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EC number: 225-691-8 | CAS number: 5012-29-3
- 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
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- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
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- 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
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
An Ames test was performed to investigate the potential of Pigment-Additiv FGR to induce gene mutations according to the plate incorporation test (experiment I) with and without rat S9 mix and the pre-incubation test (experiment II) with and without hamster S9 mix using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.
The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations in both experiments:
3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Minor toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed in strain WP2 uvrA with S9 mix at 5000 µg/plate in experiment I. No toxic effects occurred in the remaining strains with and without metabolic activation in experiment I and II.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Pigment-Additiv FGR at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
In a chromosomal aberrtion study using the analogue Pigment Red 170 100 metaphases per culture were scored for structural chromosome aberrations, except for the positive control in Experiment II at preparation interval 28 hours without metabolic activation, where only 50 metaphases were scored.
The highest applied concentration in the pre-test on toxicity (250 µg/mL; approx. 0.6 mM) was chosen with regard to the ability to formulate a homogeneous suspension of the test item in an appropriate solvent.
In this study no toxic effects indicated by reduced mitotic indices or reduced cell numbers were observed at the scored concentrations. The evaluation of higher concentrations was impossible due to strong test item precipitation.
In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item.
No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls.
Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.
The test item did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro.
Gene mutations at the HPRT locus in V79 cells of the Chinese hamster using the analogue Pigment Red 170.
The study was performed in three independent experiments. In the first experiment the treatment period was 4 hours with and without metabolic activation. To verify test results observed in the first experiment with metabolic activation (both cultures) a repeat experiment IA was performed under identical general experimental conditions with slightly modified concentrations.
The second experiment was solely performed in the absence of metabolic activation with a treatment time of 24 hours.
The highest applied concentration (250 μg/mL) was limited by the solubility properties of the test item in DMSO and aqueous media.
The tested concentrations ranged from 2 to 250 µg/mL.
No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiments.
Appropriate reference mutagens were used as positive controls and showed a distinct increases in induced mutant colonies confirming the sensitivity of the test system and the activity of the S9 mix.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2016 -03-08 till 2016-04-05
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guideline-conform study under GLP without deviations
- 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:
- other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital/ß-Naphthoflavone induced rat liver S9 (experiment I) and non-induced hamster liver (experiment II)
- Test concentrations with justification for top dose:
- 3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate / pre-experiment/experiment I
3, 10, 33; 100; 333; 1000; 2500; and 5000 µg/plate / experiment II - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Best suitable solvent - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- congo red
- other: sodium azide; 4-nitro-o-phenylene-diamine; methyl methane sulfonate, 2-aminoanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation); preincubation;
DURATION
- Preincubation period: 30 minutes
- Exposure duration: 72 hours
NUMBER OF REPLICATIONS: 3 plates
DETERMINATION OF CYTOTOXICITY
A reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.
- Evaluation criteria:
- A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant. - Statistics:
- According to the OECD guideline 471, a statistical analysis of the data is not mandatory.
- Species / strain:
- other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- exp. I strain WP2 uvrA with S9mix
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Water solubility: not soluble
- Precipitation:The test item precipitated in the overlay agar in the test tubes from 333 to 5000 µg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 333 to 5000 µg/plate. The undissolved particles had no influence on the data recording.
- Other confounding effects:
COMPARISON WITH HISTORICAL CONTROL DATA: In experiment II, the data in the negative control of strains TA 1535 with S9 mix and TA1537 with S9 mix were slightly above our historical control range. Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies.
ADDITIONAL INFORMATION ON CYTOTOXICITY: No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains with and without metabol¬ic activation in the first experiment. Only in experiment I minor toxic effects were observed in strain WP2 uvrA with S9 mix at 5000 µg/plate. - Remarks on result:
- other: other: reverse mutation assay
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used with and without rat and hamster S9.
- Executive summary:
Discussion von Bericht
This study was performed to investigate the potential of Pigment-Additiv FGR to induce gene mutations according to the plate incorporation test (experiment I) with and without rat S9 mix and the pre-incubation test (experiment II) with and without hamster S9 mix using theSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100, and theEscherichia colistrain WP2 uvrA.
