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Genetic toxicity in vitro

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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:
no data given
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well documented guideline study. GLP certificate missing.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Only 2 bacterial strains were tested instead of 4.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
other: rfa; uvrB; ampicillin resistence (R factor plamid pKM 101); and a modified postreplication DNA repair system, which increases the mutation rate by inducing a defective repair in the DNA
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
other: rfa; uvrB; ampicillin resistence (R factor plamid pKM 101); and a modified postreplication DNA repair system, which increases the mutation rate by inducing a defective repair in the DNA
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
0, 20, 100, 500, 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Parallel with each experiment with and without S9-mix a solvent control is carried out.
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2.5 µg), dissolved in DMSO
Remarks:
with metabolic activation; strains TA 98 and TA 100
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Parallel with each experiment with and without S9-mix a solvent control is carried out.
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-methyl-N'-nitro-N-nitroso-guanidine (5 µg), dissolved in DMSO
Remarks:
without metabilic activation; strain TA 100
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Parallel with each experiment with and without S9-mix a solvent control is carried out.
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylendiamine (10 µg), dissolved in DMSO
Remarks:
without metabolic activation; strain TA 98
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 minutes at 37°C
- Exposure duration: After incubation at 37°C for 48 hours in the dark, the bacterial colonies (his+ revertants) were counted.

NUMBER OF REPLICATIONS: 3 test plates per dose and test or 3 plates per control

DETERMINATION OF CYTOTOXICITY
- Method: Titer determination
In general, the titer is determined only in the experiments with S9-mix both without test substance (solvent only) and after adding the two highest amounts of substance.

No further details are given.
Evaluation criteria:
In general, a substance to be characterised as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose response relationship
- reproducibility of the results
Statistics:
According to the OECD guideline a statistical analysis of the data is not mandatory.
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
An increase in the number of his+ revertants was not observed both in the standard plate test and in the preincubation test either without S9-mix or after the addition of metabolic activation system.
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
No bacteriotoxic effect was observed.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
An increase in the number of his+ revertants was not observed both in the standard plate test and in the preincubation test either without S9-mix or after the addition of metabolic activation system.
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
No bacteriotoxic effect was observed.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Solubility of the test substance: No precipitation of the test substance was found.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

According to the results of the present study, the test substance Sicocer F Gelb 2214 is not mutagenic in the Ames test under the experimental conditions chosen here.
Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-08-28 to 2013-10-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
other: OECD 487 "In vitro mammalian cell micronucleus test", adopted July 22, 2010
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2013-04-11
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
not applicable
Species / strain / cell type:
lymphocytes: mammalian (human; female)
Details on mammalian cell type (if applicable):
- blood samples were drawn from healthy non-smoking donors not receiving medication.
- blood was collected from a female donor (33 years old) for the first experiment and from a 35 year-old female donor for Experiment II.
- the lymphocytes of the respective donors have been shown to respond well to stimulation of proliferation with phytohemeagglutinine (PHA) and to positive control substances.
- all donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes.
- bood cultures were established by preparing a 11 % mixture of whole blood in medium within 30 hrs after blood collection. The culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 µg/mL), the mitogen PHA (3 µg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL).

