2-[[4,5-dihydro-3-methyl-5-oxo-1-[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]-1H-pyrazol-4-yl]azo]naphthalene-1,5-disulphonic acid, potassium sodium salt

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Aug - 26 Sept 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Version / remarks:

April 1984

GLP compliance:

yes

Type of assay:

other: chromosome analysis

Test material

Test material information

Constituent 1

Reference substance name:

2-[[4,5-dihydro-3-methyl-5-oxo-1-[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]-1H-pyrazol-4-yl]azo]naphthalene-1,5-disulphonic acid, potassium sodium salt

EC Number:

288-950-4

EC Name:

2-[[4,5-dihydro-3-methyl-5-oxo-1-[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]-1H-pyrazol-4-yl]azo]naphthalene-1,5-disulphonic acid, potassium sodium salt

Cas Number:

85940-63-2

Molecular formula:

C22H17N4Na3O13S4

IUPAC Name:

2-[[4,5-dihydro-3-methyl-5-oxo-1-[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]-1H-pyrazol-4-yl]azo]naphthalene-1,5-disulphonic acid, potassium sodium salt

Test material form:

solid: particulate/powder

Test animals

Species:

hamster, Chinese

Strain:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SAVO med. Versuchstierzuchten GmbH; Kisslegg im Allgäu
- Age at study initiation: 10 - 14 weeks
- Weight at study initiation: male: average 32.6g range 27 - 38g; female: average 30.3g range: 25 - 34g
- Assigned to test groups randomly: [no/yes, under following basis: ] yes; randomization schedule 697 + 698/90
- Housing: in fully air-conditioned rooms in Makolon cages (Type1) on soft wood granulate, one animal per cage
- Diet (e.g. ad libitum): Altromin 7010 hamster diet, ad libitum
- Water (e.g. ad libitum): tap water in plastic bottles, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2°C
- Humidity (%): 55 +/- 10%
- Photoperiod (hrs dark / hrs light): 12h light daily

Administration / exposure

Route of administration:

oral: unspecified

Vehicle:

- Vehicle(s)/solvent(s) used: destiled water
- Concentration of test material in vehicle: 25.0% (w/v) for dose rate 5000 mg/kg bw

Duration of treatment / exposure:

For the dose of 5000 mg/kg bw the administered volume of 20 mL/kg bw was divided into two equal parts of 10ml/kg bw and these were administered within two hours.

Frequency of treatment:

once

Post exposure period:

Killing after 12, 24 and 48 hours

Doses / concentrations

Dose / conc.:

5 000 mg/kg bw (total dose)

No. of animals per sex per dose:

per dose and control: 5 animals per sex and killing time

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide - Endoxan (R)
- Route of administration: oral
- Doses: 50 mg/kg bw
- killing time: 24 h

Examinations

Tissues and cell types examined:

bone marrow

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION:
After killing, both femora were removed and the bones completely stripped of muscle tissue. After removal of the epiphyses, the bone marrow was flushed in alternate directions out of the diaphysis into a centrifuge tube by means of a syringe containing Hanks solution (2 mL/femur) at a temperature of 37°C. This suspension was mixed and centrifuged for 5 minutes at 1000 rpm. All but one drop of the supernatant was drawn off by pipette.
For hypotonic treatment, approximately 5 mL of 0.075 M potassium chloride solution at 37°C was quickly added and suspended. This suspension was then allowed to incubate for 10 minutes in a water bath at 37°C. 1.5 mL fixative (methanol : glacial acetic acid 3 + 1) was added and bubbly mixed with air.
After re-centrifuging for five minutes at 1000 rpm, all but one drop of the supernatant was drawn off by pipette. The sediment was carefully covered with a layer composed of 2.5 mL fixative. After 20 minutes, the fixation was carefully removed with a pipette and suspended in 2.5 mL fixative. After another 30 minutes, the mixture was centrifuged, after which the liquid was removed by pipette and fresh fixative added. The tubes were covered and kept for at least 12 hours (overnight) in a refrigerator at 4°C.
After re-centrifuging for 5 minutes at 1000 rpm, all but one drop of the liquid was removed by pipette and a new suspension formed with a small quantity of freshly prepared fixative. A few drops of this suspension were placed with a pasteur pipette onto clean microscopic slides which had been stored in distilled water at 4°C, the drops were then briefly passed through a Bunsen flame and air-dried for 24 hours. Staining was performed as follows:
- staining for 10 minutes in 2 % orcein solution
- rinsing 3 times in distilled water
- rinsing twice in acetone
- brief rinsing in acetone/xylene
- 2 minutes in acetone/xylene - 5 minutes in xylene
- 10 minutes in xylene
- embedding in EntellanR or EukittR

2-5 slides were prepared from each animal.


METHOD OF ANALYSIS:
After the slides had been coded (coding Scheme 700/9ß), 50 metaphases per animal were examined. The set of chromosomal aberrations was examined for completeness and the various chromosomal aberrations were assessed. Only metaphases with 22 chromosomes are included in the analysis. The metaphases were examined for the following aberrations: gap (g), is-gap (ig), break (b), iso-break (ib), fragment (f), iso-fragment (if), minute (m), iso-minute (im), deletion (d), iso-deletion (id), exchanges including intrachenages (ex), dicentrics (di), chromosome disintegration (cd), ring foramtion (ri) and polyploidy (pp). In addition, metaphases with 5 and more aberrations were classified separately as multiple aberrations (ma).
After the metaphases had been evaluated, the code was lifted. The values from control groups were compared with the results from the dose groups and the positive control.

Evaluation criteria:

The evaluation of the results was performed as follows:
- the test substance is classified as mutagenic if it induces a significantly increased aberration rate as compared with the negative controls with one of the concentrations tested. The significance is obvious either by an enhancement of the rate clearly exceeding the control range or it is proven by adequate biometry (binominal statistic with Fisher's exact test)
- the test substance is classified as mutagenic if there is a concentration related increase in the aberration rate.
- the test substance is classified as non mutagenic, if no chromosomal aberrations were diagnosed.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 and 5000mg/kg bw
- Clinical signs of toxicity in test animals: 2000mg/kg bw: no clinical signs; 5000mg/kg bw: after 24hours urine yellow coloured, after 48 hours no clinical signs
- Other: observation at 24 and 48h; no mortality occurred, each dose: 3 animals per sex

Any other information on results incl. tables

Percentages of metaphases with aberration per trail group (5 animal per group, 50 metaphases per animal)

Trial groupe	Dose [mg/kg bw]	Killing time [hours after admin.]	Methaphases with aberrations inclusive gaps [%]		Methaphases with aberrations exclusive gaps [%]		Mitotic index [arithmetic mean]
			5 male	5 female	5 male	5 female	male	female
negative control	0	12	0.0	0.8	0.0	0.0	8.9	10.1
Remazol Brillantgelb GL FWTR	5000	12	3.2	1.2	0.8	0.4	11.3	11.4
negative control	0	24	1.2	1.2	0.0	0.4	5.6	6.3
Remazol Brillantgelb GL FWTR	5000	24	1.6	0.8	0.4	0.0	9.8	10.9
Endoxan®	50	24	12.0	15.6	11.6	14.8	4.2	4.1
negative control	0	48	0.4	0.4	0.0	0.0	9.0	10.4
Remazol Brillantgelb GL FWTR	5000	48	1.6	1.6	0.0	0.0	7.7	9.5

Applicant's summary and conclusion

Conclusions:

Under given test conditions, the test itm is not mutagenic in the in vivo cytogenetic test using bone marrow cells of Chinese hamster.