A 2:1:1 mixture of: trisodium N(1')-N(2):N(1''')-N(2'')-η-6-[2-amino-4-(or 6)-hydroxy-(or 4-amino-2-hydroxy)phenylazo]-6''-(1-carbaniloyl-2-hydroxyprop-1-enylazo)-5',5'''-disulfamoyl-3,3''-disulfonatobis(naphthalene-2,1'-azobenzene-1,2'-diolato-O(1),O(2'))-chromate; trisodium N(1')-N(2):N(1''')-N(2'')-η-6,6''-bis(1-carbaniloyl-2-hydroxyprop-1-enylazo)-5',5'''-disulfamoyl-3,3''-disulfonatobis(naphthalene-2,1'azobenzene-1,2'-diolato-O(1),O(2'))-chromate; trisodium N(1')-N(2):N(1''')-N(2'')-η-6,6''-bis[2-amino-4-(or 6)-hydroxy-(or 4-amino-2-hydroxy)phenylazo]5',5'''-disulfamoyl-3,3''-disulfonatobis(naphthalene-2,1'azobenzene-1,2'-diolato-O(1),O(2'))-chromate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 02. Nov. to 30. Nov. 1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1983

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

A 2:1:1 mixture of: trisodium N(1')-N(2):N(1''')-N(2'')-η-6-[2-amino-4-(or 6)-hydroxy-(or 4-amino-2-hydroxy)phenylazo]-6''-(1-carbaniloyl-2-hydroxyprop-1-enylazo)-5',5'''-disulfamoyl-3,3''-disulfonatobis(naphthalene-2,1'-azobenzene-1,2'-diolato-O(1),O(2'))-chromate; trisodium N(1')-N(2):N(1''')-N(2'')-η-6,6''-bis(1-carbaniloyl-2-hydroxyprop-1-enylazo)-5',5'''-disulfamoyl-3,3''-disulfonatobis(naphthalene-2,1'azobenzene-1,2'-diolato-O(1),O(2'))-chromate; trisodium N(1')-N(2):N(1''')-N(2'')-η-6,6''-bis[2-amino-4-(or 6)-hydroxy-(or 4-amino-2-hydroxy)phenylazo]5',5'''-disulfamoyl-3,3''-disulfonatobis(naphthalene-2,1'azobenzene-1,2'-diolato-O(1),O(2'))-chromate

EC Number:

402-850-1

EC Name:

A 2:1:1 mixture of: trisodium N(1')-N(2):N(1''')-N(2'')-η-6-[2-amino-4-(or 6)-hydroxy-(or 4-amino-2-hydroxy)phenylazo]-6''-(1-carbaniloyl-2-hydroxyprop-1-enylazo)-5',5'''-disulfamoyl-3,3''-disulfonatobis(naphthalene-2,1'-azobenzene-1,2'-diolato-O(1),O(2'))-chromate; trisodium N(1')-N(2):N(1''')-N(2'')-η-6,6''-bis(1-carbaniloyl-2-hydroxyprop-1-enylazo)-5',5'''-disulfamoyl-3,3''-disulfonatobis(naphthalene-2,1'azobenzene-1,2'-diolato-O(1),O(2'))-chromate; trisodium N(1')-N(2):N(1''')-N(2'')-η-6,6''-bis[2-amino-4-(or 6)-hydroxy-(or 4-amino-2-hydroxy)phenylazo]5',5'''-disulfamoyl-3,3''-disulfonatobis(naphthalene-2,1'azobenzene-1,2'-diolato-O(1),O(2'))-chromate

Molecular formula:

not applicable for UVCB substance

IUPAC Name:

trichromium(3+) nonasodium 6-[2-(2-amino-4-hydroxyphenyl)diazen-1-yl]-2-[2-(2-oxido-5-sulfamoylphenyl)diazen-1-yl]-3-sulfonatonaphthalen-1-olate 6-[2-(2-amino-6-hydroxyphenyl)diazen-1-yl]-2-[2-(2-oxido-5-sulfamoylphenyl)diazen-1-yl]-3-sulfonatonaphthalen-1-olate 6-[2-(4-amino-2-hydroxyphenyl)diazen-1-yl]-2-[2-(2-oxido-5-sulfamoylphenyl)diazen-1-yl]-3-sulfonatonaphthalen-1-olate tris(6-{2-[(1Z)-2-hydroxy-1-(phenylcarbamoyl)prop-1-en-1-yl]diazen-1-yl}-2-[2-(2-oxido-5-sulfamoylphenyl)diazen-1-yl]-3-sulfonatonaphthalen-1-olate)

