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Genetic toxicity in vitro

Description of key information

Non genotoxic

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
no
Remarks:
pre GLP
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium, other: TA1535, TA1537, TA1538, TA100 and TA98
Details on mammalian cell type (if applicable):
- Storage: at 4 °C in deep soft nutrient agar.
- Preparation of inoculum: the day before the test, fresh cultures were prepared by inoculating 10 ml of nutrient broth with each strain, which were incubated overnight at 37 °C. - Checked for histidine requirementt: perfromed.
- Checked for biotin requirement: perfromed.
- Checked for rfa character: perfromed.
- Checked for uvrB character: perfromed.
- Checked for presence or absence of the pKM101 plasmid: perfromed.
- Checked for spontaneous reversion rate to histidine prototrophy: perfromed.
Mutagenesis tests were not taken into consideration, if any check was outside the limits of the expected range
Metabolic activation:
with
Metabolic activation system:
rat liver S9
Test concentrations with justification for top dose:
In view of the lack of antibacterial activity at the 1000 µg/plate level, test item was tested for mutagenic activity at the doses of 1000, 100 and 10 µg/plate.
Vehicle / solvent:
The substance was dissolved 10 mg/ml in sterile distilled water and dimethylsulfoxide (DMSO) 1:1 V/V and further diluted in the same solvent according to the need. DMSO, which does not interfere with the result of Ames' test, was choosen as a solvent because it gives obviously sterile solutions.
Untreated negative controls:
yes
Remarks:
spontaneous reversion rate
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
other: 2-aminofluorene and diazoacetylglycinamide
Details on test system and experimental conditions:
METHOD OF APPLICATION
0.1 ml of DMSO containing the required amount of dye, 0.1 ml of an overnight culture of each tester strain containing 10^7 bacteria and 0.5 ml of S9-mix were added in sequence to each top agar tube. The contents of each tube were quickly mixed and poured on the surface of a VB plate.

INCUBATION PERIOD: revertant colonies were counted after 2 days of incubation at 37 °C and compared with revertants obtained from controls prepared in the same way, dye omitted.

REPLICATE: tests were carried out in triplicate.

MEDIA
Vogel-Bonner E agar medium (VB) has the following composition: MgSO4 × 7H2O 0.2 g/l, citric acid × H2O 2 g/l, K2PO4 10 g/l, NaNH4HPO4 × 4 H2O, glucose 2 g/l, Agar 15 g/l.
It was prepared by mixing 750 ml of 2 % molten autoclave sterilized water agar with 250 ml of 4 times concentrated salt solution and 10 ml of 20 % glucose solution autoclaved separately. For the antibacterial test histidine was also added to give a final concentration.of 10 µg/ml.
10 cm plastic petri dishes were prepared containing 20 ml each of VB agar and used within 3 days from preparation.
Top agar was prepared by mixing 100 ml of autoclave sterilized 0.6 % agar and 0.5 % NaCl in water with 10 ml of a millipore filter sterilized 0.5 mM histidine, 0.5 mM biotin solution. Top agar was dispensed in 2 ml amounts into 16 × 100 mm glass tubes kept at 42 °C.

CYTOTOXICITY TEST
0.1 ml of DMSO containing 1000 µg of test item, 0.1 ml of a 10^-5 dilution of an overnight culture of TA1535 or TA98 containing between 100 and 500 colony forming units (CFU) and 0.5 ml of S9-mix were added in sequence to each top agar tube.
The contents of each tube were quickly mixed and poured on the surface of a histidine containing VB plate. Controls received exactly the same except that dye was omitted.
Colonies were counted after 24 hours of incubation at 37 °C. Tests were carried out in triplicate.

