Registration Dossier
Registration Dossier
Data platform availability banner - registered substances factsheets
Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.
The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.
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: 266-867-4 | CAS number: 67674-28-6
- 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:
- March 05 to April 11, 2001
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 001
- Report date:
- 2001
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- OECD Guideline For Testing Of Chemicals, 476 "Genetic Toxicology: In vitro Mammalian Cell Gene Mutation Test". Adopted: July 21st, 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- EEC-Direktive 2000/32/EC, L 136, Annex 4E, B.17: "Mutagenicity -In Vitro Mammalian Cell Gene Mutation Test". Adopted: May 19th, 2000
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- Version / remarks:
- U.S. Environmental Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.5300, In Vitro Mammalian Cell Gene Mutation Test, August 1998
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Test material
- Reference substance name:
- Sodium m-[[4-[[p-[bis(2-hydroxyethyl)amino]phenyl]azo]-1-naphthyl]azo]benzenesulphonate
- EC Number:
- 266-867-4
- EC Name:
- Sodium m-[[4-[[p-[bis(2-hydroxyethyl)amino]phenyl]azo]-1-naphthyl]azo]benzenesulphonate
- Cas Number:
- 67674-28-6
- Molecular formula:
- C26H25N5O5S.Na
- IUPAC Name:
- sodium m-[[4-[[p-[bis(2-hydroxyethyl)amino]phenyl]azo]-1-naphthyl]azo]benzenesulphonate
- Test material form:
- solid: particulate/powder
- Details on test material:
- Acid Red 299
Constituent 1
Method
- Target gene:
- HPRT locus
Species / strain
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- Source of cells: cell bank of "Genetic Toxicology", Aventis Pharma Deutschland GmbH, ProTox
Test organism: cell line V79 of Chinese hamster lung fibroblasts
Cell culture medium: MEM (minimal essential medium) with Hanks-salts and 25 mM Hepes-buffer - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix
- Test concentrations with justification for top dose:
- The compound was suspended in cell culture medium and tested at the following concentrations:
without S9-mix: 160, 500, 1600 and 5000 μg/ml
with S9-mix: 160, 500, 1600 and 5000 μg/ml
In a preliminary experiment for solubility Telon Rubin A5B 01 was studied with respect to its solubility in hepatocyte attachment medium. The highest evaluable concentration was found to be 5000 ug/ml. Accordingly, the preliminary toxicity study was carried out using a maximum concentration of 5000 ug/ml and a range of lower dose levels down to 10 ug/ml. Following treatment in the presence and in the absence of S9 metabolic activation, moderate toxicity was observed at the highest concentration of 5000 ug/ml. On the basis of these results, a concentration of 5000 ug/ml was selected for the main assays and three lower dose levels down to 160 ug/ml were included in the treatment series. - Vehicle / solvent:
- Formulation of test compound: suspended in cell culture medium at appropriate concentrations immediately before use.
Formulation of reference compounds:
EMS dissolved in cell culture medium on the day of treatment, final concentration: 1.0 mg/ml = 8 mM.
DMBA dissolved in DMSO and frozen in small portions. Aliquot thawed on the day of treatment,
final concentration in cell culture medium: 7.7 μg/ml = 30 μM
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9,10-dimethylbenzanthracene
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- Toxicity experiments and dose range finding
A preliminary toxicity test was undertaken in order to select appropriate dose levels for the mutation assay. In this test a wide range of dose levels of test compound was used.
Cell cultures were subjected to the same treatment conditions as in mutation assays, and the survival of the cells was subsequently determined.
The test included the following treatments:
Solvent control: the maximum final concentration of organic solvents will not exceed approx. 1 % (v/v).
Test compound: the highest dose level for the preliminary toxicity test was determined by the solubility of the test compound up to the maximum of 10 mM or 5000 μg/ml.
Treatments were performed both in the presence and absence of S9 metabolic activation system using duplicate doses at each test point.
Test procedure
In preliminary toxicity experiments approximately 1500 cells were seeded in each well of a microliter plate, allowed to attach overnight and then exposed to the test and control compound for four hours.
For each concentration at least 6 wells were used. After 4 or 5 days, the cells were fixed and stained with crystal violet.
