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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay/Ames test: negative (OECD 471; GLP)

In vitro mammalian cell micronucleus test: negative (OECD 487; GLP)

In vitro mammalian cell gene mutation test: negative (OECD 490; GLP)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2015-12-15 to 2016-01-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997-07-21
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2014-05-14
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at +10°C to +25°C, in a tightly closed container and store in a cool, dry and well-ventilated place.
Target gene:
TA1537: his C 3076
TA98: his D 3052
TA1535 & TA100: his G 46
TA102: his G428
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (containing 5 % S9): 0.4 M MgCl2 + 1.65 M KCl salt solution (2 mL); glucose-6-phosphate (141.0 mg); NADP (306.5 mg; phosphate buffer (pH 7.4; 50 mL); fill up to a total of 100.0 mL with sterile aqua ad iniectabilia
Test concentrations with justification for top dose:
Plate incorporation test: 31.6, 100, 316, 1000, 3160, and 5000 µg/plate (with and without metabolic activation)
Preincubation test: 31.6, 100, 316, 1000, 3160, and 5000 µg/plate (with and without metabolic activation)
The test concentrations were chosed based on a preliminary cytotoxicity test, which indicted no cytotoxicity up to a concentration of 5000 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: 0.05 M HCl solution
- Justification for choice of solvent/vehicle: the test item was not soluble in any of the solvents recommended: water, dimethylsulfoxide (DMSO), ethanol or acetone. The test item was completely soluble at 100 μg/mL 0.05 M HCl solution. Hence, the test item was suspended in 0.05 M HCl solution for concentrations of 31.6, 100, 316, 1000, 3160, or 5000 μg/plate. For concentrations lower than 10.0 μg/plate, which were used in the preliminary cytotoxicity test, the test item was completely dissolved. Fresh preparations of the test item were used for the treatment in all experimental parts.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
0.05 M HCl solution
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
mitomycin C
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation; independent experiment 1) and preincubation (independent experiment 2)

MAIN EXPERIMENT (two independent experiments with and without metabolic activation)
1) plate incorporation method (independent experiment 1)
2 mL of top agar were distributed into culture tubes. 0.1 mL of Salmonella cell suspension (containing approximately 10^8 viable cells in the late exponential or early stationary phase) 0.1 mL of test item (or 0.1 mL solvent or 0.1 mL positive control) and 0.5 mL of S9 mix were added to these culture tubes. In the assay without metabolic activation, the S9 mix was substituted with 0.5 mL phosphate buffer.
The test components were mixed and then poured onto a minimal glucose agar plate (Minimal Glucose Agar medium E).
The plates were inverted and placed in a dark 37°C incubator for 48 to 72 hours. The revertant colonies on the test plates and on the control plates were counted with a colony counter.

2) Preincubation method (independent experiment 2)
The test item was preincubated with the test strain (containing approximately 10^8 viable cells in the late exponential or early stationary phase) and sterile buffer (0.5 mL) or the metabolic activation system (0.5 mL) for 20 minutes at 37°C prior to mixing with the overlay agar and pouring onto the surface of a minimal agar plate. 0.1 mL of test item (or 0.1 mL solvent or 0.1 mL positive control), 0.1 mL of bacteria, and 0.5 mL of S9 mix or sterile buffer, were mixed with 2 mL of overlay agar. Tubes were aerated during preincubation by using a shaker. The remaining steps were the same as described for the plate incorporation method.

NUMBER OF REPLICATIONS: 3 replicates for each experimental point

DETERMINATION OF CYTOTOXICITY
Prior to the main test two preliminary cytotoxicity tests (plate incorporation test, with and without metabolic activation) were carried out in test strain TA100. The following concentrations were tested: 0.316, 1.0, 3.16, 10.0, 31.6, 100, 316, 1000, 3160, and 5000 µg/plate
Rationale for test conditions:
The test concentrations were chosen based on a preliminary cytotoxicity test, which indicted no cytotoxicity up to a concentration of 5000 µg/plate. Precipitation was observed for a concentration of 1000 µg/plate and higher.
Evaluation criteria:
A test item is considered to show a positive response if
- the number of revertants is significantly increased (p ≤ 0.05, U-test according to MANN and WHITNEY) compared to the solvent control to at least 2-fold of the solvent control for TA98, TA100, TA1535 and TA1537 and 1.5-fold of the solvent control for TA102 in both independent experiments.
- in addition, a significant (p ≤ 0.05) concentration (log value)-related effect (Spearman’s rank correlation coefficient) is observed;
- positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking random samples on histidine-free agar plates.
Biological relevance of the results should be considered first.
A test item for which the results do not meet the above mentioned criteria is considered as non-mutagenic in the AMES test.
Statistics:
U-test according to MANN and WHITNEY and Spearman’s rank correlation coefficient
Key result
Species / strain:
other: TA98, TA100, TA102, TA1535, and TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
please refer to the field "Additional information on results" below
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
please refer to the field "Additional information on results" below
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
- test item precipitation was noted at concentrations of 1000 to 5000 μg/plate in both experiments.
- no signs of cytotoxicity were noted.

