Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a guideline study according to OECD TG 471 under GLP conditions the test item showed no mutagenic activity in both preincubation experiments with and without metabolic activation (uninduced hamster liver S9, i.e. required Prival modification for azo-dyes).

In a guideline study according to OECD TG 473 under GLP conditions the test item did not induce structural chromosome abberations in V79 cells with and without metabolic activation.

The test item does not have the potential to induce gene mutation in CHO-K1 cells at the tested concentrations and under the conditions of testing employed.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 13 OCT 1999 to 03 NOV 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
with the required modification for azo-dyes (Prival modifications)
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 JUL 1997
Deviations:
yes
Remarks:
1) Each batch of S9 is characterized with congo red prior to usage. 2) To evaluate the toxicity of the test item a pre-experiment was performedwith strains TA1535, TA1537, TA98, TA100 and WP2uvrA.
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
29 DEC 1992
Deviations:
yes
Remarks:
see above
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
uninduced hamster liver S9 mix
Test concentrations with justification for top dose:
0, 33, 100, 333, 1000, 2500, 5000 µg/plate
5000 µg/plate represents the highest concentration to be tested as indicated in the OECD guideline.
In a pre-experiment 3-5000 µg/plate were were tested. 5000 µg/plate were chosen as the top concentration since toxic effects were only observed at some of the strains at high concentrations. The pre-experiment is reported as experiment 1).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility properties of the solvent and its relative non-toxicity to the bacteria
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
congo red
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation assay without and with non-induced hamster liver S9 mix for Experiment 1) and 2)

DURATION
- Preincubation period: 30° C for 30 minutes
- Exposure duration: 48 h at 37° C

NUMBER OF REPLICATIONS: 2 Experiments with 3 replicates per concentration
Rationale for test conditions:
Since the test substance is an azo-dye, the Ames test was performed with the preincubation method and uninduced hamster liver S9 mix.
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, WP2 uvrA) or thrice (strains TA 1535, TA 1537) the colony count of the corresponding solvent colony 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 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:
Arithmetic means and standard deviation of the counted colonies were calculated.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
For details see table below
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
for details see table below
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
for details see table below
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: results from experiment I and II

Toxic effects, evident as a reduction in the number of revertants, were observed at the following concentrations:

Strain

Experiment I [µg/plate]

Experiment II [µg/plate]

 

Without S9 mix

With S9 mix

Without S9 mix

With S9 mix

TA1535

2500-5000

/

5000

2500-5000

TA1537

5000

/

/

/

TA98

5000

/

1000-5000

/

TA100

/

/

/

/

WP2uvrA

/

/

/

/

(/ = no relevant toxic effects observed)

Conclusions:
In a guideline study according to OECD TG 471 under GLP conditions the test item showed no mutagenic activity in both preincubation experiments with and without metabolic activation (uninduced hamster liver S9, i.e. required Prival modification for azo-dyes).
Executive summary:

In a guideline study according to OECD TG 471 under GLP conditions mutagenic activity of the test item was investigated in Salmonella typhimurium strains TA 1535, TA 1537, TA98 and TA100 as well as Escherichia coli strain WP2 uvrA with (uninduced hamster liver S9 mix) and without metabolic activation at concentrations of 0, 33, 100, 333, 1000, 2500 and 5000 μg/plate. Due to the test items characteristic as an azo-dye the test was conducted using the Prival modification, i.e. testing the above mentioned bacterial strains in the preincubation assay without and with hamster liver S9 mix for metabolic activation.

The test item did not reveal any mutagenic activity under the conditions tested. The appropriate reference mutagenes showed distinct positive mutagenic effects.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 22 SEP 1999 to 14 JAN 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
21 JUL 1997
Deviations:
yes
Remarks:
Additionally, two cultures per test item and solvent control treatment group not treated with colcemid, were set up in parallel. Used for micoscopic determination of cell number within 10 defined fields per slide.
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
29 DEC 1992
Deviations:
yes
Remarks:
see above
GLP compliance:
yes (incl. certificate)
Type of assay:
other: Chromosome aberration assay in mammalian cells
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Laboratory for Mutagenicity testing, LMP, Technical University Darmstadt, Germany
- Suitability of cells: yes
- doubling time: 12 h
- Number of passages if applicable: Paasage 5-15 after thawing was used for the assay
- Methods for maintenance in cell culture if applicable: 80 cm² plastic flask, 37 °C
- Modal number of chromosomes: 22