S9 mix
A liver supernatant fraction was prepared from Wistar male rats (170-250 g). Three rats each received a single i.p. injection of Aroclor 1254 (200 mg/ml in corn oil, 500 mg/kg). Five days after injection the rats were killed by cervical dislocation having been deprived of food for the last 16 h of life. The livers washed with 0.15 M KCl ice cold solution were weighed and transferred to a beaker containing 3 ml of KCl 0.15 M solution per gram of net weight of liver, minced with sterilesgssors and homogenized in a Potter-Evenh-jem apparatus with a teflon pestle. The homogenate was centrifuged for 10 min at 9000 g and the supernatant decanted and stored at -60 °C.
100 ml of S9-mix were prepared by mixing 10 ml of liver homogenate, 4 ml of nicotinamide adenine dinucleotide phosphate (NADP) 0.1 M, 0.5 ml of glucose-6-phosphate (G6P) 1 M, 2 ml of MgCl2 0.4 M, 2 ml of KCl 1.65 M, 50 ml of phosphate buffer pH 7.4 0.2 M, 31.5 ml of sterile distilled water NADP and G6P stock solutions were millipore filter sterilized and stored at -20 °C. The stock salt solutions were autoclave sterilized and stored at 4 °C.
Each batch of S9-mix was tested for sterility and activity.
Species / strain:
S. typhimurium, other: TA1535, TA1537, TA1538, TA100 and TA98
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
It can be concluded that test item is without any mutagenic activity within the limits of the test system used.
For a better evaluation of the limits of validity of the above statement, it may be taken into consideration that 1 µg of 2-aminofluorene gave a relevant increase of revertants with strains TA1538 and TA98, while no revertants were caused by 1000 µg of test item.
Conclusions:
It can be concluded that test item is without any mutagenic activity within the limits of the test system used.
Executive summary:

The study was performed to investigate the potential of test item to induce gene mutations using the Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98, and TA 100. The assay was performed with liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations: 10, 100 and 1000 μg/plate.

No toxic effects occurred in the preliminary investigation. The test item resulted to be without any mutagenic activity within the limits of the test system used.

For a better evaluation of the limits of validity of the above statement, it may be taken into consideration that 1 µg of 2-aminofluorene gave a relevant increase of revertants with strains TA1538 and TA98, while no revertants were caused by 1000 µg of test item.

Conclusion

It can be concluded that test item is without any mutagenic activity within the limits of the test system used.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From January 10th to March 06th, 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Justification for type of information:
The read across approach is detailed into the document attached to the IUCLID section 13.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Cells: are characterised by their high proliferation rate (10-12 h doubling time of the BSL BIOSERVICE stock cultures) and their cloning efficiency, usually more than 50 %. The cells obtain a near diploid karyotype. They are heterozygous at the Thymidine Kinase (TK) locus in order to detect mutation events at the TK- locus. To prevent high backgrounds arising from spontaneous mutation, cells lacking TK can be eliminated by culturing cells in RPMI 1640 supplemented with: 9.0 µg/ml hypoxanthine; 15.0 µg/ml thymidine; 22.5 µg/ml glycine; 0.1 µg/ml methotrexate
The cells are resuspended in medium without methotrexate but thymidine, hypoxanthine and glycine for 1-3 days.
- St6orage: large stock cultmes of the cleansed L5l78Y cell line are stored over liquid nitrogen (vapour phase) in the cell bank of testing laboratory.
- Periodically checked: each cell batch is routinely checked for mycoplasma infection.
Thawed stock cultures are maintained in plastic culture flasks in RPMI l640 complete medium and subcultured three times per week.
Metabolic activation:
with and without
Metabolic activation system:
Mammalian Microsomal Fraction S9 Homogenate
Test concentrations with justification for top dose:
Experiment I, with and without metabolic activation: 10, 50, 100, 200, 400, 600, 1800 and 5000 µg/ml
Experiment II, with metabolic activation: 30, 70, 150, 350, 700, 2100, 3600 and 5000 µg,/ml
Experiment II, without metabolic activation: 10, 20, 50, 100, 200, 600, 1200 and 2500 µg/ml
Vehicle / solvent:
- Vehicle used: no vehicle. The test item was not soluble in any of the investigated vehicles. Therefore the test item was suspended in cell culture medium (RPMI + HS).
- Justification for choice of solvent/vehicle: a solubility test was performed with different solvents and vehicles up to the maximum recommended concentration of 5 mg/ml.
Untreated negative controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without metabolic activation
Untreated negative controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without metabolic activation
Untreated negative controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With metabolic activation
Details on test system and experimental conditions:
PRE-EXPERIMENT for TOXICITY
The toxicity of the test item was determined in pre-experiments up to a maximum concentration of 5 mg/ml. For pre-experiment l six concentrations [156, 313, 625, 1250, 2500, 5000 µg/ml] were tested with and without metabolic activation. For pre-experiment ll without metabolic activation (24 h long·term exposure) six concentrations [10, 100, 300, 800, 2000, 5000 µg/ml] were tested. The experimental conditions in these pre—experiments were the same as described below in the paragraph experimental performance.