All incubations were done at 37 °C with 5.5 % CO2 in humidified air.
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-naphthoflavone induced rat liver S9 was used as the metabolic activation system (protein concentration: 38.4 mg/mL).
Test concentrations with justification for top dose:
Experiment I: 0.2, 0.5, 1.3, 3.3, 8.4, 20.9, 52.2, 130.6, 326.4, 816.0, 2040.0 and 5100 µg/mL (with and without metabolic activation)
Experiment II: 0.2, 0.5, 1.3, 3.3, 8.4, 20.9, 52.2, 130.6, 326.4, 816.0, 2040.0 and 5100 µg/mL (with and without metabolic activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionised water
Stock formulations of the test item and serial dilutions were made in the vehicle. The final concentration of deionised water in the culture medium was 10 %.
All formulations were prepared freshly before treatment and used within two hours of preparation. The formulation was assumed to be stable for this period unless specified otherwise.
- Justification for choice of solvent/vehicle: the solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Positive controls without metabolic activation: pulse treatment; purity: ≥ 95 %; vehicle: deionised water; concentration: 2.0 µg/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Demecolcin
Remarks:
Positive controls without metabolic activation: continuous treatment; purity: ≥ 98 %; vehicle: deionised water; concentration: 100.0 µg/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Positive controls with metabolic activation: purity: ≥ 98 %; vehicle: Saline (0.9 % NaCl [w/v]); concentration: 15.0 µg/mL
Details on test system and experimental conditions:
PRE-TEST (EXPERIMENT I):
5100.0 µg/mL of Colorante Amarillo were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations between 0.2 and 5100.0 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test for toxicity, precipitation of the test item was observed at the end of treatment at 1.3 µg/mL and above. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.
Using a reduced Cytokinesis-block proliferation index (CBPI) as an indicator for toxicity in Experiment I, no cytotoxic effects were observed after 4 hours treatment in the absence and presence of S9 mix. Therefore, 5100.0 µg/mL was chosen as top treatment concentration for Experiment II.
Cytotoxicity is characterized by the percentages of reduction in the CBPI in comparison with the controls (% cytostasis) by counting 500 cells per culture in duplicate. The experimental conditions in this pre-experimental phase were identical to those required and described below for the mutagenicity assay.
All cell cultures were set up in duplicate. Exposure time was 4 hrs (with and without S9 mix). The preparation interval was 40 hrs after start of the exposure.

CYTOGENETIC EXPERIMENT
- puls exposure: about 48 hrs after seeding 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test item concentration. The culture medium was replaced with serum-free medium containing the test item. For the treatment with metabolic activation 50 µL S9 mix per mL culture medium was added. After 4 hrs the cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded and the cells were resuspended in and washed with "saline G" (pH 7.2, containing 8000 mg/L NaCl, 400 mg/L KCl, 1100 mg/L glucose • H2O, 192 mg/L Na2HPO4 • 2 H2O and 150 mg/L KH2PO4). The washing procedure was repeated once as described. The cells were resuspended in complete culture medium with 10 % FBS (v/v) and cultured for a 16-hour recovery period. After this period Cytochalasin B (4 µg/mL) was added and the cells were cultured another approximately 20 hours until preparation.
- continuous exposure (without S9 mix): about 48 hrs after seeding 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test item concentration. The culture medium was replaced with complete medium (with 10 % FBS) containing the test item. After 20 hours the cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded and the cells were re-suspended in and washed with "saline G". The washing procedure was repeated once as described. After washing the cells were re-suspended in complete culture medium containing 10 % FBS (v/v). Cytochalasin B (4 µg/mL) was added and the cells were cultured another approximately 20 hours until preparation.

PREPARATION REPARATION OF MICRONUCLEI
The cultures were harvested by centrifugation 40 hrs after beginning of treatment. The cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded and the cells were re-suspended in approximately 5 mL saline G and spun down once again by centrifugation for 5 minutes. Then the cells were resuspended in 5 mL KCl solution (0.0375 M) and incubated at 37 °C for 20 minutes. 1 mL of ice-cold fixative mixture of methanol and glacial acetic acid (19 parts plus 1 part, respectively) was added to the hypotonic solution and the cells were resuspended carefully. After removal of the solution by centrifugation the cells were resuspended for 2 x 20 minutes in fixative and kept cold. The slides were prepared by dropping the cell suspension in fresh fixative onto a clean microscope slide. The cells were stained with Giemsa.

EVALUATION OF CYTOTOXICITY AND CYTOGENETIC DAMAGE
Evaluation of the slides was performed using NIKON microscopes with 40 x objectives. The micronuclei were counted in cells showing a clearly visible cytoplasm area. The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976)*. The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus. At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. The frequency of micronucleated cells was reported as % micronucleated cells. To describe a cytotoxic effect the CBPI was determined in approximately 500 cells per culture and cytotoxicity is expressed as % cytostasis. A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis.