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: 4.5% CO2, MEM with fetal calf serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
induced rat liver S9 mix (induced with phenobarbital and β-naphthoflavone)
Test concentrations with justification for top dose:
Experiment I:

With and Without S9 mix:
Preparation interval: 18 h, exposure period: 4 h: 0, 3.1, 6.3, 12.5, 25, 50 and 100 µg/mL

Experiment II:
Without S9 mix:
Preparation interval: 18 h, exposure period: 18 h: 0, 3.1, 6.3, 12.5, 25, 50 and 100 µg/mL
Preparation interval: 28 h, exposure period: 28 h: 0, 12.5, 25, 50 and 100 µg/mL

With S9 mix:
Preparation interval: 28 h, exposure period: 4 h: 0, 3.1, 6.3, 12.5, 25, 50 and 100 µg/mL

100 µg/mL was chosen as the highest concentration with regard to the ability to formulate the test item in an appropriate solvent with respect to the current OECD Guideline 473.
Vehicle / solvent:
Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: In DMSO a homogenous suspension was established at a concentration of 20 mg/mL resulting in a final concentration of 100 µg/mL culture medium. In Ethanol or water no stable suspension could be established.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding: 1 - 6 x 10exp4 cells/chamber

DURATION
- Preincubation period: Experiment I: 18 h, experiment II: 18 or 28 h
- Exposure duration: Experiment I: 4 h, experiment II: 18, 28 or 4 h

SPINDLE INHIBITOR (cytogenetic assays): colcemid

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: two slides per concentration group and experiment were prepared

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: 16 h and 26 h, respectively after the start of the treatment colcemid was added (0.2 µg/ml culture medium) to the cultures. 2 h later, 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 3 + 1 methanol + glacial acetic acid. Per experirnent both slides per group were prepared. After preparation the cells were stained with Giemsa (E. Merck, D-64293 Darmstadt).

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 200 (100 per culture), only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik" (9)) using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.

DETERMINATION OF CYTOTOXICITY
- mitotoc index and cell number
Two cultures per test item and solvent control treatment group, not treated with Colcemid, were set up in parallel. These cultures were stained in order to determine microscopically the cell number within 10 defined fields per slide. The toxicity of the test item is given as reduction of % cells as compared to the solvent control.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
Rationale for test conditions:
Study performed according to OECD TG 473
Evaluation criteria:
Evaluation of Results
A test item is classified as non-mutagenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups are in the range of the laboratotys historical control data (0.0 - 4.0 % aberrant cells exclusive gaps). and/or
- no significant increase of the number of structural chromosome aberrations is observed.
A test item is classified as mutagenic if
- the number of induced structural chromosome aberrations are not in the range of our historical control data (0.0 - 4.0 % aberrant cells exclusive 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 Fischer's exact test (p < 0.05). However, both biological and statistical significance should be considered together. If the a.m. criteria 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 perforrned.
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
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no effect observed
- Effects of osmolality: no effect observed
- Precipitation: yes, starting at 12.5 µg/mL with and without metabolic activation

RANGE-FINDING/SCREENING STUDIES: In a range finding pre-test on toxicity cell numbers 24 h after start of treatment were scored as indicator for cytotoxicity. Concentrations between 0.8 and 100 µg/mL were applied. No toxic effects were observed after treatment up to the highest evaluated concentration of the test item.

The evaluated experimental points were the following:
Experiment I: without metabolic activation: 3.1, 6.3 and 12.5 µg/mL, with metabolic activation: 6.3, 12.5 and 25 µg/mL
Experiment II: without metabolic activation (18 h preparation and exposure period): 3.1, 6.3 and 12.5 µg/mL, 28 h preparation and exposure period: 12.5 µg/mL; with metabolic activation: 3.1, 6.3, 12.5 and 25 µg/mL
Remarks on result:
other: results from eperiment I and II

In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. In the absence and in the presence of S9 mix, the aberration rates of the cells after treatment with the test item (exp. I: 0.5 % - 2.0 %; exp. II: 0.0 % - 1.0 %) were near to the range of the solvent control values (exp. I: 0.0 % - 1.5 %; exp. II: 0.0 % - 1.0 %) and clearly within the range of the historical control data: 0.0 % - 4.0 %. However, a single significant increase in experiment I after 4 h exposure with 6.3 µg/mL was observed. This not dose related increase (2.0 % aberrant cells exclusive gaps) within our historical control data range (0.0 % - 4.0 %) was caused by a low solvent control value. Therefore, the significance has to be regarded as being biologically irrelevant.

