Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a study according to OECD TG 471 under GLP conditions, the substance was not mutagenic in five strains (TA98, TA100, TA1535, TA1537 and WP2 uvrA) with and without metabolic activation.

The genotoxicity of the substance was investigated in an in vitro micronucleus test according to OECD test guideline 487 under GLP-conditions. As the non-cytotoxic substance did not increase the proportion of binucleated cells with micronuclei as compared to control for the concentrations of 0.5, 1 and 2 mg/mL, the substance was considered as not genotoxic under the test conditions.

The mutagenicity of the substance was investigated in an in vitro mammalian cell gene mutation test using the HPRT genes according to OECD test guideline 476 under GLP-conditions. In experiment 1 (4 hour treatment with and without metabolic activation) and experiment 2a (24 hour treatment without metabolic activation) the non-cytotoxic substance increased mutant colony numbers, which was primarily caused by one of two replicates, without a concentration-response. These results were considered to be not biologically relevant. To confirm this conclusion, experiment 2a was (24 hour treatment without metabolic activation) conducted, which was clearly negative for all test concentration. Therefore, the substance was considered as non-mutagenic in this HPRT test.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
July 21, 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Product no. B4429, Lot no. SLBK5235V
- Expiration date of the lot/batch: 30 April 2018
- Purity test date: na

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
First and second experiment: 16, 50, 160, 500, 1600, 5000 µg/plate, where 5000 µg/plate is the recommended maximum test concentration for soluble non-cytotoxic substances
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ultrapure water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
no
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 4-Nitro-1,2-phenylenediamine and 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) (first experiment); preincubation (second experiment)

DURATION
- Preincubation period: 20 min
- Exposure duration: 48h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: number of revertant colonies (with TA98 and TA100 with and without S9)

Evaluation criteria:
reproducible increase of revertant colonies per plate (factor >=2 for TA100 and factor >= 3 for TA98m TA1535, TA1537 and WP2)
Statistics:
none
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
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 100
Metabolic activation:
with and without
Genotoxicity:
negative
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
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 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS: not available

RANGE-FINDING/SCREENING STUDIES: no precipitation and no cytotoxicity observed up to 5000 µg/plate

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%): not available

ADDITIONAL INFORMATION ON CYTOTOXICITY: none

In a study according to OECD TG 471 under GLP conditions, the substance was not mutagenic in five strains (TA98, TA100, TA1535, TA1537 and WP2 uvrA) with and without metabolic activation.

pH of the test substance stock solutions and the pH of the overlay (at highest concentration of 50 mg/mL) was measured as 9.39 and 9.18, respectively. The acidic solutions had only a slight effect on the buffered overlay system that did not impair the validity of the experiment and the results.

Detailed test results:

Initial experiment (plate incorporation) without and with metabolic activation

 control/

mutation rate per test strain: without metabolic activation        

mutation rate per test strain: with metabolic activation   

 concentration (µg/plate)

 TA98

TA100

TA1535 

TA1537 

WP2uvrA 

 TA98

TA100 

TA1535 

TA1537 

WP2uvrA 

 untreated control

 0.98

0.81

0.96 

1.63

1.24 

0.94 

1.17 

1.09 

1.00 

1.00 

 DMSO control

 1.00

 -

 -

1.00

-

 1.00

1.00 

1.00 

1.00 

1.00 

 ultrapure water control

 1.00

1.00

1.00 

1.00 

1.00 

1.00 

1.00 

1.00 

1.00 

1.00 

test substance: 5000

 1.02

 0.90

 1.36

 0.94

 1.07

 1.02

 1.02

 1.31

 0.88

 1.27

test substance: 1600

1.21

 0.87

 1.39

 0.88

 0.91

 1.26

 0.99

 1.19

 1.50

 1.07

test substance: 500

 1.00

 0.98

 1.54

 1.31

 1.11

 0.98

 0.98

 1.22

 1.00

1.26

test substance: 160

 0.85

 1.07

 1.36

 0.94

 1.30

 1.07

 1.06

 1.22

 1.25

 1.30

test substance: 50

 1.02

 0.97

 1.18

 0.88

 0.93

0.97

 1.10

 0.94

 1.38

 0.96

test substance: 16

 1.04

 0.91

 0.93

 0.81

 1.13

 0.91

 1.10

 0.88

 0.94

 1.32

 NPD (4 µg)

 27.12

 

 

 

 