MAIN STUDY (2 independent experiments)
1) Test-specific confounding factors:
- Precipitation: test item precipitation was noted in both experiments, each carried out without and with metabolic activation, at concentrations of 1000 μg/plate and higher in all test strains.

2) Cytotoxicity:
- cytotoxicity (reduction of the number of revertants by more than 50%) was noted in both experiments with metabolic activation in test strain TA1537 at 5000 μg/plate.

3) Genotoxicity:
- no increase in revertant colony numbers as compared with control counts was observed for the test item in any of the two independent experiments without and with metabolic activation.
- positive control items showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system.

Please also refer to the field "Attached background material" below.

HISTORICAL CONTROL DATA
- Positive historical control data / Negative (solvent/vehicle) historical control data: please refer to Table 1 in the field "Any other information on results incl. tables" below.

Table 1: History profile of negative and positive control values of the years 2013 to 2015 (n=83 studies) - Data obtained from plate incorporation and preincubation tests

Negative Reference Item

Strain

S9-Mix

TA 98

TA 100

TA 102

TA 1535

TA 1537

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Mean

29.9

31.3

148.5

149.2

275.5

279.8

19.4

19.2

6.4

6.5

SD

5.7

5.9

18.1

18.2

15.5

16.2

4.4

4.4

1.8

1.9

Min

19

14

103

106

240

245

10

8

2

0

Max

49

49

191

195

319

319

34

42

10

10

 

Positive Reference Item

Strain

S9-Mix

TA 98

TA 100

TA 102

TA 1535

TA 1537

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

2-nitrofluorene

Benzo [a] pyrene

Sodium azide

2-aminoanthracene

Mitomycin C

Benzo [a] pyrene

Sodium azide

2-amino-anthracene

9-amino-acridine

Benzo [a] pyrene

Mean

148.4

147.3

928.7

937.9

1014.6

1008.9

133.2

133.3

81.1

81.5

SD

33.6

33.7

107.8

99.8

121.4

109.5

28.9

29.5

28.8

28.9

Min

91

91

463

703

756

757

51

49

26

28

Max

305

315

1209

1181

1637

1571

220

271

178

189

 

 

 

