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Genetic toxicity in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
uvrB
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: essential amino acid requiring strains
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: essential amino acid requiring strains
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 mix from male Wistar rats treated with phenobarbital and beta-Naphthoflavone for enzyme induction.
Test concentrations with justification for top dose:
Experiment 1 (Plate Incorporation Test): 33, 100, 333, 1000, 2500, 5000 µg/plate
Experiment 2 (Pre-Incubation Test): 33, 100, 333, 1000, 2500, 5000 µg/plate
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
Positive control substances for tests with metabolic activation (S9 mix)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: sodium azide, 4.nitro-o-phenylene-diamine, methyl methane sulfonate
Remarks:
Positive control substances for tests without metabolic activation (S9 mix). All of them are well established reference mutagens.
Details on test system and experimental conditions:
Standard Plate Incorporation Tests were performed in both experiments (Experiments 1 and 2) and both experiments were conducted without and with metabolic activation (S9 mix).

The following positive controls were used to check mutability of the bacteria and activity of the S9 mix:

Without metabolic activation (S9 mix):

Sodium azide, NaN3:
- 10 μg/plate, dissolved in water deionised: - strains: TA 1535, TA 100

4-nitro-o-phenylene-diamine, 4-NOPD::
- 10 μg/plate, dissolved in DMSO: - strain: TA 98
- 50 µg/plate, dissolved in DMSO: - strain: TA 1537

Mehyl methane sulfonate, MMS:
- 5 μg/plate, dissolved in water deionised: - strain: WP2 uvrA

With metabolic activation (S9 mix):

2-Aminoanthracene, 2-AA:
- 2.5 μg/plate, dissolved in DMSO: - strains: TA 1535, TA 1537, TA 98, TA 100
- 10 μg/plate, dissolved in DMSO: - strain: WP2 uvrA
Evaluation criteria:
The test chemical is considered to exhibit mutagenic activity in this assay if the following criteria are met:
A reproducible increase in revertant colony number, (i.e. at least twice for strains TA 100, TA 98 and WP2 uvrA and at least three times for strains TA 1535 and TA 1537 the concurrent vehicle controls), with some evidence of a positive dose-response relationship. Such positive response in at least one tester strain without or with metabolic activation (S9 mix.) is sufficient for concluding mutagenic activity.

A test substance is considered non-mutagenic in this test if:
Exposure to a test substance does not produce a reproducible increase in revertant colony numbers.
Statistics:
not required
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
in both experiments (Experiment 1 and 2)
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
in both experiments (Experiment 1 and 2)
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:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
in both experiments (Experiment 1 and 2)
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:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
in both experiments (Experiment 1 and 2)
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:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
in both experiments (Experiment 1 and 2)
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
Additional information on results:
See tables on results and viability attached as background material.

Conclusions:
negative with metabolic activation
ambiguous without metabolic activation

The test item is considered as non-mutagenic in the bacterial reverse mutation assay. Based on these results, the test item does not have to be classified according to CLP (Regulation (EC) No. 1272/2008).
Executive summary:

The test article was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation (S9 mix). Each concentration and the controls were tested in triplicate. The test article was tested at the following concentrations: 33, 100, 333, 1000, 2500, and 5000 µg/plate. The plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.

No relevant toxic effects, evident as a reduction in the number of revertants, occured in experiment I with and without metabolic activation. In experiment II, toxic effects were observed from 100 µg/plate up to 5000 µg/plate without S9 mix and from 33 µg/plat up to 5000 µg/plate wit S9 mix in strain TA 100.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test article at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
The protein concentration in the S9 preparation in the pre-experiment was slightly higher the usual value (50.4 mg/ml compared to 20 - 45 mg/ml). This deviation had no detrimental impact on the outcome of the study.
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
The protein concentration in the S9 preparation in the pre-experiment was slightly higher the usual value (50.4 mg/ml compared to 20 - 45 mg/ml). This deviation had no detrimental impact on the outcome of the study.
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 mix from male Wistar rats treated with phenobarbital and beta-naphthoflavone for enzyme induction.
Test concentrations with justification for top dose:
EXPERIMENT I:
- without S9 mix (18 h preparation interval): (7.5), (15.0), 30.0, 60.0, (80.0), 100.0 µg/ml
- with S9 mix (18 h preparation interval): (3.1), (6.3), 12.5, 25.0, 50.0, 100.0 µg/ml

EXPERIMENT II:
- without S9 mix (18 h preparation interval): (3.1), (6.3), (12.5), 25.0, 50.0, 100.0 µg/ml
- without S9 mix (28 h preparation interval): (12.5), (25.0), (50.0), 100.0 µg/ml
- with S9 mix (28 h preparation interval): (3.1), (6.3), (12.5), 25.0, 50.0, 100.0µg/ml

Concentration in brackets indicate experiments that were not evaluated with regard to cytogenetic damage.
Vehicle / solvent:
DMSO

JUSTIFICATION FOR CHOICE OF VEHICLE:
The test article could be dissolved best in DMSO.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Cyclophosphamide, CPA, dissolved in nutrient medium, final concentration: 0.71 µg/ml = 2.5 µM, with metabolic activation (S9 mix)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Ehtylmethane sulphonate, EMS, dissolved in nutrient medium, final concentration: 600 µg/ml = 4.8 mM (continous exposure) and 1205 µg/ml = 9.6 mM (4 h exposure), without metabolic activation (S9 mix)
Details on test system and experimental conditions:
SEEDING OF THE CULTURES:
Exponentially growing stock cultures more than 50% confluent were treated with trypsin at 37 deg-C for approx. 5 minutes. Then the enzymatic digestion was stopped by adding complete culture medium and a single cell suspension was prepared. The trypsin concentration was 0.2 % in Ca-Mg-free salt solution. Prior to the tryprsin treatment, the cells were rinsed with Ca-Mg-free salt solution containing 200 mg/l EDTA.

TREATMENT:
EXPOSURE PERIOD 4 HOURS
The culture medium of exponentially growing cell cusltures was replaces with serum-free medium (for treatment with S9 mix) or complete medium (for treatment without S9 mix) with 10 % FCS (v/v), containing the test article. For the treatment with metabolic activation 50 µl/ml S9 mix per ml culutre medium were added. After 4 h the cultures were washed twice with "Saline G" and the the cells were cultured in complete medium for the remaining culture time. The "Saline G" solution contained: 8,000 mg NaCl, 400 mg KCl, 1,100 mg Glucose, 290 mg Na2HPO4x7H2O, 150 mg KH2PO4.

