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Genetic toxicity in vitro

Description of key information

Non mutagenic

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Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From January to February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted 29 July, 2016
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Test material information:
Composition 1
Species / strain:
Chinese hamster Ovary (CHO)
Details on mammalian cell lines (if applicable):
CELLS USED
- Source of cells: ECACC (European Collection of Cell Cultures).
- Cells: Sub-line (K1) of Chinese hamster ovary cell line CHO. The CHO cell line was originally derived from the ovary of a female Chinese hamster (Kao and Puck, 1967). The CHO K1 is a sub-line of CHO cell line.
- Methods for maintenance in cell culture if applicable: the cell stocks are kept in liquid nitrogen. For each experiment the cells were thawed rapidly, the cells diluted in Ham's F12 medium containing 10 % foetal bovine serum and incubated at 37 ± C in a humidified atmosphere of 5 % CO2 in air. Growing cells were subcultured in an appropriate number of flasks.
- Culturing: the CHO K1 cells for the study were grown in Ham's F12 medium (F12-10) supplemented with 1 % Antibiotic-antimycotic solution (containing 10000 U/ml penicillin, 10 mg/ml streptomycin and 25 µg/ml amphotericin-B) and heat-inactivated bovine serum (final concentration 10 %).

MEDIA USED
- Periodically checked for Mycoplasma contamination: each batch of frozen cells was purged of HPRT mutants and was free for mycoplasma infections, tested by Central Agricultural Office, National Animal Health Institute, Budapest, Hungary.
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Test concentrations with justification for top dose:
MAIN EXPERIMENT: 250, 500, 1000 and 2000 µg/ml, with and without metabolic activation.
PRELIMINARY EXPERIMENT for CYTOTOXICITY: 125, 250, 500, 1000, 1500 and 2000 µg/ml, with and without metabolic activation.
Vehicle:
- Solvent used: DMSO
- Justification for choice of solvent/vehicle: the solvent is compatible with the survival of the CHO cells and the S9 activity and was chosen based on the results of the preliminary Solubility Test. The suitability was confirmed with the available laboratory’s historical database.
Negative controls:
yes
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and conditions:
PROCEDURES
- Preparation of the dishes: on the day of treatment the culture medium of exponentially growing cell cultures were replaced with medium (F12-5) containing the test item.
- Treatment: a 5-hour treatment in the presence and absence of S9-mix was performed.
- Number of cells: 5 x10^6 cells were each placed in sterile dishes.
- Incubation conditions: dishes were incubated for approximately 24 hours before treatment at 37 °C in a humidified atmosphere of 5 % CO2.
- Post-exposure incubation: following the exposure period the cells were washed with F12-5 medium and incubated in fresh F12-10 medium for 19 hours.
- Count: after the 19-hour incubation period, cells were washed twice with F12-10 medium and suspended by treatment with trypsin-EDTA solution and counted using a Bürker chamber.
- Precipitation check: solubility of the test item in the cultures was assessed by the naked eye, at the beginning and end of treatment.
- Adjustement of cell number: in samples where sufficient cells survived, cell number was adjusted to 10^5 cells/ml. Throughout the expression period, cells were transferred to dishes for growth or diluted to be plated for survival.

REPLICATES: duplicate cultures were used at each test item concentration, for negative (solvent) controls and the positive controls for treatment without and with S9-mix.

PLATING SURVIVAL
Following adjustment of the cultures to 2 x 10^5 cells/ml, samples from these cultures were diluted to 40 cells/ml.
A total of 5 ml (200 cells/dish) of the final concentration of each culture was plated into 3 parallel dishes (diameter is approx. 60 mm).
The dishes were incubated at 37 °C in a humidified atmosphere of 5 % CO2 in air for 4-6 days for growing colonies.
Then, colonies were fixed with methanol, stained with Giemsa and counted. Survivals were assessed by comparing the cloning efficiency of the test item treated groups to the negative (solvent) control.

EXPRESSION OF MUTANT PHENOTYPE: during the phenotypic expression period the cultures were subcultured. Aliquots of approximately 2x10^6 cells were taken on days 1, 3, 6 and evaluated on day 8.

SELECTION OF THE MUTANT PHENOTYPE: at the end of the expression period, cultures from each dose level were adjusted to 2 x 10^5 cells / dish (4 x five dishes) in selection medium (hypoxanthine Ham's F12-SEL medium) containing 3.4 µg/ml of thioguanine (6-TG).

PLATING OF VIABILITY: at the end of the expression period cell number in the samples was adjusted to 2 × 10^5 cells/ml. Cells were plated in 3 parallel dishes (diameter is approx. 60 mm) for a viability test as described in “Plating for Survival“ section for the survival test.