Survival was determined by measurement of the crystal violet extinction.
In the main mutation experiments the cultures for assessing toxicity were prepared and treated with the test compound in the same way as for the preliminary experiment. 24 hours after seeding of approx. 1500 cells per well in a microtiter plate, the medium was replaced with serum-reduced (5 % v/v) medium containing the test compound to which either buffer or S9-mix was added as appropriate. After 4 hours the treatment medium was removed and the cells were rinsed twice with normal medium. Thereafter normal medium was added to the wells. The cultures were stained with crystal violet and survival was determined after an incubation period of 4 or 5 days.
Mutagenicity test
Two independent mutation tests were performed.
Exponentially growing cultures which were more than 50% confluent were trypsinated by an approx. 0.25 % (v/v) trypsin ready for use (mfr. Gibco).
A single cell suspension was prepared.
Subsequently the cells were replated to determine the mutation frequency and plating efficiency. The procedures used were as described in Test Procedure
The treatment schedule of the mutagenicity test is described below:
Day 1: Subculturing of an exponentially growing culture
a) Approx. 1500 cells in each well of a micro titer plate for determination of the plating efficiency.
b) 6E+5 – 1E+6 cells in 182 cm2 flasks with 30 ml medium for the mutagenicity test, one flask per experimental point.
Day 2: Treatment of a) and b) with the test compound in the presence and absence of S9-mix (final protein concentration: approx. 0.15 mg/ml) for 4 hours.
Day 5 or 6: Fixation and staining of the cells in a) for the determination of the plating efficiency Subculturing of b) in 182 cm2 flasks
Day 9: Subculturing of b) in five 75 cm2 flasks with culture medium containing 6-thioguanine:
Mutant selection (about 300 000 cellss/flask); subculturing of b) in two 25 cm2 flasks for plating efficiency (about 400 cells per flask)
Day 16: Fixation and staining of colonies of b) - from subcultures seeded on day 9.
All incubations were carried out at approx. 37 °C and 4 % CO2.
Staining was performed with approx. 10 % (v/v) methylene blue in approx. 0.01 % (w/v) KOH solution.
Only colonies with more than 50 cells were counted. - Rationale for test conditions:
- In accordance with test guidelines.
- Evaluation criteria:
- Criteria for a valid assay
The assay is considered valid if the following criteria are met:
- the solvent control data are within the laboratory's normal control range for the spontaneous mutant frequency
- the positive controls induced increases in the mutation frequency which were both statistically significant and within the laboratory's normal range
- the plating efficacy for the solvent control was greater than 50 %
Criteria for a positive response
The test compound is classified as mutagenic if:
- it reproducibly induces with one of the test compound concentrations a mutation frequency that is three times higher than the spontaneous mutant frequency in this experiment.
- there is a reproducible concentration-related increase in the mutation frequency. Such an evaluation may be considered independently from the enhancement factor for induced mutants.
- survival of the responding dose group is at least 30 %.
However, in a case by case evaluation both decisions depend on the level of the corresponding negative control data. - Statistics:
- The biometry of the results for the test compound is performed off-line with the MANN WHITNEY-U-TEST
Results and discussion
Test results
- Key result
- 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
- Additional information on results:
- Solubility and toxicity
In a preliminary experiment for solubility Acid Red 299 was studied with respect to its solubility in hepatocyte attachment medium. The highest evaluable concentration was found to be 5000 μg/ml.
Accordingly, the preliminary toxicity study was carried out using a maximum concentration of 5000 μg/ml and a range of lower dose levels down to 10 μg/ml.
Following treatment in the presence and in the absence of S9 metabolic activation, moderate toxicity was observed at the highest concentration of 5000 μg/ml.
On the basis of these results, a concentration of 5000 μg/ml was selected for the main assays and three lower dose levels down to 160 μg/ml were included in the treatment series.
Mutagenicity
Experimental design
Two independent mutation assays to examine resistance to 6-thioguanine were performed.
In the presence and in the absence of S9 metabolic activation dose levels of 160, 500, 1600 and 5000 μg/ml were used.
Before treatment, the pH values and osmolality of the treatment media were determined. The addition of test compound solutions did not have any effect on these parameters.