Conclusions:
The substance tested non-mutagenic under the conditions of the study.
According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance should not be considered to have a mutagenic potential.
Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2015-12-16 to 2016-01-27
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
2014-09-26
Deviations:
yes
Remarks:
no harvest at the end of treatment period after continuous treatment (harvest was conducted 20 hours after test item removal); number and sex of donors unknown
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2014-05-14
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at +10 °C to +25 °C, in a tightly closed container and stored in a cool, dry and well-ventilated place.
Target gene:
not applicable
Species / strain / cell type:
lymphocytes: human (peripheral)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: healthy human donors (non-smoking, no known recent exposures to genotoxic chemicals or radiation)
- Sex, age and number of blood donors: approx. 18 - 35 years of aga/ male or female individuals
- Whether whole blood or separated lymphocytes were used: small innocula of whole blood (0.5 mL) were added to tubes containing chromosome complete culture medium with phytohemagglutinin and penicillin/streptomycin. The tubes are sealed and incubated at 37 °C, and shaken occasionally to prevent clumping.
Cytokinesis block (if used):
5 µg/mL cytochalasin B (CytoB)(exposure duration: 20 hours)
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (components per 15 mL): rat liver S9 (1.5 mL); 0.4 M MgCl2 + 1.65 M KCl salt solution (0.3 mL); glucose-6-phosphate (5.41 mM; 21.1 mg); NADP (3.89 mM;45.9 mg; 20 mM Hepes buffer (5.7 mL; pH 7.4); phosphate buffer (7.5 mL)
Test concentrations with justification for top dose:
4 hour exposure (without metabolic activation): 6.25, 12.5, 25, 50 or 100 μg/mL medium
24 hour exposure (without metabolic activation): 12.5, 25, 50 or 100 μg/mL medium
4 hour exposure (with metabolic activation): 12.5, 25, 50 or 100 μg/mL medium
In the preliminary experiment cytotoxicity was noted at concentrations of 100 and 316.2 μg test item/mL medium in the experiment without and with metabolic activation (24-hour or 4-hour exposure, respectively). Test item precipitation was noted at the top concentration of 316.2 μg test item/mL in both experiments. Hence, 100 μg/mL were employed as the top concentration for the genotoxicity tests without and with metabolic activation.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: 0.05 M HCl solution
- Justification for choice of solvent/vehicle: the test item was not soluble in any of the solvents recommended: aqueous media, dimethylsulfoxide, ethanol or acetone. However, the test item was completely soluble as 100 μg/mL 0.05 M HCl solution. Hence, 3.162 mg of the test item were suspended in 1 mL 0.05 M HCl solution. This suspension was diluted 1:10 with culture medium to obtain a final concentration of 316.2 μg/mL medium for the highest tested dose in the preliminary test for determination of cytotoxicity. In the main study 1 mg of the test item was suspended in 1 mL 0.05 M HCl solution. This suspension was diluted 1:10 with culture medium to obtain a final concentration of 100 μg/mL which was completely dissolved in the medium. This solution was then further diluted to the appropriate concentrations. Fresh preparations of the test item were prepared on the day of the experiment and used for the treatment in all experimental parts.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
0.05 M HCl solution
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: colchicine
Details on test system and experimental conditions:
1) RANGE-FINDING/SCREENING STUDIES:
In a preliminary experiment without and with metabolic activation concentrations of 1.0, 3.162, 10.0, 31.62, 100 or 316.2 μg test item/mL medium were employed. Based on the outcome of this study the concentrations for the main test were selected.
At least 500 cells/replicate cell culture (one culture per concentration) were scored and classified as mononucleates, binucleates or multinucleates to estimate the proliferation index as a measure of toxicity. The evaluation of cytotoxicity was based on the Cytokinesis-Block Proliferation Index (CBPI) or the Replicative Index (RI).

2) MAIN TEST:
Cell treatment and harvest times for the used human lymphocytes line are as follows:
1) 4 and 24 hour exposure (without S9 mix)
- freshly prepared blood was seeded with chromosome complete culture medium with phytohemagglutinin and penicillin/streptomycin.
- after 48 hours the medium was replaced by fresh Ham’s F10 medium with fetal calf serum (FCS).
- vehicle control and test item treatments were added at a volume of 500 μL and the positive control at a volume of 50 μL (to 4.95 mL medium) to obtain the corresponding target concentrations.
- cultures were then incubated for 4 or 24 hours at +37°C.
- afterwards the medium was removed and the cultures were washed twice with Ham’s F10 medium.
- after addition of chromosome medium containing Cytochalasin B the cultures were incubated for further 20 hours at 37°C.

2) 4 hour exposure (with S9 mix)
- freshly prepared blood lymphocytes were seeded with chromosome complete culture medium with phytohemagglutinin and penicillin/streptomycin.
- after 48 hours the medium was removed and replaced by Ham’s F10 medium with FCS and 0.5 mL S9 Mix.
- vehicle control and test item treatments were added at a volume of 500 μL and the positive control at a volume of 50 μL (to 4.95 mL medium) to obtain the corresponding target concentrations.
- cultures were then incubated for 4 hours at +37°C.
- afterwards the medium was removed and the cultures were washed twice with Ham’s F10 medium.
- After addition of chromosome medium containing Cytochalasin B the cultures were incubated for further 20 hours at 37°C.

- precipitation of the test item was checked using a light microscopy at the beginning and end of treatment.
- cells were sampled at a time equivalent to about 1.5 times the normal cell cycle length either after the beginning or at the end of treatment.

Number of replicates: 2 replicate cultures/test item concentration

Methods of slide preparation and staining technique used:
- each culture was harvested and processed separately.
- after an incubation of 20 hours with Cytochalasin B, the cultures were centrifuged, the supernatant was discarded and the cells resuspended in KCl.
- after incubation, the cell suspensions were centrifuged.
- supernatant was discarded and freshly prepared fixative (3 parts methanol : 1 part glacial acetic acid v/v) added.
- cells were left in fixative for 30 minutes followed by centrifugation.
- supernatant was removed and discarded, and the cell pellet was resuspended in about fresh fixative and 30% glacial acetic acid by repeated aspiration through a Pasteur pipette.
- two drops of this cell suspension were dropped onto a microscope slide and left to air-dry at room temperature.
- slides were then stained using 10% Giemsa.