In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (exp. I: 1.5 % - 5.9 %; exp. II: 0.8 % - 3.6 %) as compared to the rates of the solvent controls (exp. I: 2.6 % - 3.4 %; exp. II: 2.3 % ­3.2 %).

In both experiments, ethylmethanesulfonate (600 and 1000 µg/mL, respectively, without metabolic activation) and cyclophosphamide (0.71 µg/mL, with metabolic activation) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Conclusions:
In a guideline study according to OECD TG 473 under GLP conditions the test item did not induce structural chromosome abberations in V79 cells with and without metabolic activation.
Executive summary:

In a guideline study (OECD 473 as of 1997, GLP conditions) the test item, suspended in DMSO, was assessed for its potential to induce structural chromosome aberrations in Chinese hamster V79 cells with and without metabolic activation (induced rat liver S9 mix).

Prior to the chromosomal aberration test, a cell growth inhibition test was performed to determine test concentrations for the chromosomal aberration test. No cytotoxicity was noted at 100 µg/mL, the highest concentration tested. Precipitations were noted at concentrations of 12.5 µg/mL and higher.

For the chromosomal aberration test two experiments were performed: In Experiment I concentrations of 0, 3.1, 6.3, 12.5, 25, 50 and 100 µg/mL were tested with and without metabolic activation (preparation time: 18 h, exposure period 4 h). In a second experimet (Experiment II, with and without metabolic activation) the same concentrations were tested with preparation intervals of 18 or 28 h and exposure times of 18, 28 or 4 h.

In total 200 metaphases per dose were evaluated for structural chromosome aberrations in each experiment. In both independent experiments, no biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed alter treatment with the test item. One single statistical significancy in experiment I in the presence of S9 mix must be regarded as being biologically irrelevant (not dose related increase, only due to low solvent control value). Appropriate mutagens were used as positive controls. They produced markedly positive results.

It was concluded that the test substance is not clastogenic in Chinese hamster V79 cells under the experimental conditions described in the report.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
other: In vitro Mammalian cell gene mutation test using the Hprt Gene
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: American Type Culture Collection, P.O.Box 1549, Manassas, VA 20108
USA

- Suitability of cells: Test approaches currently accepted under the OECD for the assessment of mammalian cell gene mutation involve the use of Chinese Hamster Ovary (CHO) cell line. This cell line has been demonstrated to be sensitive to the mutagenic activity of a variety of chemicals.
Established CHO cell line is useful in in vitro gene mutation testing because it is easily cultured in standard medium, has a small number of large chromosomes each with a more or less distinctive morphology and a relatively short cycle time.

- Cell cycle length, doubling time or proliferation index: Chinese Hamster (Cricetulus griseus) ovary cell line CHO-K1, (ATCC CCL-61, Lot 4765275) with a modal chromosome number 20 and a population doubling time of 12 to 14 hours was used.
This cell line is capable of developing resistance to 6-thioguanine (6TG) resulting from lack of hypoxanthine guanine phosphoribosyl transferase (hprt) enzyme activity as a result of mutation at the X chromosomes.
The cell line was tested for mycoplasma in the test facility. The karyotype analysis of this cell line is periodically performed and documented.
Cells were grown in tissue culture flasks at 37 ± 1 °C in a humidified carbon dioxide incubator (5 ± 0.2 % CO2 in air).

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Ham’s F-12 medium supplemented with sodium bicarbonate, antibiotics, and L-glutamine was the basic medium.

Basic medium supplemented with 10% fetal bovine serum (FBS) was the complete medium and was used for the growth and multiplication of cells as well as in detaching and diluting the cells.

Basic medium supplemented with 5% fetal bovine serum (FBS) was the treatment medium and was used for target cell exposure to the test item and controls.

Cloning medium was basic medium supplemented with 20 % FBS and was used for the determination of cell viability or plating/cloning efficiency.

Selective medium was basic medium supplemented with 20 % FBS and the selective agent 6-Thioguanine (6-TG) at 35 µM and was used for the selection of mutants.