Survival after treatment
In the presence and in the absence of 89 metabolic activation survival was slightly reduced in the second mutation experiment with the concentration of 5000 μg/ml.
In the presence of S9-mix a decrease in the survival rate was also observed with the concentration of 500 μg/ml in both mutation experiments.
Mutation results
The test compound was assessed for its mutagenic potential in vitro in the HPRT-test in two independent experiments without metabolic activation and two independent experiments with metabolic activation.
In the presence of metabolic activation with the toxic concentration of 500 μg/ml a slight enhancement of the mutation rate over the range of the solvent control was induced, which did not fulfil the criteria for a positive response.
In addition, this effect was based mainly on one of the duplicate cultures. Beside this no concentration-related increases were observed and therefore the effect is considered to be without biological relevance.
No relevant reproducible increases in the mutant colonies or mutant frequency over the range of the solvent control was found with any other of the investigated concentrations, either with or without metabolic activation by S9-mix.
The sensitivity of the test system was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control compounds.
Any other information on results incl. tables
Toxicity Data (Preliminary experiment)
|
Dose μg/ml |
S9-mix |
Extinction in microwell plates mean less blank values |
Standard deviation |
Relative survival* |
Solvent control |
0 |
- |
2.403 |
0.10 |
100.0 |
Telon Rubin |
10 50 100 250 500 1000 2500 5000 |
- - - - - - - - |
2.315 2.248 2.295 2.396 2.325 2.452 2.349 1.512 |
0.15 0.12 0.09 0.05 0.06 0.09 0.10 0.17 |
96.3 § 93.5 #§ 95.5 #§ 99.7 #§ 96.6 #§ 102.0 #§ 97.8 #§ 62.9 #§ |
|
Dose μg/ml |
S9-mix |
Extinction in microwell plates mean less blank values |
Standard deviation |
Relative survival* |
Solvent control |
0 |
+ |
2.416 |
0.06 |
100.0 |
Telon Rubin |
10 50 100 250 500 1000 2500 5000 |
+ + + + + + + + |
2.415 2.360 2.364 2.076 1.973 2.355 2.357 1.777 |
0.04 0.06 0.07 0.20 0.33 0.06 0.06 0.10 |
99.9 § 97.7 #§ 97.8 #§ 85.9 #§ 81.7 #§ 97.5 #§ 97.5 #§ 73.5 #§ |
# macroscopic precipitate§ microscopic precipitate
*= relative survival (mean value/mean value corresponding control x 100)
Solvent = MEM
Toxicity Data (First main mutation experiment)
|
Dose μg/ml |
S9-mix |
Extinction in microwell plates mean less blank values |
Standard deviation |
Relative survival* |
Solvent control |
0 |
- |
2.143 |
0.07 |
100.0 |
Positive control |
1000 |
- |
1.784 |
0.06 |
83.2 |
Telon Rubin |
160 500 1600 5000 |
- - - - |
2.158 2.135 2.002 1.717 |
0.12 0.12 0.08 0.13 |
100.7 #§ 99.7 #§ 93.4 #§ 80.1 #§ |
|
Dose μg/ml |
S9-mix |
Extinction in microwell plates mean less blank values |