The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations in both experiments:
3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
The test item precipitated in the overlay agar in the test tubes from 333 to 5000 µg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 333 to 5000 µg/plate. The undissolved particles had no influence on the data recording.
The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.
Minor toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed in strain WP2 uvrA with S9 mix at 5000 µg/plate in experiment I. No toxic effects occurred in the remaining strains with and without metabolic activation in experiment I and II.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Pigment-Additiv FGR at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.
In experiment II, the data in the negative control of strains TA 1535 with S9 mix and TA1537 with S9 mix were slightly above our historical control range. Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2008
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- 2/1998
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- other: In vitro Chromosome Aberration Test in Chinese Hamster V79 Cells
- Specific details on test material used for the study:
- Batch No.: 35455.055.MH516.001 or DEBF 042800
Aggregate state at room temperature: Solid
Colour: Red
Molecular weight: 454.49 g/mol
Purity: > 98.5 % (w/w) The purity was calculated by the amount of the by-compounds.
Certificate of Analysis: AZ 175/e3
Solubility (estimated): < 1 mg/L in water (room temperature)
10 - 100 mg/L in DMSO (room temperature)
Stability: > 72 h in DMSO at room temperature
Storage: At room temperature
Expiration Date: September 21, 2009 (Statement of producer) - Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- The V79 cell line has been used successfully for many years in in vitro experiments. The
high proliferation rate (doubling time of clone V79/D3 in stock cultures: 12 hours, determined
on January 02, 2006) and a reasonable plating efficiency of untreated cells (as a rule more
than 70 %) both necessary for the appropriate performance of the study, support the use of
this cell line. The cells have a stable karyotype with a modal chromosome number of 22. - Cytokinesis block (if used):
- Colcemid (0.2 Mg/mL culture medium)
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital / ß-Naphthoflavone induced rat liver S9
- Vehicle / solvent:
- DMSO
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- other: EMS; ethyl-methanesulfonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium;
- Cell density at seeding (if applicable): 1 x 104 - 6 x 104 cells per chamber
DURATION
- Preincubation period:
- Exposure duration: 15.5 hours and 25.5 hours
- Selection time (if incubation with a spindle inhibitor): 2.5 hours
- Fixation time (start of exposure up to fixation or harvest of cells):
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa
NUMBER OF REPLICATIONS: 2
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The cells on the slides were treated in the chambers with hypotonic solution (0.4 % KCl) for 20 min at 37° C. After incubation in the hypotonic solution the cells were fixed with a mixture of methanol and glacial acetic acid (3:1 parts, respectively).
NUMBER OF CELLS EVALUATED: 100 per group
NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 2 x 100
DETERMINATION OF CYTOTOXICITY
- Method: cell numbers, mitotic index
OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: not specified - Rationale for test conditions:
- Standard test conditions
- Evaluation criteria:
- Acceptability of the Test
The chromosome aberration test performed in our laboratory is considered acceptable if it meets the following criteria:
a) The number of structural aberrations found in the solvent controls falls within the range of the laboratory’s historical control data: 0.0 - 4.0 % aberrant cells, excluding gaps.
b) The positive control substances should produce significant increases in the number of cells with structural chromosome aberrations, which are within the range of the laboratory’s historical control data:
Evaluation of Results
A test item is classified as non-clastogenic if:
-the number of induced structural chromosome aberrations in all scored dose groups is in the range of the laboratory’s historical control data range (0.0 - 4.0 % aberrant cells, excluding gaps).
and/or
-no significant increase of the number of structural chromosome aberrations is observed.
A test item is classified as clastogenic if:
the number of induced structural chromosome aberrations is not in the range of the
laboratory’s historical control data range (0.0 - 4.0 % aberrant cells, excluding gaps).
and
either a concentration-related or a significant increase of the number of structural
chromosome aberrations is observed. - Statistics:
- Statistical significance was confirmed by means of the Fisher’s exact test (9) (p < 0.05).