Dulbecco’s Phosphate buffered saline (PBS) (pH: 7.4)
Trypsin: EDTA solution

- Properly maintained: [yes]
- Periodically checked for Mycoplasma contamination: [yes]
- Periodically checked for karyotype stability: [yes]
Metabolic activation:
with and without
Metabolic activation system:
S9 homogenate was prepared from male Wistar rats induced with a single intra-peritoneal injection of Aroclor 1254 (0.7 mL/rat ready to use solution), 5 days prior to sacrifice. Batches are stored in a deep freezer at -68 to -86 ºC.
Test concentrations with justification for top dose:
Based on the results of the preliminary cytotoxicity test, the following test concentrations were selected for testing in the gene mutation test:
Experiments 1 and 2 (Presence and Absence of Metabolic Activation, respectively)

A) 250 B) 500 C) 1000 and D) 2000 µg/mL (factor of 2)
Vehicle / solvent:
One hundred fifty microliters (150 micro L) of Dimethyl sulphoxide (DMSO) was used per 15 mL of treatment medium as the vehicle control in each of the experiments.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; Exponentially growing CHO-KI cells were plated in two replicates and each replicate has two flasks containing 15 mL of complete medium at a density of approximately 4.5 x 106 cells / 75 cm2 flask and incubated for approximately 24 hours.
Two parallel cultures were also kept along with the vehicle control and treatment groups. Cell counts were made from these cultures at the 0-hour treatment to obtain the baseline cell count for estimation of Relative Survival (RS).

- Cell density at seeding (if applicable): 4.5 x 106 cells / 75 cm2

DURATION
- Period: All test item and positive control concentrations were prepared immediately before use in sterile test tubes. The target cells were exposed to the vehicle, positive control and various concentrations of the test item for 3 hours in the presence and absence of metabolic activation.

The medium from each target cells flask was removed by aspiration and replaced with 13.5 mL and 15 mL of treatment medium for the experiment in the presence and absence of metabolic activation, respectively. For the experiment incorporating metabolic activation, 1.5 mL of S9 mix was added to give a final concentration of 1 % S9 (v/v) in the test medium.

One hundred fifty microliters (150 µL) each of the vehicle control, the positive control, dilution of the test item were transferred to respective flasks and gently mixed and kept for incubation to expose the cells to treatment.

- Exposure duration: 3 hours
- Expression time (cells in growth medium): 9 days
- Selection time (if incubation with a selection agent): 10 days


SELECTION AGENT (mutation assays): Replicate cultures from controls and each treatment level were trypsinized, and the cells suspended in 10 mL complete medium, pooled, and counted using a hemocytometer.
For selection of the 6-Thioguanine (6-TG) resistant phenotype, cells from each of the replicate cultures were plated in to 5 flasks at a density of approximately 2 x 105 cells/25 cm2 flask (total of 106 cells/replicate) in selective medium and incubated for 10 days.
For cloning efficiency determination at the time of selection, cells from each of the replicate cultures were plated approximately at 200 cells/25 cm2 flask in triplicate in cloning medium and incubated for 10 days.

STAIN (for cytogenetic assays): The colonies were stained with 0.5 % methylene blue and counted for both cloning efficiency and mutant selection after 10 days of incubation.

NUMBER OF REPLICATIONS: Two

Evaluation criteria:
When all the validity criteria are fulfilled:

1. A test chemical is considered to be clearly positive if, in any of the experimental conditions examined:

• At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control
• The increase is concentration-dependent when evaluated with an appropriate trend test
• Any of the results are outside the distribution of the historical vehicle control data

When all of these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.

2. A test chemical is considered to be clearly negative if, in all experimental conditions examined:

• None of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control
• There is no concentration-related increase when evaluated with an appropriate trend test
• All results are inside the distribution of the historical vehicle control data

The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.
Statistics:
A power transformation procedure (Snee and Irr, 1981) was used with which, the observed mutant frequency was transformed using the formula:

Y = (X + A) B

where,
Y = transformed mutant frequency
X = observed mutant frequency
and A, B = constants.

Statistical analysis of the experimental data was carried out using validated copies of SYSTAT Statistical package version 12.0. In cases where analysis of variance was significant at p < 0.05, a Dunnett’s test was conducted, comparing each treatment group and the positive control to the vehicle control (p < 0.05).
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no mutagenic potential (based on QSAR/QSPR prediction)

Summary Results of the Gene Mutation Assay in the Presence of Metabolic Activation (Experiment 1)

Treatment

µg/mL

Mutation Assay Flasks

Cloning Efficiency of Mutant Colonies

Cloning Efficiency Flasks

6-TG Mutants

per 106Clonable Cells

(MF)