 

 

 

 

 

 SAZ (2 µg)

 

 14.08

 104.93

 

 

 

 

 

 

 

9AA (50 µg) 

 

 

 

 112.17

 

 

 

 

 

 

 MMS (2 µL)

 

 

 

 34.30

 

 

 

 

 

 2AA (2 µg)

 

 

 

 

 

 52.66

 9.19

 19.93

 22.04

 

 2AA (50 µg)

 

 

 

 

 

 

 

 

 

 13.50

Confirmatory experiment (pre-incubation) without and with metabolic activation

 control/

mutation rate per test strain: without metabolic activation        

mutation rate per test strain: with metabolic activation   

 concentration (µg/plate)

 TA98

TA100

TA1535 

TA1537 

WP2uvrA 

 TA98

TA100 

TA1535 

TA1537 

WP2uvrA 

 untreated control

 1.09

0.86 

1.00 

1.47 

1.04 

1.02 

0.97 

1.04 

1.18 

0.75 

 DMSO control

 1.00

 -

 -

1.00

-

 1.00

1.00 

1.00 

1.00 

1.00 

 ultrapure water control

 1.00

1.00

1.00 

1.00 

1.00 

1.00 

1.00 

1.00 

1.00 

1.00 

test substance: 5000

 1.33

 1.14

 0.78

 1.60

 1.36

 0.78

 0.84

 1.11

1.24

 1.00

test substance: 1600

 1.07

 1.01

 1.05

 1.40

 1.56

 0.88

 0.85

 0.96

 1.29

 0.92

test substance: 500

 1.23

 0.95

 0.95

 1.60

 1.45

 0.78

 0.88

 0.96

 0.82

0.83

test substance: 160

 1.35

 1.10

 0.95

 0.80

 1.40

 1.02

0.88

 1.00

 0.94

 0.74

test substance: 50

 1.37

 0.99

 1.34

 1.40

 1.15

 0.87

 0.83

 0.86

 1.06

 0.74

test substance: 16

 1.05

 1.09

 0.88

 1.40

 1.25

 0.77

 0.95

 1.36

 1.06

 0.71

 NPD (4 µg)

 22.03

 

 

 

 

 

 

 

 

 

 SAZ (2 µg)

 

 9.87

 78.49

 

 

 

 

 

 

 

9AA (50 µg) 

 

 

 

 112.25

 

 

 

 

 

 

 MMS (2 µL)

 

 

 

 

 52.36

 

 

 

 

 

 2AA (2 µg)

 

 

 

 

 

 40.73

 11.83

 12.07

 15.16

 

 2AA (50 µg)

 

 

 

 

 

 

 

 

 

 4.59

Executive summary:

The mutagenic potential of the substance was investigated in a bacterial reverse mutation assay according to OECD TG 471 under GLP. The substance was soluble and non-cytotoxic up to 5000 µg/plate, i.e. the recommended maximum concentration. The substance was tested in five strains (TA98, TA100, TA1535, TA1537 and WP2 uvrA) with and without metabolic activation (S9).

In a first experiment, six concentration (16, 50, 160, 500, 1600 and 5000 µg/plate) were tested for 48 hours in the plate incorporation method. In the second experiment the same concentrations were tested (48 h) in the pre-incubation method. The number of colonies were evaluated by eye.

While the positive controls were clearly mutagenic, the substance did not increase the number of colonies in any strain in any experiment. Therefore, the substance is considered to be not mutagenic.

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:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
minor, not affecting validity and results
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: Product no. B4429, Lot no. SLBK5235V
- Expiration date of the lot/batch: 30 April 2018
- Purity test date: na

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: CLS (Eppelheim, Germany)
- Suitability of cells: analysis of modal chromosome number
- Cell cycle length, doubling time or proliferation index: cell cycle time was determined in experiment and within the normal range; doubling time: 10-12 h
- Sex, age and number of blood donors if applicable: not applicable
- Whether whole blood or separated lymphocytes were used if applicable: not applicable
- Number of passages if applicable: no information
- Methods for maintenance in cell culture if applicable: no information
- Modal number of chromosomes: 22
- Normal (negative control) cell cycle time: no information

