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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three in vitro tests are available.

Ames study:

The test item was evaluated for its mutagenic potential by a reverse mutation test with four strains of Salmonella typhimurium (TA100, TA98, TA1535 and TA1537) and one strain of Escherichia coli (WP2 uvrA) based on OECD 471. The test item was not mutagenic under the test conditions.

 

In Vitro Mammalian Chromosome Aberration Test:

An in vitro chromosomal aberration test of test item was conducted using cultured cells derived from Chinese hamster lung (CHL/IU) according to OECD 473. The test item has no potential to induce chromosomal aberration in CHL/IU cells under the test conditions.

 

In vitro mammalian cell gene mutation test:

An in vitro mammalian cell gene mutation test was performed to investigate the potential of test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster in compliance with OECD 476. The test item is considered to be non-mutagenic in this HPRT assay.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 2010-02-25 to 2010-03-04
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:
adopted in Jul 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Criteria for Mutagenicity Test in Bacterial Systems (Ministry of Labour, Japan, Notification No. 77, September 1, 1988; Notification No. 67, June 2, 1997)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese Ministry of Health, Labor and Welfare; Ministry of Economy, Trade and Industry; and Ministry of the Environment; Heisei 15.11.21 Yakushokuhatsu Notification No. 1121002, Heisei 15.11.13 Seikyoku Notific. No. 2, Kanpokihatsu Notific. No. 031121002
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Purity: 100%
- Lot No.: 10SC8156928-2-2
Target gene:
his operon (S. typhimurium strains)
trp operon (E. coli strain)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
- Selection reason: because they have high sensitivity to detect mutagens and are widely used in the reverse mutation test
-Source and: The Salmonella strains were obtained from Dr. B. N. Ames (University of California, Berkeley, U.S.A.). The Escherichia strain was obtained from Dr. T. Matsushima (Institute of Medical Science, the University of Tokyo, Tokyo, Japan).
- Preparation: A culture of the test strain was mixed with DMSO using a ratio of 8 parts cell culture stock and 0.7 parts DMSO by volume and frozen in an acetone-dry ice bath. These frozen stocks were kept in a deep freezer at -80℃ until use.
The stock cultures of each strain were checked for their phenotypic characteristics [the amino acid requirement, UV sensitivity and presence of rfa and R factors]. The results confirmed that all strains retained their own phenotypic characteristics. These strains also yielded solvent and positive control substances-induced revertants within the frequency ranges expected from the historical control data. Each bacterial frozen stock culture was thawed and 0.1 mL of bacterial suspension was added to 60 mL of nutrient broth medium in a flask of 300 mL volume and cultured for 10 hours at 37℃ with shaking.
- Composition of nutrient broth medium (per 1000 mL of purified water):
Nutrient broth (Bacto Nutrient Broth): 8g; NaCl: 5g
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : purchased from Oriental Yeast Co., Ltd. (Tokyo, Japan)
- method of preparation of S9 mix: cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and 5,6-benzoflavone.
- concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL S9 mix
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): The S9 used in this study was characterised with mutagen requiring metabolic activation, based on the assay data certificate of S9 batch used.
Test concentrations with justification for top dose:
Range-finding experiment (all strains): 1.22, 4.88, 19.5, 78.1, 313, 1250, 5000 μg/plate with and without metabolic activation
Main experiment (all strains): 156, 313, 625, 1250, 5000 μg/plate with and without metabolic activation
Since neither precipitation of the test substance nor cytotoxicity to bacteria was observed in the dose-finding assay, the dose of 5000 μg/plate was selected as the highest dose in the main assay as specified in the guidelines.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: the test substance has a water solubility of ≥ 50 mg/mL and is stable at this concentration for 24 h in room temperature
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see 'Remarks' field
Remarks:
2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2), Sodium azide (NaN3), 9-aminoacridine (9-AA), 2-Aminoanthracene (2-AA) (see 'Details on test system and conditions' for concentrations)
Details on test system and experimental conditions:
DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 2 replications each in 2 independent experiments

METHOD OF TREATMENT/ EXPOSURE:
Test substance solution (0.1 mL), either 100 mM Na-phosphate buffer (0.5 mL, pH 7.4) or S9 mix (0.5 mL), and bacterial suspension (0.1 mL) were mixed in a small test tube and incubated for 20 minutes at 37℃ with shaking. After addition of 2.0 mL of the top agar solution, the mixture was poured onto a minimal glucose agar plate, spread evenly and solidified at room temperature. All plates were incubated for 48 hours at 37℃.


METHODS FOR MEASUREMENTS OF GENOTOXICIY
The number of revertant colonies on a plate was counted using an automatic colony counter.

DETERMINATION OF CYTOTOXICITY
- Method: examination of bacterial backgroud lawn under stereomicroscope.

