5-(benzoylamino)-4-hydroxy-3-[[1-sulpho-6-[[2-(sulphooxy)ethyl]sulphonyl]-2-naphthyl]azo]naphthalene-2,7-disulphonic acid, sodium salt

Genetic toxicity: in vivo

Administrative data

Endpoint:

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

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: 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)

GLP compliance:

yes

Type of assay:

chromosome aberration assay

Test material

Test material information

Constituent 1

Reference substance name:

5-(benzoylamino)-4-hydroxy-3-[[1-sulpho-6-[[2-(sulphooxy)ethyl]sulphonyl]-2-naphthyl]azo]naphthalene-2,7-disulphonic acid, sodium salt

EC Number:

287-842-4

EC Name:

5-(benzoylamino)-4-hydroxy-3-[[1-sulpho-6-[[2-(sulphooxy)ethyl]sulphonyl]-2-naphthyl]azo]naphthalene-2,7-disulphonic acid, sodium salt

Cas Number:

85586-40-9

Molecular formula:

C29H19N3Na4O17S5 C29H23N3O17S5.xNa

IUPAC Name:

5-(Benzoylamino)-4-hydroxy-3-((1-sulpho-6-((2-(sulphooxy)ethyl)sulphonyl)-2-naphthyl)azo)naphthalene-2,7-disulphonic acid, sodium salt

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Remazol Brillantrot F3B FWTR

Test animals

Species:

hamster, Chinese

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SAVO med. Versuchstierzuchten GmbH, 7964 Kisslegg im Allgau
- Age at study initiation: 10 - 14 weeks
- Weight at study initiation: males: mean = 31.6 g (26 - 37 g)
females: mean = 29.5 (24 - 34 g)
- Assigned to test groups randomly: yes, computer based randomization scheme
- Fasting period before study: no data
- Housing: one animal per cage
- Diet: Altromin 7010 hamster diet (Altromin GmbH, Lage/lippe), ad libitum
- Water: tap water in plastic bottles ad libitum
- Acclimation period:at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24
- Humidity (%): 45 to 65
- Air changes (per hr): -
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 08. Aug to 26. Sep 1990

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: destilled water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: 25% (w/v) in water
Dosing volume: 20 mL/kg bw divided in two portions 2 hours apart

Duration of treatment / exposure:

single dose divided in two portions 2 hours apart

Frequency of treatment:

day one

Post exposure period:

12, 24 and 48 hours

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
5000 mg/kg body weight
Basis:
nominal in water

Doses / concentrations 2

Remarks:

Doses / Concentrations:
250 mg/mL
Basis:
nominal conc.

No. of animals per sex per dose:

5 males and 5 females per killing time point
Positive control: 5 males and 5 females

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide (Endoxan)
- Justification for choice of positive control(s): availability of historical data
- Route of administration: oral
- Doses / concentrations: 50 mg/kg body weight (5 mg/mL)

Examinations

Tissues and cell types examined:

bone marrow cells from femur: 50 metaphases/animal

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: MTD based on preliminary study


TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The test substance was administered orally by gavage to the test animals at a single dose of 5000 mg/kg bodyweight. Sesame oil was administered in the same way to the negative control group. A positive control group, introduced after exactly 24 hours to run parallel with the negative control and the dose groups, received Endoxan at an oral dose of 50 mg/kg bodyweight. Two hours before killing by carbon dioxide asphyxation (12, 24, or 48 hours after treatment ), the hamsters each received an intraperitoneal injection of 3.3 mg demecolcin (Colcemid) per kg bodyweight.

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 the temperature of 37°C, mixed and centrifuged for 5 minutes at 1000 rpm after which all but a few drops of the supernatant was drawn off by pipette and sediment resuspended by tapping.
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. Addition of 1.5 mL fixative (methanol : glacial acetic acid 3 + 1) and flow through with air. After re-centrifugation 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 removed carefully 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 Entellan or Eukitt


METHOD OF ANALYSIS:

After the slides had been coded, 50 metaphases per animal were examined. The set of chromosomes was examined for completeness and the various chromosomal aberrations were assessed. The chromosomal aberrations were classified. The metaphases were examined for the following aberrations: gap (g), break (b), fragment (f), minute (m), deletion (d), exchanges including intrachanges (ex), dicentricity (di), chromosome disintegration (cd) ring (ri) and polyploidy (pp). In addition, metaphases with 5 and more aberrations were classified separatly as multiple aberrations (ma).
After the metaphases had been evaluated, the code was lifted. The values for the control group were compared at each killing time 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 chromsomal aberrations were diagnosed.

Statistics:

Comparison of no of aberrations of treated and control groups by binominal statistic with Fisher's exact test - was not necessary as all mean chromosome aberration rates after treatment with the test article were in the range of the negative control values.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

24h after treatment 2 males and 1 female out of 5 animals died before demecolcin-injection. These animals were replaced and the replacements survived until scheduled study end.
The following signs of toxicity were observed after treated with 5000 mg per kg bwt.: urin rose coloured, faeces red coloured, dyschromia of the skin.
The dissection of the animals revealed the following macroscopic findings: red tissue, intestinum, stomage.

A slight indication of cytotoxicity as reduction of the mitotic index was observed for females in the 12 hour group and for males and females in the 48 hour group as compared with the negative controls.
The positive control substance EndoxanR was administered in a dose of 50 mg/kg bodyweight and produced a marked increase in the aberration rate among the animals killed after 24 hours as compared with the values for the negative controls. Endoxan caused aberrations inclusive gaps in 12.0 - 15.6 % of the metaphases and exclusive gaps in 11.6 - 14.8 % of the metaphases. The positive control substance shows various types of aberrations and several aberrations per metaphase. So the sensitivity of the test system was demonstrated by the enhanced mutation frequency.

Summarizing, it can be stated that, under the conditions described, administration of Remazol-Brillantrot F3B FWTR did not lead to a substantial increase of chromosome aberrations.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
The test substance is not mutagenic in the in vivo chromosome aberration test in bone marrow cells of the Chinese hamster.

Executive summary:

The test substance was administered orally by gavage in a single dose of 5000 mg/kg bodyweight to male and female Chinese hamsters. This dose had been shown in a preliminary study to be the maximum tolerated dose.

A positive control group, induced exactly 24 hours later to run parallel with the negative control and the dose group, received Endoxan in an oral dose of 50 mg/kg bodyweight.

Animals from each group were killed 12, 24 and 48 hours after treatment by carbon dioxide asphyxiation. 5 males and 5 females from each group were killed at each of these times.

The bone marrow obtained from femora of the animals was prepared, placed on microscopic slides and stained, after which 50 metaphases per animal were evaluated. The completeness in the number of chromosomes and the various chromatic and chromosomal aberrations were assessed.

Under the conditions of the present study, the test substance caused no significant increase in the aberration rate in the bone marrow cells of the treated animals as compared with the control group.

Endoxan however produced a marked increase in the aberration rate in the test animals.

The results indicate that, under the conditions of the present study, the test substance is not mutagenic in the in vivo chromosome aberration test in bone marrow cells of the Chinese hamster.