However, both biological and statistical significance should be considered together. If the criteria mentioned above for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.
Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. The following criterion is valid:
A test item can be classified as aneugenic if the number of induced numerical aberrations is not in the range of the laboratory’s historical control data range (0.0 – 5.2 % polyploid cells - Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Conclusions:
- Under the experimental conditions reported, the test item did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line) when tested up to precipitating or the highest evaluable concentrations.
- Executive summary:
The test item, suspended in DMSO, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments. The following study design was performed:
Without S9 mix With S9 mix Exp. I Exp. II Exp. II Exp. I Exp. II Exposure period 4 hrs 18 hrs 28 hrs 4 hrs 4 hrs Recovery 14 hrs 14 hrs 24 hrs Preparation interval 18 hrs
18 hrs
28 hrs
18 hrs
28 hrs
In each experimental group two parallel cultures were set up. Per culture 100 metaphases were scored for structural chromosome aberrations, except for the positive control in Experiment II at preparation interval 28 hours without metabolic activation, where only 50 metaphases were scored.
The highest applied concentration in the pre-test on toxicity (250 Mg/mL; approx. 0.6 mM) was chosen with regard to the ability to formulate a homogeneous suspension of the test item in an appropriate solvent. Dose selection for the cytogenetic experiments was performed considering the toxicity data and the occurrence of precipitation.
In this study no toxic effects indicated by reduced mitotic indices or reduced cell numbers were observed at the scored concentrations. The evaluation of higher concentrations was impossible due to strong test item precipitation.
In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item.
No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls.
Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.
Conclusion
Under the experimental conditions reported, the test item did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro.
Therefore, the test item is considered non-clastogenic in this chromosome aberration test with and without S9 mix when tested up to precipitating concentrations or to the highest evaluable concentration.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2008
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- 2002
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- other: HGPRT
- Target gene:
- hypoxanthine-guanine phosphoribosyl transferase
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital/β-Naphthoflavone induced rat liver S9
- Test concentrations with justification for top dose:
- 2 - 250 µg/mL
The test concentration was limited by precipitation of the test item. - Vehicle / solvent:
- DMSO
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- Large stocks of the V79 cell line (supplied by Laboratory for Mutagenicity Testing; Technical University; D-64287 Darmstadt) are stored in liquid nitrogen in the cell bank of RCC-CCR allowing the repeated use of the same cell culture batch in experiments. Before freezing, the level of spontaneous mutants was depressed by treatment with HAT-medium. Each batch is screened for mycoplasma contamination and checked for karyotype stability and spontaneous mutant frequency. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells.
Thawed stock cultures are propagated at 37 °C in 80 cm² plastic flasks (Greiner, D-72632 Frickenhausen). About 5×105 cells are seeded into each flask with 15 mL of MEM (minimal essential medium; Seromed, D-12247 Berlin) supplemented with 10 % fetal calf serum (FCS; PAA Laboratories GmbH, D-35091 Cölbe). The cells are subcultured twice weekly. The cell
cultures are incubated at 37 °C in a 4.5 % carbon dioxide atmosphere (95.5% air).
For the selection of mutant cells the medium is supplemented with 11 μg/mL thioguanine (6TG, Sigma GmbH, D-82041 Deisenhofen). - Rationale for test conditions:
- The V79 cell line as well as the conditions applied have been used successfully in in vitro experiments for many years. Especially the high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50 %) both necessary for the appropriate performance of the study, recommend the use of this cell line. The cells have a stable karyotype with a modal chromosome number of 22.
In the range finding pre-experiment test item concentrations between 2.0 and 250 μg/mL
were used to evaluate toxicity in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation. The maximum concentration formed a homogeneous suspension in DMSO. - Evaluation criteria:
- Acceptability of the Assay
The gene mutation assay is considered acceptable if it meets the following criteria:
- the numbers of mutant colonies per 106 cells found in the negative and/or solvent controls fall within the laboratory historical control data range of 2001 – 2006.