CBPI = ((MONC x 1) + (BINC x 2) + (MUNC x 3)) / n

CBPI: cytokinesis-block proliferation index
n: total number of cells
MONC: mononucleate cells
BINC: binucleate cells
MUNC: multinucleate cells

Cytostasis% = 100 - 100 [(CBPIt - 1) / (CBPIc - 1)]
T: test item
C: solvent control

OTHER EXAMINATIONS:
- pH: the effect of the test item on pH in the medium was measured.
- Osmolarity: the effect of the test item on osmolarity in the medium was measured.
The osmolarity and pH-value were determined in the solvent control and the maximum concentration without metabolic activation.

ACCEPTABILITY CRITERIA
The micronucleus assay will be considered acceptable if it meets the following criteria:
a) The rate of micronuclei in the solvent controls falls within the historical laboratory control data range.
b) The rate of micronuclei in the positive controls is statistically significant increased.
c) The quality of the slides must allow the evaluation of a sufficient number of analyzable cells.

* Reference:
Countryman, P.I. and Heddle, J.A. (1976) The production on micronuclei from chromosome aberrations in irradiated cultures of human lymphocytes. Mutation Research, 41, 321-332.

Evaluation criteria:
The current historical data range together with the statistical significance, confirmed by the Chi square test (α < 0.05), should be considered for classification of the test item.
A test item can be classified as non-clastogenic and non-aneugenic if:
- the number of micronucleated cells in all evaluated dose groups is in the range of the historical laboratory control data and
- no statistically significant or concentration-related increase of the number of micronucleated cells is observed in comparison to the respective solvent control.
A test item can be classified as clastogenic and aneugenic if:
- the number of micronucleated cells is not in the range of the historical laboratory control data and
- either a concentration-related increase in three test groups or a statistically significant increase in the number of micronucleated cells is observed.

If the above mentioned criteria for the test item are not clearly met, the test item will be classified as equivocal or a confirmatory experiment may be performed.
An increase in the number of micronucleated mononucleate cells may indicate that the test item has aneugenic potential.
Statistics:
Please refer to the field "Evaluation criteria" above.
Species / strain:
lymphocytes: mammalian (human; female)
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 examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:
No relevant influence on pH value was observed.
Solvent control (experiment I): pH-value: 7.7
Solvent control (experiment II): pH-value: 7.8
5100 µg/mL (experiment I): pH-value: 7.7
5100 µg/mL (experiment II): pH-value: 7.8
- Effects of osmolality:
No relevant influence on osmolaritywas observed.
Solvent control (experiment I): osmolarity: 282
Solvent control (experiment II): osmolarity: 280
5100 µg/mL (experiment I): osmolarity: 287
5100 µg/mL (experiment II): osmolarity: 290
- Precipitation: in Experiment I in the absence and presence of S9 mix and in Experiment II in the presence of S9 mix, visible precipitation of the test item in the culture medium was observed at 1.3 µg/mL and above. In Experiment II in the absence of S9 mix precipitation was observed at 3.3 µg/mL and above at the end of treatment.

CYTOTOXICITY:
In Experiment I in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment II in the absence and presence of S9 mix no cytotoxicity was observed up to the highest evaluable concentration. Evaluation was limited by severe test item precipitation on the slides

In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying micronuclei was observed. The micronucleus rates of the cells after treatment with the test item (0.25 - 1.05 % micronucleated cells) were within the range of the solvent control values (0.25 - 1.10 % micronucleated cells) and within the range of the laboratory historical control data.