FIXATION AND STAINING OF COLONIES: after the selection period, the colonies were fixed with methanol for five minutes, stained with Giemsa and counted for either mutant selection or cloning efficiency determination.

DETERMINATION OF CYTOTOXICITY
Treatment concentrations for the mutation assay were selected on the basis of the result of a Pre-test on cell toxicity. During the Pre-test on Toxicity (cytotoxicity assay), proliferating cells were trypsinised and cell suspensions was prepared in Ham's F12-10 medium. Cells were seeded into petri dishes (tissue cultures quality: TC sterile) at 2.5x10^6 cells each and incubated with culture medium.
After 24 hours the cells were treated with the suitable concentrations of the test item in absence or in presence of S9 mix (50 ml/ml) and incubated at 37 °C for 5 hours.
After the treatment cells were washed and incubated in fresh Ham's F12-10 medium for 19 hours. 24 hours after the beginning of treatment, the cultures were washed with Ham's F12-5 medium and the cells were covered with trypsin-EDTA solution, counted and the cell concentration will be adjusted to 40 cells/ml with Ham's F12-10 medium. For each concentration of test solution or control solution, 5 ml will be plated in parallel into 3 sterile dishes (diameter is approx. 60 mm). The dishes were incubated at 37 °C in a humidified atmosphere of 5 % CO2 in air for 5-7 days for colony growing. Colonies were then be fixed with methanol and will be stained with Giemsa and the colonies will be counted. In order to determine cytotoxicity, survivals were assessed by comparing the colony forming ability of the treated groups to the negative (solvent) control.
Precipitation of the test item in the final culture medium was visually examined at beginning and end of the treatments.

PRELIMINARY SOLUBILITY TEST
A non GLP Preliminary Solubility Test was performed suspending the test item in DMSO. A homogeneous suspension was obtained up to a concentration of 100 mg/ml.

METABOLIC ACTIVATION FRACTION
The S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver was provided by Trinova Biochem GmbH (Rathenau Strasse 2; D-35394 Giessen, Germany; Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA).

The S9 Mix (with Rat Liver S9)
Before adding to the culture medium the S9 Mix will be kept in an ice bath.
S9 mix preparation (concentration in the mix): HEPES 0.2 ml/ml, KCl 0.1 ml/ml, MgCl2 0.1 ml/ml, NADP 0.1 ml/ml, D-Glucose-6-phosphate (Monosodium salt) 0.1 ml/ml, F12 medium 0.1 ml/ml and S9 0.3 ml/ml.

ACCEPTANCE CRITERIA
The assay was considered valid as all the following criteria were met:
- The mutant frequency of concurrent negative controls is within the 95% control limits of the distribution of the laboratory’s historical negative control database.
- The positive control chemicals induced a statistically significant and biologically relevant increase in mutant frequency compared to the concurrent negative control. The increases are compatible with the laboratory historical positive control data base.
- Adequate number of cells and concentrations were analysable.
- Two experimental conditions with and without metabolic activation were tested.
- The highest concentration is adequate.
- The cloning efficiency of the negative controls is between the range of 60 % to 140 % on Day 1 and 70 % to 130 % on Day 8.
Evaluation criteria:
Providing that all acceptability criteria are fulfilled, a test item 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 negative control,
- any of the results are outside the distribution of the laboratory historical negative control data (based 95 % control limit),
- the increase of mutant frequency is concentration-related when evaluated with an appropriate trend test.

Providing that all acceptability criteria are fulfilled, a test item is considered clearly negative if, in all experimental conditions examined:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend test,
all results are inside the distribution of the historical negative control data (based 95 % control limit).
Statistics:
Statistical Analysis was performed with SPSS PC+ software for the following data:
- mutant frequency between the negative (solvent) control group and the test item or positive control item treated groups.
- mutant frequency between the laboratory historical negative (solvent) control group and concurrent negative (solvent) control, the test item or positive control item treated groups.
- The lower and upper 95 % confidence intervals of historical control were calculated with C-chart.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Cytotoxicity:
no
Remarks on result:
other: test still in progress

Preliminary experiment for cytotoxicity (5 hours treatment)