EXPOSURE PERIOD 18 AND 28 HOURS
The culture medium of exponentially growing cell cutltures was replaced with complete medium (10 % FCS) containing different concentrations of the test article without S9 mix. The medium was not changed until preparation of the cells. All cells were incubated at 37 deg-C in a humidified atmosphrere with 4.5 % CO2 (95.5 % air).

PREPARATION OF THE CULTURES:
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 deg-C. After incubation in the hypotonic solution the cells were fixed with 3 + 1 methanol + glacial acetic acid. Per experiment two slides per group were prepared. After preparation the cells were stained with Giemsa.

Additionally, two cultures per 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 substance is evaluated as reduction of % cells as compared to the solvent control.

ANALYSIS OF METAPHASE CELLS:
Evaluation of the cultures was performed microscopically. 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. 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides. Only methaphases with characteristic chromosome numbers 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 was determined (% polyploid methaphases, in the case of this aneuploid cell line polyploid means a near tetraploid karyotype).
Evaluation criteria:
ACCEPTABILITY OF THE ASSAY:
The chromosome aberration assay perfomred is considered acceptable if it meets the following critera:
- The number of structural aberrations found in the negative and/or solvent controls falls within the range of the historical laboratorycontraol data: 0.00 % - 4.00 %.
- The positive control substances should produce significant increases in the number of cells with structural chromosome aberratons, which are within the range of the laboratory's historical control data: 9.0 - 39.0 % (without S9 mix, EMS 600 µg/ml) or 7.5 - 49.5 % (with S9 mix, CPA 0.47 - 0.93 µg/ml).

VALIDITY CRITERIA
A test article is classified as non-mutagenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups are in the range of the historical control data of the laboratory performing the study (0.0 - 4.0 % aberrant cells exclusive gaps).
- no significant increase of the number of structural chromosome aberrations are observed.

A test article is classified as mutagenic if:
- the number of induced structural chromosome aberrations are not in the range of the historical control data of the laboratory performing the study (0.0 - 4.0 % aberrant cells exclusive gaps).
- either a concentration-related or a significant increase of the number of structural chromosome aberrations are observed.
Statistics:
Statistical significance was confirmed by means of the Fischer's exact test (p < 0.05).
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 influence of test article detected in any experiment
- Effects of osmolality: No influence of test article detected in any experiment
- Precipitation: Test article precipitated in culture medium at highest dose level of 100 µg/ml

GENERAL OBSERVATION:
- Neither reduced mitotic indices nor reduced cell numbers (< 50 %) could be observed up to the highest concentration evaluated.
- 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 both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test article.
- in both experiments, EMS (600 and 1,205 µg/ml) and CPA (0.71 µg/ml) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

See tables on toxicity and mutations attached as background material

EXPERIMENT I:

Table 1: Number of polyploid cells and mitotic index; preparation interval 18 h with and without S9 mix; exposure period 4 h

 Group  conc./ml  S9 mix  prep. inter.  polyploid cells           mitotic index         
         culture 1  culture 2  total  %  absolute 1  absolute 2  mean  %
 Neg. ctrl.    -  18 h  13  10  23  2.3  8.7  7.5  8.1  100.0
 Solv. ctrl.  0.5 %  -  18 h  10  14  24  2.4  7.1  6.1  6.6  100.0
 Pos. ctrl.  1205 µg  -  18 h  16  12  28  2.8  4.8  6.1  5.5  67.3
 Test artcl.  30.0 µg  -  18 h  17  8  25  2.5  7.5  6.0.  6.8  102.3
   60.0 µg  -  18 h  10  16  26  2.6  10.3  10.7  10.5  159.1
   100.0 µg  -  18 h  18  10  28  2.8  5.4  6.2  5.8  87.9
 Neg. ctrl.    +  18 h  14  16  30  3.0  7.0  7.3  7.2  100.0
 Solv. ctrl.  0.5 %  +  18 h  15  16  31  3.1  10.0  8.9  9.5  100.0
 Pos. ctrl.  0.71 µg  +  18 h  14  4  18  1.8  2.6  2.4  2.5  35.0
 Test artcl.  12.5 µg  +  18 h  6  7  13  1.3  5.1  7.8  6.5  68.3
   25.0 µg  +  18 h  11  12  23  2.3  9.8  10.8  10.3  109.0
   50.0 µg  +  18 h  16  11  27  2.7  9.2  10.3  9.8  103.2
   100.0 µg  +  18 h  14  12  26  2.6  7.7  7.7  7.7  81.5

The number of polyploid cells was determined of each test group in a sample of 500 cells per culture. The mitotic index was determined in a sample of 1000 cells per culture of each test group. For the positive control groups, the relative values of teh mititic index are related to the negative controls; for the test article treatment groups the values are related to the solvent controls.

Table 2: Structural chromosome aberrations; exposure period 4 h; peparation interval 18 h, without S9 mix

 slide no.  cells scored  % aberrant       cells      aberrations                           
     incl. gaps  excl. gaps  with exchanges  gaps     chromatid type           chromosome type           other   
           g  ig  b  f  d  ex  ib  if  id  cx  ma  cd
 negative control                                                
 1  100        1  0  2  0  0  0  0  1  0  0  0  0
 2  100        0  0  0  0  0  0  0  0  0  1  0  0
 1 + 2  200  2.5  2.0  0.5  1  0  2  0  0  0  0  1  0  1  0  0
 solvent control: DMSO 0.5 %                                                
 1  100        1  0  1  1  0  0  0  0  0  0  0  0
 2  100        1  0  0  0  0  0  0  0  0  1  1  0
 1 + 2  200  3.0  2.0  0.5  2  0  1  1  0  0  0  0  0  1  1  0
 positive control: EMS 1205 µg/ml                                                
 1  100        1  0  7  3  0  14  6  1  0  0  2  0
 2  100        4  0  16  1  0  18  7  2  0  0  1  0
 1 + 2  200  26.5  25.5  13.0  5  0  23  4  0  32  13  3  0  0  3  0
 test article: 30 µg/ml                                                
 1  100        1  0  1  0  0  0  1  0  0  0  0  0
 2  100        3  0  0  0  0  1  1  1  0  0  0  0
 1 + 2  200  4.5  2.5  0.5  4  0  1  0  0  1  2  1  0  0  0  0
 test article: 60 µg/ml                                                
 1  100        3  0  1  3  0  0  1  0  0  0  0  0
 2  100        0  0  1  0  0  0  1  4  0  0  0  0
 1 + 2  200  4.0  2.5  0.0  3  0  2  3  0  0  2  4  0  0  0  0
 test article: 100 µg/ml                                                
 1  100        2  0  1  1  0  0  1  0  0  0  0  0
 2  100        1  0  0  0  0  0  0  0  0  0  0  0
 1 + 2  200  3.0  1.5  0.0  3  0  1  1  0  0  1  0  0  0  0  0