OTHER:
Positive control concentrations: AF-2 (0.01 μg/plate in DMSO, -S9, TA100 and WP2 uvrA; 0.1 μg/plate in DMSO, -S9, TA98); NaN3 (0.5 μg/plate in water, -S9, TA1535); 9-AA (80 μg/plate in DMSO, -S9, TA 1537); 2-AA (1.0 μg/plate in DMSO, +S9, TA100; 2.0 μg/plate in DMSO, +S9, TA1535 and TA1537; 0.5 μg/plate in DMSO, +S9, TA98; 10.0 μg/plate in DMSO, +S9, WP2 uvrA)
Evaluation criteria:
When the test substance shows a dose-dependent increase in the number of revertant colonies to at least twice as many as that of the solvent control, the response is judged to be positive. The study director judges the result with scientific consideration of biological relevance. When the positive response is reproducible, the test substance is judged to have mutagenic potential.
Statistics:
Mean values were calculated at each concentration for the two experiments. For the historical control data, mean values and standard deviation were calculated for each test strain. Any statistical analysis was not applied to evaluate the test results.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no precipitation was observed.
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES:
Neither precipitation of the test substance nor cytotoxicity to bacteria was observed. The results of the range-finding study were evaluated together with the main study.

COMPARISON WITH HISTORICAL CONTROL DATA:
Yes, see Table 1 under 'Any other information on materials and methods incl tables'.

Table 1. Results of dose range-finding assay (preincubation method)

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 2 plates)

Base-pair substitution type

Frameshift type

TA 100

TA1535

WP2 uvrA

TA98

TA1537

Sterilised purified water

104

15

24

37

11

1.22

87

9

22

28

11

-

4.88

92

12

32

32

10

19.5

92

11

21

39

10

-

78.1

97

16

22

39

10

312

88

11

20

44

11

-

1250

84

12

23

39

10

5000

94

14

23

42

12

Positive controls, –S9

Name

AF-2

NaN3

AF-2

AF-2

9-AA

Concentrations

(μg/plate)

0.01

0.5

0.01

0.1

80

Mean No. of colonies/plate

(average of 2 plates)

594

356

102

335

540

+

Sterilised purified water

93

9

20

38

15

+

1.22

93

8

30

40

15

+

4.88

105

8

28

38

12

+

19.5

80

13

20

42

15

+

78.1

85

12

30

39

18

+

313

94

7

22

36

17

+

1250

88

9

23

43

9

+

5000

86

7

23

33

14

Positive controls, +S9

Name

2AA

2AA

2AA

2AA

2AA

Concentrations

(μg/plate)

1

2

10

0.5

2

Mean No. of colonies/plate

(average of 2 plates)

760

206

570

214

133

AF-2: 2-(2 -furyl)-3-(5-nitro-2-furyl)acrylamide

NaN3: sodium azide

9-AA: 9-aminoacridine

2-AA: 2 -aminoanthracene

Table 2. Results of main assay (preincubation method)

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 2 plates)

Base-pair substitution type

Frameshift type

TA 100

TA1535

WP2 uvrA

TA98

TA1537

Sterilised purified water

96

10

25

28

13

156

90

7

21

19

11

-

313

98

9

23

26

12

625

94

7

18

27

13

-

1250

90

12

23

26

13

2500

95

9

17

30

9

-

5000

98

11

26

31

10

Positive controls, –S9

Name

AF-2

NaN3

AF-2

AF-2

9-AA

Concentrations

(μg/plate)

0.01

0.5

0.01

0.1

80

Mean No. of colonies/plate

(average of 2 plates)

696

311

110

317

535

+

Sterilised purified water

82

10

25

35

19

+

156

100

10

29

30

21

+

313

83

10

23

28

11

+

625

86

5

18

27

15

+

1250

85

12

29

30

19

+

2500

86

5

31

37

18

+

5000

103

11

29

35

11

Positive controls, +S9

Name

2AA

2AA

2AA

2AA

2AA

Concentrations

(μg/plate)

1

2

10

0.5

2

Mean No. of colonies/plate

(average of 2 plates)

729

193

480

216

120

AF-2: 2-(2 -furyl)-3-(5-nitro-2-furyl)acrylamide

NaN3: sodium azide

9-AA: 9-aminoacridine

2-AA: 2 -aminoanthracene

Conclusions:
The test item is not mutagenic under the test conditions.
Executive summary:

The test item was evaluated for its mutagenic potential by a reverse mutation test with four strains of Salmonella typhimurium (TA100, TA98, TA1535 and TA1537) and one strain of Escherichia coli (WP2 uvrA) based on OECD 471. The test was conducted in the presence and the absence of a rat liver drug metabolizing enzyme system (S9 mix).