METHOD OF APPLICATION: RPMI medium; short-term experiment: 11 ml with 5 % horse serum; long-term experiment: 25 ml with 7.5 % horse serum

DURATION
Short-term
- Exposure duration: 4 hours
- Removal of test item: by centrifugation; 200 x g, 10 min
- Expression time: 2 days at 37 °C in 5 % CO2/95 % humidified air
- Cell density: detrmined each day and adjusted to 3×10^5 cell/ml in a total culture volume of 20 ml, if necessary.
- Determination of cells number: by automated cell counting.

Long-term
- Exposure duration: 24 hours
- Removal of test item: by centrifugation; 200 × g, 10 min
- Expression time: 2 days at 37 °C in 5 % CO2/95 % humidified air.
- Cell density: detrmined each day and adjusted to 3×10^5 cell/ml in a total culture volume of 20 ml, if necessary
- Determination of cells number: by automated cell counting.

DETERMINATION of CLONING EFFICIENCY (CE)
Detrmined by seeding a statistical number of 1.6 cell/well in two 96-well plates
- Incubation: Cells were incubated for at least 6 days at 37 °C in a humidified atmosphere with 5 % CO2
- Selective medium: additionally, cultures were seeded in selective medium. Cells from each experimental group were seeded in four 96-well plates at a density of approximately 2000 cells/well in 200 µl selective medium with TFT. The plates were scored after an incubation period of -14 days at 37 °C in 5 % CO2/95 % humidified air.
- Mutant frequency determination: calculated dividing the number of TFT resistant colonies by number of cells plated for selection, corrected for the plating efficiency of cells from the same culture grown in the absence of TFT.

SUSPENSION GROWTH (SG)
Reflects the number of times the cell number increases from the starting cell density. The relative total growth (RTG) is the product of the relative suspension growth (RSG calculated by comparing the SG of the dose groups with the SG of the control) and the relative cloning efficiency (RCE) for each culture: RTG = RSG × RCE /100. The mutant frequencies obtained from the experiments are compared with the Global Evaluation Factor (GEF). To arrive at a GEF was analyzed distributions of negative/vehicle mutant frequencies of the MLA. The GEF is defined as the mean of the negative/vehicle mutant frequency plus one standard deviation. The non-parametric Mann-Whitney test is applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative/solvent controls.

MAMMALIAN MICROSOMAL FRACTION S9 HOMOGENATE
The S9 liver microsomal fraction was prepared at testing laboratory. Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and B-naphthotlavone (100 mg/kg bw) for three consecutive days by oral route.
The following quality control determinations were performed:
a) Biological activity in:
· the Salmonella typhimurium assay using 2-aminoanthracene
- the mouse lymphoma assay using benzo[a]pyrene
- the chromosome aberration assay using cyclophosphamide.
b) Sterility Test
A stock of the supernatant containing the microsomes was frozen in aliquots of 2 and 4.5 ml and stored at ≤ -75 °C.
The protein concentration in the S9 preparation was 39 mg/ml.

S9 mix
An appropriate quantity of the S9 supematant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/ml in the cultures. Cofactors were added to the S9 mix to reach the following concentrations: 8 mM MgCl2, 33 mM KCI, 5 mM Glucose-6·phosphate
5 mM NADP in 100 mM sodium-phosphate-buffer pH 7.4. During the experiment the S9 mix was stored on ice.