Number of cells evaluated: the micronucleus frequencies were analysed in at least 2000 binucleated cells/concentration (at least 1000 binucleated cells per culture; two cultures per
concentration). Care was taken not to score binucleate cells with irregular shapes or where the two nuclei differ greatly in size; neither would binucleate cells be confused with poorly spread multi-nucleate cells. Cells containing more than two main nuclei were not analysed for micronuclei, as the baseline micronucleus frequency might be higher in these cells. Scoring of mononucleate cells is acceptable if the test item is shown to interfere with CytoB activity.

Determination of cytotoxicity:
- Method: mitotic index; cloning efficiency; relative total growth; other:
At least 500 cells/replicate cell culture (two cultures per concentration) were scored and classified as mononucleates, binucleates or multinucleates to estimate the proliferation index as a measure of toxicity. The evaluation of cytotoxicity was based on the Cytokinesis-Block Proliferation Index (CBPI) or the Replicative Index (RI).

Other examinations:
- pH values and osmolality measurements: pH and osmolality of the negative control and all test item formulations in the medium were determined for each experiment.

Rationale for test conditions:
The concentrations employed were chosen based on the results of a cytotoxicity study. Cytotoxicity was noted at concentrations of 100 and 316.2 μg test item/mL medium in the experiment without and with metabolic activation (24-hour or 4-hour exposure, respectively). Test item precipitation was noted at the top concentration of 316.2 μg test item/mL in both experiments. Hence, 100 μg/mL were employed as the top concentration for the genotoxicity tests without and with metabolic activation.
Evaluation criteria:
A test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
• at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
• the increase is dose-related in at least one experimental condition when evaluated with an appropriate trend test
• any of the results are outside the distribution of the historical negative control data (Poisson-based 95% control limits)

A test chemical is considered clearly negative if, in all experimental conditions examined:
• none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
• there is no concentration-related increase when evaluated with an appropriate trend test,
• all results are inside the distribution of the historical negative control data (Poisson-based 95% control limits).
Statistics:
Poisson-based 95% control limits
Chi²-test
Key result
Species / strain:
lymphocytes: human (peripheral)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
100 μg test item/mL medium (with and without metabolic activation)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
1) RANGE-FINDING/SCREENING STUDIES:
- test item precipitation was noted at the top concentration of 316.2 μg test item/mL (with and without metabolic activation).
- cytotoxicity was noted at concentrations of 100 and 316.2 μg test item/mL medium in the experiment without and with metabolic activation (24-hour or 4-hour exposure, respectively).

2) MAIN STUDY
Test without metabolic activation (4- and 24-hour exposure):
Test item:
- micronucleus frequencies of cultures treated with the concentrations of 6.25 and/or 12.5, 25, 50 or 100 μg test item /mL medium in the absence of metabolic activation (4- and 24-hour exposure, respectively) ranged from 4.0 to 11.5 micronucleated cells per 1000 binucleated cells.
- no dose-related increase in micronuclei up to the top concentration of 100 μg test item/mL medium.
- frequency of micronucleated cells was within the historical control range of the untreated and vehicle controls.
Vehicle control:
- in this test the following frequencies were observed: vehicle control: 7.0 or 7.5 micronucleated cells per 1000 binucleated cells for the 4-hour and 24-hour exposure, respectively.
- vehicle results were within the historical control ranges. However, the mean frequency of the 24-hour exposure experiment was slightly above the upper limit of the 95% confidence interval of the historical background data at the laboratory. However, the vehicle control in this study gave reproducibly low and consistent micronuclei frequencies without extreme outliers. Therefore, the slight exceeding of the upper limit of 95% confidence interval was judged as not biologically relevant.
Positive control:
- micronucleus frequencies were increased to 31.0 or 45.5 micronucleated cells per 1000 binucleate cells for the 4-hour and 24-hour exposure, respectively.

Test with metabolic activation (4-hour exposure)
Test item:
- micronucleus frequencies of cultures treated with the concentrations of 12.5, 25, 50 or 100 μg test item/mL medium (4-h exposure) in the presence of metabolic activation ranged from 2.5 to 7.0 micronucleated cells per 1000 binucleated cells.
- no dose-related increase in micronuclei up to the top concentration of 100 μg test item/mL medium.
- frequency of micronucleated cells was within the historical control range of the untreated and vehicle controls.
Vehicle control:
- in this test a mean frequency of 2.5 micronucleated cells per 1000 binucleated cells was observed.
- vehicle result was within the historical control ranges. However, the vehicle control data were slightly below the lower limit of the 95% confidence interval of the historical background data at the laboratory. However, the vehicle control in this study gave reproducibly low and consistent micronuclei frequencies without extreme outliers.
Positive control:
- micronucleus frequency was increased to 30.5 micronucleated cells per 1000 binucleate cells for the 4-hour exposure.