In a dose-finding assay, the highest dose of test item was set at 5000 μg/plate, and the test was conducted at dose levels ranging from 5000 to 1.22 μg/plate in duplicate plating. Neither precipitation of the test substance nor cytotoxicity to bacteria was observed.

In the main assay, the test item was tested at doses ranging from 5000 to 156 μg/plate for all five test strains with and without S9 mix in duplicate plating based on the results of the dose-finding assay.

Throughout the tests, the test item did not show any statistically significant dose-dependent increase in the number of revertant colonies to at least twice as many as that of the solvent control with or without S9 mix in any of the strains. Positive control substances showed marked increases in the number of revertant colonies.

Based on these results, it is concluded that test item is not mutagenic under the test conditions.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 2012-09-24 to 2013-02-08
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)
Version / remarks:
July 21 , 1997
Deviations:
yes
Remarks:
In Experiment 1, slides could not be prepared for three analyzable levels of concentrations. In 1st re-test, microbial contamination occurred. 2nd re-test was performed. This did not adversely affect the reliability of the test.
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
- Purity: 100.0%
- Lot No.: KNC110310
Species / strain / cell type:
Chinese hamster lung (CHL/IU)
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: Cells derived from Chinese hamster lung (CHL/IU) were obtained on June 29, 1987 from Dainippon Pharmaceutical Co., Ltd (Osaka, Japan).

For cell lines:
- Absence of Mycoplasma contamination: The stocked cells have been confirmed to be free from mycoplasma contamination.
- Number of passages if applicable: between 10 and 23
- Methods for maintenance in cell culture: The stock was thawed and continuously cultivated to be used for the test. Cells were grown as monolayer in Eagle's MEM supplemented with 10% of bovine serum in a plastic dish.
- Doubling time: 15 hours (latest measured value: approximately 14 hours)
- Modal number of chromosomes: 25

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: Eagle's MEM (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) supplemented with 10% of bovine serum (Life Technologies, Inc., USA; Lot No. 8203309) in a plastic dish under a humidified atmosphere of 5% CO2 in air at 37℃.
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : purchased from Oriental Yeast Co., Ltd. (Tokyo, Japan)
- method of preparation of S9 mix: The S9 fraction was mixed with cofactors to prepare S9 mix just before use.
The composition of S9 mix: S9 fraction: 30%; KCl: 33 mM; MgCl2: 5mM; HEPES buffer (pH 7.2): 4 mM; Glucose-6-phosphoric acid: 5 mM; NADPH: 4mM.
- concentration or volume of S9 mix and S9 in the final culture medium: 1 mL S9 mix
Test concentrations with justification for top dose:
- Experiment 1 (short-term treatment):
Cytotoxicity test: 1720, 860, 430, 215, 108, 53.8, 26.9, 13.4, 6.72 μg/mL, the highest concentration was set according to the test guidelines
Chromosomal aberration test: In the absence of S9 mix: 50.0, 60.0, 70.0, 80.0, 90.0 μg/mL, 1st and 2nd re-test: 30.0, 40.0, 50.0, 60.0, 70.0, 80.0 μg/mL; In the presence of S9 mix: 87.5, 175, 350 μg/mL
The highest concentration was set at the dose at which the growth rate was expected to be inhibited to 50% or less both in the presence and absence of S9 mix.

- Experiment 2 (Continuous treatment and confirmation test):
Cytotoxicity test: 1720, 860, 430, 215, 108, 53.8, 26.9, 13.4, 6.72 μg/mL, the highest concentration was set according to the test guidelines
Chromosomal aberration test: In the absence of S9 mix: 24-hour continuous treatment: 10.0, 12.5, 15.0, 17.5, 20.0, 22.5 μg/mL; 48-hour continuous treatment: 0.840, 1.68, 3.36, 6.72 μg/mL.
Based on the results of the cytotoxicity test, the highest dose was set at the dose at which the growth rate was expected to be inhibited to 50% or less both in 24-hour and 48-hour treatment in the absence of S9 mix.

- Experiment 3 (Confirmation test):
Chromosomal aberration test: In the presence of S9 mix: 87.5, 175, 350 μg/mL.
The highest concentration was set in the same manner as in Experiment 1.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: physiological saline

- Justification for choice of solvent/vehicle: As the test substance was soluble in water, physiological saline was selected for the vehicle of the test substance. The test substance was soluble in physiological saline up to 172 mg/mL (approximately 1 M).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : 3

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): approximately 3 E+03 cells/mL
- Test substance treatment:
The test substance was added to solvent to prepare the test substance solution at the highest concentration. The solution was serially diluted to prepare test substance solution at individual concentrations.
In the absence of S9 mix, the test substance solution (50 μL) was added to 5.0 mL of culture. In the presence of S9 mix, the test substance solution (60 μL) and S9 mix (1 mL) was added to 5.0 mL of culture. In the group with a recovery culture, cells were washed with fresh medium at the end of exposure period, and cultured in 5mL of fresh medium until the designated harvest time. In the control group, cells were treated with solvent or positive control substance solution in the same manner as in the test substance-treated group.