In both experiments, either Demecolcin (100.0 ng/mL), mitomycin C (2.0 µg/mL) or cyclophosphamide (15.0 µg/mL) were used as positive controls and showed distinct increases in cells with micronuclei.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.
Therefore, Colorante Amarillo is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to precipitating concentrations.
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-08-27 to 2013-10-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted 1997-07-21
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2013-04-11
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
Stocks of the V79 cell line (supplied by Laboratory for Mutagenicity Testing; Technical University, Germany) are stored in liquid nitrogen. Before freezing, the level of spontaneous mutants was depressed by treatment with HAT-medium. Each batch is checked for spontaneous mutant frequency.
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Thawed stock cultures are propagated at 37 °C in 75 cm² plastic flasks. About 5x10^5 cells were seeded into each flask with 15 mL of MEM (minimal essential medium) containing Hank’s salts supplemented with 10 % foetal bovine serum (FBS; except during 4 hour treatment), neomycin (5 µg/mL) and amphotericin B (1 %). The cells were sub-cultured twice weekly. The cell cultures were incubated at 37°C in a 1.5 % carbon dioxide atmosphere (98.5 % air).

Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-naphthoflavone induced rat liver S9 (protein concentration: 38.4 mg/nL)
Test concentrations with justification for top dose:
Pre-test: 39.8, 79.7, 159.4, 318.8, 637.5, 1275.0, 2550.0 and 5100.0 µg/mL
Experiment I:
- 10.0, 20.0, 40.0, 80.0, 160.0 and 320.0 µg/mL (4 hour treatment; without metabolic activation)
- 20.0, 40.0, 80.0, 160.0, 320.0 and 640.0 µg/mL (4 hour treatment; with metabolic activation)
Experiment II:
- 10.0, 20.0, 40.0, 80.0, 160.0 and 320.0 µg/mL (24 hour treatment; without metabolic activation)
- 20.0, 40.0, 80.0, 160.0, 320.0 and 640.0 µg/mL (4 hour treatment; with metabolic activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionised water
A sample of the test item was sterilized by dry heat (approximately 180 °C for 3 hours) prior to each experiment. On the day of the experiment (immediately before treatment), the sterilized test item was suspended in the vehicle. The final concentration of deionised water in the culture medium was 10% (v/v).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Positive control without metabolic acitvation: purity: ≥ 98 %; vehicle: nutrient medium; final concentration: 0.15 mg/mL = 1.2 mM (experiment I)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
Positive control with metabolic acitvation: purity: ≥ 95 %; vehicle: dimethylsulfoxide (final concentration in nutrient medium 0.5%); final concentration: 1.1 µg/mL = 4.3 µM
Details on test system and experimental conditions:
RANGE FINDING EXPERIMENT
The range finding pre-experiment was performed using a concentration range of 39.8 to 5100 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 (5100 µg/ml) was equal to 5 mg/mL of the pure substance. The dose range of the main experiments was set according to the data generated in the pre-experiment.

MAIN EXPERIMENTS
- Seeding:
Two to three days after sub-cultivation stock cultures were trypsinized at 37 °C for 5 minutes. Then the enzymatic digestion was stopped by adding complete culture medium with 10 % FBS and a single cell suspension was prepared. The trypsin concentration for all sub-culturing steps was 0.2 % in PBS.
Prior to the trypsin treatment the cells were rinsed with PBS buffer containing 200 mg/l EDTA (ethylene diamine tetraacetic acid). Approximately 1.5x10^6 (single culture) and 5x10^2 cells (in duplicate) were seeded in plastic culture flasks. The cells were grown for 24 hours prior to treatment.
- Treatment:
After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 µl/mL S9 mix. Concurrent solvent and positive controls were treated in parallel. After 4 hours this medium was replaced with complete medium following two washing steps with "saline G". In the second experiment the cells were exposed to the test item for 24 hours in complete medium, supplemented with 10 % FBS, in the absence of metabolic activation.
The pH was adjusted to 7.2.
The colonies used to determine the cloning efficiency (survival) were fixed and stained approx. 7 days after treatment as described below.
Three or four days after treatment 1.5x10^6 cells per experimental point were sub-cultivated in 175 cm² flasks containing 30 mL medium. Following the expression time of 7 days five 80 cm² cell culture flasks were seeded with about 3 - 5x10^5 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability.
The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 for about 8 days. The colonies were stained with 10 % methylene blue in 0.01 % KOH solution.
The stained colonies with more than 50 cells were counted.