Standard deviation |
Relative survival* |
Solvent control |
0 |
+ |
2.081 |
0.19 |
100.0 |
Positive control |
7.7 |
+ |
1.882 |
0.07 |
90.4 |
Telon Rubin |
160 500 1600 5000 |
+ + + + |
2.146 1.67 1.731 1.688 |
0.08 0.19 0.07 0.09 |
103.1 #§ 56.1 #§ 83.2 #§ 81.1 #§ |
# macroscopic precipitate§ microscopic precipitate
*= relative survival (mean value/mean value corresponding control x 100)
Solvent = MEM
Positive control without S9-mix = EMS
Positive control with S9-mix = DMBA
Toxicity Data (Second main mutation experiment)
|
Dose μg/ml |
S9-mix |
Extinction in microwell plates mean less blank values |
Standard deviation |
Relative survival* |
Solvent control |
0 |
- |
2.210 |
0.10 |
100.0 |
Positive control |
1000 |
- |
1.694 |
0.147 |
76.6 |
Telon Rubin A5B 01 |
160 500 1600 5000 |
- - - - |
2.235 2.197 2.091 1.625 |
0.15 0.07 0.15 0.15 |
101.1 #§ 99.4 #§ 94.6 #§ 73.6 #§ |
|
Dose μg/ml |
S9-mix |
Extinction in microwell plates mean less blank values |
Standard deviation |
Relative survival* |
Solvent control |
0 |
+ |
2.030 |
0.34 |
100.0 |
Positive control |
7.7 |
+ |
2.000 |
0.12 |
98.5 |
Telon Rubin A5B 01 |
160 500 1600 5000 |
+ + + + |
0.1940 0.246 2.149 1.295 |
0.12 0.04 0.12 0.27 |
95.6 #§ 12.1 #§ 105.8 #§ 63.8 #§ |
# macroscopic precipitate§ microscopic precipitate
*= relative survival (mean value/mean value corresponding control x 100)
Solvent = MEM
Positive control without S9-mix = EMS
Positive control with S9-mix = DMBA
Mutagenicity Data – Part 1 (First main mutation experiment)
|
Dose μg/ml |
S9-mix |
Number of cells per flask |
Factor* calculated |
Cells** seeded |
Cells*** survived |
|||
Seeded |
Found |
Mean |
|||||||
I / II |
I |
II |
|||||||
Solvent control 1 (MEM) |
0.0 |
- |
396 |
254.0 |
257.5 |
255.8 |
0.65 |
302250 |
195203 |
Solvent control 2 (MEM) |
0.0 |
- |
399 |
254.0 |
259.5 |
256.8 |
0.64 |
330150 |
212446 |
Positive control (EMS) |
1000.0 |
- |
404 |
239.0 |
247.0 |
243.0 |
0.60 |
315000 |
189468 |
Telon Rubin A5B 01 |
160.0 |
- |
403 |
328.3 |
316.5 |
322.4 |
0.80 |
280800 |
224640 |
160.0 |
- |
402 |
217.5 |
218.0 |
217.8 |
0.54 |
327150 |
177206 |
|
500.0 |
- |
399 |
279.0 |
280.5 |
279.8 |
0.70 |
318900 |
223590 |
|
500.0 |
- |
398 |
278.5 |
267.0 |
272.8 |
0.69 |
290400 |
199012 |
|
1600.0 |
- |
400 |
263.0 |
271.7 |
267.4 |
0.67 |
299700 |
200312 |
|
1660.0 |
- |
399 |
293.0 |
283.0 |
288.0 |
0.72 |
321150 |
231808 |
|
5000.0 |
- |
395 |
253.5 |
248.0 |
250.8 |
0.63 |
278100 |
176541 |
|
5000.0 |
- |
393 |
251.5 |
259.5 |
255.5 |
0.65 |
351900 |
228780 |
|
|
|||||||||
Solvent control 1 (MEM) |
0.0 |
+ |
400 |
246.7 |
245.7 |
246.2 |
0.62 |
328350 |
202099 |
Solvent control 2 (MEM) |
0.0 |
+ |
407 |
223.0 |
222.0 |
222.5 |
0.55 |
345900 |
189098 |
Positive control (DMBA) |
7.7 |
+ |
401 |
226.0 |
213.5 |
219.8 |
0.55 |
311400 |