- the positive control substances must produce a significant increase in mutant colony frequencies (see Annex II, Historical data).
- the cloning efficiency II (absolute value) of the negative and/or solvent controls must exceed 50 %.
The data of this study comply with the above mentioned criteria
Evaluation of Results
A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
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 non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations 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.
However, in a case by case evaluation this decision depends on the level of the corresponding negative control data. If there is by chance a low spontaneous mutation rate in the range normally found (0.5 – 31.8 mutants per 106 cells) a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of negative and solvent controls within all experiments of this study was also taken into consideration. - Statistics:
- A linear regression was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT® statistics software. The number of mutant colonies obtained in 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 should be considered together.
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- Under the experimental conditions reported, the test item did not induce gene mutations at the HPRT locus in V79 cells.
- Executive summary:
The study was performed to investigate the potential of the test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.
The study was performed in three independent experiments. In the first experiment the treatment period was 4 hours with and without metabolic activation. To verify test results observed in the first experiment with metabolic activation (both cultures) a repeat experiment IA was performed under identical general experimental conditions with slightly modified concentrations.
The second experiment was solely performed in the absence of metabolic activation with a treatment time of 24 hours.
The highest applied concentration (250 μg/mL) was limited by the solubility properties of the test item in DMSO and aqueous media.
The tested concentrations ranged from 2 to 250 µg/mL.
No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiments.
Appropriate reference mutagens were used as positive controls and showed a distinct increases in induced mutant colonies confirming the sensitivity of the test system and the activity of the S9 mix.
Referenceopen allclose all
Summary Tabellen
Table1 Summary of Experiment I
Study Name: 1755506 |
Study Code: Envigo 1755506 |
Experiment: 1755506 VV Plate |