OTHER EXAMINATIONS:
- pH-value: pH was determined in culture medium of the solvent control and of the maximum concentration in the pre-experiment without metabolic activation.
Solvent control: 7.43
5100 µg/mL: 7.41
- Osmolarity: osmolarity was determined in culture medium of the solvent control and of the maximum concentration in the pre-experiment without metabolic activation
Solvent control: 284 mOsm
5100 µg/mL: 280 mOsm

ACCEPTABILITY OF THE ASSAY:
The gene mutation assay is considered acceptable if it meets the following criteria:
a) the numbers of mutant colonies per 10^6 cells found in the solvent controls fall within the laboratory historical control data range.
b) the positive control substances should produce a significant increase in mutant colony frequencies and remain within the historical control range of positive controls.
c) the cloning efficiency II (absolute value) of the solvent controls must exceed 50 %.
The data of this study comply with the above mentioned criteria.
Evaluation criteria:
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 fre¬quency at least at one of the concen¬trations 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 corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.

Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were 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 and statistical significance were considered together.
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 examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING EXPERIMENT:
No relevant toxic effect occurred up to the maximum concentration tested with and without metabolic activation following 4 and 24 hours of treatment.
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. Precipitation occurred at 159.4 µg/mL and above after 4 hours treatment without metabolic activation and at 318.8 µg/mL and above after 4 hours treatment with metabolic activation. Following 24 hours treatment without metabolic activation, precipitation was noted at 79.7 µg/mL and above.
There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.

MAIN EXPERIMENTS
Following the expression phase the cultures at the highest concentration in experiment I with and without metabolic activation were not continued to avoid analysis of too many precipitating concentrations. In experiment II the cultures at the lowest concentration with and without metabolic activation were not continued since a minimum of only four concentrations is required by the guidelines.

Precipitation of the test item was observed at 40.0 µg/mL and above in the first experiment without metabolic activation and at 160.0 µg/mL and above with metabolic activation. In the second experiment precipitation was noted at 80.0 µg/mL and above without metabolic activation and at 160.0 µg/mL and above with metabolic activation.

No relevant toxic effects occurred up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment. The mutation frequency remained well within the historical range of solvent controls. Still, the induction factor exceeded the threshold of three times the mutation frequency of the corresponding solvent control in the first culture of the second experiment at 40.0 and 80.0 µg/mL with metabolic activation. This effect was judged as biologically irrelevant as it is based on the low solvent control (4.8 mutant colonies per 10x6 cells) and was neither reproduced in the parallel culture nor dose dependent as indicated by the lacking statistical significance.
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 4.8 up to 45.7 mutant colonies per 10^6 cells; the range of the groups treated with the test item was from 6.4 up to 30.5 mutant colonies per 10^6 cells.
The highest solvent control (45.7 mutant colonies/10^6 cells) slightly exceeded the historical range of solvent controls (2.6-43.5 mutant colonies/106 cells). However, this effect was observed in culture II only. The mean of both parallel cultures (31.6 mutant colonies/10^6 cells) remained well within the range of historical solvent control data.

Ethylmethane sulfonate and 7,12-dimethylbenz(a)anthracene were used as positive controls and showed a distinct increase in induced mutant colonies.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

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, Colorante Amarillo is considered to be non-mutagenic in this HPRT assay.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The chemical and physiological properties of the pigment zirconium praseodymium yellow zircon are characterised by low to very low solubility because of the specific synthetic process (calcination at high temperatures, approximately1000°C), rendering the substance to be of a unique, stable crystalline structure in which all atoms are tightly bound and not prone to dissolution in environmental and physiological media. This manufacturing process leads to a very low bioaccessibility of the elements contained in the pigment. This has been investigated experimentally in vitro by simulating dissolution under physiological conditions considered to mimic the most relevantexposure routes (oral, dermal and inhalation), as follows:

 

1.) Gamble’s solution (GMB, pH 7.4) which mimics the interstitial fluid within the deep lung under normal health conditions,

2.) phosphate-buffered saline (PBS, pH 7.2), which is a standard physiological solution that mimics the ionic strength of human blood serum,

3.) artificial sweat (ASW, pH 6.5) which simulates the hypoosmolar fluid, linked to hyponatraemia (loss of Na+ from blood), which is excreted from the body upon sweating,

4.) artificial lysosomal fluid (ALF, pH 4.5), which simulates intracellular conditions in lung cells occurring in conjunction with phagocytosis and represents relatively harsh conditions and

5.) artificial gastric fluid (GST, pH 1.7), which mimics the very harsh digestion milieu of high acidity in the stomach.

