Benzenesulfonic acid, 2-[2-[5-(aminocarbonyl)-1-ethyl-1,6-dihydro-2-hydroxy-4-methyl-6-oxo-3-pyridinyl]diazenyl]-4-[[4-chloro-6-[[4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]amino]-1,3,5-triazin-2-yl]amino]-, sodium salt (1:2)

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:

19 April 1993 to 19 May 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to OECD test guidance in compliance with GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

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

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= 29.1 g (24 - 34 g)
females: x= 23.5 g (20 - 27 g)
Acclimatisation: 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 °C
Relative humidity: approx. 30%
Lighting time: 12 hours daily
Animal identification: fur-marking with KMn04 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-66 923 FW were weight in a beaker, mixed with starch mucilage, washed out in a 25 ml flask and topped up to the calibration mark. A solution was formed.

Details on exposure:

2 dose groups (0 and 2000 mg/kg bwt.) and 1 positive control group

Duration of treatment / exposure:

Up to 72 hours exposure

Frequency of treatment:

Once

Post exposure period:

None

Doses / concentrations

Remarks:

Doses / Concentrations:
2000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

Group Dose (mg/kg bw) Conc. (%) (w/v) Volume (ml/kg bw) Number of animals and sex Killing time (hours p.a.)
1 0 0 10 5 males 24
5 females
4 2000 20.0 10 5 males 24
5 females
5* 50 0.5 10 5 males 24
5 females
1 0 0 10 5 males 48
5 females
4 2000 20.0 10 5 males 48
5 females
1 0 0 10 5 males 72
5 females
4 2000 20.0 10 5 males 72
5 females
*: Endoxan® (positive control)
Hours p.a.: hours after administration

Control animals:

yes

Positive control(s):

Endoxan® (50 mg/kg bodyweight p.o.)

For the Endoxan®stock solution, 5 ml distilled water were added to 100 mg Endoxan® 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:

Rationale for dose selection
Oral administration of 2000 mg Reaktiv-Gelb F-66 923 FY kg bodyweight did not cause any toxic effects in male and female mice. This dose level was selected as the limit dose for the main study.

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 1M 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:

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:

No data

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:

All animals survived after application of Reaktiv-Gelb F-66 923 FW. No signs of toxicity were observed.
The dissection of the animals revealed no test substance related macroscopic 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-66 923 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 nonnochromatlc 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.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Summarising it can be stated that administration of Reaktiv-Gelb F-66 923 FW did not lead to a substantial increase of micronucleated polychromatic erythrocytes and is not mutagenic in the micronucleus test.

Executive summary:

The study was performed according to the OECD guideline No. 474 and was conducted in compliance with the principles of Good Laboratory Practice (GLP).

Reaktiv-Gelb F-66 923 FW was tested in the micronucleus test. The test compound was dissolved in starch mucilage and dosed once orally at 2000 mg per kg body-weight 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 micronuclei was not increased. The ratio of polychromatic/normochromatlc erythrocytes in both male and female animals remained unaffected by the treatment with Reaktiv-Gelb F-66 923 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-66 923 FW is not mutagenic in the micronucleus test.