ACCEPTABILITY OF THE ASSAY
A mutation assay is considered acceptable if it meets the criteria mentioned in current international guidelines and the current recommendations of the IWGT (11, 12, 13, 14, 1 5):
- At least three out of four 96-well plates from the TFT resistance-testing portion of the experiment are scorable.
- The cloning efficiency of the negative and/or solvent controls is in the range 65 - 120 %.
- The spontaneous mutant frequency in the negative and/or solvent controls is in the range 50-170 per 10 cells
- The cell number of the negative/solvent controls should undergo 8-32 fold increase during a 2 day growth period (short—term treatment) or 32-180 fold increase during a 3 day growth period (long—term treatment).
- The clastogenic positive controls (MMS and B[a]P) have to produce an induced mutant frequency (total mutant frequency minus concurrent negative control mutant frequency) of at least 300 per 10^6 cells with at least 40 % of the colonies being small colonies or with an induced small colony mutant frequency of at least 150 per 10^6 cells The RTG must be greater than 10 %.
Evaluation criteria:
The test item is considered mutagenic if following criteria are met:
- The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 per 10^6 cells
- A dose-dependent increase in mutant frequency is detected.
Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (240 % of total colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations.
According to the OECD guideline, the biological relevance is considered first for the interpretation of results. Statistical methods might be used as an aid in evaluation the test result.
A test item is considered to be negative if the induced mutant frequency is below the GEF and the trend test is negative.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: in experiment I with and without metabolic activation precipitation of the test item was noted from concentrations of 50 µg/rnl and higher; in experiment II with metabolic activation precipitation was noted from concentrations of 30 µg/ml and higher; in experiment II without metabolic activation from concentrations of I0 µg/ml and higher.

In experiment I with metabolic activation all validity criteria were met.
The negative controls showed mutant frequencies within the acceptance range of 50-170 mutants/10^6 cells, according to the IWGT criteria. The mutant frequencies of the negative controls were 123.3 and 128.8 mutants/10^6 cells, the positive control B[a]P induced a distinct increase in mutant frequency with 627.7 mutants/10^6 cells. The mutant frequencies induced by the test item did not show a biologically relevant increase. The GEF of 126 was not exceeded in any of the dose groups showing induced mutant values between -5.6 and 69.1 mutants/10^6 cells. A statistical analysis confirmed that most of the mutant values were significantly different to the mutant frequencies of the negative controls, but there was no evidence for a dose-response relationship.

ln experiment I without metabolic activation all validity criteria were met.
The negative controls showed mutant frequencies within the acceptance range of 50-170 mutants/10^6 cells, according to the IWGT criteria. The mutant frequencies of the negative controls were 135.2 and 163.3 mutants/10^6 cells, the positive controls EMS and MMS induced a distinct increase in mutant frequency with 625.8 and 529.7 mutants/10^6 cells. The mutant frequencies induced by the test item did not show a biologically relevant increase. The GEF of 126 was not exceeded in any of the dose groups showing induced mutant frequencies between -61.5 and 68.5 mutants/10^6 cells. A statistical analysis continned that most of the mutant frequencies were significantly different to the mutant frequencies of the negative controls, but there was no evidence for a dose-response relationship.

ln experiment II with metabolic activation all validity criteria were met.
The negative controls showed mutant frequencies within the acceptance range of 50-170 mutants/10^6 cells, according to the IWGT criteria. The mutant frequencies of the negative controls were 149.0 and 150.1 mutants/10^6 cells, the positive control B[a]P induced a distinct increase in mutant frequency with 696.9 mutants/10^6 cells. The mutant frequencies induced by the test item did not show a biologically relevant increase. The GEF of 126 was not exceeded in any of the dose groups showing induced mutant frequencies between -32.6 and 34.2 mutants/10^6 cells. None of the observed mutant frequencies were statistically significantly increased over those from the negative controls.
Conclusions:
Under the experimental conditions, the test item is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5l78Y cells.
Executive summary:

The test item was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

The selection of the concentrations used in the main experiments was based on data from the pre-experiments. In experiment I 5000 µg/ml (with and without metabolic activation) was selected as the highest concentration. In experiment ll 5000 µg/ml (with metabolic activation) and 2500 µg/ml (without metabolic activation) were selected as the highest concentrations. Experiment 1 with and without metabolic activation and experiment ll with metabolic activation were performed as a 4 h short-term exposure assay. Experiment II without metabolic activation was performed as a 24 h long-term exposure assay.