Test group S9-mix Treatment/time/ hour Number of colonies/200cells/dish Mean  Relativeasurvival (%)
Untreated Control  - 206 201 203 203.3 101
Solvent Control (DMSO) 5 202 200 204 202.0 100
Test item, 125µg/ml 5 203 199 203 201.7 100
Test item, 250µg/ml 5 205 202 203 203.3 101
Test item, 500µg/ml 5 199 203 204 202.0 100
Test item, 1000µg/ml 5 203 202 204 203.0 100
Test item, 1500µg/ml 5 194 200 197 197.0 98
Test item, 2000µg/ml 5 194 199 198 197.0 98
Untreated Control + - 201 201 203 201.7 103
Solvent Control (DMO) + 5 195 196 196 195.7 100
Test item, 125µg/ml + 5 191 190 195 192.0 98
Test item, 250µg/ml + 5 194 192 192 192.7 98
Test item, 500µg/ml + 5 152 158 157 155.7 80
Test item, 1000µg/ml + 5 135 139 140 138.0 71
Test item, 1500µg/ml + 5 128 132 127 129.0 66
Test item, 2000µg/ml + 5 124 126 122 124.0 63

* Relative to Solvent Control

Conclusions:
Substance not cytotoxic; main test still ongoing
Executive summary:

The substance was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The test item was dissolved in DMSO and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study without and with metabolic activation: 125, 250, 500, 1000, 1500 and 2000 µg/ml, with and without S9-mix.

In the performed Mutation Assay the concentration levels were chosen mainly based on the maximum recommended concentration. The maximum recommended concentration for soluble, lower -cytotoxic substances is 2000 µg/ml (based on the updated OECD Guideline 476 (2016)). Phenotypic expression was evaluated up to 8 days following exposure.

Conclusion

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Reference:
Composition 0
Principles of method if other than guideline:
Escherichia coli strain WP2 uvrA- were tested in order to assess the test item mutagenicity activity.
A first experiments has been performed to evaluate the antibacterial activity potential at the concentration of 5000 µg/plate; after that, the mutagenicity assay has been performed at concentrations between 5000 - 10 µg/plate.
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Species / strain:
E. coli WP2 uvr A
Details on mammalian cell lines (if applicable):
Escherichia coli strain WP2 uvrA- was kept at 4 °C in deep soft nutrient agar.
The day before the test, a fresh culture was prepared in nutrient broth and incubated overnight at 37 °C.
Concurrently with each series of test, the above cultre was checked for relevant characters, namely:
- tryptophan requirement
- uvrA character
- number of spontaneous tryptophan prototrophe revertants
- number revertants induced by methylmethane sulphonate (a known mutagen of the base pair-substitution type).
Metabolic activation:
with and without
Metabolic activation system:
liver supernatant fraction (rats)
Test concentrations with justification for top dose:
ANTIBACTERIAL ACTIVITY: 5000 µg/plate
MUTAGENESIS TEST: 5000, 1000, 500, 100, 50, 10 µg/plate
Vehicle:
Test item was dissolved 50 mg/ml in dimenthylsulfoxide (DMSO) and further diluted in the same solvent according the need.
It was determined that the amounts of solvent added to the plates did not interfere with the results of mutagenesis or antibacterial test.
Negative controls:
yes
Solvent controls:
yes
Remarks:
0.1 ml of the solvent mixture
Positive controls:
yes
Positive control substance:
N-dimethylnitrosamine
Details on test system and conditions:
MEDIA
Semi-enriched medium (SEM) had the following composition: K2HPO4 7 g/l, KH2PO4 3 g/l, Sodio citrato 0.5 g/l, MgSO4 x 7H2O 0.1 g/l, (NH4)2SO4 1 g/l, Nutrient broth 0.3 g/l, Glucose 2 g/l, Agar 15 g/l.
It was prepared by adding 750 rnl of 2 % molten autoclave sterilized water agar with 250 ml of 4 times con centrated salt solution, 10 ml of 20 % glucose solution and 12.5 ml of Oxoid N. 2 nutrient broth autoclaved separately. 10 cm plastic petri dishes were prepared cogtaining 20 ml each of SEM agar and used within 3 days from preparation.
Top agar (0.6 % agar in distilled water) was dispersed in 2 ml amounts into 16 x 100 mm glass tube kept at 42 °C.

ANTIBACTERIAL ACTIVITY TEST
For testing the antibacterial activity 0.1 ml of solvent containing the required amount of substance, 0.1 ml of a 10^-5 dilution of an overnight culture of WP2 uvrA containing between 100 and 500 colony forming units (CFU) and, if requested, 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 MM plate.

MUTAGENESIS TEST
In view of the results of antibacterial activity, the substance was tested for mutagenic activity at the doses of 5000 to 10 µg/plate.
0.1 ml of solvent containing the required amounts of substance, 0.1 ml of overnight culture of WP2 uvrA containing 10^7 bacteria and, if required, 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 SEM plate.

NUMBER OF REPLICATIONS
Tests were carried out in triplicate.

CELLS COUNT
Antibacterial activity: colonies were counted after 24 hours of incubation at 37°C.
Mutagenesis test: revertant colonies were counted after 2 days of incubation at 37 °C.