TG Colonies/Flask

No. of Colonies/Flask

1

2

3

4

5

Total

1

2

3

CE*

DMSO

2

2

2

0

2

19

0.0000095

189

188

185

0.925

10.27

3

2

2

2

2

183

182

183

250

2

1

1

2

2

15

0.0000075

180

181

178

0.901

8.32

2

1

1

1

2

183

180

179

500

2

2

1

1

1

16

0.000008

178

174

175

0.876

9.13

3

2

0

2

2

172

176

176

1000

2

2

2

1

1

16

0.000008

165

169

175

0.849

9.42

2

1

2

2

1

171

174

165

2000

2

2

1

3

2

17

0.0000085

175

174

170

0.848

10.02

2

1

1

1

2

169

165

164

3-MCA

36

31

33

34

37

339

0.0001695

144

151

149

0.730

232.19+

31

33

36

34

34

148

139

145

Positive Control: 8 µg/mL 3-MCA           Vehicle Control: DMSO              CE: Cloning Efficiency              MF: Mutant Frequency

* calculated from the mean values of the replicates of each group and rounded off to three decimal places

  +:Mutant frequency of 6-TG mutants is significantly higher than the concurrent vehicle control value (p < 0.05)

 

CE

=

Total No. of colonies

 

MF

=

CE of mutant colonies in selective medium

x 106

No. of cells plated

 

CE in non-selective medium

Summary Results of the Gene Mutation Assay in the Absence of Metabolic Activation (Experiment 2)

Treatment

µg/mL

Mutation Assay Flasks

Cloning Efficiency of Mutant Colonies

Cloning Efficiency Flasks

6-TG Mutants

per 106Clonable Cells

(MF)

TG Colonies/Flask

No. of Colonies/Flask

1

2

3

4

5

Total

1

2

3

CE*

DMSO

2

2

2

1

2

20

0.00001

189

182

186

0.928

10.78

3

2

3

1

2

185

188

184

250

1

1

3

1

2

15

0.0000075

180

173

180

0.890

8.43

1

2

2

1

1

176

177

182

500

1

2

2

1

2

15

0.0000075

171

165

169

0.836

8.97

2

2

1

1

1

167

163

168

1000

2

1

1

1

1

15

0.0000075

159

151

160

0.795

9.43

2

2

2

2

1

162

159

163

2000

2

2

1

1

2

16

0.000008

154

159

151

0.782

10.23

2

1

1

2

2

155

161

158

Vehicle Control: DMSO            CE: Cloning Efficiency      MF: Mutant Frequency                                       

* calculated from the mean values of the replicates of each group and rounded off to three decimal places

 

 

CE

=

Total No. of colonies

 

MF

=

CE of mutant colonies in selective medium

x 106

No. of cells plated

 

CE in non-selective medium

 

 

Conclusions:
It is concluded that the test item, PV-Echtgelb H9G VP 2430 does not have the potential to induce gene mutation in CHO-K1 cells at the tested concentrations and under the conditions of testing employed.
Executive summary:

The genotoxic potential of the test item PV-Echtgelb H9G VP 2430 to induce gene mutation in mammalian cells was evaluated using Chinese Hamster ovary (CHO) cells.

 

The study consisted of a preliminary cytotoxicity test and a definitive gene mutation test. The gene mutation test comprised of two independent experiments, one each in the presence and absence of metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver).

 

PV-Echtgelb H9G VP 2430 formed a free flowing suspension in dimethyl sulphoxide (DMSO) at 200 mg/mL.

 

In a preliminary cytotoxicity test for the selection of test concentrations for the gene mutation assay, the Relative Survival was 54 and 57 % at the 2000 µg/mL, in the presence and absence of metabolic activation, respectively. There was slight precipitation of the test item in the test medium at 160 and 320 µg/mL and moderate precipitation of the test item at and above 640 µg/mL. There was no appreciable change in the pH and osmolality of test medium. Based on these observations a maximum of 2000 µg/mL was tested in the gene mutation assay. 

 

In the gene mutation test, CHO-K1 cells were exposed to the test item in duplicate at concentrations of 250, 500, 1000 and 2000 µg/mL of the medium for 3 hours in the presence (Experiment 1) and absence (Experiment 2) of metabolic activation. In a similar way, a concurrent vehicle control (DMSO) and a positive control, 3-methylcholanthrene (Experiment 1) were also tested in duplicate.

 

There was no evidence of induction of gene mutations in any of the test item treated cultures either in the presence or absence of metabolic activation. The positive control in experiment 1 produced a statistically significant increase in the frequencies of mutants, under identical conditions.

 

The results of the forward gene mutation test at thehprtlocus with PV-Echtgelb H9G VP 2430 indicated that the test item was non-mutagenic under the conditions of this study.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

No classification

No mutagenic effects were observed in several in vitro studies.