The test item was investigated at the following concentrations: Experiment I with and without metabolic activation 10, 50, 100, 200, 400, 600, 1800 and 5000 µg/ml; Experiment ll with metabolic activation 30, 70, 150, 350, 700, 2100, 3600 and 5000 µg/ml; and without metabolic activation 10, 20, 50, 100, 200, 600, 1200 and 2500 µg/ml.

Precipitation of the test item was noted in all experiments.

Growth inhibition was observed in experiment I and ll without metabolic activation.

ln experiment I with metabolic activation the relative total growth (RTG) was 93.7 % for the highest concentration (5000 µg/ml) evaluated; the highest concentration evaluated without metabolic activation was 5000 µg/ml with a RTG of 65.0 %. In experiment II with metabolic activation the relative total growth (RTG) was 71.5 % for the highest concentration (5000 µg/ml) evaluated; the highest concentration evaluated without metabolic activation was 2500 µg/ml with a RTG of 12.9 %.

In experiment I and ll no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). The Global Evaluation Factor (GEF; defined as the mean of the negative/vehicle mutant frequency plus one standard deviation) was not exceeded by the induced mutant frequency at any concentration.

No dose~response relationship was observed.

Additionally, in experiment I and II colony sizing showed no clastogenic effects induced by the test item under the experimental conditions (with and without metabolic activation).

EMS, MMS and B[a]P were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. Additionally, MMS and B[a]P significantly increased the number of small colonies, thus proving the efficiency of the test system to indicate potential clastogenic effects.

Conclusion

In the described mutagenicity test under the experimental conditions reported, the test item is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Justification for type of information:
The read across approach is detailed into the document attached to the IUCLID section 13.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
On the day of the experiment, the test article was dissolved in saline G solution.
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
At 7 h 1.0 mg/ml; at 18 h 0.5, 2.5, 5.0 mg/ml; at 28 h 1.0 mg/ml
Vehicle / solvent:
- Vehicle used: physiol. saline G.
Untreated negative controls:
yes
Positive controls:
yes
Remarks:
Dissolved in nutrient medium; final concentration of 0.72 mg/ml = 5.76 mM.
Positive control substance:
ethylmethanesulphonate
Remarks:
without methabolic activation
Untreated negative controls:
yes
Positive controls:
yes
Remarks:
Dissolved in nutrient medium; final concentration of 1.40 µg/ml = 5.00 µM.The stability of CPA at room temperature is good. At 20 °C only 1 % of CPA is hydrolysed per day in aqueous solution.
Positive control substance:
cyclophosphamide
Remarks:
with methabolic activation
Details on test system and experimental conditions:
CELL CULTURES
Large stocks of the V79 cell line (supplied by LMP; D-6100 Darmstadt, F.R.G.) are stored in liquid nitrogen in the cell bank allowing the repeated use of the same cell culture batch in experiments. Before freezing, each batch is screened for mycoplasma contamination and checked for karyotype stability. 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 cm^2 plastic flasks. Seeding is done with about 5 x 10^5 cells per flask in 15 ml of MEM (minimal essential medium) supplemented with 10 % fetal calf serum. The cells are subcultured twice weekly. The cell cultures are incubated at 37 °C and 4.5 % carbon dioxide atmosphere.

MAMMALIAN MICROSOMAL FRACTION S9 MIX
- S9 preparation: the S9 liver microsomal fraction was obtained from the liver of 8 - 12 weeks old male Wistar rats, strain WU (Savo-Ivanovas, med; weight ca. 150 - 200 g) which received a single i.p. injection of 500 mg/kg b.w. Aroclor 1254 in olive oil 5 days previously.
After cervical dislocation the livers of the animals were removed, washed in 150 mM KCl and homogenised. The homogenate, diluted 1:3 in KCl was centrifuged cold at 9000 g for 10 minutes. A stock of the supernatant containing the microsomes was frozen in ampoules of 2 or 5 ml and stored at -70 °C. Small
numbers of the ampoules were kept at -20 °C for only several weeks before use. The standardization of the protein content was made using an analysis kit. The protein concentration in the S9 preparation is usually between 20 and 45 mg/ml.