Date Plated: 08/03/2016 |
Assay Conditions: |
Date Counted: 11/03/2016 |
Metabolic Activation |
Test Group |
Dose Level (per plate) |
|
Revertant Colony Counts (Mean ±SD) |
||||
|
|
|
|
|
|
|
|
|
|
|
|
|
TA 1535 |
TA 1537 |
TA 98 |
TA 100 |
WP2 uvrA |
|
|
|
|
|
|
|
|
|
Without Activation |
DMSO |
|
|
16 ± 1 |
9 ± 6 |
25 ± 7 |
186 ± 15 |
51 ± 4 |
Untreated |
|
|
13 ± 4 |
8 ± 2 |
22 ± 7 |
194 ± 15 |
51 ± 3 |
|
Pigment- |
3 µg |
|
11 ± 2 |
12 ± 6 |
29 ± 3 |
191 ± 7 |
47 ± 12 |
|
Additiv FGR |
10 µg |
|
13 ± 3 |
12 ± 4 |
31 ± 4 |
197 ± 17 |
50 ± 16 |
|
|
33 µg |
|
14 ± 7 |
9 ± 2 |
22 ± 2 |
165 ± 8 |
45 ± 12 |
|
|
100 µg |
|
15 ± 2 |
11 ± 3 |
30 ± 9 |
178 ± 13 |
40 ± 10 |
|
|
333 µg |
|
16 ± 5P |
10 ± 2P |
34 ± 7P |
178 ± 2P |
49 ± 9P |
|
|
1000 µg |
|
11 ± 3P |
10 ± 1P |
26 ± 6P |
199 ± 15P |
48 ± 7P |
|
|
2500 µg |
|
11 ± 3P |
7 ± 1P |
24 ± 6P |
185 ± 12P |
45 ± 4P |
|
|
5000 µg |
|
13 ± 3P |
11 ± 2P |
32 ± 10P |
199 ± 17P |
46 ± 4P |
|
NaN3 |
10 µg |
|
1205 ± 83 |
|
|
2418 ± 75 |
|
|
4-NOPD |
10 µg |
|
|
|
407 ± 22 |
|
|
|
4-NOPD |
50 µg |
|
|
83 ± 14 |
|
|
|
|
MMS |
2.0 µL |
|
|
|
|
|
1056 ± 53 |
|
|
|
|
|
|
|
|
|
|
With Activation |
DMSO |
|
|
12 ± 4 |
16 ± 3 |
39 ± 3 |
196 ± 33 |
51 ± 14 |
Untreated |
|
|
14 ± 6 |
11 ± 3 |
39 ± 14 |
175 ± 26 |
61 ± 10 |
|
Pigment- |
3 µg |
|
11 ± 6 |
15 ± 2 |
39 ± 8 |
203 ± 34 |
55 ± 1 |
|
Additiv FGR |
10 µg |
|
12 ± 3 |
13 ± 4 |
40 ± 12 |
212 ± 53 |
49 ± 8 |
|
|
33 µg |
|
12 ± 5 |
13 ± 2 |
43 ± 14 |
210 ± 51 |
54 ± 14 |
|
|
100 µg |
|
14 ± 3 |
15 ± 1 |
32 ± 4 |
193 ± 14 |
56 ± 4 |
|
|
333 µg |
|
12 ± 4P |
13 ± 3P |
31 ± 11P |
178 ± 23P |
50 ± 3P |
|
|
1000 µg |
|
14 ± 3P |
13 ± 1P |
40 ± 3P |
176 ± 7P |
57 ± 14P |
|
|
2500 µg |
|
9 ± 1P |
15 ± 2P |
31 ± 7P |
186 ± 24P |
64 ± 2P |
|
|
5000 µg |
|
11 ± 5P |
9 ± 4P |
32 ± 5P |
192 ± 19P |
22 ± 5P M |
|
2-AA |
2.5 µg |
|
539 ± 19 |
92 ± 13 |
4980 ± 327 |
3152 ± 55 |
|
|
2-AA |
10.0 µg |
|
|
|
|
|
476 ± 25 |
|
|
|
|
|
|
|
|
|
|
Key to Positive Controls |
Key to Plate Postfix Codes |
||
|
|
||
NaN3 2-AA 4-NOPD MMS |
sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate |
P M |
Precipitate Manual count |
Table2 Summary of Experiment II
Study Name: 1755506 |
Study Code: Envigo 1755506 |
Experiment: 1755506 HV2 Pre |
Date Plated: 31/03/2016 |
Assay Conditions: |
Date Counted: 05/04/2016 |
Metabolic Activation |
Test Group |
Dose Level (per plate) |
|
Revertant Colony Counts (Mean ±SD) |
||||
|
|
|
|
|
|
|
|
|
|
|
|
|
TA 1535 |
TA 1537 |
TA 98 |
TA 100 |
WP2 uvrA |
|
|
|
|
|
|
|
|
|
Without Activation |
DMSO |
|
|
12 ± 2 |
11 ± 3 |
28 ± 10 |
151 ± 15 |