g = gap; ig = iso-gap; gaps are achromatic lesions of chromatid or chromosome type where no or only a minimal misalignment of chromosomal material is visible; b 0 breack; ib = iso-break; f = fragment; if 0 iso-fragment; d = deletion; id = iso-deletion; ma = multiple aberration (= more than 4 events in one cell [excl. gaps]); ex = chromatid type exchange; cx = chromosome type exchange; cd = chromosomal disintegrations (= pulverization)

Table 3: Structural chromosome aberrations; exposure period 4 h; peparation interval 18 h, with S9 mix

 slide no.  cells scored  % aberrant       cells      aberrations                           
     incl. gaps  excl. gaps  with exchanges  gaps     chromatid type           chromosome type           other   
           g  ig  b  f  d  ex  ib  if  id  cx  ma  cd
 negative control                                                
 1  100        0  0  1  0  0  2  0  0  0  0  1  0
 2  100        0  0  3  0  0  1  2  1  0  0  0  0
 1 + 2  200  4.0  4.0  1.5  0  0  4  0  0  3  2  1  0  0  1  0
 solvent control: DMSO 0.5 %                                                
 1  100        2  0  0  2  0  0  0  1  0  0  0  0
 2  100        6  0  0  0  0  0  0  0  0  0  1  0
 1 + 2  200  6.0  2.0  0.0  8  0  0  2  0  0  0  1  0  0  1  0
 positive control: CPA 0.71 µg/ml                                                
 1  100        2  0  1  3  0  5  3  1  0  0  3  0
 2  100        1  0  3  0  0  8  4  2  1  0  0  0
 1 + 2  200  12.0  11.5  6.0  3  0  4  3  0  13  7  3  1  0  3  0
 test article: 12.5 µg/ml                                                
 1  100        0  0  3  1  0  1  0  1  0  2  0  0
 2  100        3  0  0  0  0  0  0  0  0  0  0  0
 1 + 2  200  4.5  3.0  1.0  3  0  3  1  0  1  0  1  0  2  0  0
 test article: 25.0 µg/ml                                                
 1  100        0  0  0  1  0  1  0  0  0  0  0  0
 2  100        0  0  4  0  0  2  1  0  0  0  0  0
 1 + 2  200  3.5  3.5  1.0  0  0  4  1  0  3  1  0  0  0  0  0
 test article: 50.0 µg/ml                                                
 1  100        0  0  1  1 0  0  1  0  0  0  0  0
 2  100        3  0  1  0  0  2  0  0  0  0  0  0
 1 + 2  200  4.5  3.0  1.0  3  0  2  1  0  2  1  0  0  0  0  0
 test article: 100.0 µg/ml                                                
 1  100        2  0  1  1  0  1  0  0  0  0  0  0
 2  100        2  0  1  1  0  1  0  0  0  0  1  0
 1 + 2  200  4.5  3.0  1.0  4  0  2  2  0  2  0  0  0  0  1  0

g = gap; ig = iso-gap; gaps are achromatic lesions of chromatid or chromosome type where no or only a minimal misalignment of chromosomal material is visible; b 0 breack; ib = iso-break; f = fragment; if 0 iso-fragment; d = deletion; id = iso-deletion; ma = multiple aberration (= more than 4 events in one cell [excl. gaps]); ex = chromatid type exchange; cx = chromosome type exchange; cd = chromosomal disintegrations (= pulverization)

EXPERIMENT II:

Table 4: Number of polyploid cells and mitotic index; preparation interval 18 and 28 h without S9 mix; exposure period 18 and 28 h; preparation interval 28 h with S9 mix; exposure period 4 h

 Group  conc./ml  S9 mix  prep. inter.  polyploid cells           mitotic index         
         culture 1  culture 2  total  %  absolute 1  absolute 2  mean  %
 Neg. ctrl.    -  18 h  38  39  77  7.7  12.5  11.6  12.1  100.0
 Solv. ctrl.  0.5 %  -  18 h  32  28  60  6.0  11.8  14.7  13.3  100.0
 Pos. ctrl.  600 µg  -  18 h  8  8  16  1.6  6.7  6.6  6.7  55.2
 Test artcl.  25.0 µg  -  18 h  20  16  36  3.6  12.7  13.9  13.3  100.4
   50.0 µg  -  18 h  9  34  43  4.3  9.9  13.3  11.6  87.5
   100.0 µg  -  18 h  32  24  56  5.6  13.3  11.2  12.3  92.5
 Solv. ctrl.  0.5 %  -  28 h  28  28  56  5.6  15.4  16.8  16.1  100.0
 Test artcl.  100.0 µg  -  28 h  33  22  55  5.5  19.6  17.4  18.5  114.9
 Neg. ctrl.    +  28 h  16  30  46  4.6  13.4  11.3  12.4  100.0
 Solv. ctrl.  0.5 %  +  28 h  8  22  30  3.0  12.8  13.3  13.1  100.0
 Pos. ctrl.  0.71 µg  +  28 h  12  12  24  2.4  13.1  15.1  14.1  114.2
 Test artcl.  25.0 µg  +  28 h  40  28  68  6.8  12.8  12.0  12.4  95.0
   50.0 µg  +  28 h  28  14  42  4.2  10.7  15.3  13.0  99.6
   100.0µg  +  28 h  28  11  39  3.9  14.3  16.1  15.2  116.5

The number of polyploid cells was determined of each test group in a sample of 500 cells per culture. The mitotic index was determined in a sample of 1000 cells per culture of each test group. For the positive control groups, the relative values of teh mititic index are related to the negative controls; for the test article treatment groups the values are related to the solvent controls.