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: DMEM (from Biochrom AG) wad used as a basis for complete culture medium with medium DMEM with 5% HS (5% horse serum, 1% Penicillin/Streptomycin, 1% phytohaemagglutinin solution) and selection medium (5% horse serum, 1% Penicillin/Streptomycin, 2 µg/mL 6-thioguanine (1 mg/mL))
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
concentration in experiment: 0.03, 0.06, 0.13, 0.25, 0.5, 1, 2 mg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMEM
- Justification for choice of solvent/vehicle: no effects on cell viability and good solubility of test substance
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium;
- Cell density at seeding (if applicable): 1000000/dish (10 cm diameter)

DURATION
- Preincubation period: none
- Exposure duration: Experiment 1: 4h; Experiment 2: 24h
- Expression time (cells in growth medium): at least 168h
- Selection time (if incubation with a selection agent): 7-8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 14-16 days

SELECTION AGENT (mutation assays): 6-thioguanine

SPINDLE INHIBITOR (cytogenetic assays): not applicable

STAIN (for cytogenetic assays): not applicable

NUMBER OF REPLICATIONS: 2 cultures per control and test concentration

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: counting of cell colonies

NUMBER OF CELLS EVALUATED: not applicable

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): not available

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: not applicable

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency
- Any supplementary information relevant to cytotoxicity: none

OTHER EXAMINATIONS:
- Determination of polyploidy: no
- Determination of endoreplication: no
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): not applicable

- OTHER: Metabolic activation: S9-mix
Experiment 1: with and without
Experiment 2: without
Rationale for test conditions:
The conditions of experiment 1 are according to the recommendations of the OECD TG 476.
The second experiment was performed to confirm the findings of experiment 1.
Evaluation criteria:
According to EOCD TG 476 (version of July 2016)
Statistics:
Chi-square test with a significance level of 5%
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Evaporation from medium: no information
- Water solubility: the substance was highly soluble
- Precipitation: no
- Definition of acceptable cells for analysis: no information
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES:
- cytotoxicity pre-test: highest recommended concentration (2 mg/mL) not cytotoxic, determined by cloning efficiency) and not precipitating after 4h exposure with and without metabolic activation

CYTOKINESIS BLOCK (if used): not applicable

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data (number of mutant colonies per 1000000 cells):
With S9 (4h treatment): DMBA: range: 100 - 461; mean: 261; sd: 66.6
Without S9 (4h treatment): EMS: range: 71 - 207; mean: 128; sd: 33.1
Without S9 (24h treatment): EMS: range: 35 - 517; mean: 238; sd: 105.8
- Negative (solvent/vehicle) historical control data(number of mutant colonies per 1000000 cells):
With S9 (4h treatment): DMEM: range: 4 - 35; mean: 16; sd: 10.0
With S9 (4h treatment): DMSO: range: 2 - 39; mean: 14; sd: 8.9
Without S9 (4h treatment): DMEM: range: 2- 31; mean: 14; sd: 9.1
Without S9 (24h treatment): DMEM: range: 4 - 27; mean: 12; sd: 6.1

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: cloning efficiency
- Other observations when applicable: [complete, e.g. confluency, apoptosis, necrosis, metaphase counting, frequency of binucleated cells]: none

The mutagenicity of the substance was investigated in an in vitro mammalian cell gene mutation test using the HPRT genes according to OECD test guideline 476 under GLP-conditions. In experiment 1 (4 hour treatment with and without metabolic activation) and experiment 2a (24 hour treatment without metabolic activation) the non-cytotoxic substance increased mutant colony numbers, which was primarily caused by one of two replicates, without a concentration-response. These results were considered to be not biologically relevant. To confirm this conclusion, experiment 2a was (24 hour treatment without metabolic activation) conducted, which was clearly negative for all test concentration. Therefore, the substance was considered as non-mutagenic in this HPRT test.

Exp. 1 without S9 (4h treatment):

Treatment      culture          rel. survival       mutant frequency per 1000000 cells       mean mutant frequency per 1000000 cells

DMEM              1                    -                                         16                                               20

(test substance)   2                    -                                         24    

DMEM              1                    -                                         28                                               23

(EMS)               2                     -                                         19

EMS                 1                     86.2%                               106**                                         114**

2                     95.7%                                122**

Test item

2 mg/mL          1                    92.8%                                   3                                                43

2                    79.8%                                   83**                                                 

1 mg/mL          1                    60.0%                                   55**                                          50**

2                    84.2%                                   46**                                                 

0.5 mg/mL       1                    64.3%                                   31                                              29

2                    82.8%                                   27                       

0.25 mg/mL      1                   75.7%                                   57**                                          44