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 mglml in corn oil, 500 mg/kg). Five days after injection the rats were killed by cervical dislocation. The livers washed with 0.15 KCl ice cold solution were weighted and transferred to a beaker containing 3 ml of KCl 0.15 M solution per gram of net weight of liver, minced with steril scissors and homogenized in a Potter-Evenhjem apparatus with a teflon pestle.
The homogenate was centrifuged for 10 min at 9000 g and the supernatant decanted and stored at -80 °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 KCl 1.65 M, 2 ml of MgCl2 0.4 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:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Remarks:
no antibacterial activity
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
MUTAGENESIS TEST
None of the tested doses of test item increases the number of revertants over the spontaneous levels. It may be concluded that the substance is without any mutagenic activity on the bacterial strain used in the test.

ANTIBACTERIAL ACTIVITY
Result show that test item is without antibacterial activity up tn the dose of 5000 µg/plate: counts of CFUs in the control plates and in plates containing the highest dose of compound did not differ significantly.

Mutagenicity

Strain Dose
µg/plate
Revertant colonies + S9-mix Revertant colonies - S9-mix
Mean R* Mean R*
WP2 uvrA- 5000 21 < 2 19 < 2
1000 22 < 2 18 < 2
500 24 < 2 16 < 2
100 24 < 2 20 < 2
50 22 < 2 18 < 2
10 17 < 2 17 < 2
0 22 17

* times of increase over the spontaneous reversion rate. Values less than 2 were considered not significant of mutagenic activity.

Antibacterial activity

Strain Dose
µg/plate
Colony counts + S9-mix Colony counts - S9-mix
CFU/plate Mean % survival CFU/plate Mean % survival
WP2 uvrA- 0 184 196 220 200 99 190 201 214 202 100
5000 187 189 215 197 201 207 220 209

CFU: colony forming units

Control of E. coli WP2 uvrA-

Grown in MM -
Grown in MM + tryptophan +
uvrA character highly UV sensitive
Spontaneous reversion rate* 9, 11, 12
Reversion by 1 mg of MMS 250, 318, 356

MM: minimal medium

MMS: methylmethanesulphonate

*Number of revertant colonies per plate

Control of S9 -mix

Sterility OK
Activity Number of revertants/plate
WP2 uvrA- 9, 11, 12
WP2 uvrA- + S9-mix 20, 25, 26
WP2 uvrA- + DMNA (5 µg) 8, 10, 14
WP2 uvrA- + DMNA (5 µg) + S9-mix 352, 411, 428

DMNA = dimethylnitrosamine by liquid test.

Conclusions:
The substance did not showed any mutagenic activity on the bacterial strain used in the test, with and without metabolic activation.
Executive summary:

Escherichia coli strain WP2 uvrA- were tested in order to assess the test item mutagenicity activity.

A first experiments has been performed to evaluate the antibacterial activity potential at the concentration of 5000 µg/plate; after that, the mutagenicity assay has been performed at concentrations between 5000 - 10 µg/plate.

Result show that test item is without antibacterial activity up to the dose of 5000 µg/plate: counts of CFUs in the control plates and in plates containing the highest dose of compound did not differ significantly.

None of the tested doses of test item increases the number of revertants over the spontaneous levels.

Conclusion

The substance did not showed any mutagenic activity on the bacterial strain used in the test, with and without metabolic activation.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Reference:
Composition 0
Principles of method if other than guideline:
Ames strains of Salmonella typhimurium TA1535, TA1537, TA1538, TA100 and TA98 were tested in order to assess the test item mutagenicity activity. A first experiments has been performed on TA1535 and TA98 to evaluate the antibacterial activity potential of the substance; then, the mutagenicity assay has been performed on all strains.
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Species / strain:
other: TA1535, TA1537, TA1538, TA100 and TA98
Details on mammalian cell lines (if applicable):
Ames strains of Salmonella typhimurium TA1535, TA1537, TA1538, were kept at 4 °C in deep soft nutrient agar. Strains TA100 and TA98 were kept at -80 °C in nutrient broth plus ampicillin (20 µg/ml) and dimethylsufoxide (80 µl/ml).

