Registration Dossier
Registration Dossier
Diss Factsheets
Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 287-574-8 | CAS number: 85536-87-4
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data

Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2013-09-12 until 2013-11-13
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 014
- Report date:
- 2014
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Principles of method if other than guideline:
- first experiment 4 hours treatment with and without metabolic activation
second experiment 24 hours treatment without metabolic activation, 4 hours treatment with metabolic activation - GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Test material
- Reference substance name:
- 7-acetamido-4-hydroxy-3-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]naphthalene-2-sulphonic acid, sodium salt
- EC Number:
- 287-574-8
- EC Name:
- 7-acetamido-4-hydroxy-3-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]naphthalene-2-sulphonic acid, sodium salt
- Cas Number:
- 85536-87-4
- Molecular formula:
- C20H19N3O11S3.xNa C20H(19-x)N3NaxO11S3
- IUPAC Name:
- 7-acetamido-4-hydroxy-3-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]naphthalene-2-sulphonic acid, sodium salt
- Test material form:
- solid: particulate/powder
- Details on test material:
- Identification: Reactive Orange 72-78
Constituent 1
Method
- Target gene:
- HPRT
Species / strain
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: MEM
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital/Beta-Naphtoflavone induced Rat liver S9
- Test concentrations with justification for top dose:
- Experiment I:
without metabolic activation: 568.8; 1137.5; 2275.0; 4550.0; 6825.0 µg/mL
with metabolic activation: 568.8; 1137.5; 2275.0; 4550.0; 9100.0 µg/mL
Experiment II:
without metabolic activation: 71.1; 142.2; 284.4; 568.8; 716.2 µg/mL
with metabolic activation: 568.8; 1137.5; 2275.0; 4550.0; 9100.0 µg/mL - Vehicle / solvent:
- Deionised water. The final concentration of deionised water in the culture medium was 10% (v/v).
Controlsopen allclose all
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: Experiment I: 4 hours with and without metabolic activation, Experiment II: 24 hours without metabolic activation, 4 hours with metabolic activation
- Expression time (cells in growth medium): 72 hours
- Selection time (if incubation with a selection agent): 10 days
SELECTION AGENT (mutation assays): 6-Thioguanine
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: >1,5x10exp. 6
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency - Evaluation criteria:
- A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces reproducibly with one of the concen¬trations a mutation frequency that is three times higher than the spontaneous mutation fre¬quency in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
In a case by case evaluation this decision depends on the level of the correspon¬ding solvent control data. - Statistics:
- A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT(R)11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological relevance and statistical significance were considered together.
Results and discussion
Test resultsopen allclose all
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not effected (pH 7.42 in the solvent control versus pH 7.43 at 9100 µg/mL) measured in the range finding pre-experiment
- Effects of osmolality: No relevant increase (281 mOsm in the solvent control versus 340 mOsm at 9100 µg/mL, and 320 mOsm at 4550 µg/mL) measured in the pre-experiment
- Evaporation from medium: Not examined
- Water solubility: Soluble
- Precipitation: No precipitation of the test item was observed up to the maximum concentration in all experiments.
- Other confounding effects: None
RANGE-FINDING/SCREENING STUDIES:
The range finding pre-experiment was performed using a concentration range of 71.1 to 9100 µg/mL to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24-hours treatment) of metabolic activation. The highest applied concentration in the pre-test on toxicity (9100 µg/ml) was equal to 5 mg/mL of the pure substance.
Relevant toxic effect occurred after the 4-hour treatment at 4550 µg/mL and above without metabolic activation. Following the 24-hour treatment without metabolic activation toxic effects were noted at 568.8 µg/mL and above. Another low value of the cloning efficiency was noted at 568.8 µg/mL following 4-hour treatment without metabolic activation. This effect was judged as irrelevant fluctuation rather than a true cytotoxic effect however, as the relative cloning efficiency remained above 50% at the next two higher concentrations.
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. No precipitation or phase separation was observed up to the maximum concentration with and without metabolic activation following 4 and 24 hours treamtment.
There was no relevant shift of the pH of the medium even at the maximum concentration of the test item. The osmolarity was increased from 283 to 340 mOsm at 9100 µg/mL and 320 mOsm at 4550 µg/mL. Both levels of osmolarity are sufficiently close to the physiological level of approximately 300 mOsm.
The dose range of the first experiment was set according to the cytotoxicity data generated in the pre-experiment. The dose range of the second experiment was adjusted to data produced in the pre-experiment (without metabolic activation) and in the first experiment (with metabolic activation). The individual concentrations were generally spaced by a factor of 2.0. A narrower spacing was used at higher concentrations to cover the cytotoxic range more closely.
To overcome problems with possible deviations in toxicity the main experiments were started with more than four concentrations.
