Reaction mass of disodium 4-amino-6-[[4-[(2,4-diaminophenyl)diazenyl]phenyl]]-3-((E)-[4-((E)-(2,4-diaminophenyl)diazenyl]phenyl )diazenyl)-5-hydroxynaphthalene-2,7-disulphonate and disodium 4-amino-6-((E)-(4-aminophenyl)diazenyl)-3-((E)-(4-((E)-(2,4-diaminophenyl)diazenyl)phenyl)diazenyl)-5-hydroxynaphthalene-2,7-disulfonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Not mutagenic

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

other: experimentakresult on similar substance

Adequacy of study:

key study

Study period:

1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

Pre-incubation test. Screening program: strain TA98 placed in standard plates. Evaluation without metabolic activation.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 98

Metabolic activation:

without

Metabolic activation system:

No

Test concentrations with justification for top dose:

Range 20-5000 µg/plates: 20, 100, 500, 2500, 5000 µg/plate

Untreated negative controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

NPD

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: 2500-5000 µg/plate

	Revertant/plate			Quotient
Dose µg/plate	- S9	M	DS	-S9
Negative control	29 29 18	25	6	1.0
20	19 13 24	19	6	0.7
100	26 26 26	26	0	1.0
500	31 29 28	29	2	1.2
2500	31 30 24	28	4	1.1
5000	32 31 33	32	1	1.3
Positive control	608 572 666	615	47	24.3

Conclusions:

Negative

Executive summary:

Non mutagen; no increase of revertants has been observed.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

other: experimental resulta on similar substance

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Chinese Hamster ceU line V79, clone 65/3, Dr. D. Wild, Freiburg, Germany

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

no data

Test concentrations with justification for top dose:

In the preliminary toxicity test with and without metabolic activation 12 concentrations of the tested substance were tested. The concentrations selected ranged from 0.24 to 500.0 µg/ml and separated by 2-fold intervals.
In the part with metabolic activation the highest concentration produced an acute growth inhibition of 62.70%. In the part without metabolic activationthe substance exerted a complete growth inhibitory effect at the highest concentration of 500.0 µg/ml. The next lower concentrationsof 250.0 and 125.0 µg/ml revealed an acute growth inhibitory effect of 98.46 and 72.56% respectively.

Accordingly, four concentrations were selected for the original experiment ranging from 18.52 to 500.0 µg/ml and from 4.63 to 125.0 µg/ml in the presence and absence of metabolic activation, respectively.
In the part with metabolic activation, the growth inhibition determined after treatment at the highest concentration of 500.0 µg/ml showed a mean value of 6.91%. After expression growth was inhibited by 13.51%.
In the absence of metabolic activation no significant growth inhibitory effect was seen after treatment and expression.
The highest concentration of 500.0 µg/ml tested in the original experiment with activation was determined to be the highest suitable concentration due to solubility limitations in the vehicle. Therefore the same concentration range was tested in the respective confirmatory experiment, although no severe toxiticy was reached. In the part without metabolic activation a concentration range of 9.26 to 250.0 µg/ml was selected for the confirmatory experiment in order to reach a more pronounced toxicity at the highest concentration. In the presence of metabolic activation no significant acute cytotoxicity was obtained at the highest concentration. After expression growth was inhibited by 9.54%.
In the part without activation, the mean growth inhibition at the highest concentration of 250.0 µg/ml was 27.91% in spite of the increased concentration.

Vehicle / solvent:

No data

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

not specified

Evaluation criteria:

Cytotoxicity is determined my measuring the relative cloning efficiency (survival) of the cultures after the treatment period.
Mutant frequency is determined by seeding known numbers of cells in medium containing the selective agent to detect mutant cells, and in medium without selective agent to determine the cloning efficiency (viability).
After the incubation time, colonies are counted. The mutant frequency is derived from the number of mutant colonies in selective medium and the number of colonies in non-selective medium.

Statistics:

No data

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: strain/cell type: cell line V79, clone 65/3

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation
negative with metabolic activation

Based on the results of the performed experiments and under the given experimental conditions, it is concluded that the tested substance and its metabolites did not show any mutagenic activity in this forward mutation system.

Executive summary:

Direct Black 19 was tested for mutagenicity, in details for the evalutation of properties to induce gene mutations.

The mutagenicity studies were conducted following OECD 476, gene mutation test with Chinese Hamster cell V79.