Table 5: Structural chromosome aberrations; exposure period 18 h; peparation interval 18 h, without S9 mix

 slide no.  cells scored  % aberrant       cells      aberrations                           
     incl. gaps  excl. gaps  with exchanges  gaps     chromatid type           chromosome type           other   
           g  ig  b  f  d  ex  ib  if  id  cx  ma  cd
 negative control                                                
 1  100        3  0  0  0  0  0  0  0  0  0  0  0
 2  100        0  0  0  0  0  0  0  0  0  0  0  0
 1 + 2  200  1.0  0.0  0.0  3  0  0  0  0  0  0  0  0  0  0  0
 solvent control: DMSO 0.5 %                                                
 1  100        1  0  1  0  0  0  0  1  0  0  0  0
 2  100        1  0  0  0  0  0  0  0  0  0  0  0
 1 + 2  200  2.0  1.0  0.0  2  0  1  0  0  0  0  1  0  0  0  0
 positive control: EMS 600 µg/ml                                                
 1  100        6  0  5  3  0  14  1  1  0  0  2  0
 2  100        1  0  14  2  0  27  10  1  0  0  0  0
 1 + 2  200  27.0  26.0  15.0  7  0  19  5  0  41  11  2  0  0  2  0
 test article: 25.0 µg/ml                                                
 1  100        2  0  1  0  0  0  0  0  0  0  0  0
 2  100        1  0  0  0  0  0  0  0  0  0  0  0
 1 + 2  200  2.0  0.5  0.0  3  0  1  0  0  0  0  0  0  0  0  0
 test article: 50.0 µg/ml                                                
 1  100        1  0  0  0  0  0  0  0  0  0  0  0
 2  100        1  0  0  0  0  0  0  0  0  0  0  0
 1 + 2  200  1.0  0.0  0.0  2  0  0  0  0  0  0  0  0  0  0  0
 test article: 100.0 µg/ml                                                
 1  100        0  0  1  0  0  0  0  0  0  0  0  0
 2  100        1  0  0  0  0  0  0  0  0  0  0  0
 1 + 2  200  1.0  0.5  0.0  1  0  1  0  0  0  0  0  0  0  0  0

g = gap; ig = iso-gap; gaps are achromatic lesions of chromatid or chromosome type where no or only a minimal misalignment of chromosomal material is visible; b 0 breack; ib = iso-break; f = fragment; if 0 iso-fragment; d = deletion; id = iso-deletion; ma = multiple aberration (= more than 4 events in one cell [excl. gaps]); ex = chromatid type exchange; cx = chromosome type exchange; cd = chromosomal disintegrations (= pulverization)

Table 6: Structural chromosome aberrations; preparation interval 28 h; exposure period 28 h without S9 mix

 slide no.  cells scored  % aberrant       cells      aberrations                           
     incl. gaps  excl. gaps  with exchanges  gaps     chromatid type           chromosome type           other   
           g  ig  b  f  d  ex  ib  if  id  cx  ma  cd
 solent control: DMSO 0.5 %
 1  100        5  0  3  0  0  0  0  0  0  0  0  0
 2  100        3  0  0  0  0  0  0  0  0  0  0  0
 1 + 2  200  5.5  1.5  0.0  8  0  3  0  0  0  0  0  0  0  0  0
 test article: 100.0 µg/ml
 1  100        0  0  0  0  0  0  0  0  0  0  0  0
 2  100        1  0  0  1  0  0  0  0  0  1  0  0
 1 + 2  200  1.5  1.0  0.5  1  0  0  1  0  0  0  0  0  1  0  0

g = gap; ig = iso-gap; gaps are achromatic lesions of chromatid or chromosome type where no or only a minimal misalignment of chromosomal material is visible; b 0 breack; ib = iso-break; f = fragment; if 0 iso-fragment; d = deletion; id = iso-deletion; ma = multiple aberration (= more than 4 events in one cell [excl. gaps]); ex = chromatid type exchange; cx = chromosome type exchange; cd = chromosomal disintegrations (= pulverization)g = gap; ig = iso-gap; gaps are achromatic lesions of chromatid or chromosome type where no or only a minimal misalignment of chromosomal material is visible; b 0 breack; ib = iso-break; f = fragment; if 0 iso-fragment; d = deletion; id = iso-deletion; ma = multiple aberration (= more than 4 events in one cell [excl. gaps]); ex = chromatid type exchange; cx = chromosome type exchange; cd = chromosomal disintegrations (= pulverization)

Table 7: Structural chromosome aberrations; preparation interval 28 h; exposure period 4 h with S9 mix

 slide no.  cells scored  % aberrant       cells      aberrations                           
     incl. gaps  excl. gaps  with exchanges  gaps     chromatid type           chromosome type           other   
           g  ig  b  f  d  ex  ib  if  id  cx  ma  cd
 negative control                                                
 1  100        0  0  0  0  0  0  1  0  0  0  0  0
 2  100        2  0  0  0  0  0  1  0  0  0  0  0
 1 + 2  200  2.0  1.0  0.0  2  0  0  0  0  0  2  0  0  0  0  0
 solvent control: DMSO 0.5 %                                                
 1  100        1  0  0  0  0  0  0  1  0  0  0  0
 2  100        0  0  0  0  0  1  0  0  0  1  0  0
 1 + 2  200  2.0  1.5  1.0  1  0  0  0  0  1  0  1  0  1  0  0
 positive control: CPA 0.71 µg/ml                                                
 1  100        0  0  3  4  0  0  1  0  0  0  0  0
 2  100        0  0  0  2  0  0  1  3  0  4  2  0
 1 + 2  200  8.0  8.0  2.0  0  0  3  6  0  0  2  3  0  4 2  0
 test article: 25.0 µg/ml                                                
 1  100        1  0  1  0  0  0  0  0  0  0  0  0
 2  100        1  0  0  0  0  0  0  0  0  0  0  0
 1 + 2  200  1.5  0.5  0.0  2 0  1  0  0  0  0  0  0  0  0  0
 test article: 50.0 µg/ml                                                
 1  100        5  0  1  0  0  0  0  0  0  0  0  0
 2  100        1  0  0  0  0  0  0  0  0  0  0  0
 1 + 2  200  3.5  0.5  0.0  6  0  1  0  0  0  0  0  0  0  0  0
 test article: 100.0 µg/ml                                                
 1  100        4  0  0  0  0  0  1  0  1  0  0  0
 2  100        0  0  0  1  0  0  0  0  0  0  0  0
 1 + 2  200  2.5  1.0  0.0  4  0  0  1  0  0  1  0  1  0  0  0

g = gap; ig = iso-gap; gaps are achromatic lesions of chromatid or chromosome type where no or only a minimal misalignment of chromosomal material is visible; b 0 breack; ib = iso-break; f = fragment; if 0 iso-fragment; d = deletion; id = iso-deletion; ma = multiple aberration (= more than 4 events in one cell [excl. gaps]); ex = chromatid type exchange; cx = chromosome type exchange; cd = chromosomal disintegrations (= pulverization)

Conclusions:
negative without and with metabolic activation

The test item did not induce structural chromosome aberration in V79 cells and is therefore considered negative. Based on these results, the test item does not have to be classified according to GHS/CLP (Regulation (EC) No. 1272/2008).
Executive summary:

The test article, dissolved in DMSO, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments. In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations.