Test material form:

solid: particulate/powder

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Zentralinstitut für Versuchstierzucht, 3000 Hannover, DE
- Age at study initiation: minimum 10 weeks
- Weight at study initiation: approximately 30 g
- Assigned to test groups: randomly
- Fasting period before study: 18 hours
- Housing: singularly in Makrolon Type I cages with wire mesh top (EBCO, 4620 Castrop-Rauxel, DE) with granulated soft wood bedding (ALTROMIN, 4937 Lage/Lippe, DE)
- Diet: ad libitum; pelleted standard diet (ALTROMIN, 4937 Lage/Lippe, DE)
- Water: ad libitum; tap water (Südhessische Gas- und Wasser AG, 6100 Darmstadt, DE)
- Acclimation period: at least 5 days after assessment for healthy condition

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 3 °C
- Humidity: not regulated
- Air changes: not reported
- Photoperiod (hrs dark / hrs light): artificial light 06:00 to 18:00

JUSTIFICATION FOR TEST ANIMAL SELECTION
The mouse is an animal which has been used for many years as suitable experimental animal in cytogenetic investigations. There are many data available from such investigations which may be helpful in the interpretation of results from the micronucleus test. In addition, the mouse is an experimental animal in many physiological, pharmacological and toxicological studies. Data from such experiments also may be useful for the design and the performance of the micronucleus test.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle: aqua dest.
- Justification for choice of vehicle: the vehicle was chosen to its nontoxicity for the animals
- Dose level: 2000 mg/kg bw
- Justification for dose: the dose used in this study was the maximum attainable dose
- Volume in vehicle: 20 ml/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Preparation: dissolved in vehicle on the day of the experiment

Duration of treatment / exposure:

Single administration

Frequency of treatment:

Single administration

Post exposure period:

Group 1: 24 hours
Group 2: 48 hours
Group 3: 72 hours

No. of animals per sex per dose:

Total number of animals: 84 (42 males and 42 females)
Test Group 1: 12 animals (6 males and 6 females); sampled 24 hours after dosing
Test Group 2: 12 animals (6 males and 6 females); sampled 48 hours after dosing
Test Group 3: 12 animals (6 males and 6 females); sampled 72 hours after dosing
Negative Control Group 1: 12 animals (6 males and 6 females); sampled 24 hours after dosing
Negative Control Group 2: 12 animals (6 males and 6 females); sampled 48 hours after dosing
Negative Control Group 3: 12 animals (6 males and 6 females); sampled 72 hours after dosing
Positive Control Group 1: 12 animals (6 males and 6 females); sampled 24 hours after dosing

Control animals:

yes, concurrent vehicle

Positive control(s):

- Substance: cyclophosphamide (CPA)
- Source: SERVA, 6900 Heidelberg, DE
- Justification for choice of positive control:
- Route of administration: oral gavage
- Concentration: 30 mg/kg bw
- Vehicle: physiological saline
- Volume: 10 ml/kg bw

Examinations

Tissues and cell types examined:

Polychromatic erythrocytes (PCE) of the NMRI mouse femor bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: it is generally recommended to use the máximum tolerated dose or the highest dose that can be formulated and administered reproducibly. The volume to be administered should be compatible with the physiological space available. A preliminary study on acute toxicity was performed with the same strain and under identical conditions as in the mutagenicity study.

TREATMENT AND SAMPLING TIMES: approximately 18 hours before treatment with the test article the animals received no food but water ad libitum. At the beginning of the treatment, the animals were weighed and the individual volume to be administered was adjusted to the animal's body weight. The animals received the test article once. Twelve animals, six males and six females, were treated per dose group. Sampling of the bone marrow was done 24, 48 and 72 hours after treatment. The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with 2.0 ml fetal calf serum, using a 5 ml syringe, into 1 ml fetal calf serum.