Table 1 Doses applied in the gene mutation assay with Reactive Orange 72-78
Experiment I
4 hours treatment without S9 mix: 142.2; 284.4; 568.8; 1137.5; 2275.0; 4550.0; 6825.0 µg/mL
4 hours treatment with S9 mix: 284.4; 568.8; 1137.5; 2275.0; 4550.0; 9100.0 µg/mL
Experiment II
24 hours treatment without S9 mix: 17.8; 35.6; 71.1; 142.2; 284.4; 568.8; 716.2 µg/mL
4 hours treatment with S9 mix: 284.4; 568.8; 1137.5; 2275.0; 4550.0; 9100.0 µg/mL
In experiment I and II the cultures at the two lowest concentrations without metabolic activation and the culture at the lowest concentration with metabolic activation were not continued since a minimum of only four concentrations is required by the guidelines.
COMPARISON WITH HISTORICAL CONTROL DATA: Complies
ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant cytotoxic effects, indicated by a relative cloning efficiency I or a relative cell density at first subcultivation of less than 50% in both parallel cultures, occurred in the first experiment at 4550 µg/mL and above without metabolic activation. In the second experiment relevant cytotoxic effects as described above were noted at 568.8 µg/mL and above without metabolic activation.
Any other information on results incl. tables
Summary Table
relative | relative | relative | mutant | relative | relative | relative | mutant | |||||
conc. | S9 | cloning | cell | cloning | colonies/ | induction | cloning | cell | cloning | colonies/ | induction | |
µg/mL | mix | efficiency I | density | efficiency II | 106cells | factor | efficiency I | density | efficiency II | 106cells | factor | |
% | % | % | % | % | % | |||||||
Column | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
Experiment I / 4 h treatment | culture I | culture II | ||||||||||
Solvent contro with water | - | 100.0 | 100.0 | 100.0 | 17.2 | 1.0 | 100.0 | 100.0 | 100.0 | 15.4 | 1.0 | |
Positive control (EMS) | 150.0 | - | 96.4 | 58.2 | 108.2 | 167.6 | 9.8 | 97.5 | 108.8 | 98.1 | 168.3 | 10.9 |
Test item | 142.2 | - | 99.3 | culture was not continued# | 85.4 | culture was not continued# | ||||||
Test item | 284.4 | - | 82.0 | culture was not continued# | 83.1 | culture was not continued# | ||||||
Test item | 568.8 | - | 91.2 | 69.4 | 111.4 | 14.5 | 0.8 | 95.2 | 135.9 | 102.2 | 11.5 | 0.7 |
Test item | 1137.5 | - | 100.7 | 53.2 | 100.5 | 19.8 | 1.2 | 91.1 | 117.4 | 101.3 | 21.3 | 1.4 |
Test item | 2275.0 | - | 80.4 | 57.1 | 99.0 | 13.2 | 0.8 | 79.9 | 112.1 | 100.6 | 13.1 | 0.8 |
Test item | 4550.0 | - | 32.0 | 56.3 | 98.2 | 20.2 | 1.2 | 28.3 | 108.2 | 95.6 | 20.9 | 1.4 |
Test item | 6825.0 | - | 4.9 | 30.4 | 97.6 | 21.7 | 1.3 | 4.1 | 73.2 | 103.6 | 19.1 | 1.2 |
Solvent contro with water | + | 100.0 | 100.0 | 100.0 | 6.3 | 1.0 | 100.0 | 100.0 | 100.0 | 7.5 | 1.0 | |
Positive control (DMBA) | 1.1 | + | 99.2 | 80.3 | 103.2 | 159.0 | 25.4 | 93.6 | 108.4 | 75.9 | 214.1 | 28.6 |
Test item | 284.4 | + | 100.0 | culture was not continued# | 98.1 | culture was not continued# | ||||||
Test item | 568.8 | + | 103.9 | 90.9 | 76.8 | 14.9 | 2.4 | 94.4 | 109.5 | 66.3 | 11.8 | 1.6 |
Test item | 1137.5 | + | 96.4 | 82.5 | 93.8 | 12.2 | 1.9 | 91.7 | 133.5 | 90.2 | 9.6 | 1.3 |
Test item | 2275.0 | + | 97.5 | 80.9 | 77.0 | 6.6 | 1.1 | 103.1 | 110.7 | 81.4 | 11.9 | 1.6 |
Test item | 4550.0 | + | 83.2 | 73.4 | 80.2 | 10.4 | 1.7 | 83.9 | 114.1 | 81.9 | 19.5 | 2.6 |
Test item | 9100.0 | + | 52.9 | 79.8 | 80.6 | 15.3 | 2.4 | 48.9 | 86.9 | 87.7 | 9.2 | 1.2 |
Experiment II / 24 h treatment | culture I | culture II | ||||||||||
Solvent contro with water | - | 100.0 | 100.0 | 100.0 | 15.5 | 1.0 | 100.0 | 100.0 | 100.0 | 19.8 | 1.0 | |
Positive control (EMS) | 150.0 | - | 94.5 | 89.8 | 89.8 | 269.9 | 17.4 | 89.0 | 85.3 | 97.5 | 318.3 | 16.1 |
Test item | 17.8 | - | 101.8 | culture was not continued# | 99.9 | culture was not continued# | ||||||