170649 |
Telon Rubin A5B 01 |
160.0 |
+ |
404 |
298.0 |
296.8 |
297.4 |
0.74 |
342600 |
252201 |
160.0 |
+ |
399 |
273.0 |
275.5 |
274.3 |
0.69 |
252500 |
242289 |
|
500.0 |
+ |
399 |
258.0 |
252.5 |
255.3 |
0.64 |
336600 |
215331 |
|
500.0 |
+ |
397 |
280.3 |
282.0 |
281.2 |
0.71 |
353250 |
250167 |
|
1600.0 |
+ |
397 |
271.5 |
265.0 |
268.3 |
0.68 |
318150 |
214972 |
|
1600.0 |
+ |
402 |
269.5 |
285.7 |
277.6 |
0.69 |
327300 |
226016 |
|
5000.0 |
+ |
400 |
283.0 |
294.0 |
288.5 |
0.72 |
351750 |
253700 |
|
5000.0 |
+ |
402 |
291.0 |
293.0 |
292.0 |
0.73 |
342900 |
249072 |
* factor calculated: mean value / number of cells per flask seeded
** cells seeded in 6-thioguanine (TG) containing medium
*** cells survived after plating in (TG) containing medium (cells seeded x factor calculated)
Mutagenicity Data – Part 1 (Second main mutation experiment)
|
Dose μg/ml |
S9-mix |
Number of cells per flask |
Factor* calculated |
Cells** seeded |
Cells*** survived |
|||
Seeded |
Found |
Mean |
|||||||
I / II |
I |
II |
|||||||
Solvent control 1 (MEM) |
0.0 |
- |
400 |
308.8 |
308.0 |
308.4 |
0.77 |
356850 |
275131 |
Solvent control 2 (MEM) |
0.0 |
- |
397 |
310.8 |
292.0 |
301.4 |
0.76 |
289650 |
219901 |
Positive control (EMS) |
1000.0 |
- |
400 |
280.0 |
282.0 |
281.0 |
0.70 |
315150 |
221393 |
Telon Rubin A5B 01 |
160.0 |
- |
400 |
266.5 |
285.0 |
275.8 |
0.69 |
330750 |
228011 |
160.0 |
- |
399 |
307.3 |
308.0 |
307.7 |
0.77 |
351600 |
271102 |
|
500.0 |
- |
401 |
411.0 |
397.5 |
404.3 |
1.01 |
320850 |
323450 |
|
500.0 |
- |
402 |
314.8 |
306.0 |
310.4 |
0.77 |
326850 |
252374 |
|
1600.0 |
- |
398 |
271.5 |
268.0 |
269.8 |
0.68 |
340200 |
230575 |
|
1660.0 |
- |
395 |
230.0 |
227.0 |
228.5 |
0.58 |
296100 |
171288 |
|
5000.0 |
- |
407 |
285.0 |
273.0 |
279.0 |
0.69 |
368500 |
184058 |
|
5000.0 |
- |
399 |
286.5 |
288.0 |
287.3 |
0.72 |
340200 |
244918 |
|
|
|||||||||
Solvent control 1 (MEM) |
0.0 |
+ |
397 |
225.5 |
213.0 |
219.3 |
0.55 |
326850 |
180508 |
Solvent control 2 (MEM) |
0.0 |
+ |
404 |
325.0 |
314.0 |
319.5 |
0.79 |
332250 |
262757 |
Positive control (DMBA) |
7.7 |
+ |
399 |
270.0 |
274.0 |
272.0 |
0.68 |
350700 |
239074 |
Telon Rubin A5B 01 |
160.0 |
+ |
402 |
287.0 |
287.0 |
287.0 |
0.71 |
325200 |
232170 |
160.0 |
+ |
395 |
267.0 |
265.0 |
266.0 |
0.67 |
309750 |
208591 |
|
500.0 |
+ |
402 |
292.3 |
304.0 |
298.2 |
0.74 |
337350 |
250201 |
|
500.0 |
+ |
402 |
268.0 |
271.0 |
269.5 |
0.67 |
344850 |
231197 |
|
1600.0 |
+ |
402 |
317.5 |
315.5 |
316.5 |
0.79 |
328650 |
258751 |
|
1600.0 |
+ |
400 |
294.5 |
274.7 |
284.6 |
0.71 |
325350 |
231487 |
|
5000.0 |
+ |
403 |
264.5 |
254.0 |
259.3 |
0.64 |
332100 |
213640 |
|
5000.0 |
+ |
407 |
299.9 |
295.0 |
297.5 |
0.73 |
297750 |
217606 |
* factor calculated: mean value / number of cells per flask seeded