The concentrations applied in the pre-test on toxicity were chosen with regard to the current OECD Guideline 473. The maximum concentration (100 µg/ml) was limited by the solubility properties of the test article. Dose selection of the cytogenetic experiments was performed considering the toxicity data and the occurrence of precitpitation.

Using reduction of cell numbers and mitotic indices as an indicator for toxicity, no relevant toxic effects were observed.

No biologically relevant increase in the number of cells carrying structural chromsomal aberrations was observed after treatment with the test article.

No increase in the frequencies of polyploid metaphases was found after treatment with the test article as compared to thte frequencies of the controls.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 mix from male Wistar rats treated with phenobarbital and beta-Naphthoflavone for enzyme induction.
Test concentrations with justification for top dose:
PRE-TEST FOR TOXICITY:
Toxicity of the test item was determined in pre-experiments. Five concentrations (50, 200, 1000, 2500, 5000 µg/ml) were tested with and without metabolic activation. The experimental conditions in these pre-experiments were the same as for the main mutation experiments with short-term exposure of 4 h. For the 20 h long-term exposure assay (experiment II, only without metabolic activation), six concentrations (2, 5, 10, 25, 50, 100 µg/ml) were tested. The experimental conditions in this pre-experiment were the same as for the long-term exposure mutation experiment.

MUTATION EXPERIMENTS
The selcetion of the concentrations was based on data from pre-experiments. In experiment I 1000 µg/ml without metabolic activation and 4750 µg/ml with metabolic activation were selected as the highest concentrations. In experiment II 40 µg/ml without metabolic activation and 4000 µg/ml with metabolic activation were selected as the highest concentrations. Experiment II without metabolic activation was performed as 20 h long time exposure assay.

The test item was investigated at the following concentrations:

Experiment I:
- without S9 mix: 5, 10, 20, 50, 100, 250, 500, 1000 µg/ml
- with S9 mix: 500, 1000, 2500, 2900, 3300, 3800, 4300, 4750 µg/ml

Experiment II:
- without S9 mix: 1, 5, 15, 20, 22.5, 27.5, 35, 40 µg/ml
- with S9 mix: 125, 500, 1400, 1800, 2200, 2600, 3000, 4000 µg/ml

According to OECD guidelines, at least 8 concentrations of the test item were set up in the mutation experiments with and without metabolic activation.
Vehicle / solvent:
DMSO

JUSTIFICATION FOR USING VEHICLE:
The test item showed a good solubility in DMSO. Furthermore, DMSO was compatible with the survival of the cells and the S9 activity.
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Ethylmethansulfonate, EMS, dissolved in Medium, final concentration: 300 µg/ml, without S9 mix
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
7,12-dimethylbenz(a)anthracene, DMBA, dissolved in DMSO, final concentration: 1.5 µg/ml, with S9 mix
Details on test system and experimental conditions:
SEEDING OF THE CULTURES:
Two or three days old exponentially growing stock cultures (more than 50 % confluent) were trypsinised at 37 deg-C for 5 minutes. Then the enzymatic digestion was stopped by adding complete cultur medium and a single cell suspension was prepared. The trypsine concentration for all subculturing steps was 0.05 %.

Approx. 1 x 10E6 cells per concentration, solvent/negative and positive control, were seeded in complete culture medium (Minimum Essential Medium, MEM, supplemented with 10 % Fetal Bovine Serum, FBS) in a culture flask, respectively.

TREATMENT:
Approx. 24 h after seeding the cells were exposed to designated concentrations of the test item either in the presence or absence of metabolic activation in the mutation experiments. After 4 h (short-term exposure) or 20 h (long-term exposure) the treatment medium containing the test item was removd and the cells were washed twice with Phosphate Buffered Saline (PBS). Subsequently complete medium (MEM supplemented with 10 % FBS) was added. During the following experession period the cells of the logarithmic growing culture were subcultured 48 to 72 h after treatment. Additionally, the cell density was measured for toxicity criteria and adjusted to 1 x 10E6 cells/ml.

At the end of the expression period for selection the mutants, about 4 x 10E5 cells from each treatment group were seeded in cell culture petri dishes with selection medium containing 11 µg/ml thioguanine for further incubation (about one week). At the end of the selection period, colonies were fixed and stained for counting.

The cloning efficiencies were determined in parallel to the selection of mutants. For each treatment group, two flasks were seeded with approx. 200 cells to determine cloning efficiencies. After incubation for an appropriate time, colonies were fixed, stained and counted.

METHOD OF APPLICATION: in medium

DURATION
- Expression time (cells in growth medium): 48 to 72h
- Selection time (if incubation with a selection agent): about one week

SELECTION AGENT (mutation assays): thioguanine

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
ACCEPTABILITY OF THE ASSAY:
A mutation assay is considered acceptable if it meets the following criteria:
- Negative and/or solvent controls fall within the performing laboratory's historical control data range: 1 - 39 mutants/10E6 cells
- The absolute cloning efficiency of the negative and/or solvent controls is > 50 %
- The spontaneous mutant frequency in the negative and/or solvent controls is in the range of the performing laboratory's historical control data
- The positive controls induce significant increases (at least 3-fold increase of mutant frequencies related to the comparable negative control value and higher than the historical range of negative controls) in the mutant frequencies.

EVALUATION OF RESULTS:
A test is considered to be negative if there is no biological relevant increase in the number of mutants.