Test item | 35.6 | - | 100.3 | culture was not continued# | 99.1 | culture was not continued# | ||||||
Test item | 71.1 | - | 103.6 | 117.2 | 89.3 | 21.2 | 1.4 | 97.9 | 73.6 | 101.7 | 11.7 | 0.6 |
Test item | 142.2 | - | 97.3 | 74.7 | 92.3 | 18.9 | 1.2 | 98.7 | 89.4 | 102.8 | 15.0 | 0.8 |
Test item | 284.4 | - | 102.6 | 89.5 | 113.9 | 13.1 | 0.9 | 96.1 | 110.4 | 104.7 | 18.4 | 0.9 |
Test item | 568.8 | - | 21.7 | 92.0 | 98.3 | 12.0 | 0.8 | 19.0 | 131.0 | 103.9 | 15.8 | 0.8 |
Test item | 716.2 | - | 0.8 | 102.2 | 77.0 | 10.9 | 0.7 | 3.3 | 107.0 | 104.6 | 31.7 | 1.6 |
Experiment II / 4 h treatment | culture I | culture II | ||||||||||
Solvent contro with water | + | 100.0 | 100.0 | 100.0 | 11.8 | 1.0 | 100.0 | 100.0 | 100.0 | 14.2 | 1.0 | |
Positive control (DMBA) | 1.1 | + | 88.1 | 91.0 | 95.8 | 77.7 | 6.6 | 92.7 | 147.4 | 89.7 | 92.5 | 6.5 |
Test item | 284.4 | + | 83.7 | culture was not continued# | 112.5 | culture was not continued# | ||||||
Test item | 568.8 | + | 78.5 | 132.2 | 122.4 | 14.0 | 1.2 | 91.9 | 129.3 | 92.5 | 14.5 | 1.0 |
Test item | 1137.5 | + | 76.2 | 167.0 | 110.2 | 12.2 | 1.0 | 101.5 | 142.1 | 93.8 | 16.2 | 1.1 |
Test item | 2275.0 | + | 80.9 | 166.2 | 117.0 | 13.9 | 1.2 | 94.1 | 127.8 | 92.8 | 15.5 | 1.1 |
Test item | 4550.0 | + | 95.5 | 182.7 | 102.7 | 28.8 | 2.4 | 103.4 | 159.2 | 82.4 | 17.8 | 1.3 |
Test item | 9100.0 | + | 72.2 | 78.8 | 121.8 | 2.9 | 0.2 | 94.9 | 133.2 | 91.8 | 6.9 | 0.5 |
# culture was not continued since a minimum of only four analysable concentrations is required
Applicant's summary and conclusion
- Conclusions:
- Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.
Therefore, Reactive Orange 72/78 is considered to be non-mutagenic in this HPRT assay. - Executive summary:
The test item was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.
The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.
The main experiments were evaluated at the following concentrations:
exposure
periodS9
mixconcentrations
in µg/mLExperiment I
4 hours
-
568.8
1137.5
2275.0
4550.0
6825.0
4 hours
+
568.8
1137.5
2275.0
4550.0
9100.0
Experiment II
24 hours
-
71.1
142.2
284.4
568.8
716.2
4 hours
+
568.8
1137.5
2275.0
4550.0
9100.0
No precipitation of the test item was observed up to the maximum concentration in all experiments.
Relevant cytotoxic effects, indicated by a relative cloning efficiency I or a relative cell density at first subcultivation of less than 50% in both parallel cultures, occurred in the first experiment at 4550 µg/mL and above without metabolic activation. In the second experiment relevant cytotoxic effects as described above were noted at 568.8 µg/mL and above without metabolic activation.
No relevant and reproducible increase in mutant colony numbers/106cells was observed in the main experiments up to the maximum concentration. The mutation frequency did not exceed the historical range of solvent controls.
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.
In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 6.3 up to 19.9 mutants per 106cells; the range of the groups treated with the test item was from 2.9 up to 31.7 mutants per 106cells.
EMS (150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies. The positive control with DMBA in the first culture of the second experiment with metabolic activation did not quite reach the historical range of positive controls (77.7 versus 91.4 - 2666.3 colonies per 106cells). The positive control was valid however, as the induction factor was 6.6 and the positive control of the parallel culture remained within the historical range of positive controls.
In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test item is considered to be non-mutagenic in this HPRT assay.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