** cells seeded in 6-thioguanine (TG) containing medium
*** cells survived after plating in (TG) containing medium (cells seeded x factor calculated)
Mutagenicity Data – Part 2 (First main mutation experiment)
|
Dose μg/ml |
S9-mix |
Number of mutant colonies |
Standard deviation |
Mutation frequency |
Stat. sig. |
||||||
I |
II |
III |
IV |
V |
Mean |
|
ẍ |
|||||
Solvent control 1 (MEM) |
0 |
- |
4 |
3 |
3 |
3 |
5 |
3.6 |
0.89 |
18.4 |
21.5 |
|
Solvent control 2 (MEM) |
0 |
- |
6 |
3 |
4 |
4 |
9 |
5.2 |
2.39 |
24.5 |
|
|
Positive control (EMS) |
1000 |
- |
213 |
195 |
209 |
203 |
215 |
207.0 |
8.12 |
1092.5 |
|
* |
Telon Rubin A5B 01 |
160 |
- |
2 |
7 |
5 |
5 |
1 |
4.0 |
2.45 |
17.8 |
28.7 |
|
160 |
- |
7 |
4 |
8 |
3 |
13 |
7.0 |
3.94 |
39.5 |
|
||
500 |
- |
3 |
3 |
3 |
1 |
5 |
3.0 |
1.41 |
13.4 |
11.7 |
|
|
500 |
- |
1 |
4 |
2 |
1 |
2 |
2.0 |
1.22 |
10.0 |
|
||
1600 |
- |
0 |
3 |
3 |
7 |
2 |
3.0 |
2.55 |
15.0 |
22.2 |
|
|
1600 |
- |
6 |
10 |
7 |
4 |
7 |
6.8 |
2.17 |
29.3 |
|
||
5000 |
- |
3 |
5 |
4 |
6 |
7 |
5.0 |
1.58 |
28.3 |
23.4 |
|
|
5000 |
- |
4 |
4 |
4 |
7 |
2 |
4.2 |
1.79 |
18.4 |
|
||
|
||||||||||||
Solvent control 1 (MEM) |
0 |
+ |
7 |
4 |
6 |
4 |
6 |
5.4 |
1.34 |
26.7 |
24.5 |
|
Solvent control 2 (MEM) |
0 |
+ |
5 |
3 |
4 |
4 |
5 |
4.2 |
0.84 |
22.2 |
|
|
Positive control (DMBA) |
7.7 |
+ |
28 |
24 |
20 |
36 |
28 |
27.2 |
5.93 |
159.4 |
|
* |
Telon Rubin A5B 01 |
160 |
+ |
4 |
5 |
1 |
6 |
4 |
4.0 |
1.87 |
15.9 |
22.0 |
|
160 |
+ |
6 |
7 |
6 |
9 |
6 |
6.8 |
1.30 |
28.1 |
|
||
500 |
+ |
10 |
15 |
10 |
16 |
15 |
13.2 |
2.95 |
61.3 |
41.5 |
|
|
500 |
+ |
5 |
7 |
6 |
6 |
3 |
5.4 |
1.52 |
21.6 |
|
||
1600 |
+ |
3 |
8 |
3 |
4 |
2 |
4.0 |
2.35 |
18.6 |
17.3 |
|
|
1600 |
+ |
5 |
4 |
5 |
3 |
1 |
3.6 |
1.67 |
15.9 |
|
||
5000 |
+ |
4 |
6 |
5 |
5 |
5 |
5.0 |
0.71 |
19.7 |
17.5 |
|
|
5000 |
+ |
5 |
6 |
3 |
3 |
2 |
3.8 |
1.64 |
15.3 |
|
Mutation frequency (mutant colonies per 1 million cells): mean value / cells surviving
*Statistical significant (p<0.05) Mann-Whitney-U-Test
Mutagenicity Data – Part 2 (Second main mutation experiment)
|
Dose μg/ml |
S9-mix |
Number of mutant colonies |
Standard deviation |
Mutation frequency |
Stat. sig. |
||||||
I |
II |
III |
IV |
V |
Mean |
|
ẍ |
|||||
Solvent control 1 (MEM) |
0 |
- |
6 |
4 |
7 |
8 |
1 |
5.2 |
2.77 |
18.9 |
21.3 |
|
Solvent control 2 (MEM) |
0 |
- |
6 |
6 |
3 |
5 |
6 |
5.2 |
1.30 |
23.6 |
|
|
Positive control (EMS) |
1000 |
- |
110 |
97 |
118 |
131 |
114 |
114.0 |
12.35 |
514.9 |
|
* |
Telon Rubin A5B 01 |
160 |
- |
7 |
2 |
5 |
4 |
5 |
4.6 |
1.82 |
20.2 |
13.4 |
|
160 |
- |
0 |
2 |
1 |
3 |
3 |
1.8 |
1.30 |
6.6 |
|
||
500 |
- |
1 |
1 |
6 |
4 |
1 |
2.6 |
2.30 |
8.0 |
10.4 |
|
|
500 |
- |
1 |
3 |
5 |
4 |
3 |
3.2 |
1.48 |
12.7 |
|
||
1600 |
- |
8 |
4 |
4 |
9 |
5 |
6.0 |
2.35 |
26.0 |
18.3 |
|
|
1600 |
- |
2 |
1 |
1 |
2 |
3 |
1.8 |
0.84 |