- S9 Mix: before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.3 mg/ml in the cultures. The composition of the cofactor solution was concentrated to yield the following concentrations in the S9 mix:
8 mM MgCla
33 mM KCl
5 mM glucose-6Tphosphate
5 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath.

PRE-EXPERIMENT FOR TOXICITY AND DOSE SELECTION
The toxicity of the test article was determined in a pre-experiment in order to establish a concentration dependent plating efficiency relationship. The experimental conditions in this preexperiment were the same as described below for the experiment.

According to the results from pre-experiment six concetrations (18 h interval) to be applied in the chromosomal aberration assay were chosen.
The highest dose level used was 10 mM unless limited by the solubility of the test article or that producing some indication of cytotoxicity (reduced plating efficiency and/or partial inhibition of mitosis).
If toxic effects were produced the highest dose level should reduce the plating efficiency to approximately 20-50 %. In addition, this concentration should suppress if possible mitotic activity (% cells in mitosis) by approximately 50 %, but not so great a reduction that insufficient scorable mitotic cells can be found.
Treatment was performed with the following concentrations:
without S9 mix:
7 h: 0.50; 1.00 mg/ml
18 h: 0.10; 0.25; 0.50; 1.00; 2.50; 5.00 mg/ml
28 h: 0.50; 1.00 mg/ml

with S9 mix:
7 h: 0.50; 1.00 mg/ml
18 h: 0.10; 0.25; 0.50; 1.00; 2.50; 5.00 mg/ml
28 h: 0.50; 1.00 mg/ml
One (7 h and 28 h) and three concentrations (18 h) were selected to evaluate metaphases for cytogenetic damage.
In the pre-experiment for toxicity the colony forming ability of the V79 cells was clearly reduced after treatment with 5.00 mg/ml in the absence and presence of S9 mix.
In the cytogenetic experiment at fixation interval 18 h with and without metabolic activation samples after treatment with 5.00 mg/ml as highest concentration were evalutated. In a previous experiment, at fixation interval's 7 h and 28 h in the samples treated with 2.50 mg/ml and 5.00 mg/ml not enough scorable cells could be found. Therefore, in the main experiment cultures were treated only with 0.50 mg/ml and 1.00 mg/ml at these intervals. Samples treated with 1.00 mg/ml could be scored for cytogenetic demage.

EXPERIMENTAL PERFORMANCE
- Seeding of the culture: two days old logarithmically growing stock cultures more than 50 % confluent were trypsinised and a single cell suspension
was prepared. The trypsin concentration was 0.2 % in Ca-Mg-free salt solution. The Ca-Mg-free salt solution was composed as follows (per litre): NaCl 8000 mg, KCl 400 mg, Glucose 1000 mg, NaHCO3 350 mg
The cells were seeded into Quadriperm dishes which contained microscopic slides (4 chambers per dish and test group). In each chamber 5 × 10^4 - 1 x 10^5 cells were seeded with regard to preparation time. The medium was MEM + 10 % FCS.

- Treatment: after 48 h (7 h, 28 h preparation interval) and 55 h (18 h preparation interval) the medivim was replaced with serum-free medium containing the test article, either without S9 mix or with 20 µL/mL S9 mix. After 4 h this medium was replaced with normal medium after rinsing twice with "saline G".
The "saline G" solution is composed as follows (per litre): NaCl 8000 mg, KCl 400 mg, Glucose 1100 mg, Na2HPO4.7H20 290 mg, KH2PO4 150 mg, ph was adjusted at 7.2
All incubations were done at 37 °C in a humidified atmosphere with 4.5 % CO2.

- Preparation of the Cultures: 5, 15.5 and 25.5 h after the start of the treatment colcemid was added (0.2 µg/ml culture medium) to the cultures. 2.0 h (7 h
interval) or 2.5 h later, (18 h and 28 h interval) the cells were treated on the slides in the chambers with hypotonic solution (0.4 % KCl) for 20 min at 37 °C.After incubation in the hypotonic solution the cells are fixed with 3 + 1 absolute methanol + glacial acetic acid. All four slides per group were prepared.
After fixation the cells were stained with giemsa.