DETAILS OF SLIDE PREPARATION: the cell suspension was centrifuged at 1000 rpm for 5 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald/Giemsa. Cover slips were mounted with EUKITT (KINDLER, 7800 Freiburg, DE). At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS: evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. 1000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect, the ratio between polychromatic and normochromatic erythrocytes was determined in same sample and expressed in normochromatic erythrocytes per 1000 PCEs. The analysis was performed with coded slides.

OTHER: five animals per sex and group were evaluated as described. The remaining animal of each test group was evaluated in case an animal had died in its test group spontaneously or due to gavage error.

Evaluation criteria:

The frequencies of micronucleated PCEs in the groups treated with the test article are compared with their corresponding negative controls. This is achieved by means of the non-parametric Mann-Whitney test. Positive responses are those in which an increase of micronucleated PCEs can be confirmed statistically significant at the five per cent level (p < 0.05). However, both biological and statistical significance should be considered together in the evaluation.
To describe a cytotoxic effect due to the treatment with the test item, the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reportad as the number of NCE per 1000 PCE.

Statistics:

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Animals: 16 total (2 males and 2 females per dose level)
- Dosing: single dose by oral gavage
- Dose range: 750, 1000, 1500 and 2000 mg/kg bw
- Vehicle: in aqua dest.
- Volume in vehicle: 20 ml/kg bw
- Rationale for exposure: based on findings of preliminary toxicity study; limited by application volume in rodents
- Harvest times: 24, 48 and 72 hours after exposure
- Clinical signs of toxicity in test animals:
- At 750 mg/kg bw, none of the animals expressed toxic reations.
- At 1000 mg/kg bw, the animals expressed toxic reactions: reduction of spontaneous activity, eyelid closure, apathy.
- At 1500 mg/kg bw, the animals expressed toxic reactions: reduction of spontaneous activity, eyelid closure, apathy, dyspnoea, and one male died within 24 h after application, though propably due to a gavage error.
- At 2000 mg/kg bw, the animals expressed toxic reactions: reduction of spontaneous activity, eyelid closure, apathy, dyspnoea; additionally, the males expressed pilo erection.
- Conclusions: higher dosing was not attainable as appropriate solutions could be obtained only up to 100 mg/ml, and application volumes higher than 20 ml/kg bw were not justifiable for the rodents used. Based on this preliminary experiment, a dose of 2000 mg/kg bw was selected as an appropriate dose for the main study.

RESULTS OF MAIN STUDY
- Compared to negative controls, no substantial enhancement in detectad micronuclei frequency was detected at any preparation interval after application of the test item.
- An appropriate reference mutagen was used as positive control which showed a distinct increase of induced micronucleus frequency.
- During the study described and under the experimental conditions reportad, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

Applicant's summary and conclusion

Conclusions:

The substance is not considered mutagenic in this micronucleus assay.

Executive summary:

Potential to induce micronuclei in polychromatic erythrocytes in the femoral bone marrow of the mouse was evaluated in an in vivo experimental study according to the OECD Guideline 474 (1983).

The test item was dissolved in aqua dest.. This solvent was used as negative control. The volume administered orally was 20 ml/kg b.w.. 24 h, 48 h and 72 h after a single application of the test item, the bone marrow cells were collected for micronuclei analysis.

Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei.

To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 1000 PCE.

The following dose level of the test article was investigated: 24 h, 48 h, and 72 h preparation interval: 2000 mg/kg b.w.

In a pre-experiment this dose level was estimated to be the maximum attainable dose. The animals expressed toxic reactions.

In addition, after treatment with the test article the ratio between PCEs and NCEs was shifted as compared to the corresponding negative controls at preparation interval 24 h and 48 h, thus, indicating cytotoxic effects. In comparison with the corresponding negative controls there was no substantial enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test item.

An appropriate reference mutagen was used as positive control which showed a distinct increase of induced micronucleus frequency.

In conclusion, it can be stated that during the study described and under the experimental conditions reportad, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the substance is not considered mutagenic in this micronucleus assay.