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. Concurrently with each series of test, cultures were checked for relevant characters, namely:
- histidine requirement
- biotin requirement
- uvrB character
- rfa character
- presence or absence of the pKM101 plasmid
Metabolic activation:
with and without
Metabolic activation system:
liver supernatant fraction (rats)
Test concentrations with justification for top dose:
ANTIBACTERIAL ACTIVITY: 5000 µg/plate
MUTAGENESIS TEST: 5000, 1000, 500, 100, 50, 10 µg/plate
Vehicle:
Test item was dissolved 50 mg/ml in dimenthylsulfoxide (DMSO) and further diluted in the same solvent according the need.
It was determined that the amounts of solvent added to the plates did not interfere with the results of mutagenesis or antibacterial test.
Negative controls:
yes
Solvent controls:
yes
Remarks:
0.1 ml of the solvent mixture
Positive controls:
yes
Positive control substance:
9-aminoacridine
ethylmethanesulphonate
methylmethanesulfonate
other: 2-aminofluorene 10 µ + S9-mix
Details on test system and conditions:
MEDIA
Vogel-Bonner E agar medium (VB) has the following composition: MgSO4 x 7H2O 0.2 g/l, Citric acid x H2O 2 g/l, K2HPO4 10 g/l, NaNH4HPO4 x 4H2O 3.5 g/l, 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 em plastic petri dished 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 16x100 mm glass tubes kept at 42 °C.

ANTIBACTERIAL ACTIVITY TEST
For testing the antibacterial activity 0.1 ml of solvent containing the required amount of substance, 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, if requested, 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.

MUTAGENESIS TEST
In view of the results of antibacterial activity, the substance was tested for mutagenic activity at the doses of 5000 to 10 µg/plate.
0.1 ml of solvent containing the required amounts of substance, 0.1 ml of overnight culture of each strain containing 10^7 bacteria and, if required, 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.

NUMBER OF REPLICATIONS
Tests were carried out in triplicate.

CELLS COUNT
Antibacterial activity: colonies were counted after 24 hours of incubation at 37°C.
Mutagenesis test: revertant colonies were counted after 2 days of incubation at 37 °C.

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 mglml in corn oil, 500 mg/kg). Five days after injection the rats were killed by cervical dislocation. The livers washed with 0.15 KCl ice cold solution were weighted and transferred to a beaker containing 3 ml of KCl 0.15 M solution per gram of net weight of liver, minced with steril scissors and homogenized in a Potter-Evenhjem apparatus with a teflon pestle.
The homogenate was centrifuged for 10 min at 9000 g and the supernatant decanted and stored at -80 °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 KCl 1.65 M, 2 ml of MgCl2 0.4 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:
other: TA1535, TA1537, TA1538, TA100 and TA98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Remarks:
no antibacterial activity
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
MUTAGENESIS TEST
None of the tested doses of test item increases the number of revertants over the spontaneous levels. It may be concluded that the substance is without any mutagenic activity on the bacterial strains used in the test.

ANTIBACTERIAL ACTIVITY
Result show that test item is without antibacterial activity up tn the dose of 5000 µg/plate: counts of CFUs in the control plates and in plates containing the highest dose of compound did not differ significantly.

Mutagenicity

Strain Dose
µg/plate
Revertant colonies + S9-mix Revertant colonies - S9-mix
Mean R* Mean R*
TA1535 5000 14 < 2 14 < 2
1000 11 < 2 16 < 2
500 14 < 2 13 < 2
100 12 < 2 13 < 2
50 13 < 2 15 < 2
10 13 < 2 11 < 2
0 12 14
TA1537 5000 7 < 2 6 < 2
1000 7 < 2 6 < 2
500 8 < 2 6 < 2
100 6 < 2 6 < 2
50 6 < 2 6 < 2
10 5 < 2 7 < 2
0 7 6
TA1538 5000 23 < 2 11 < 2
1000 25 < 2 11 < 2
500 20 < 2 9 < 2
100 30 < 2 12 < 2
50 30 < 2 12 < 2
10 24 < 2 10 < 2
0 24 10
TA100 5000 108 < 2 98 < 2
1000 105 < 2 99 < 2
500 102 < 2 102 < 2
100 100 < 2 99 < 2
50 105 < 2 106 < 2
10 113 < 2 102 < 2
0 103 101
TA198 5000 29 < 2 19 < 2
1000 30 < 2 19 < 2
500 32 < 2 22 < 2
100 36 < 2 21 < 2
50 34 < 2 24 < 2
10 37 < 2 23 < 2
0 38 23

* times of increase over the spontaneous reversion rate. Values less than 2 were considered not significant of mutagenic activity.