- Analysis of Metaphase Cells: evaluation of the slides were performed using NIKON microscopes, with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. At least 100 well spread metaphases per slide were scored for cytogenetic damage on coded slides. Only metaphases with characteristic chromosome number of 22 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined. In addition, the number of polyploid cells (% polyploid metaphases; in case of this aneuploid cell line polyploid means a near tetraploid karyotype) was scored.

DATA RECORDING
The data generated were recorded in the laboratory protocol. The results are presented in tabular form, including experimental groups with the test article, negative and positive controls.
Evaluation criteria:
The chromosomal aberration assay is considered acceptable if it meets the following criteria:
a) the number of aberrations found in the negative and/or solvent controls fall within the laboratory historical control, data range: 0.00 % - 4.00 %.
b) the positive control substances should produce significant increases in the number of cells with structural chromosome aberrations.

EVALUATION OF RESULTS
A test article is classified as mutagenic if it induces either a significant dose-related increase in the number of structural chromosomal aberrations or a significant and reproducible positive response for at least one of the test points.
A test article producing neither a significant dose-related increase in the number of structural chromosomal aberrations nor a significant and reproducible positive response at any one of the test points,is considered non-mutagenic in this system.
However, both biological and statistical significance should be considered together.
Statistics:
Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the chi-square test. Evaluation was performed only for cells carrying aberrations exclusive gaps.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
highest dose levels
Vehicle controls validity:
not specified
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
PRE-EXPERIMENT
In the absence and presence of S9 mix after treatment with concentration of 5.00 mg/ml the colony forming ability was clearly reduced as compared to the solvent controls.

MAIN EXPERIMENT
At fixation interval 18 hours,.the mitotic index was clearly reduced after treatment with the highest dose levels in the presence of S9 mix, indicating that substance had cytotoxic properties. In some cases, (intervals 7 h and 28 h; with S9 mix and 18 h; without S9 mix) slight reductions of the mitotic indices could be observed.
Both, in the absence and presence of S9 mix the test article did not increase either significantly or biologically relevant the frequency of cells with aberrations at any fixation interval. The aberration rates of the cells after treatment with the- test article (0.75 % - 3.33 %) were in or very near to the range of the control values: 1.50 % - 3.25 %.

CONTROLS
EMS (0.72 mg/ml) and CPA (1.40 µg/ml) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.
Conclusions:
It can be stated that in the described study and under the experimental conditions reported, the test article did not induce structural chromosome aberrations in the V79 Chinese hamster cell line.
Executive summary:

The test article was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro, in the absence and presence of metabolic activation by S9 mix, according to the OECD guideline 473.

Preparation of chromosomes was done 7 h (high dose), 18 h (low, medium and high dose) and 28 h (high dose) after start of the treatment with the test article. The treatment interval was 4 h. In each experimental group, except the positive controls, four parallel cultures were used. Per culture 100 metaphases were scored for structural chromosome aberrations.

The following dose levels were evaluated, without S9 mix: with 89 mix: at 7 h 1.0 mg/ml, at 18 h 0.5, 2.5 and 5.0 mg/ml, at 28 h 1.0 mg/ml.

In the pre-experiment on toxicity (colony forming ability) in the absence and presence of S9 mix after treatment with concentration of 5.0 mg/ml the colony forming ability was clearly reduced as compared to the solvent controls. In the main experiment, at fixation interval 18 hours, the mitotic index was clearly reduced after treatment with the highest dose levels in the presence of S9 mix, indicating that test item had cytotoxic properties. In some cases, (intervals 7 h and 28 h; with 89 mix and 18 h; without 89 mix) slight reductions of the mitotic indices could be observed. Both, in the absence and presence of S9 mix the test article did not increase either significantly or biologically relevant the frequency of cells with aberrations at any fixation interval. The aberration rates of the cells after treatment with the test article (0.75 - 3.33 %) were in or very near to the range of the control values: 1.50 - 3.25 %. Positive controls showed distinct increases in cells with structural chromosome aberrations.