2                    98.2%                                   31                                          

0.13 mg/mL      1                   81.1%                                    7                                              33

2                    91.6%                                   60**    

0.006 mg/mL    1                   68.1%                                    47**                                         52**

2                   105.8%                                   57**

Exp. 1 with S9 (4h treatment):

Treatment      culture          rel. survival       mutant frequency per 1000000 cells       mean mutant frequency per 1000000 cells

DMEM              1                    -                                         18                                               27

(test substance)   2                    -                                         35    

DMSO              1                    -                                         28                                               26

(DMBA)            2                     -                                        23

DMBA               1                     90.5%                               206**                                         172**

2                     96.6%                               138**

Test item

2 mg/mL          1                    112.3%                                   25                                               28

2                    129.9%                                   31                                                 

1 mg/mL          1                    99.1%                                    45**                                           36

2                   153.7%                                    27                                                 

0.5 mg/mL       1                    130.8%                                    22                                              34

2                    166.9%                                    45                       

0.25 mg/mL      1                   125.9%                                    24                                             27

2                    164.1%                                   30                                          

0.13 mg/mL      1                   112.3%                                    24                                              22

2                    200.8%                                   20    

0.006 mg/mL    1                   104.1%                                    53**                                          29

2                    158.8%                                    4

Exp. 2a without S9 (24h treatment):

Treatment      culture          rel. survival       mutant frequency per 1000000 cells       mean mutant frequency per 1000000 cells

DMEM              1                    -                                         18                                                17

(test substance)   2                    -                                         16    

DMEM              1                    -                                         27                                               35

EMS                   2                     -                                        42

EMS                   1                     74.7%                               338**                                         312**

2                     133.3%                             285**

Test item

2 mg/mL          1                         48.3%                               30                                               51**

2                        64.5%                                73**                                                 

1 mg/mL          1                        38.3%                                  13                                            15

2                        61.0%                                  18                                                 

0.5 mg/mL       1                       38.4%                                  25                                            38**

2                        52.1%                                   50**                       

0.25 mg/mL      1                       50.5%                                    40**                                       29

2                        93.5%                                   19                                          

0.13 mg/mL      1                        42.0%                                    n/a                                          n/a

2                        67.8%                                   n/a   

0.006 mg/mL    1                        40.6  %                                 n/a                                            n/a

2                        82.1%                                    n/a

Legend: **: p < 0.01; n/a: not evaluated (as only 4 concentrations need to be evaluated)

Exp. 2b without S9 (24h treatment):

Treatment      culture          rel. survival       mutant frequency per 1000000 cells       mean mutant frequency per 1000000 cells

DMEM              1                    -                                         10                                                10

(test substance)   2                    -                                         9    

DMEM              1                    -                                         7                                                7

EMS                   2                     -                                        8

EMS                   1                     89.5%                               383                                         378**

2                     116.2%                             372

Test item

2 mg/mL          1                         112.6%                              12                                              11

2                        93.0%                                 9                                                 

1 mg/mL          1                        99.1%                                   9                                            12

2                        102.3%                                14                                                 

0.5 mg/mL       1                       126.6%                                  10                                             7

2                        113.4%                                  5                       

0.25 mg/mL      1                       126.3%                                  12                                        11

2                        97.8%                                    11                                          

0.13 mg/mL      1                        119.4%                                   8                                          8

2                        105.6%                                   8   

0.006 mg/mL    1                        126.2%                                 10                                             11

2                        112.1%                                   12

Legend: **: p < 0.01; n/a: not evaluated (as only 4 concentrations need to be evaluated)

Executive summary:

The mutagenicity of the substance was investigated in an in vitro mammalian cell gene mutation test using the HPRT genes according to OECD test guideline 476 under GLP-conditions. In experiment 1 (4 hour treatment with and without metabolic activation) and experiment 2a (24 hour treatment without metabolic activation) the non-cytotoxic substance increased mutant colony numbers, which was primarily caused by one of two replicates, without a concentration-response. These results were considered to be not biologically relevant. To confirm this conclusion, experiment 2a was (24 hour treatment without metabolic activation) conducted, which was clearly negative for all test concentration. Therefore, the substance was considered as non-mutagenic in this HPRT test.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
version of July 29, 2016
Deviations:
yes
Remarks:
without effect on study validity and results
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Product no. B4429, Lot no. SLBK5235V
- Expiration date of the lot/batch: 30 April 2018
- Purity test date: na