The criteria for determining a positive result are as follows:
- A reproducible 3-fold higher mutation frequency than the solvent control for at least one of the concentrations
- A concentration related increase of the mutation frequency; such an evaluation may be considered also in the case that a 3-fold increase of the mutant frequency is not observed
Statistics:
According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results. A statistical evaluation of the results is not regarded as necessary.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in both experiments without S9, detailed in tables 1 - 3
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
See tables on toxicity and mutations attached as background material

PRE-EXPERIMENTS

Table 1: Pre-Experiment for cytotoxicity without metabolic activation (S9 mix), 4 h exposure time

 Group  Test item conc. [µg/ml]  Cell density [cells/ml]  Relative growth [%]
 Neg. ctrl.  0  837000  98.8
 Neg. ctrl.  0  702000  82.8
 Solv. ctrl.  0  857000  100.0
 Solv. ctrl.  0  838000  100.0
 Test item  50 (P)  710000  83.8
 Test item  200 (P)  635000  74.9
 Test item  1000 (P)  242000  28.6
 Test item  2500 (P)  114000  13.5
 Test item  5000 (P)  601000  70.9

Cell density and relative growth at 1st subcultivation; (P) = Precipitation of test item

Table 2: Pre-Experiment for cytotoxicity with metabolic activation (S9 mix), 4 h exposure time

 Group  Test item conc. [µg/ml]  Cell density [cells/ml]  Relative growth [%]
 Neg. ctrl.  0  1290000  107.9
 Neg. ctrl.  0  1270000  106.3
 Solv. ctrl.  0  1220000  100.0
 Solv. ctrl.  0  1170000  100.0
 Test item  50 (P)  847000  70.9
 Test item  200 (P)  749000  62.7
 Test item  1000 (P)  776000  64.9
 Test item  2500 (P)  551000  46.1
 Test item  5000 (P)  932000 78.0

Cell density and relative growth at 1st subcultivation; (P) = Precipitation of test item

Table 3: Pre-Experiment for cytotoxicity without metabolic activation (S9 mix), 20 h exposure time

 Group  Test item conc. [µg/ml]  Cell density [cells/ml]  Relative growth [%]
 Neg. ctrl.  0  1960000  113.3
 Neg. ctrl.  0  1980000  114.5
 Solv. ctrl.  0  1740000  100.0
 Solv. ctrl.  0  1720000  100.0
 Test item  2  1680000  97.1
 Test item  5  1980000  114.5
 Test item  10  1730000  100.0
 Test item  25  430000  24.9
 Test item  50 (P)  67600 3.9
 Test item  100 (P)  88300  6.1

Cell density and relative growth at 1st subcultivation; (P) = Precipitation of test item

MUTATION EXPERIMENT I

Table 4: Toxicity and mutagenicity data, cell survival, without S9 mix

 Group  Conc. [µg/ml]  Cell dens. [cells/ml]  Relative growth [%]  No. of cells / flask*        Factor**  Cells seeded  Cells survived
         I  II  mean      
 Neg. ctrl.  0  2130000  117.9  180  180  180  0.90  400000  360000
 Neg. ctrl.  0  1880000  104.1  164  183  174  0.87  400000  347000
 Solv. ctrl.  0  1970000  100.0  154  175  165  0.82  400000  329000
 Solv. ctrl.  0  1642000  100.0  141  135  138  0.69  400000  276000
 Test item  5  1906000  105.5  148  148  148  0.74  400000  296000
 Test item  10  1802000  99.8  155  173  164  0.82  400000  328000
 Test item  20  1742000  96.5  148  178  163  0.82  400000  326000
 Test item  50 (P)  1518000  84.1  150  163  157  0.78  400000  313000
 Test item  100 (P)  1531000  84.8  151  157  154  0.77  400000  308000
 Test item  250 (P)  791000  43.8  178  149  164  0.82  400000  327000
 Test item  500 (P)  282000  15.6  149  157  153  0.77  400000  306000
 Test item  1000 (P)  183  10.1  126  148  137  0.69  400000  274000
 Pos. ctrl.  300  1357000  75.1  111  117  114  0.57  400000  228000

Cell density and relative growth at 1st subcultivation; (P) = Precipitation of test item

*: mean value of cells per flask / 200

**: cloning efficiency x cells seeded

Table 5: Mutagenicity data, mutation rates, without S9 mix

 Group  Conc. [µg/ml]  No. of mutant colonies / flask              Mean  SD  Mutant colonies per 10E6 cells*  Mutation factor
     I  II  III  IV  V        
 Neg. ctrl.  0  5  5  5  7  2  4.8  1.60  13.33  
 Neg. ctrl.  0  8  4  1  6  4  4.6  2.33  13.26  
 Solv. ctrl.  0  6  2  8  8  4  5.6  2.33  17.02  
 Solv. ctrl.  0  4  2  3  2  3  2.6  0.80  9.42  
 Test item  5  3  1  5  8  7  4.8  2.56  16.22  1.23
 Test item  10  5  1  6  4  4  4.0  1.67  12.20  0.92
 Test item  20  3  0  3  4  3  2.6  1.36  7.98  0.60
 Test item  50 (P)  5  3  3  6  3  4.0  1.28  12.78  0.97
 Test item  100 (P)  2  1  0  1  0  0.8  0.75  2.60  0.20
 Test item  250 (P)  3  0  0  3  1  1.4  1.36  4.28  0.32
 Test item  500 (P)  1  3  2  6  4  3.2  1.72  10.48  0.79
 Test item  1000 (P)  1  1  3  0  0  1.0  1.10  3.65  0.28
 Pos. ctrl.  300  97  125  108  113  109  110.4  9.02  484.21  36.62

(P) = Precipitation of test item

*: mean value x 10E6 / value of survied cells

Table 6: Toxicity and mutagenicity data, cell survival, with S9 mix

 Group  Conc. [µg/ml]  Cell dens. [cells/ml]  Relative growth [%]  No. of cells / flask*        Factor**  Cells seeded  Cells survived
         I  II  mean      
 Neg. ctrl.  0  2014000  124.3  157  163  160  0.80  400000  320000
 Neg. ctrl.  0  1798000  111.0  168  138  153  0.77  400000  306000
 Solv. ctrl.  0  1650000  100.0  184  178  181  0.91  400000  362000
 Solv. ctrl.  0  1590000  100.0  170  170  170  0.85  400000  340000
 Test item  500 (P)  1830000  113.0  179  204  192  0.96  400000  383000
 Test item  1000 (P)  1640000  101.2  184  201  193  0.96  400000  385000
 Test item  2500 (P)  1730000  106.8  181  183  182  0.91  400000  364000
 Test item  2900 (P)  1540000  95.1  192  175  183  0.92  400000  367000
 Test item  3300 (P)  1530000  94.4  183  179  181  0.91  400000  362000
 Test item  3800 (P)  1350000  83.3  166  162  164  0.82  400000  328000
 Test item  4300 (P)  1630000  100.6  163  185  174  0.87  400000  348000
 Test item  4750 (P)  1670000  103.1  172  193  183  0.91  400000  365000
 Pos. ctrl.  1.50  1677000  103.5  122  138  130  0.65  1400000  260000