Conclusion

In conclusion, it can be stated that, in the described study and under the experimental conditions reported, the test article did not induce structural chromosome aberrations in the V79 Chinese hamster cell line.

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

Additional information

Investigation on the genetic toxicity has been performed with the integrated evaluation of the in vitro AMES assay performed on Direct Orange 118, in vitro gene mutation on mammalian cells performed on the structural analogue Similar Substance 01 and in vitro chromosomal aberration conducted on Similar Substance 03.

The read across approach can be considered as reliable and suitable for the purpose; details and explanations are detailed in the report attached to the IUCLID section 13.

IN VITRO GENE MUTAITON IN BACTERIA

The study was performed using the Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98, and TA 100, in presence and absence of liver microsomal activation system. The test article was tested at the following concentrations: 10, 100 and 1000 μg/plate. No toxic effects occurred in the preliminary investigation. The test item resulted to be without any mutagenic activity within the limits of the test system used. For a better evaluation of the limits of validity, it may be taken into consideration that 1 µg of 2-aminofluorene gave a relevant increase of revertants with strains TA1538 and TA98, while no revertants were caused by 1000 µg of test item. Thus, it was concluded that test item is without any mutagenic activity within the limits of the test system used (Monti-Bracadin, 1975).

IN VITRO GENE MUTAITON IN MAMMALIAN CELLS

The Similar Substance 01 was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The selection of the concentrations used in the main experiments was based on data from the pre-experiments. Precipitation of the test item was noted in all experiments. Growth inhibition was observed in experiment I and ll without metabolic activation. ln experiment I with metabolic activation the relative total growth (RTG) was 93.7 % for the highest concentration (5000 µg/ml) evaluated; the highest concentration evaluated without metabolic activation was 5000 µg/ml with a RTG of 65.0 %. In experiment II with metabolic activation the relative total growth (RTG) was 71.5 % for the highest concentration (5000 µg/ml) evaluated; the highest concentration evaluated without metabolic activation was 2500 µg/ml with a RTG of 12.9 %. In experiment I and ll no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). The Global Evaluation Factor (GEF; defined as the mean of the negative/vehicle mutant frequency plus one standard deviation) was not exceeded by the induced mutant frequency at any concentration. No dose~response relationship was observed. Additionally, in experiment I and II colony sizing showed no clastogenic effects induced by the test item under the experimental conditions (with and without metabolic activation).In conclusion, under the experimental conditions reported, the test item was considered to be non-mutagenic (Trenz, 2012).

IN VITRO CHROMOSOMA ABERRATION

The Similar Substance 03 was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster, in the absence and presence of metabolic activation by S9 mix, according to the OECD guideline 473. In the pre-experiment on toxicity (colony forming ability) in the absence and presence of S9 mix after treatment with concentration of 5.0 mg/ml the colony forming ability was clearly reduced as compared to the solvent controls. In the main experiment, at fixation interval 18 hours, the mitotic index was clearly reduced after treatment with the highest dose levels in the presence of S9 mix, indicating that test item had cytotoxic properties. In some cases, (intervals 7 h and 28 h; with 89 mix and 18 h; without 89 mix) slight reductions of the mitotic indices could be observed. Both, in the absence and presence of S9 mix the test article did not increase either significantly or biologically relevant the frequency of cells with aberrations at any fixation interval. The aberration rates of the cells after treatment with the test article (0.75 - 3.33 %) were in or very near to the range of the control values: 1.50 - 3.25 %. In conclusion, it was stated that, in the described study and under the experimental conditions reported, the test article did not induce structural chromosome aberrations in the V79 Chinese hamster cell line (Heidemann and Volkner, 1990).

Justification for classification or non-classification

According to the CLP Regulation (EC) No 1272/2008, for the purpose of the classification for germ cell mutagenicity, substances are allocated in one of two categories in consideration of the fact that they are:

- substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans or substances known to induce heritable mutations in the germ cells of humans or

- substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

The available information on the substance under investigation and on the structural analogues suggest that the substance did not show any reasons of concern from the genotoxicity point of view.

 

In conclusion, the substance does not meet the criteria to be classified for genetic toxicity according to the CLP Regulation (EC) No 1272/2008.