Antibacterial activity

Strain Dose
µg/plate
Colony counts + S9-mix Colony counts - S9-mix
CFU/plate Mean % survival CFU/plate Mean % survival
TA1535 0 123 156 170 150 100 130 136 144 137 98
5000 125 144 186 155 131 133 140 155
TA98 0 210 226 240 225 99 209 240 244 231 98
5000 207 231 233 223 215 222 241 225

CFU: colony forming units

Control of Salmonella strains

Growth in TA1535 TA1537 TA1538 TA100 TA98
VB - - - - -
VB + his - - - - -
VB + bio - - - - -
VB + his + bio + + + + +
VB + his + bio + amp - - - + +
rfa charactera) 20 22 20 21 20
uvrB characterb) high high high high high
Spontaneous reversion ratec) 10, 14, 16 4, 4, 7 8, 16, 17 95, 100, 171 9, 15, 31
Reversion by
2-aminofluorene 10 µg + S9-mix 11, 13, 17 46, 48, 54 > 1000d) > 1000d) > 1000d)
methylmethanesulfonate 1000 µg 10, 11, 17 3, 7, 8 19, 12, 15 > 1000d) 18, 22, 27
ethylmethanesulfonate 5000 µg > 1000d) 4, 5, 7 20, 22, 23 > 1000d) 18, 22, 34
9-aminoacridine 10 µg 12, 13, 14 237, 315, 322 9, 15, 18 122, 138, 160 9, 21, 24

a) mm of the diameter of inhibition zone caused by 10 µg of crystal violet

b) high or low sensibility to ultraviolet irradiation

c) number of revertant colonies per plate

d) result from three different plates

Control of S9 -mix

Sterility OK
Activity Number of revertants/plate
TA1538 28, 11, 32
TA1538 + S9-mix 42, 40, 51
TA1538 + AF (10 µg) 34, 42, 47
TA1538 + AF (10 µg) + S9-mix > 1000, > 1000, > 1000

AF = 2-aminofluorene

Conclusions:
The substance did not showed any mutagenic activity on the bacterial strains used in the test, with and without metabolic activation.
Executive summary:

Ames strains of Salmonella typhimurium TA1535, TA1537, TA1538, TA100 and TA98 were tested in order to assess the test item mutagenicity activity.

A first experiments has been performed on TA1535 and TA98 to evaluate the antibacterial activity potential at the concentration of 5000 µg/plate; after that, the mutagenicity assay has been performed on all strains at concentrations between 5000 - 10 µg/plate.

Result show that test item is without antibacterial activity up to the dose of 5000 µg/plate: counts of CFUs in the control plates and in plates containing the highest dose of compound did not differ significantly.

None of the tested doses of test item increases the number of revertants over the spontaneous levels.

Conclusion

The substance did not showed any mutagenic activity on the bacterial strains used in the test, with and without metabolic activation.

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

Genetic toxicity in vivo

Description of key information

Non mutagenic

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From March 10th to May 06, 1986
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:
Justification for Read Across is detailed in the IUCLID section 13.
Reason / purpose:
read-across source
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
May 26, 1983
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
no
Type of assay:
mammalian erythrocyte micronucleus test
Test material information:
Composition 1
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: KIeintierfarm Madoerin AG, CH 4414 Fuellinsdorf/Switzerland.
- Age at study initiation: 9 - 10 weeks
- Weight at study initiation: males 31 - 38 g, females 25 - 32 g.
- Housing: n Makrolon type-3 cages with wire mesh top and granulated softwood bedding.
- Diet: peIIeted standand Kliba no. 343 mouse diet, Batch 33/85 (Klingentalmuehle), ab Iibitum.
- Water: tap water, ad libitum.
- Acclimation period: 7 days under laboratory conditions, after veterinary examination.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidit: 40 - 70 %
- Photoperiod: 12 hours artificial fluorescent light / 12 hours dark.
Route of administration:
oral: gavage
Vehicle:
4 %, w/v carboxymethylcellulose sodium salt in distilled water
Details on exposure:
- Volume administrated: 20 ml/kg body weight

FORMULATION OF THE TEST ARTICLE
A suspension was prepared immediately before application by adding the test article to distilled water containing CMC (4 % w/v).
Homogeneity of the suspension was maintained during application using a magnelic stirrer.

DOSE SELECTION
The maximum tolerated dose based on acute oral toxicity data. A preliminany acute oral LD50 study (Iimit test; 1 dose, 3 males and 3 females per dose) performed with the same mouse strain as used in this study showed the following result after 15 days observation: 5000 mg/kg body weight, 0 mortality in 6 animals. The 5000 mg/kg hody weight was used in the current study as the maximurn tolerated dose.
Frequency of treatment:
Single application
Dose / conc.:
5 000 mg/kg bw/day
No. of animals per sex per dose:
three groups, each containing 18 males and 18 females
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Vehocle: dissolved in 0.9 %, w/v saline solution.
- Dose: 50 mg/kg body weight
Tissues and cell types examined:
One thousand polychromatic erythrocytes (PCE) per animal were scored for micronuclei. The ratio of polychromatic to normochromatic erythrocytes (NCE) was used to assess the toxicity of the test article by counting a total of 1000 erythrocytes.
Details of tissue and slide preparation:
SACRIFICE
24, 48 and 72 hours after treatment, six mice per sex and group were sacrificed and bone marrow, was removed from the femora for examination. The first five animals were used for evaluation. AII mice were sacrificed by cervical dislocation. The femora were removed from each mouse and freed of adherent tissue.