Cell density and relative growth at 1st subcultivation; (P) = Precipitation of test item

*: mean value of cells per flask / 200

**: cloning efficiency x cells seeded

Table 7: Mutagenicity data, mutation rates, with S9 mix

 Group  Conc. [µg/ml]  No. of mutant colonies / flask              Mean  SD  Mutant colonies per 10E6 cells*  Mutation factor
     I  II  III  IV  V        
 Neg. ctrl.  0  5  5  3  3  0  3.2  1.83  10.0  
 Neg. ctrl.  0  2  3  4  2  2  2.6  0.80  8.50  
 Solv. ctrl.  0  3  8  5  4  3  4.6  1.85  12.71  
 Solv. ctrl.  0  4  4  4  3  2  3.4  0.80  10.00  
 Test item  500 (P)  4  4  5  6  9  5.6  1.85  14.62  1.29
 Test item  1000 (P)  0  5  6  4  4  3.8  2.04  9.87  0.87
 Test item  2500 (P)  6  8  6  4  5  5.8  1.33  15.93  1.40
 Test item  2900 (P)  6  7  7  3  5  5.6  1.50  15.26  1.34
 Test item  3300 (P)  2  3  2  5  4  3.2  1.17  8.84  0.78
 Test item  3800 (P)  6  8  2  12  6  6.8  3.25  20.73  1.83
 Test item  4300 (P)  3  4  4  5  5  4.2  0.75  12.07  1.06
 Test item  4750 (P)  2  5  3  3  2  3.0  1.10  8.22  0.72
 Pos. ctrl.  1.50  62  52  61  58  59  58.4  3.50  224.62  19.78

(P) = Precipitation of test item

*: mean value x 10E6 / value of survied cells

MUTATION EXPERIMENT II

Table 8: Toxicity and mutagenicity data, cell survival, without S9 mix

 Group  Conc. [µg/ml]  Cell dens. [cells/ml]  Relative growth [%]  No. of cells / flask*        Factor**  Cells seeded  Cells survived
         I  II  mean      
 Neg. ctrl.  0  3030000  117.0  151  148  150  0.75  400000  299000
 Neg. ctrl.  0  2890000  111.6  175  135  155  0.78  400000  310000
 Solv. ctrl.  0  2470000  100.0  138  133  136  0.68  400000  271000
 Solv. ctrl.  0  2710000  100.0  157  164  161  0.80  400000  321000
 Test item  1.00  2400000  92.7  153  138  146  0.73  400000  291000
 Test item  5.00  2000000  77.2  126  140  138  0.69  400000  276000
 Test item  15.00  1100000  42.5  148  139  144  0.72  400000  287000
 Test item  20.00  489000  18.9  166  183  175  0.87  400000  349000
 Test item  22.50  603000  23.3  131  150  141  0.70  400000  281000
 Test item  27.50  435000  16.8  128  136  132  0.66  400000  264000
 Test item  35.00  333000  12.9  142  146  144  0.72  400000  288000
 Test item  40.00  426000  16.4  176  187  182  0.91  400000  363000
 Pos. ctrl.  300  1660000  64.1  89  94  92  0.46  400000  183000

Cell density and relative growth at 1st subcultivation; (P) = Precipitation of test item

*: mean value of cells per flask / 200

**: cloning efficiency x cells seeded

Table 9: Mutagenicity data, mutation rates, without S9 mix

 Group  Conc. [µg/ml]  No. of mutant colonies / flask              Mean  SD  Mutant colonies per 10E6 cells*  Mutation factor
     I  II  III  IV  V        
 Neg. ctrl.  0  1  2  2  4  0  1.8  1.33  6.02  
 Neg. ctrl.  0  1  4  1 4  4  2.8  1.47  9.03  
 Solv. ctrl.  0  3  2  0  1  1  1.4  1.02  5.17  
 Solv. ctrl.  0  4  6  8  4  4  5.2  1.60  16.20  
 Test item  1.00  0  4  2  3  3  2.4  1.36  8.25  0.77
 Test item  5.00  3  3  4  5  5  4.0  0.89  14.49  1.36
 Test item  15.00  1  2  1  2  1  1.4  0.49  4.88  0.46
 Test item  20.00  4  10  4  4  7  5.8  2.40  16.62  1.56
 Test item  22.50  1  2  1  2  4  1.6  0.49  5.69  0.53
 Test item  27.50  3  5  4  6  6  4.8  1.17  18.18  1.70
 Test item  35.00  1  2  2  1  5  2.2  1.47  7.64  0.72
 Test item  40.00  5  10  11  11  5  8.4  2.80  23.14  2.17
 Pos. ctrl.  300  106  127  103  126  110  114.4  10.13  625.14  58.52

(P) = Precipitation of test item

*: mean value x 10E6 / value of survied cells

Table 10: Toxicity and mutagenicity data, cell survival, with S9 mix

 Group  Conc. [µg/ml]  Cell dens. [cells/ml]  Relative growth [%]  No. of cells / flask*        Factor**  Cells seeded  Cells survived
         I  II  mean      
 Neg. ctrl.  0  2000000  94.8  172  169  171  0.85  400000  341000
 Neg. ctrl.  0  2220000  105.2  158  185  172  0.86  400000  343000
 Solv. ctrl.  0  2120000  100.0  160  155  158  0.79  400000  315000
 Solv. ctrl.  0  2100000  100.0  171  165  168  0.84  400000  336000
 Test item  125 (P)  1920000  91.0  177  177  177  0.89  400000  354000
 Test item  500 (P)  1950000  92.4  176  187  182  0.91  400000  363000
 Test item  1400 (P)  1660000  78.7  188  162  175  0.88  400000  350000
 Test item  1800 (P)  1720000  81.5  184  170  177  0.89  400000  354000
 Test item  2200 (P)  1400000  66.4  188  172  180  0.90  400000  360000
 Test item  2600 (P)  1540000  73.0  180  190  185  0.93  400000  370000
 Test item  3000 (P)  1630000  77.3  200  185  193  0.96  400000  385000
 Test item  4000 (P)  1920000  91.0  162  174  168  0.84  400000  336000
 Pos. ctrl.  1.50  1220000  57.8  153  158  156  0.78  400000  311000

