Disodium [N-[7-hydroxy-8-[(2-hydroxy-5-mesylphenyl)azo]-1-naphthyl]acetamidato(2-)][2-hydroxy-3-[(2-hydroxy-1-naphthyl)azo]-5-nitrobenzenesulphonato(3-)]chromate(2-)

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Justification for read across is detailed in the report attached to the IUCLID section 13.

Cross-reference

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Justification for read across is detailed in the report attached to the IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

None

GLP compliance:

yes

Type of assay:

mammalian germ cell cytogenetic assay

Specific details on test material used for the study:

None

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

None

Sex:

male/female

Details on test animals or test system and environmental conditions:

Strain: NMRISource: BRL, CH-4414 FüllinsdorfNumber of Animals: 72 (36 males/36 females)Initial Age at Start of Acclimatization: 8-12 weeksAcclimatization: minimum 5 daysInitial Body Weight at Start of Treatment: males mean value 31.5 g (SD ± 3.2 g)females mean value 25.1 g (SD ± 1.8 g)

Route of administration:

oral: gavage

Vehicle:

Vehicle: Deionised water

Details on exposure:

It is generally recommended to use the maximum tolerated dose or the highest dose that canbe formulated and administered reproducibly or 2000 mg/kg as the upper limit for non-toxictest articles.The maximum tolerated dose level is determined to be the dose that causes toxic reactionswithout having major effects on survival within 48 hours.The volume to be administered should be compatible with physiological space available.Three adequate spaced dose levels extending over a single log range were applied at thecentral sampling interval 24 h after treatment. For the highest dose level an additionalsample was taken at 48 h after treatment.

Duration of treatment / exposure:

48 hours

Frequency of treatment:

once

Post exposure period:

None

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

6 animals per sex per dose

Control animals:

yes

Positive control(s):

Cyclophosphamide; - Justification for choice of positive control(s): recommended by the guidelines - Route of administration: oral - Doses : 10 mg/kg b.w.- Frequency: once

Tissues and cell types examined:

The animals were sacrificed by cervical dislocation: The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 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-Griinwald (MERCK, D-64293 Darmstadt)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-79110 Freiburg). At least one slide was made from each bone marrow sample.

Details of tissue and slide preparation:

Analysis of Cells:Evaluation of the slides was performed using NIKON microscopes with lOOx oil immersion objectives. At least 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 the same sample and expressed in normochromatic erythrocytes per 1000 the PCEs. The analysis was performed with coded slides.Five animals per sex and group were evaluated as described. The remaining 6th animal of each test group was evaluated in case an animal had died in its test group.

Evaluation criteria:

The study is considered valid if the following criteria are met:- the vehicle controls are in the range of our historical control data (0.03 - 0.26 % PCEs with micronuclei).- the positive controls show substantially increased values- more than 80 % of animals are évaluable

Statistics:

None

Key result

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:

None

None

Conclusions:

non-mutagenic in in vivo micronucleus assay.

Executive summary:

An in vivo study was performed to investigate the potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test article was formulated in deionised water . Deionised water was used as vehicle control. The volume administered orally was 20 ml/kg b.w.. 24 h and 48 h after a single administration of the test article 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 article 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 levels of the test article were investigated: 24 h preparation interval: 200, 670 and 2000 mg/kg b.w.. 48 h preparation interval: 2000 mg/kg b.w.. The highest guideline-recommended dose (2000 mg/kg) was estimated by a pre-experiment to be suitable. None of the animals expressed toxic reactions.

After treatment with the highest dose of test article the number of NCEs was increased as compared to the corresponding vehicle controls thus indicating that the Similar substance 01 had cytotoxic effectiveness in the bone marrow. In comparison to the corresponding vehicle controls there was no significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test article and with any dose level used. 40 mg/kg b.w. cyclophosphamide administered per os was used as positive control which showed a statistically significant increase of induced micronucleus frequency.

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the Similar substance 01 is considered to be non-mutagenic in this micronucleus assay.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

None

GLP compliance:

yes

Type of assay:

mammalian germ cell cytogenetic assay

Test material

Test material information

Constituent 1

Reference substance name:

Similar substance 01 of Acid Black 213

IUPAC Name:

Similar substance 01 of Acid Black 213

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

None

Test animals

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

None

Sex:

male/female

Details on test animals or test system and environmental conditions:

Strain: NMRISource: BRL, CH-4414 FüllinsdorfNumber of Animals: 72 (36 males/36 females)Initial Age at Start of Acclimatization: 8-12 weeksAcclimatization: minimum 5 daysInitial Body Weight at Start of Treatment: males mean value 31.5 g (SD ± 3.2 g)females mean value 25.1 g (SD ± 1.8 g)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Vehicle: Deionised water

Details on exposure:

It is generally recommended to use the maximum tolerated dose or the highest dose that canbe formulated and administered reproducibly or 2000 mg/kg as the upper limit for non-toxictest articles.The maximum tolerated dose level is determined to be the dose that causes toxic reactionswithout having major effects on survival within 48 hours.The volume to be administered should be compatible with physiological space available.Three adequate spaced dose levels extending over a single log range were applied at thecentral sampling interval 24 h after treatment. For the highest dose level an additionalsample was taken at 48 h after treatment.

Duration of treatment / exposure:

48 hours

Frequency of treatment:

once

Post exposure period:

None

Doses / concentrations

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

6 animals per sex per dose

Control animals:

yes

Positive control(s):

Cyclophosphamide; - Justification for choice of positive control(s): recommended by the guidelines - Route of administration: oral - Doses : 10 mg/kg b.w.- Frequency: once

Examinations

Tissues and cell types examined:

The animals were sacrificed by cervical dislocation: The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 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-Griinwald (MERCK, D-64293 Darmstadt)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-79110 Freiburg). At least one slide was made from each bone marrow sample.

Details of tissue and slide preparation:

Analysis of Cells:Evaluation of the slides was performed using NIKON microscopes with lOOx oil immersion objectives. At least 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 the same sample and expressed in normochromatic erythrocytes per 1000 the PCEs. The analysis was performed with coded slides.Five animals per sex and group were evaluated as described. The remaining 6th animal of each test group was evaluated in case an animal had died in its test group.

Evaluation criteria:

The study is considered valid if the following criteria are met:- the vehicle controls are in the range of our historical control data (0.03 - 0.26 % PCEs with micronuclei).- the positive controls show substantially increased values- more than 80 % of animals are évaluable

Statistics:

None

Results and discussion

Test results

Key result

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:

None

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:

non-mutagenic in in vivo micronucleus assay.

Executive summary:

An in vivo study was performed to investigate the potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test article was formulated in deionised water . Deionised water was used as vehicle control. The volume administered orally was 20 ml/kg b.w.. 24 h and 48 h after a single administration of the test article 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 article 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 levels of the test article were investigated: 24 h preparation interval: 200, 670 and 2000 mg/kg b.w.. 48 h preparation interval: 2000 mg/kg b.w.. The highest guideline-recommended dose (2000 mg/kg) was estimated by a pre-experiment to be suitable. None of the animals expressed toxic reactions.

After treatment with the highest dose of test article the number of NCEs was increased as compared to the corresponding vehicle controls thus indicating that the Similar substance 01 had cytotoxic effectiveness in the bone marrow. In comparison to the corresponding vehicle controls there was no significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test article and with any dose level used. 40 mg/kg b.w. cyclophosphamide administered per os was used as positive control which showed a statistically significant increase of induced micronucleus frequency.

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the Similar substance 01 is considered to be non-mutagenic in this micronucleus assay.