PREPARATION OF SUSPENSION
The proximal end of the femur was cut with scissors; the distal end was left intact. The needle of a plastic syringe containing 0.2 ml calf serum was inserted into the proximar part of the marrow canal.
The bone marrow cells were dispersed in 1.5 ml of calf serum as a homogeneous suspension. The tube containing the bone marrow cells of both femora was centrifuged at 1000 RPM (ca. 180-190 G) for 5 minutes.
The supernatant was removed, leaving a thin layer of serum. The cells of the sediment were suspended by aspiration in a siliconized pasteur pipette. A small drop of the marrow, serum suspension was smeared on the slide, which was allowed to dry overnight. Two slides per animal were prepared. One to three days later, the smears were stained using the panoptic stain method developed by Pappenheim.

METHOD OF ANALYSIS AND EVALUATION
The slides were coded before microscopic analysis. If macroscopic evaluation revealed technical imperfections, the first slide was replaced by the second slide prepared. From each animal, one thousand (1000) polychromatic erythrocytes (PCE) were scored under the microscope (magnification 1000x), for the incidence of micronuclei. Micronuclei were evaluated according to the characteristics which have been described by Schmid. Some characteristics of micronuclei are round features of a sharp contour, sometimes slightly oval or ring-shaped, average size of 1/20 to 1/5 of the erythrocyte size and darkly blue-stained, comparable with the staining quality of nucleic material. Structures of irregular or fibrillar shape, of divergent staining properties of other characteristics not typical for a micronucleus (e.g. atypical focus properties) were not considered to be micronuclei.
Multiple micronuclei in one cell were registered and calculated as one micronucleated cell.
The calculated ratio of polychromatic to normochromatic erythrocytes (PCE/NCE), based on 1000 erythrocytes scored per slide, measured the toxic efficacy of the test article.
Polychromatic erythrocytes (PCE) can be differentiated from normochromatic erythrocytes (NCE) by their basophilic, blue- grey color compared to the acidophilic orange-pink colon of NCE. PCE also tend to be somewhat larger and have more diffuse boundaries.
Additional information chromatic erythrocytes could be obtained for micronuclei.
Evaluation criteria:
The frequencies of micronucleated polychromatic erythrocytes of the treated male and female groups were compared with those of the negative control groups at each sampling time. A regression model assuming a Poisson distribution was applied.
Estimation and testing were performed by maximum likelihood method . If a test article produced no statistically significant positive response at any one of the test points, it was considered to be non-mutagenic in this system.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
in males
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
After a single application of the test article at 5000 mg/kg body weight by gavage no significant test article-related increase of micronucleated polychromatic erythrocytes was observed in either male of female treated groups when compared with corresponding negative control groups. These results were found at the three examination times , 24, 48 and 72 hours post-application, respectively.
Reduced PCE/NCE ratios were observed in bone marrow preparations of male animals 24, 48 and 72 hours post-application. In female animals, no toxic effect of the test article was observed.

POSITIVE CONTROL
The positive control groups receiving cyclophosphamide exhibited a significant and clear increase in the number of micronucleated polychromatic erythrocytes 24 and 48 hours post-application and thus validated the test. The dose used for this reference mutagen exhibited a toxic effect as should by the reduced PCE/NCE ratio.

CLINICAL EXAMINATION
No death occurred during the application period. Yellowish-coloured urine and faces as well as diarrhea was observed in all test article-treated animals up to 48 hours post-application. In male animals, slight sedation was observed 4 hours and ruffled fun 48 hours post-application.
The animals were free of clinical findings 56 hours post-application and later.
Conclusions:
The test item is not considered to be mutagenic in the in vivo mouse micronucleus assay performed.
Executive summary:

The study with mice was performed to assess the potential mutagenic activity, induced by test item, through damage to the chromosomes on to the mitotic apparatus, according to OECD guideline 474.

The experiment was performed with three groups, each containing 18 males and 18 females, for a total of 108 mice. The negative control group received the test article vehicle, i.e. 4 %, w/v carboxymethylcellulose sodium salt in distilled water. The test groups received 5000 mg/kg body weight as the maximum tolerated dose of the test article. The positive control groups received 50 mg/kg body weight cyclophosphamide dissolved in 0.9 %, w/v saline solution. All animals were given a single application of 20 ml/kg body weight by gavage.

