
Statistics:
As detailed above under "Evaluation Criteria".

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:
Rats were treated twice at an interval of 24 hours with 2000 mg Reaktiv Gelb BZK 5934 per kg body weight to study the induction of micronuclei in bone marrow cells.

All animals survived after treatment. No signs of toxicity were observed in the main study.

The dissection of the animals revealed no test substance related macroscopic findings.
The bone marrow smears were examined for the occurrence of micronuclei in red blood cells.

A very slight increase of micronucleated polychromatic erythrocytes (mean value) was observed in the animals treated with 2000 mg/kg of the test compound. However, the incidence of micronucleated polychromatic erythrocytes in this dose group was within the historical range of the negative control groups (mean of micronucleated polychromatic erythrocytes per 2000 cells: 1.7-4.9). Moreover, the increase was not statistically significant. It was therefore considered as incidental finding without biological relevance.

The ratio of polychromatic erythrocytes to total erythrocytes remained essentially unaffected by the test compound and differed less than 20% from the control values.

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

Any other information on results incl. tables

SUMMARY TABLES AND STATISTICS

 

Test compound: Reaktiv Gelb BZK 5934

 

Sex

Dose

mg/kg b.w.

Killing time

Number of animals

Poly / animal counted

Poly/Ery

 

Mean

Poly/Ery SD Mean

Poly with MN

 

Mean

Poly with MN [%]

Mean

Poly with MN SD

Mean

Male

0 – Control

24 h

5

2000

0.48

0.08

2.4

0.12

0.08

Male

2000

24 h

5

2000

0.48

0.04

2.6

0.18

0.04

Male

40 – Endoxan

24 h

5

2000

0.43

0.05

28.2*

1.41

0.70

Female

0 – Control

24 h

5

2000

0.50

0.04

2.4

0.12

0.08

Female

2000

24 h

5

2000

0.47

0.04

3.4

0.17

0.10

Female

40 – Endoxan

24 h

5

2000

0.40

0.05

18.8*

0.94

0.24

 

Sex

Dose

mg/kg b.w.

Killing time

Number of animals

Poly / animal counted

Poly/Ery

 

Mean

Poly/Ery SD Mean

Poly with MN

 

Mean

Poly with MN [%]

Mean

Poly with MN SD

Mean

Mut. I.

Pooled

0 – Control

24 h

10

2000

0.49

0.06

2.40

0.1

0.07

1.0

Pooled

2000

24 h

10

2000

0.48

0.04

3.50

0.2

0.07

1.5

Pooled

40 – Endoxan

24 h

10

2000

0.42

0.05

23.5*

1.2

0.55

9.8

 

Mut. I. = Mutagenic Index

Control = Vehicle (deionised water)

* = Significantly different from control (p < 0.05)

 

A cross comparison of individual data and pooled data may show discrepancies since the values are rounded.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The results lead to the conclusion that Reaktiv Gelb BZK 5934 did not cause a substantial increase in micronucleated polychromatic erythrocytes and is not clastogenic in the micronucleus test in vivo under the conditions described in this report.
Executive summary:

The micronucleus test was carried out with Reaktiv Gelb BZK 5934 (Batch No. BZK5934). The test compound was suspended in deionised water and was given twice at an interval of 24 hours as oral doses of 2000 mg per kg body weight to male and female rats (Hsd:Sprague Dawley), based on the results of a previous dose range finding assay. Endoxan® was used as positive control substance and was administered once orally at a dose of 40 mg per kg body weight.

 

The number of polychromatic erythrocytes containing micronuclei in all dose groups was not significantly increased compared with the control. The ratio of polychromatic erythrocytes to total erythrocytes in both male and female animals remained unaffected by the treatment with Reaktiv Gelb BZK 5934 and differed less than 20% from the control value.

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

 

Under the conditions of the present study the results indicate that Reaktiv Gelb BZK 5934 is not clastogenic in the micronucleus test in vivo.