38 ± 6 |
Untreated |
|
|
13 ± 2 |
8 ± 2 |
26 ± 9 |
182 ± 28 |
51 ± 4 |
|
Pigment- |
3 µg |
|
12 ± 1 |
11 ± 2 |
32 ± 8 |
141 ± 4 |
42 ± 4 |
|
Additiv FGR |
10 µg |
|
11 ± 4 |
13 ± 2 |
29 ± 4 |
148 ± 15 |
44 ± 1 |
|
|
33 µg |
|
12 ± 5 |
10 ± 1 |
27 ± 5 |
141 ± 17 |
42 ± 2 |
|
|
100 µg |
|
11 ± 2 |
11 ± 3 |
24 ± 4 |
138 ± 21 |
38 ± 6 |
|
|
333 µg |
|
12 ± 1P |
11 ± 1P |
23 ± 6P |
153 ± 17P |
41 ± 1P |
|
|
1000 µg |
|
14 ± 2P |
10 ± 2P |
25 ± 11P |
158 ± 12P |
37 ± 4P |
|
|
2500 µg |
|
11 ± 2P |
8 ± 1P |
20 ± 3P |
153 ± 17P |
39 ± 8P |
|
|
5000 µg |
|
12 ± 4P |
6 ± 1P |
21 ± 4P |
116 ± 17P |
43 ± 4P |
|
NaN3 |
10 µg |
|
1211 ± 42 |
|
|
2161 ± 107 |
|
|
4-NOPD |
10 µg |
|
|
|
487 ± 52 |
|
|
|
4-NOPD |
50 µg |
|
|
80 ± 11 |
|
|
|
|
MMS |
2 µL |
|
|
|
|
|
981 ± 56 |
|
|
|
|
|
|
|
|
|
|
With Activation |
DMSO |
|
|
21 ± 4 |
24 ± 6 |
51 ± 7 |
112 ± 9 |
66 ± 15 |
Untreated |
|
|
27 ± 8 |
30 ± 4 |
53 ± 5 |
175 ± 10 |
72 ± 11 |
|
Pigment- |
3 µg |
|
25 ± 7 |
27 ± 4 |
56 ± 10 |
109 ± 3 |
60 ± 7 |
|
Additiv FGR |
10 µg |
|
27 ± 7 |
18 ± 2 |
54 ± 7 |
127 ± 6 |
68 ± 11 |
|
|
33 µg |
|
29 ± 9 |
18 ± 4 |
45 ± 8 |
132 ± 8 |
68 ± 4 |
|
|
100 µg |
|
30 ± 4 |
23 ± 3 |
58 ± 13 |
138 ± 12 |
67 ± 6 |
|
|
333 µg |
|
27 ± 4P |
29 ± 3P M |
59 ± 7P |
134 ± 18P |
61 ± 6P |
|
|
1000 µg |
|
18 ± 4P |
22 ± 3P M |
55 ± 2P |
110 ± 4P |
65 ± 11P |
|
|
2500 µg |
|
16 ± 1P |
21 ± 5P M |
52 ± 10P |
101 ± 7P |
63 ± 5P |
|
|
5000 µg |
|
15 ± 2P |
19 ± 5P M |
31 ± 4P |
94 ± 1P |
48 ± 4P |
|
2-AA |
2.5 µg |
|
|
|
|
1592 ± 269 |
|
|
2-AA |
2.5 µg |
|
328 ± 45 |
149 ± 7 |
|
|
|
|
2-AA |
10 µg |
|
|
|
|
|
953 ± 63 |
|
Congo red |
500 µg |
|
|
|
434 ± 119 |
|
|
|
|
|
|
|
|
|
|
|
|
Key to Positive Controls |
Key to Plate Postfix Codes |
||
|
|
||
NaN3 2-AA 4-NOPD Congo red MMS |
sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine Congo red methyl methane sulfonate |
P M |
Precipitate Manual count |
Table 1: Summary of results of the chromosome aberration study with Novoperm-Rot F3RK70
Exp. |
Preparation interval |
Test item concentration inµg/mL |
Polyploid cells in % |
Cell numbers in % of control |
Mitotic indices in % of control |
|
Aberrant cells in % |
|
|
|
|
|
|
|
incl. gaps* |
excl. gaps* |
With exchanges |
Exposure period 4 hrs without S9 mix |
||||||||
I |
18 hrs |
Solvent control1 |
2.6 |
100.0 |
100.0 |
3.0 |
2.0 |
0.5 |
|
|
Positive control2 |
2.9
|
n.t. |
85.2 |
12.5 |
12.5S |
7.0 |
|
|
2.0 |
2.1 |
106.0 |
96.6 |
1.5 |
1.5 |
1.0 |
|
|
3.9 |
2.0 |
94.0 |
91.6 |
3.0 |
2.5 |
0.5 |
|
|
7.8P |
2.0 |
90.7 |
82.1 |
0.5 |
0.5 |
0.0 |
Exposure period 18 hrs without S9 mix |