The studies were performed in the absence and in the presence of metabolic activation. A dose range with different doses was used. The tested substance shows no mutagenic activity.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Not mutagenic

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

other: experimental resulta on similar substance

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Mollegard Deutshland
- Number: 85 (50 male and 35 female)
- Age at study initiation: between 6 and 8 weeks
- Weight at study initiation: 27-48 g for male, 23-37 g for female
- Housing: mouse were housed in Macrolon Type 2 cages in optimal hygienic conditions.
- Diet: Granular Altromin N 1326
- Water: municipal water ad libitum
- Acclimation period:at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 55 ± 10 %
- Air changes (per hr): 10 times/h
- Photoperiod: 12 h light / 12 h dark

Route of administration:

oral: gavage

Vehicle:

Acqueous solution of ultra - amylopectin (0.7%) with the addiction of two drops of Tween 80 (polysorbate) per 10 ml of solution.

Details on exposure:

single oral administration for the tested item
single oral administration for the negative control
single intraperitoneal application for the positive control

Duration of treatment / exposure:

one single application

Frequency of treatment:

single oral administration for the tested item
single oral administration for the negative control
single intraperitoneal application for the positive control

Post exposure period:

24 h and 48 h

Remarks:

Doses / Concentrations:
1600 mg/kg
Basis:
nominal in water

Remarks:

Doses / Concentrations:
800 mg/kg
Basis:
nominal in water

Remarks:

Doses / Concentrations:
400 mg/kg
Basis:
nominal in water

Remarks:

Doses / Concentrations:
1200 mg/kg
Basis:
nominal in water

No. of animals per sex per dose:

Dose 1600 mg/kg: Observation time: 48 h, 5 male and 5 female
Dose 1600 mg/kg: Observation time: 24 h, 5 male and 5 female
Dose 800 mg/kg: Observation time: 24 h, 5 male and 5 female
Dose 400 mg/kg: Observation time: 24 h, 5 male and 5 female
Dose 1200 mg/kg: Observation time: 24 h, 5 male and 5 female

Positive control: Dose 80 mg/kg, Observation time: 24 h, 5 male and 5 female

Control animals:

yes

Positive control(s):

Name: cyclophosphamide
Supplier: SIGMA CHMIE Gmbh
- Route of administration: intraperitoneal
- Doses / concentrations: 80 mg/kg
- Application volume: 0.1 ml/10 g body weight
- Solvent: water for injection

The preparations of the positive control substance were freshly prepared before administration.

Tissues and cell types examined:

The cells suspension was obtained from the extraction of bone marrow from the medullary canal of femurs.

Details of tissue and slide preparation:

The cells suspension was treated and the specimens colored following Pappenheimer's method.
2000 polychromatic erythrocytes per animal were examined microscopically in order to determine the presence and the number o micronuclei.

Evaluation criteria:

The classification of micronuclei was performed using the following criteria:
- Micronuclei are round, rarely oval or crescent shaped.
- They have a sharp contour.
- They are uniformly colored and are approximately from 1/20 to 1/5 of the diameter of the erythrocytes.
- It is usually only a micronucleus present.
The ratio of polychromatic to normochromatic erythrocytes was determined individually for each animal by counting a total of 1000 erythrocytes.

Statistics:

The statistics of the test were riported in the study report.
The calculations were performed using the program MUTAI (SOP M/EDV/036) performed on a 386 AT DIGICOM. The program is written in PowerBASIC and is extensively tested.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

No signs of systemic intoxication were observed in female mice for the sigle oral doses of 1600, 800 and 400 mg/kg.
The highest dose of 1600 mg/kg causes the death of 2 male mice within 24h. Conseguently the dose was lowered to 1200 mg/kg. The other doses of 800 and 400 mg/kg were tolerate without symptoms.

The results show no citotoxic effect of any chromosomal damages.

Conclusions:

Interpretation of results (migrated information): negative
No cytotoxic effect of the test substance were observed.
No potential of chromosomal damages were observed.

Executive summary:

This end point was assessed following the OECD 474 and it was performed in GLP conditions. Micronuclei arise as a result of damage to the chromosomes or the spindle apparatus through a mutagenic active substance during the mitotic cell division in proliferating bone marrow cells. The results show no increased incidence of micronucleated polychromatic erythrocytes

The calculated frequency of the micronuleated polychromatic erythrocytes of the groups treated with the substance was between 0,17 and 0,42 %.The observed frequency corresponds to the data reported in the literature which is related to the spontaneous rate of occurrence of micronuclei in polychromatic erythrocytes.

Hence a potential of chromosomal damages or damage to the mitotic apparatus could be excluded for the tested substance.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Not mutagenic based on available data both in vitro and in vivo.