10.5 |
|
||
5000 |
- |
1 |
3 |
3 |
6 |
1 |
2.8 |
2.05 |
15.2 |
13.7 |
|
|
5000 |
- |
3 |
2 |
4 |
3 |
3 |
3.0 |
0.71 |
12.2 |
|
||
|
||||||||||||
Solvent control 1 (MEM) |
0 |
+ |
5 |
4 |
3 |
8 |
5 |
5.0 |
1.87 |
27.7 |
20.0 |
|
Solvent control 2 (MEM) |
0 |
+ |
4 |
4 |
3 |
1 |
4 |
3.2 |
1.30 |
12.2 |
|
|
Positive control (DMBA) |
7.7 |
+ |
17 |
25 |
28 |
25 |
20 |
23.0 |
4.42 |
96.2 |
|
* |
Telon Rubin A5B 01 |
160 |
+ |
5 |
6 |
4 |
0 |
9 |
4.8 |
3.27 |
20.7 |
16.1 |
|
160 |
+ |
3 |
1 |
1 |
3 |
4 |
2.4 |
1.34 |
11.5 |
|
||
500 |
+ |
13 |
23 |
23 |
26 |
16 |
20.2 |
5.45 |
80.7 |
46.4 |
|
|
500 |
+ |
1 |
0 |
6 |
5 |
2 |
2.8 |
2.59 |
12.1 |
|
||
1600 |
+ |
3 |
3 |
1 |
2 |
3 |
2.4 |
0.89 |
9.3 |
15.9 |
|
|
1600 |
+ |
9 |
5 |
3 |
5 |
4 |
5.2 |
2.28 |
22.5 |
|
||
5000 |
+ |
5 |
2 |
2 |
2 |
2 |
2.6 |
1.34 |
12.2 |
15.8 |
|
|
5000 |
+ |
2 |
5 |
8 |
0 |
6 |
4.2 |
3.19 |
19.3 |
|
Mutation frequency (mutant colonies per 1 million cells): mean value / cells surviving
*Statistical significant (p<0.05) Mann-Whitney-U-Test
Applicant's summary and conclusion
- Conclusions:
- Acid Red 299 did not induce gene mutation, i.e. was not mutagenic, in this HPRT-test with V79 Chinese hamster cells, either in the presence or in the absence of metabolic activation.
The substance is not classified in accordance with CLP criteria. - Executive summary:
The study was performed to investigate the potential of Acid Red 299 (Batch No. 0481 09300) to induce gene mutations at the HPRT locus in V 79 cells of the Chinese hamster in vitro.
Two independent experiments were conducted both with and without an exogenous rat liver microsomal activation system (S9-mix).
The compound was suspended in cell culture medium and tested at the following concentrations:
without S9-mix: 160, 500, 1600 and 5000 μg/ml
with S9-mix: 160, 500, 1600 and 5000 μg/ml
With all investigated concentrations microscopically and macroscopically precipitation was observed.
The concentration ranges were based on the results of preliminary tests for solubility and toxicity. The highest concentration showed slight toxic effects with and without metabolic activation.
Up to the highest investigated dose no statistically significant and reproducible increase in mutant colony numbers was obtained in two independent experiments.
Appropriate reference mutagens used as positive controls showed a distinct increase in induced mutant colonies, thus indicating the sensitivity of the assay and the efficacy of the S9-mix.
In conclusion, Acid Red 299 does not induce gene mutations in the HPRT-test with V79 Chinese hamster cells, both in the presence as well as in the absence of a metabolic activation system, under the experimental conditions described.
Acid Red 299 (Batch No. 048109300) is therefore 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.