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no
Target gene:
not applicable
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: young, non-smoking donors (26 year old female and 31 year old male)
- Suitability of cells: human peripheral lymphocytes are suitable due to their low and stable background rate of micronuclei and as they eliminate inter-species differences
- Cell cycle length, doubling time or proliferation index: Cytokinesis-block proliferation index (CBPI): approx. 1.65
- Sex, age and number of blood donors if applicable: 26 year old female and 31 year old male
- Whether whole blood or separated lymphocytes were used if applicable: whole blood
- Number of passages if applicable: not applicable
- Methods for maintenance in cell culture if applicable: not applicable
- Modal number of chromosomes: 46
- Normal (negative control) cell cycle time: 12h

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 (from Biochrom AG) wad used as a basis for complete culture medium (15% FCS, 1% Penicillin/Streptomycin, 1% phytohaemagglutinin solution) and minimal culture medium (MCM) (1% Penicillin/Streptomycin, 2% phytohaemagglutinin solution)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no information
- Periodically checked for karyotype stability: not applicable
- Periodically 'cleansed' against high spontaneous background: not applicable
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
Cytochalasin B
Metabolic activation:
with and without
Metabolic activation system:
S9 (from Trinova Biochem GmbH, Gießen, Germany, batch no. 3653)
Test concentrations with justification for top dose:
- test concentrations (in the experiment): 0.13, 0.25, 0.5, 1, 2 mg/mL
- concentrations scored for micronuclei: 0.5, 1, 2 mg/mL
- top dose according to OECD TG 487
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: minimal culture medium
- Justification for choice of solvent/vehicle: no solvent effect on cell viability and the test substance was completely soluble
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: colchicine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): not applicable

DURATION
- Preincubation period: none
- Exposure duration: Experiment 1: 4h; Experiment 2: 23.5h
- Expression time (cells in growth medium): Experiment 1: 19h; Experiment 2:-
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): exposure and expression time and 30 min fixation

SELECTION AGENT (mutation assays): not applicable

SPINDLE INHIBITOR (cytogenetic assays): not applicable

STAIN (for cytogenetic assays): not applicable

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: dropping of cell suspension onto a clean slide; staining with 10% Giemsa solution

NUMBER OF CELLS EVALUATED: 1000 binucleated cells

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): not applicable

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: scoring of binucleated cells with sufficiently distinguishable cytoplasmic boundaries and clearly visible cytoplasm

DETERMINATION OF CYTOTOXICITY
- Method: other: Cytokinesis-block proliferation index
- Any supplementary information relevant to cytotoxicity: none

OTHER EXAMINATIONS:
- Determination of polyploidy: no
- Determination of endoreplication: no
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): not applicable

- OTHER: none
Rationale for test conditions:
According to recommendation in the OECD TG 487 for lymphocytes, i.e. for the initial experiment 3-6h and for the subsequent experiment 1.5-2 cell cycles (18-24h)
Evaluation criteria:
Comparison of binucleated cells with micronuclei of each of the treatment groups with the solvent control group
Statistics:
Fisher's exact test with a significance level of 5% (for each treatment-control comparison)
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Evaporation from medium: no information
- Water solubility: the substance was highly soluble
- Precipitation: no
- Definition of acceptable cells for analysis: binucleated cells with sufficiently distinguishable cytoplasmic boundaries and clearly visible cytoplasm
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: experiment 1 was initially the range-finding experiment, but was also a valid of a full experiment

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells:
Experiment 1 (without positive controls): 39%, 56% , 6% (average across all groups)
Experiment 1 (without positive controls): 34%, 51%, 14% (average across all groups)

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture:
see 'Any other information...' below
- Indication whether binucleate or mononucleate where appropriate: no

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
Without S9: 0.3 µg/mL MMC (short exposure): range: 1.08 - 7.67; mean: 3.37; sd: 1.73
0.3 µg/mL MMC (extended exposure): range: 1.33 - 7.75; mean: 3.85; sd: 1.67
Colchicine: range: 1.24 - 10.32; mean: 5.74; sd: 2.64
With S9: CPA: range: 1.25 - 5.14; mean: 2.63; sd: 0.81
- Negative (solvent/vehicle) historical control data:
Without S9: medium: range: 0.05- 1.06; mean: 0.44; sd: 0.27
NaCl (0.9%): range: 0.05 - 1.24; mean: 0.47; sd: 0.30
With S9: medium: range: 0.10- 0.83; mean: 0.32; sd: 0.16
NaCl (0.9%): range: 0.05 - 1.11; mean: 0.36; sd: 0.22