||||||||
II
|
18 hrs |
Solvent control1 |
3.2 |
100.0 |
100.0 |
1.0 |
1.0 |
0.0 |
|
|
Positive control3 |
3. 0 |
n.t. |
97.8 |
21.0 |
20.5S |
4.5 |
|
|
0.25 |
3.4 |
77.1 |
68.6 |
1.0 |
1.0 |
0.0 |
|
|
0.5 |
3.0 |
89.1 |
63.8 |
2.5 |
0.5 |
0.0 |
|
|
1.0P |
2.6 |
93.8 |
90.6 |
0.5 |
0.5 |
0.0 |
Exposure period 28 hrs without S9 mix |
||||||||
II
|
28 hrs |
Solvent control1 |
2.7 |
100.0 |
100.0 |
0.0 |
0.0 |
0.0 |
|
|
Positive control3# |
2.8 |
n.t. |
69.1 |
38.0 |
38.0S |
11.0 |
|
|
0.5 |
2.4 |
89.7 |
83.0 |
1.5 |
1.5 |
0.5 |
|
|
1.0P |
2.7 |
122.8 |
86.1 |
0.5 |
0.5 |
0.0 |
* Inclusive cells carrying exchanges
#Evaluation of 50 metaphase plates per culture
n.t. Not tested
PPrecipitation occurred
SAberration frequency statistically significant higher than corresponding control values
1DMSO 0.5 % (v/v)
2EMS 900.0Mg/mL
3EMS 500.0Mg/mL
4CPA 1.4Mg/mL
5CPA 2.0Mg/mL
Table 1: Summary of results
|
|
|
relative cloning efficiency 1 |
relative cloning efficiency 2 |
mutant colonies / 106cells |
|
relative cloning efficiency 1 |
relative cloning efficiency 2 |
mutant colonies/ 106cells |
|
|
conc. per ml |
S9 mix |
induction factor |
induction factor |
||||||
|
|
|
% |
% |
|
|
% |
% |
|
|
column |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
Experiment I / 4 hour treatment
|
culture I |
culture II |
||||||||
Negative control -
|
|
- |
100.0 |
100.0 |
12.3 |
|
100.0 |
100.0 |
6.3 |
|
Solvent control DMSO -
|
|
- |
100.0 |
100.0 |
7.2 |
1.0 |
100.0 |
100.0 |
5.6 |
1.0 |
Pos. control with EMS
|
150.0 |
- |
83.0 |
74.9 |
127.4 |
10.4 |
92.3 |
80.3 |
119.8 |
19.0 |
Test item
|
2.0 |
- |
77.0 |
culture was not continued# |
92.0 |
culture was not continued# |
||||
Test item
|
3.9 |
- |
85.5 |
105.7 |
10.6 |
1.5 |
104.3 |
94.9 |
11.4 |
2.0 |
Test item
|
7.8 |
- |
91.8 |
91.7 |
14.3 |
2.0 |
102.4 |
79.3 |
23.6 |
4.2 |
Test item
|
15.6 |
- |
80.0 |
88.5 |
6.4 |
0.9 |
106.2 |
87.2 |
12.2 |
2.2 |
Test item
|
31.3 (p) |
- |
75.6 |
96.3 |
11.0 |
1.5 |
90.1 |
82.4 |
10.2 |
1.8 |
Test item
|
250.0 (p) |
- |
1.7 |
82.8 |
52.6 |
7.3 |
2.4 |
67.4 |
124.2 |
22.2 |
|
|
|
|
|
|
|
|
|
|
|
Negative control +
|
|
+ |
100.0 |
100.0 |
11.6 |
|
100.0 |
100.0 |
16.7 |
|
Solvent control DMSO +
|
|
+ |
100.0 |
100.0 |
6.6 |
1.0 |
100.0 |
100.0 |
13.6 |
1.0 |
Pos. control with DMBA
|
2.0 + |
+ |
67.3 |
31.0 |
2499.2 |
377.1 |
67.1 |
41.7 |
1645.6 |
120.9 |
Test item
|
2.0 + |
+ |
94.6 |
73.8 |
15.7 |
2.4 |
97.6 |
82.1 |
15.2 |
1.1 |
Test item
|
3.9 + |
+ |
95.2 |
79.9 |
17.1 |
2.6 |
91.0 |
71.6 |
25.7 |
1.9 |
Test item
|
7.8 + |
+ |
92.3 |
88.9 |
8.9 |
1.3 |
94.3 |
78.8 |