 

 

The dissolution of zirconium from the test item zirconium praseodymium yellow zircon was in range of below the L.O.D and 1.5 µg/L (pH 6.5) at a loading of 0.1g/L after 2 hours and in range of below the L.O.D and 2.4µg/L (pH 4.5 ) after24 hours. The dissolution of silicon from the test item zirconium praseodymium yellow zircon was in a range of below blank (pH 7.4 and 6.5) and 36.7µg/L(pH 4.5) after 2 hoursat a loading of 0.1 g/Land in range of below blank and 62.7 µg/L (pH 1.7) after 24 hours. Further, dissolution of praseodymium was in range of below L.O.D. and 348 µg/L (pH 1.7)at a loading of 0.1g/L after 2 hours and in range of below L.O.D. and 574 µg/L (pH 1.7).

 

In conclusion, since the dissolved Zr, Si and Pr concentrations from this pigment were below 640 µg/L even at the highest loading of 0.1g/L, corresponding to a solubility of less than 0.65 %, this pigment may reasonably be considered as low to very low soluble and virtually biologically inert.

Zirconium praseodymium yellow zircon has been tested in bacterial reverse mutation assays. According to the results of the present study, the test substance zirconium praseodymium yellow zircon (Sicocer F Gelb 2214) is not mutagenic in the Ames test under the experimental conditions chosen here.

Further, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes (OECD 487) and did not induce gene mutations at the HPRT locus in V79 cells (OECD 476).

Therefore, Colorante Amarillo is considered to be non-mutagenic. The results are in line with the considered low biological availability of the pigment.


Short description of key information:
Zirconium praseodymium yellow zircon has been tested in bacterial reverse mutation assays. According to the results of the present study, the test substance zirconium praseodymium yellow zircon (Sicocer F Gelb 2214) is not mutagenic in the Ames test under the experimental conditions chosen here.
Further, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes and did not induce gene mutations at the HPRT locus in V79 cells.

Therefore, Colorante Amarillo is considered to be non-mutagenic in the HPRT assay as well as non-mutagenic in the in vitro micronucleus test, when tested up to precipitating concentrations.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

in vitro clastogenicity

Based on the outcome of guideline-compliant studies Zirconium praseodymium yellow zircon does not induce chromosome aberrations in mammalian cells, when tested up to precipitating concentrations in two independent experiments in the absence and presence of a rat liver metabolic activation system (S9 mix).

Overall it can be concluded that Zirconium praseodymium yellow zircon does not induce chromosome aberrations in vitro in mammalian cells. Therefore the conduct of in vivo clastogenicity experiments is not required.

in vitro gene mutation

Zirconium praseodymium yellow zircon did not induce gene mutations in the Ames test and in the HPRT locus of Chinese hamster V79 cells in vitro when tested up to precipitating concentrations in two independent experiments, in the absence and presence of a rat liver metabolic activation system (S9 mix).  

Overall it can be concluded that zirconium praseodymium yellow zircon does not induce gene mutations in vitro in bacteria and somatic mammalian cells. Therefore the conduct of in vivo gene mutation experiments is not required.

None of the in vitro genotoxicity studies rated as reliable showed any effect, neither in the bacterial reverse mutation assay, in a mammalian cell gene mutation test (HPRT assay) nor in a mammalian cell chromosome aberration test (micronecleus). Hence, the classification criteria according to regulation (EC) 1272/2008 as germ cell mutagen are not met.