- Cytotoxicity:
Cytotoxicity was noted in the experiments without and with metabolic activation at the top concentration of 100 μg test item/mL medium.

Please refer to the field "Attached background material" below.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no relevant changes in pH of the test item formulations at concentrations of 1.0 to 316.2 μg/mL medium were noted.
- Effects of osmolality: no relevant changes in osmolality of the test item formulations at concentrations of 1.0 to 316.2 μg/mL medium were noted.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data / Negative (solvent/vehicle) historical control data: please refer to Table 1 in the field "Any other information on results incl. tables" below.

Table 1: Historical background data in vitro micronucleus test in cultured human peripheral lymphocytes (n = 13; background data from 2013 to 2015)

 

Micronucleus frequency per 1000 cells

 

without metabolic activation

with metabolic activation

 

Untreated control

Vehicle control

Untreated control

Vehicle control

mean

6.6

6.3

6.4

6.1

SD

2.9

3.1

2.6

4.2

range

2.0 - 17

2.0 – 18

3.0 – 13

1 – 22

95% Confidence interval

5.9 – 7.3

5.7 – 6.8

5.7 - 7.1

5.1 - 6.7

 

Mitomycin C Positive control

colchicine

 Positive control

Cyclophosphamide

Positive control

mean

46.9

25.9

44.0

SD

35.0

9.5

37.7

range

17 - 137

15 - 63

14 - 158

Conclusions:
The substance tested non-clastogenic under the conditions of the study.
According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance should not be considered to have a mutagenic potential.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2015-12-16 to 2016-02-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Version / remarks:
2015-07-28
Deviations:
yes
Remarks:
incubation time fafter addition of TFT was 11 to 14 days
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2014-05-14
Type of assay:
other: in vitro mammalian cell gene mutation test
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at +10 °C to +25 °C, in a tightly closed container and stored in a cool, dry and well-ventilated place.
Target gene:
TK gene
Species / strain / cell type:
mouse lymphoma L5178Y cells
Remarks:
clone 3.7.2C
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: ATCC (American Type Culture Collection), 0801 University Blvd., Manassas, VA 20110-2209, USA

MEDIA USED
- Type and identity of media:
1) growth medium RPMI 1640 with glutamaxTM medium supplemented with Pluronic® F68, gentamycin, amphotericin B and horse serum (10% by volume).
2) treatment medium: growth medium with a reduced horse serum content (5% by volume).
3) plating medium: growth medium with increased horse serum content but without Pluronic® F68.
4) selection medium: growth medium that contains 3 μg/mL of 5-trifluoro-thymidine (TFT).

- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
S9-mix: rat liver S9 (3.0 mL); 150 mM KCl salt solution (1.5 mL); glucose-6-phosphate (270.0 mg); NADP (37.5 mg); sterile aqua ad iniectabilia (3.0 mL); treatment medium (15 mL)
Test concentrations with justification for top dose:
Experiment 1: 1.0, 3.16, 10.0, 31.6, and 100 µg/mL (with and without metabolic activation; 3-hour exposure)
Experiment 2: 1.0, 3.16, 10.0, 31.6, and 100 µg/mL (without metabolic activation; 24-hour exposure)
Experiment 2: 1.0, 3.16, 10.0, 31.6, and 100 µg/mL (with metabolic activation; 3-hour exposure)
In a preliminary cytotoxicity study cytotoxicity was noted in the absence and in the presence of metabolic activation at concentrations of 100 μg/mL and higher. Hence the top dose for this study was selected to be 100 µg/mL.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: 0.05 M HCl solution
- Justification for choice of solvent/vehicle: the test item was not soluble in any of the solvents recommended: aqueous media, dimethylsulfoxide, ethanol or acetone. However, the test item was completely soluble at 100 μg/mL 0.05 M HCl solution. Hence, 20 mg of the test item were suspended in 1 mL 0.05 M HCl solution. This suspension was diluted 1:20 with culture medium to obtain a final concentration of 1000 μg/mL medium for the highest tested dose in the preliminary test for determination of cytotoxicity. In the main study 2 mg of the test item was suspended in 1 mL 0.05 M HCl solution. This suspension was diluted 1:20 with culture medium to obtain a final concentration of 100 μg/mL medium which led to test item precipitation. The test item was completely dissolved in the medium at a concentration of 31.6 μg/mL and further dilutions.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
0.05 M HCl solution in culture medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
methylmethanesulfonate
Details on test system and experimental conditions:
1) RANGE-FINDING/SCREENING STUDIES:
A preliminary cytotoxicity experiment was performed without and with S9 metabolic activation. The following concentrations were tested for cytotoxicity: 3.16, 10.0, 31.6, 100, 316 and 1000 μg test item/mL medium. The preliminary cytotoxicity information was then used to select concentration levels for the mutation assay.
For the evaluation of the cytotoxicity in the preliminary experiment the relative plating efficiency RPE1 obtained at the time after treatment is sufficient and was evaluated.