24, 48 and 72 hours after treatment, six mice per sex and group were sacrificed and bone marrow, was removed from the femora for examination. The first five animals were used for evaluation. One thousand polychromatic erythrocytes (PCE) per animal were scored for micronuclei. The ratio of polychromatic to normochromatic erythrocytes (NCE) was used to assess the toxicity of the test article by counting a total of 1000 erythrocytes.

The data were statistically analyzed by means of a regression model assuming a Poisson distribution. Estimation and testing were performed by maximum likelihood method.

Reduced PCE/NCE ratios were observed in bone marrow preparation of male animals 24, 48 and 72 hours post-application. In female animals, no toxic effects of the test article was observed.

After single application of the test article at 5000 mg/kg body weight by gavage, no significant test article-related increase of micronucleated polychromatic erythrocytes was observed in either male or female treated groups, when compared with corresponding negative control groups. These results were found at the three examination times, 24, 48 and 72 hours post-application, respectively. The positive control groups, which received cyclophosphamide, exhibited a significant and clear increase in the number of micronucleated polychromatic erythrocytes 24 and 48 hours post-application and thus validated the test.

Conclusion

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article induced no chromosome mutations by chromosome-breaking activity or by damage to the mitotic apparatus. Therefore, test item is not considered to be mutagenic in the in vivo mouse micronucleus assay.

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 following studies: in vitro gene mutation assay in bacteria, in vitro gene mutation on mammalian cells and in vivo chromosomal aberration assay.

The gene mutation assays in bacteria and in mammalian cells were conducted with Basic Orange 064; the in vivo chromosomal aberration test available was conducted on the structural analogous Similar Substance 02; the read across approach can be considered as appropriate and suitable to assess the property under investigation (details about the approach are reported into the IUCLID section 13).

IN VITRO BACTERIA GENE MUTATION ASSAY

Two experiments assaying the bacteria gene mutation conducted with Basic Orange 064 are available; one was conducted in the Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 100 and TA 98; the other one was conducted using Escherichia coli strain WP2 uvrA.

Result show that test item is without antibacterial activity up to the dose of 5000 µg/plate. None of the tested doses of test item increases the number of revertants over the spontaneous levels in both Salmonella typhimurium and Escherichia coli strains.

In addition, the available information on the structural analogous Similar Substance 01 have been taken into consideration. Similar Substance 01 is the acid form of Basic Orange 064 (details about the approach are reported into the IUCLID section 13). Back mutation test has been performed according to testing method described by Ames (1975) and Tajima et al. (1980). Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 1538, TA 98 and Escherichia coli WP2 uvrA strains were used. No remarkable increase in the mutation colony was recognized. Therefore, the specimen was judged not to posses mutagenicity concerning the test conducted.

IN VITRO MAMMALIAN CELL GENE MUTATION ASSAY

The substance is tested in Mammalian Gene Mutation Test in CHO-K1 cells. The test item was dissolved in DMSO and the following concentrations were selected for the main experiment, on the basis of cytotoxicity investigations made in a preliminary study: 250, 500, 1000 and 2000 µg/ml, with and without metabolic activation.

The main experiment is still running.

IN VIVO CHROMOSOMAL ABERRATION

The available information on the structural analogous Similar Substance 02 have been taken into consideration; the read across approach can be considered as appropriate and suitable to assess the property under investigation (details about the approach are reported into the IUCLID section 13).

The study with mice was performed to assess the potential mutagenic activity, induced by Similar Substance 02, through damage to the chromosomes on to the mitotic apparatus, according to OECD guideline 474. The experiment was performed with three groups, each containing 18 males and 18 females, for a total of 108 mice. The test groups received 5000 mg/kg body weight as the maximum tolerated dose of the test article.

Reduced PCE/NCE ratios were observed in bone marrow preparation of male animals 24, 48 and 72 hours post-application. In female animals, no toxic effects of the test article was observed.

After single application of the test article at 5000 mg/kg body weight by gavage, no significant test article-related increase of micronucleated polychromatic erythrocytes was observed in either male or female treated groups, when compared with corresponding negative control groups. These results were found at the three examination times, 24, 48 and 72 hours post-application, respectively. The positive control groups, which received cyclophosphamide, exhibited a significant and clear increase in the number of micronucleated polychromatic erythrocytes 24 and 48 hours post-application and thus validated the test.

DISCUSSION AND CONCLUSION

Based on the currently available information, there are noreasons of concern from the genotoxicity point of view.Since some tests are still running, the assessment and conclusion on genetic toxicity potentialwill be confirmed and, eventually, updated as soon as all the experiments will be finished.

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 suggest that test 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.