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI
- Other observations when applicable: [complete, e.g. confluency, apoptosis, necrosis, metaphase counting, frequency of binucleated cells]: none

The genotoxicity of the substance was investigated in an in vitro micronucleus test according to OECD test guideline 487 under GLP-conditions. In two experiments (experiment 1: 4 hour exposure and 19 h expression time with and without metabolic activation; experiment 2: 23.5h exposure without metabolic activation) the non-cytotoxic substance did not increase the proportion of binucleated cells with micronuclei as compared to control for the concentrations of 0.5, 1 and 2 mg/mL.

Therefore, the substance was considered as not genotoxic under the test conditions.

Exp. 1 without S9:

Treatment      culture              BINC       MBNC       total examined       % MNBC

MCM               1                    993              7                     1000                  0.70%

2                    996              4                     1000                  0.40%    

0.9% NaCl       1                    989             11                    1000                  1.10%

2                    990             10                    1000                  1.00%

MMC               1                    993             76                    1000                  7.60%

2                    996             31                    1003                  3.09%

Test item

2 mg/mL          1                    991              9                     1000                   0.90%

2                    991              9                     1000                  0.90%    

1 mg/mL          1                    992              10                    1002                 1.00%

2                    993              7                     1000                   0.70%

0.5 mg/mL        1                   992              8                    1000                   0.80%

2                    995             5                     1000                   0.50%

Exp. 1 with S9:

Treatment      culture              BINC       MBNC       total examined       % MNBC

MCM               1                    992              8                     1000                  0.80%

2                    993              7                     1000                  0.70%    

0.9% NaCl       1                    999              7                     1006                  0.70%

2                    997              5                     1002                  0.50%

CPA                 1                    966             34                    1000                  3.40%

2                    993             20                    1013                  1.00%

Test item

2 mg/mL          1                    990              10                     1000                  1.00%

2                    991              9                      1000                  0.90%    

1 mg/mL          1                    996               6                      1002                  0.60%

2                    994              6                      1000                  0.60%

0.5 mg/mL        1                   992              8                     1000                   0.80%

2                    990             10                     1000                   1.00%

Exp. 2 without S9:

Treatment      culture              BINC       MBNC       total examined       % MNBC

MCM               1                    989             11                     1000                  1.10%

2                    990             10                     1000                  1.00%    

0.9% NaCl       1                    998              2                     1000                   0.20%

2                    995              5                     1000                   0.50%

MMC               1                    981             19                    1000                   1.90%

2                    972             28                    1000                   2.80%

Colchincine      1                    957             43                    1000                   4.30%

2                    955             45                    1000                   4.50%

Test item

2 mg/mL          1                    997              3                     1000                   0.30%

2                    996              4                     1000                   0.40%    

1 mg/mL          1                    997              3                     1000                   0.30%

2                    997              3                     1000                   0.30%

0.5 mg/mL        1                   999              1                     1000                   0.10%

2                    996             4                      1000                   0.40%

Legend: BINC: binucleate cells; MBNC: binucleate cells with micronucleus

Executive summary:

The genotoxicity of the substance was investigated in an in vitro micronucleus test according to OECD test guideline 487 under GLP-conditions. Human peripheral blood lymphocytes were stimulated to divide (by adding phytohaemagglutinin) and then exposed in two experiments (experiment 1: 4 hour exposure and 19 h expression time with and without metabolic activation; experiment 2: 23.5h exposure without metabolic activation). The non-cytotoxic substance (up to 10 mM, corresponding to 1.95 mg/mL, determined with the cytokinesis-block proliferation index) did not increase the proportion of binucleated cells with micronuclei as compared to control for the concentrations of

0.5, 1 and 2 mg/mL. Therefore, the substance was considered as not genotoxic under the test conditions.

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

Additional information

Justification for classification or non-classification

As the substance was negative, i.e. non-mutagenic and non-genotoxic, in a bacterial reverse mutation assay according to OECD TG 471,

an in vitro mammalian cell gene mutation test using the HPRT genes according to OECD test guideline 476 and in an in vitro micronucleus test according to OECD test guideline 487, it does not need to be classified.