Other examinations:
- pH values and osmolality measurements: pH and osmolality of the negative control and all test item formulations in the medium were determined for each experiment.

2) MUTAGENICITY TESTING
Experiment 1:
The assay procedure used is based on that reported by COLE et al. (1990)*.
Cells were obtained from logarithmically growing laboratory stock cultures. The cells were pelleted by centrifugation, the culture medium was removed, and the cells were resuspended in treatment medium that contained 5% heat inactivated horse serum and the corresponding concentration of test substance was added with or without metabolic activation. The final cell density at start of treatment was 0.5 cells/mL. The dosed tubes were closed, mixed and placed on a roller drum at approx. 37°C for an exposure period of 3 hours. Thereafter the cells were washed and resuspended in growth medium.
Cell densities were adjusted to 2 x 10^5/mL and the cells were plated for survival and incubated for the expression period in parallel, i.e. an aliquot of the cells was diluted to 8 cells/mL and 0.2 mL of each culture were placed in two 96 well microtiter plates (192 wells, averaging 1.6 cells/well), incubated for 1 week (plating efficiency step 1) and at the end the number of viable clones was recorded.
The rest of the cells was incubated for 2 days for the expression period. Within the expression period cell densities were determined after incubation for 24 hours and adjusted to 2 x 10^5/mL and incubated for another 24 hours.
At the end of the expression period a minimum of 4 concentration levels plus positive and negative control were selected for 5-trifluoro-thymidine (TFT) resistance. The selected cultures were diluted to 1 x 10^4 cells/mL and plated for survival (plating efficiency step 2) and TFT resistance in parallel (plating efficiency for TFT resistance). The plating for survival was identical to the above described method (plating efficiency step 1 in 192 wells with average 1.6 cells/well). For the plating for TFT resistance 3 μg/mL TFT (final concentration) were added to the cultures and 0.2 mL of each suspensions placed into four 96-well microtiter plates (384 wells, averaging 2 x 10³ cells/well). The plates were incubated for 11 to 14 days and wells containing clones were identified microscopically and counted.
The mutant frequency was determined and expressed as mutants/10^6 viable cells.
The number of large and small colonies was recorded. Large colonies are defined as ≥ 1/4 and small colonies < 1/4 of the well diameter of 6 mm. The positive control 3-Methylcholanthrene only served as positive control item for mutagenicity but not for small/large colony ratios.

Experiment 2:
An exposure time of 3 hours was used for the repeat experiment with metabolic activation and an exposure time of 24 hours was used for the repeat experiment without metabolic activation to also cover long term effects. The experiments were conducted with the same concentrations as in the first experiment.

Number of replicates: single cultures/test item concentration

Determination of cytotoxicity
- Method: relative total growth (RTG) which includes the Relative Suspension Growth (RSG) during the 2 day expression period and the Relative Plating Efficiency (RPE2) obtained at the time of mutant selection.

*Reference:
- COLE, J., M. FOX, R. C. GARNER, D. B. McGREGOR and J. THACKER. Gene mutation assays in cultured mammalian cells. In, 'Basic Mutagenicity Tests': UKEMS recommended procedures, Ed.: D. J. KIRKLAND et al., Cambridge University Press, Chapter 4, pp. 81 - 114 (1990).
Rationale for test conditions:
Based on the results of the preliminary study the test item concentrations were employed in the mutagenicity test. During the preliminary test cytotoxicity and test item precipitation were noted in the absence and in the presence of metabolic activation at concentrations of 100 μg/mL and higher.
Evaluation criteria:
The interpretation relies on the use of a predefined induced mutant frequency (i.e. increase in mutant frequency above concurrent control), designated as the Global Evaluation Factor (GEF). The GEF (126 x 10^-6) is based on the analysis of the distribution of the negative control mutant frequency data from participating laboratories (M.M. Moore et al., 2006)*.
A test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in mutant frequency above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system.
A test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in mutant frequency, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system.
In cases when the response is neither clearly negative nor clearly positive as described above and/or in order to assist in establishing the biological relevance of a result the data is evaluated by expert judgement and/or further investigations.