18.0 |
1.3 |
Test item
|
15.6 (p) |
+ |
89.1 |
95.4 |
8.7 |
1.3 |
88.4 |
77.7 |
11.1 |
0.8 |
Test item
|
31.3 (p) |
+ |
91.1 |
culture was not continued## |
87.2 |
culture was not continued## |
||||
Test item
|
250.0 (p) |
+ |
75.9 |
41.9 |
56.7 |
8.6 |
80.9 |
55.2 |
47.3 |
3.5 |
Experiment IA / 4 hour treatment
|
culture I
|
culture II
|
||||||||
Negative control
|
|
+ |
100.0 |
100.0 |
14.9 |
|
100.0 |
100.0 |
6.6 |
|
Solvent control DMSO
|
|
+ |
100.0 |
100.0 |
9.2 |
1.0 |
100.0 |
100.0 |
8.1 |
1.0 |
Pos. control with DMBA
|
2.0 |
+ |
12.5 |
54.1 |
750.5 |
81.2 |
14.0 |
56.6 |
922.5 |
114.0 |
Test item
|
7.8 |
+ |
94.1 |
94.8 |
14.3 |
1.6 |
97.0 |
99.6 |
18.2 |
2.2 |
Test item
|
15.6 |
+ |
108.5 |
96.5 |
12.8 |
1.4 |
92.2 |
115.6 |
11.5 |
1.4 |
Test item
|
31.3 (p) |
+ |
89.8 |
102.5 |
6.7 |
0.7 |
103.0 |
97.4 |
12.0 |
1.5 |
Test item
|
62.5 (p) |
+ |
96.1 |
culture was not continued |
95.4 |
culture was not continued |
||||
Test item
|
125.0 (p) |
+ |
90.9 |
98.6 |
11.6 |
1.3 |
108.6 |
118.2 |
10.4 |
1.3 |
Test item
|
250.0 (p) |
+ 1 |
03.5 |
80.5 |
12.1 |
1.3 |
95.2 |
112.1 |
6.0 |
0.7 |
Experiment II / 24 hour treatment |
culture |
culture II |
||||||||
Negative control -
|
|
|
100.0 |
100.0 |
5.7 |
|
100.0 |
100.0 |
4.8 |
|
Solvent control DMSO -
|
|
|
100.0 100.0 |
13.4 |
1.0 |
|
100.0 |
100.0 |
4.7 |
1.0 |
Pos. control with EMS
|
150.0 |
- |
48.4 |
95.2 |
379. 6 |
28.3 |
46.1 |
69.5 |
504.9 |
106.8 |
Test item
|
3.9 |
- |
100.1 |
113.8 |
9.2 |
0.7 |
101.9 |
102.1 |
6.1 |
1.3 |
Test item
|
7.8 |
- |
94.2 |
111.1 |
8.5 |
0.6 |
91.3 |
124.5 |
3.5 |
0.7 |
Test item
|
15.6 (p) |
- |
96.7 |
culture was not continued## |
101.4 |
culture was not continued## |
||||
Test item
|
31.3 (p) |
- |
97.6 |
97.3 |
9.7 |
0.7 |
101.3 |
118.8 |
6.5 |
1.4 |
Test item
|
62.5 (p) |
- |
107.7 |
92.8 |
98.1 |
11.9 |
0.9 |
98.7 |
6.6 |
1.4 |
Test item
|
125.0 (p) |
- |
93.1 |
103.5 |
11.7 |
0.9 |
97.2 |
106.5 |
10.2 |
2.2 |
p precipitation visible to the naked eye.
# culture was not continued since a minimum of only four analysable concentrations are required
## culture was not continued to avoid analysis of too many test points in the precipitating range.
The bold printed values are not considered valid due to exceedingly severe cyto-toxicity
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Justification for classification or non-classification
No classification
No indications of mutagenic potential were detected in in vitro muatgenicity studies with the test item or the analogue Pigment Red 170.
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