Sodium 6-[(4-aminophenyl)azo]-5-hydroxynaphthalene-1-sulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

reductive (Prival) metabolic activation system

Test concentrations with justification for top dose:

Highest dose tested: 5000 μg/plate unless limited by cytotoxicity or solubility

Species / strain:

other: all strains

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

other: read across from similar substance

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, test procedure in accordance with OECD 476 methodes, meets generally accepted scientific principles, acceptable for assessment and GLP compliant. Justification for Read Across will be provided in section 13.

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 activation the substance exerted a complete growth inhibitory effect at the highest concentration of 500.0 µg/ml. The next lower concentrations of 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 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:

negative

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:

The substance was tested for mutagenicity 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.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

other: experimental result on similar substance

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, test procedure in accordance with OECD 471 methods, meets generally accepted scientific principles, acceptable for assessment and GLP compliant. The test has been performed on only four Salmonella strains.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Salmonella typhimurium and Escheria coli

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from rats

Test concentrations with justification for top dose:

For both mutagenicity test the treatment-levels are with and without S9 mix: from 0.005 to 5000 µg/plate

Vehicle / solvent:

water for injections (Batch EVB03A, Frenesius Kabi, Sevres, France)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA100 an TA1535 with and without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA 97 and TA1535 with and wihtout S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine (NPD)

Remarks:

TA98 without S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
S9 mix: The S9 mix consists of induced enzymatic systems contained in rat liver post-mitochondrial fraction (S9 fraction) and the cofactors necessary for their function.

Evaluation criteria:

A substance was considered positive when the number of revertants above background was at least twice the value of the historical control mean or twice the value of the current control mean, whichever was greater and a dose response curve could be generated.

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Negative
The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the tested strains.

Executive summary:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore the tested substance is considered to be non-mutagenic in this Bacterial reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

other: read across from similar substance

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The test has been performed following official guidelines and in accordance to OECD474, that completely assesses the end point. The study is well documented. GLP compliant with certificate. Justification for Read Across will be provided in section 13.

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.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

chromosomal aberration

Type of information:

other: read across from similar substance

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Guideline:

other: NTP protocol

Principles of method if other than guideline:

Mouse Bone marrow micronucleus test employs 1 to 3 treatment of the chemical under study, administered at 24hour intervals. Normally five male animals per treatment (dose) group. Doses extend up to the maximum tolerated dose. The route of administration in these short-term tests is usually either intraperitoneal injection or oral gavage. Based on the cell cycle and maturation times of the erythrocytes, harvesting of the bone marrow usually occurs 24 hours after the last dosing; this interval is indicated in the data tables as "sample collection time."
At that time, about 50% of the erythrocytes in the bone marrow are immature, newly formed erythrocytes, and these are the cell types that are checked for presence of micronuclei. The animals are euthanized by CO2 inhalation and the femurs are removed.

GLP compliance:

no

Type of assay:

micronucleus assay

Species:

mouse

Strain:

B6C3F1

Sex:

male

Route of administration:

oral: gavage

Vehicle:

phosphate buffer saline

Duration of treatment / exposure:

72h

Frequency of treatment:

3 treatments at 24 hour intervals

Post exposure period:

24 hours

Remarks:

Doses / Concentrations:
39.02, 78.125, 156.25, 312.5, 625, 1250 and 2500 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

5 males per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: gavage
- Doses / concentrations: 50 mg/kg

Tissues and cell types examined:

polychromatic erytrocytes

Details of tissue and slide preparation:

The bone marrow is flushed from the femurs and spread onto slides. The slides are air-dried, fixed, and stained with a fluorescent DNA-specific stain that easily illuminates any micronuclei that may be present.
Typically, 2000 polychromatic erythrocytes (PCEs, reticulocytes; immature erythrocytes) are scored per animal for frequency of micronucleated cells in each of 5 animals per dose group. In addition, the percentage of PCEs among the total erythrocyte population in the bone marrow is scored for each dose group as an indicator of chemical-induced toxicity.

In non-treated healthy mice and rats, the %PCE in bone marrow is usually around 50-60%. If a chemical interferes with the production of erythrocytes in the bone marrow, then the %PCE in the bone marrow may decline from the typical normal level. Conversely, if erythrocyte production is stimulated by chemical exposure, then a higher percentage of immature erythrocytes may be observed.

Statistics:

A formal statistical analysis of the data is performed that includes a trend test, to determine if there is an overall increase across all doses in the frequency of cells containing micronuclei, and a pairwise comparison of each dose group to the corresponding control, to see if any one dose group is statistically
different from the control group in frequency of micronucleated cells.
Data are typically presented as the mean number of micronucleated cells per 1,000 cells for each treatment group. A positive trend test is one in which the P value is equal to or less than 0.025. For the slide-based micronucleus data, the micronucleus frequency in any dose group is considered significantly elevated over the control group if the P value is equal to or less than 0.025 divided by the number of chemical-treatment groups.

Thus, if the number of treated groups is 3, then the required pairwise P value is 0.008. This adjustment in the pairwise P value is a correction for multiple comparisons of the same data. In the short-term studies, tests that give positive results are repeated to confirm the response.

Sex:

male

Genotoxicity:

negative

Remarks:

trend P = 0.370

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

negative

Executive summary:

The substance was tested for Micronucleus assay (short term bone marrow) on male mice. The results show that no micronucleated cells are generated after exposure to the substance by oral gavage for 72 hours.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Bacterial reverse mutation assay, Ames test, showed positive results both with and without metabolic activation in the test performed on Acid Brown 4 and on similar substances.

The results of the in vitro gene mutation test, mammalian cell gene mutation assay and the in vivo micronucleous assay, chromosome aberration test, made on the similar substances are negative.

The in vivo micronucleus (bone marrow) test on similar substance following NTP guideline, showed negative results.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

This hazard class is primarily concerned with substances that may cause mutations in the germ cells of humans that can be transmitted to the progeny. However, the results from mutagenicity or genotoxicity tests in vitro and in mammalian somatic and germ cells in vivo are also considered in classifying substances and mixtures within this hazard class.

For the purpose of classification for germ cell mutagenicity, substances are classified if there is at least positive evidence obtained from experiments in mammals and/or in some cases from in vitro experiments, obtained from:

— somatic cell mutagenicity tests in vivo, in mammals; or

— other in vivo somatic cell genotoxicity tests which are supported by positive results from in vitro mutagenicity assays.

The presented results from in vivo and the in vitro tests are negative, therefore it is concluded that Acid Brown 4 is not genotoxic with or without metabolic activation.