*Reference:
- MOORE, M.M., HONMA, M., CLEMENTS, J., BOLCSFOLDI, G., BURLINSON, B., CIFONE, M., CLARKE, J., DELONGCHAMP, R., DURWARD, R., FELLOWS, M., GOLLAPUDI, B., HOU, S., JENKINSON, P., LLOYD, M., MAJESKA, J., MYHR, B., O’DONOVAN, M., OMORI, T., RIACH, C., SAN, R., STANKOWSKI, L.F. JR., THAKUR, A.K., VAN GOETHEM, F., WAKURI, S. AND YOSHIMURA, I. (2006). Mouse Lymphoma Thymidine Kinase Gene Mutation Assay: Follow-Up Meeting of the International Workshop on Genotoxicity Tests – Aberdeen, Scotland, 2003 – Assay Acceptance Criteria, Positive Controls, and Data Evaluation, Environ. Mol. Mutagen., 47 (1):1-5.
Statistics:
- Cytotoxicity (mutagenicity testing):
RTG = RSG x RPE2
RTG = Relative Total Growth; RSG = Relative Suspension Growth; RPE2 = Relative Plating Efficiency
RSG [ SG value (treatment)/ mean SG value (control)] x 100
With SG being the suspension growth, the measure of the growth in suspension during treatment and the expression period. SG was calculated as follows:
SG = a x b x c
a = (D0 post treatment cell count/ pre-treatment cell density)
b = (D1 cell count/ cell count set up on D0 post treatment)
c = (D2 cell count/cell count set up on D1)
RPE2 [PE2 value (treatment)/mean PE2 value (control)] x 100
With PE2 being the plating efficiency obtained at the time of mutant selection. The calculations are based on P(0), the proportion of wells in which a colony
has not grown:
PE2 = [-ln P(0)/number of cells plated per well]
Where P(0) = Number of wells with no colony/Total number of wells
The cloning efficiency (CE) is defined as the plating efficiency in percent (CE=PE2 x 100).

- Mutant frequency:
mutant frequency (MF/10^6): (PEm of mutant cells / PE2 of viable cells) x 10^6
Key result
Species / strain:
mouse lymphoma L5178Y cells
Remarks:
clone 3.7.2C
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
100 μg test item/mL (with and without metabolic activation)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
1) RANGE-FINDING/SCREENING STUDIES:
Cytotoxicity and test item precipitation were noted in the absence and in the presence of metabolic activation at concentrations of 100 μg test item/mL and higher.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no relevant changes in pH of the test item formulations at concentrations of 3.16 to 1000 μg/mL medium compared to the controls were noted.
- Effects of osmolality: no relevant changes in osmolality of the test item formulations at concentrations of 3.16 to 1000 μg/mL medium compared to the controls were noted.

2) MUTAGENICITY TESTING
- cytotoxicity: cytotoxicity (decreased relative total growth (RTG)) was noted at the top concentration of 100 μg test item/mL in the absence and presence of metabolic activation

- precipitation: test item precipitation was noted at the top concentration of 100 μg test item/mL in the absence and presence of metabolic activation

- test item treatment: mutation frequencies of the cultures treated with the test item ranged from 54.34 to 159.89 per 10^6 cloneable cells (3 hours exposure) and from 64.43 to 181.75 per 10^6 cloneable cells (24 hours exposure) in the experiments without metabolic activation and from 59.83 to 155.92 per 10^6 cloneable cells (3 hours exposure, first assay) and from 52.52 to 127.08 per 10^6 cloneable cells (3 hours exposure, second assay) in the experiments with metabolic activation. These results were within the range of the negative control values and the normal range of 50 to 170 mutants per 10^6 viable cells and, hence, no mutagenicity was observed according to the criteria for assay evaluation. Only the culture treated with the cytotoxic concentration of 100 μg test item/mL marginally exceeded the range of the negative control values and the normal range of 50 to 170 mutants per 10^6 viable cells (181.75 mutants per 10^6 cloneable cells in the experiment without S9, 24 hours exposure). This increase is below the Global Evaluation Factor (GEF) of 126 x 10^-6 and, hence, not considered to be mutagenic.
No change was observed in the ratio of small to large mutant colonies, ranging from 0.44 to 1.13 for test item-treated cells and from 0.72 to 0.95 for the negative controls