Cell density and relative growth at 1st subcultivation; (P) = Precipitation of test item

*: mean value of cells per flask / 200

**: cloning efficiency x cells seeded

Table 11: Mutagenicity data, mutation rates, with S9 mix

 Group  Conc. [µg/ml]  No. of mutant colonies / flask              Mean  SD  Mutant colonies per 10E6 cells*  Mutation factor
     I  II  III  IV  V        
 Neg. ctrl.  0  2  4  3  0  3  2.4  1.36  7.04  
 Neg. ctrl.  0  0  2  2 1  0  1.0  0.89  2.92  
 Solv. ctrl.  0  2  0  4  1  3  2.0  1.41  6.35  
 Solv. ctrl.  0  4  3  1  1  1  2.0  1.26  5.95  
 Test item  125 (P)  5  1  1  1  2  2.0  1.55  5.65  0.92
 Test item  500 (P)  3  2  3  2  1  2.2  0.75  6.06  0.99
 Test item  1400 (P)  2  3  3  2  0  2.0  1.10  5.71  0.93
 Test item  1800 (P)  1  4  3  4  2  2.8  1.17  7.91  1.29
 Test item  2200 (P)  0  2  2  3  1  1.6  1.02  4.44  0.72
 Test item  2600 (P)  2  3  1  2  2  2.0  0.63  5.41  0.88
 Test item  3000 (P)  0  3 2  1  1.4  1.02  3.64  0.59
 Test item  4000 (P)  2  2  3  1  5  2.6  1.36  7.74  1.26
 Pos. ctrl.  1.50  70  78  57  70  87  72.4  9.93  232.80  37.85

(P) = Precipitation of test item

*: mean value x 10E6 / value of survied cells

Conclusions:
The test substance is consdiered to be non-mutagenic in the HPRT locus using V79 cells.
Executive summary:

The test item was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster. The main experiments were carried out without and with metabolic activation by S9 mix.

The selection of the concentrations used in the main mutation experiments was based on data from pre-experiments according to the OECD guideline 476.

In experiment I 1000 µg/ml without metabolic activation and 4750 µg/ml with metabolic activation were selected as the highest concentrations. In experiment II 40 µg/ml without metabolic activation and 4000 µg/ml with metabolic activation were selected as the highest concentrations. Experiment II without metabolic activation was performed as 20 h long-term exposure assay. The pH value detected with the test item was withing the physiological range.

The test item was investigated at the following concentrations:

EXPERIMENT I

- without metabolic activation (S9 mix): 5, 10, 20, 50, 100, 250, 500, 1000 µg/ml

- with metabolic activation (S9 mix): 500, 1000, 2500, 2900, 3300, 3800, 4300, 4750 µg/ml

EXPERIMENT II

- without metabolic activation (S9 mix): 1, 5, 15, 20, 22.5, 27.5, 35, 40 µg/ml

- with metabolic activation (S9 mix): 125, 500, 1400, 1800, 2200, 2600, 3000, 4000 µg/ml

TOXICITY

No biologically relevant growth inhibition was observed in experiment I and II with metabolic activation. In experiment I and II without metabolic activation, biologically relevant growth inhibition was noted.

In experiment I without metbolic activation the relative growth was 10.1 % for the highest concentration (1000 µg/ml) evaluated. The highest concentration evaluated with metabolic activation was 4750 µg/ml with a relative growth of 103.1 %. In experiment II without metabolic activation the relative growth was 16.4 % for the highest concentration (40 µg/ml) evaluated. The highest concentration evaluated with metabolic activation was 4000 µg/ml with a relative growth of 91.0 %.

MUTAGENICITY

In experiment I without metabolic activation mutant values of the negative and solvent controls and all test item concentratios found were withing the historical control data of the test facility (about 1 - 39 mutants per 10E6 cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the solvent controls.

Mutation frequencies of the negative control were found to be 13.33 and 13.26, for the solvent control 17.02 and 9.42 mutants/10E6 cells and in the range of 2.60 to 16.22 mutants/10E6 cells with the test item, respectively. The highest mutation rate (compared to the solvent control values) of 1.23 was found at a concentration of 5 µg/ml with a relative growth of 105.5 %.

In experiment I with metabolic activation all mutant values found were withing the historical control data of the test facility (about 2 - 28 mutants per 10E6 cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the solvent controls.

Mutation frequencies of the negative control were found to be 10.00 and 8.50, of the solvent control 12.71 and 10.00 mutants/10E6 cells and in the range of 8.22 to 20.73 mutants/10E6 cells with the test item, respectively. The highest mutation rate (compared to the solvent control values) of 1.83 was found at a concentration of 3800 µg/ml with a relative growth of 83.3 %.

In experiment II without metabolic activation all mutant values found were within the historical control data of the test facility (about 1 - 39 mutants per 10E6 cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the solvent controls.

Mutation frequenices of the negative control were found to be 6.02 and 9.03, of the solvent control 5.17 and 16.20 mutants/10E6 cells and in the range of 4.88 to 23.14 mutants/10E6 cells with the test item, respecively. The highest mutation rate (compared to the solvent control values) of 2.17 was found at a concentration of 40 µg/ml with a relative growth of 16.4 %.

In experiment II with metabolic activation all mutant values found were within the historical control data of the test facility (about 2 - 28 mutants per 10E6 cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the negative controls.

Mutation frequencies of the negative control were found to be 7.04 and 4.08, of the solvent control 6.35 and 5.95 mutants/10E6 cells and in the range of 3.64 to 7.91 mutants/10E6 cells with the test item, respectively. The highest mutation rate (compared to the solvent control values) of 1.29 was found at a concentration of 1800 µg/ml with a relative growth of 81.5 %.

7,12 -diemthylbenzantracen (DMBA, 1.5 µg/ml) and Ethylmethanesulfonate (EMS, 300 µg/ml) were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Since no classification criteria according to GHS/CLP (Regulation (EC) No. 1272/2008) are fulfilled, the substance is not classified for genetic toxicity.