- negative control: values of mutation frequencies of the negative controls ranged from 54.62 to 75.89 per 10^6 cloneable cells in the experiments without metabolic activation and from 82.51 to 105.32 per 10^6 cloneable cells in the experiments with metabolic activation (well within the historical data-range).
The calculations of suspension growth of the negative control replicates were in the range between 8 and 32 following 3-hour treatments or between 32 and 180 following 24-hour treatments. The mean cloning efficiencies (CE = PE2 x 100) of the negative controls were between the range 65% to 120%

- positive controls: methylmethanesulfonate and 3-Methylcholanthrene caused pronounced increases in the mutation frequency ranging from 785.74 to 920.93 per 10^6 cloneable cells in the case of methylmethanesulfonate and ranging from 884.41 to 1032.89 per 106 cloneable cells in the case of 3-Methylcholanthrene. The colony size ratio was moderately shifted towards an increase in small
colonies, ranging from 1.38 to 1.63 in the case of methylmethanesulfonate.
As the absolute increase in the mean total mutation frequency of at least 300 x 10^-6 (at least 40% small colony mutation frequency) and the mean relative total growth (RTG) for the positive controls was greater than 10%.

Please refer to the field "Attached background material" below.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data / Negative (solvent/vehicle) historical control data: please refer to Table 1 and Table 2 in the field "Any other information on results incl. tables" below.

Table 1: Historical data from 2012 to 2014 (n = 22)

Number of mutant colonies per 106cells

3h treatment

 

Negative control

Negative control without S9 mix: Solvent#

Positive control

 

without S9 mix

with S9 mix

without S9 mix

with S9 mix

without S9 mix

with S9 mix

 

 

 

Aqueous

Organic

Aqueous

Organic

MMS

3-MC

range:

50.2-163.5

48.5-161.54

56.8-160.5

50.2-159.41

52.6-167.32

51.8-169.51

465.1-2724.1

415.7-3510.3

mean value:

80.8

78.1

98.6

79.3

77.0

77.2

1595.3

1313.5

Standard deviation:

27.2

24.5

44.37

23.1

38.5

21.7

585.6

629.1

Number of mutant colonies per 106cells

24h treatment

 

Negative control

Negative control without S9 mix: Solvent#

Positive control

 

without S9 mix

Aqueous

Organic

without S9 mix (MMS)

range:

49.4-161.54

59.0-155.73

49.4-161.54

585.6-2939.4

mean value:

81.1

78.7

81.5

1563.9

Standard deviation:

27.0

45.2

25.9

599.0

# Aqueous solvent (n = 3): Deionised water; organic solvent: DMSO, acetone, ethanol (n = 19)

 MMS = Methylmethanesulfonate 3-MC = 3-Methylcholanthrene

Table 2: Historical data from 2012 to 2014 (n = 22)

Small : large colonies (colony size ratio)

3h treatment

 

Negative control

Negative control: Solvent#

Positive control

 

without S9 mix

with S9 mix

without S9 mix

with S9 mix

without S9 mix

with S9 mix

 

 

 

Aqueous

Organic

Aqueous

Organic

MMS

3-MC

range:

0.45-1.30

0.18-2.00

0.52-1.12

0.45-1.30

0.58-1.40

0.35-1.40

1.20-2.91

0.22-1.55

mean value:

0.88

0.92

0.90

0.88

0.95

0.88

2.04

0.81

Standard deviation:

0.21

0.27

0.07

0.22

0.26

0.24

0.37

0.31

24h treatment

 

Negative control

Negative control without S9 mix: Solvent#

Positive control

 

without S9 mix

Aqueous

Organic

without S9 mix (MMS)

range:

0.37-1.67

0.71-1.12

0.37-1.67

1.25-3.84

mean value:

0.99

0.86

0.99

2.07

Standard deviation:

0.24

0.12

0.28

0.45

# Aqueous solvent (n = 3): Deionised water; organic solvent: DMSO, acetone, ethanol (n = 19)

 MMS = Methylmethanesulfonate 3-MC = 3-Methylcholanthrene

Conclusions:
The substance tested non-mutagenic and non-clastogenic under the conditions of the study.
According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance should not be considered to have a mutagenic potential.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Genetic toxicity in vitro

The substance was not observed to be mutagenic in a reliable bacterial reverse mutation assay (OECD 471), a reliable in vitro mammalian cell micronucleus test (OECD 487), and a reliable in vitro mammalian cell gene mutation test (OECD 490).

Justification for classification or non-classification

Genetic toxicity in vitro

The substance has no mutagenic potential based on a bacterial reverse mutation assay (OECD 471), an in vitro mammalian cell micronucleus test (OECD 487) and an in vitro mammalian cell gene mutation test (OECD 490). Consequently, the substance does not require classification according to Regulation (EC) No 1272/2008 and subsequent adaptations.