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: Experiment 1 (short-term treatment): 6h with/without S9 mix; Experiment 2 (continuous treatment): 24/48 h without S9 mix;
Experiment 3 (short-term treatment): 6h with S9 mix
- Harvest time after the end of treatment (sampling/recovery times): Experiment 1 (short-term treatment): Recovery period: 18h, Harvest period: 24h; Experiment 2 (continuous treatment): Harvest period: 24/48 h for 24/48h exposure each, no recovery period; Experiment 3 (short-term treatment): Recovery period: 18h, Harvest period: 24h.
In the cytotoxicity test, medium was discarded and cells were treated with 5mL of 0.04 % trypsin solution (dissolved in phosphate buffered saline (PBS(-))). The obtained cell suspension was used for counting of cells.
In the chromosomal aberration test, colcemid was added to the culture approximately 1.5 hours prior to cell harvest (final concentration: 0.1 μg/mL) to accumulate metaphase cells. Cells were treated with 5mL of 0.04% trypsin solution (dissolved in (PBS(-))) for a few minutes at the designated harvest time to obtain a cell suspension. The cell growth rate was measured using 0.5 mL of cell suspension. Chromosome spreads were prepared using the remaining cell suspension.

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays):
Cells were collected by centrifugation (1000 rpm, 5 minutes). After the supernatant was discarded, the cells were swollen with 75 mM KCl at room temperature for 20 minutes and the fixative (acetic acid : methanol = 1 : 3, v/v) was added. The fixative was changed three times by centrifugation. Then the cells were pelleted and resuspended in a minimal amount of the fixative, and a few drops of the cell suspension were spread onto a clean slide and air-dried. After aging for at least one day, the slides were stained with 3% Giemsa solution in 1/15 M phosphate buffer (pH 6.8) for 30 minutes. The slides were then rinsed, dried, and mounted with coverslips.
The lowest dose level where the cell growth rate was decreased 50% or less was selected as the highest dose level for preparation of metaphase spreads, and metaphase spreads were not prepared for higher dose levels.
- Cytogenetic analysis:
Chromosome spreads containing sufficient amounts of observable metaphases were observed at three consecutive levels of test substance concentrations. Chromosome spreads from the solvent control group and the positive control group were also observed.
One hundred metaphases per slide were observed for structural aberrations. Structural aberrations were classified into chromatid gaps, chromosome gaps, chromatid breaks, chromatid exchanges, chromosome breaks, chromosome exchanges (such as dicentric, circular chromosomes) and fragmentation. The number of aberrations and the number of cells with aberrations were recorded. When cells with fragmentation or more than 9 chromosomal aberrations were observed, the number of such aberrations was counted as 10 instead of counting each aberration. Gaps were defined as achromatic regions smaller than the width of a chromatid. Positions of cells with aberration were recorded.
One hundred metaphases per slide were observed for numerical aberrations. The number of occurrences of polyploid cells and endoreduplicated cells were recorded to determine the incidence of cells with numerical aberrations.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Cell growth rate
- Any supplementary information relevant to cytotoxicity: The cell suspension was diluted with Isoton-II (Coulter Corporation, Florida, USA). The number of cells was counted twice per sample with a Coulter Counter (Coulter Corporation) and the mean value was obtained. The cell growth rate was expressed as percentage of the number of cells in a treated group to that of a corresponding solvent control.
Evaluation criteria:
The incidence of cells with structural aberrations excluding gaps and incidences of polyploid at each treatment concentration were classified according to the following criteria:
Negative (-): Less than 5%; Marginal (±): Not less than 5% and less than 10%; Positive (+): Not less than 10%.
Test substances were judged to induce chromosomal aberration when they satisfied both of the following criteria:
• The incidence of cells with structural aberrations excluding gaps and/or with numerical aberrations are classified as marginal or positive.
• Reproducibility or dose dependency is recognized in its activity.
Statistics:
No statistical analysis was conducted to evaluate the results of the test
Species / strain:
Chinese hamster lung (CHL/IU)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
- Experiment 1(Short-term treatment):
Cytotoxicity test:
Marked growth inhibitions where the growth rate showed 50% or less were seen both in the presence and absence of S9 mix (430 μg/mL or more). No precipitation of the test substance was observed at the beginning or at the end of treatment.
Chromosomal Aberration Test:
No precipitation of the test substance was observed at the beginning or at the end of treatment both in the presence and absence of S9 mix.
Metaphases were observable in all test substance-treated groups and no conspicuous cell-cycle delay was observed in any test substance-treated group. There was no increase found in the incidence of cells with structural or numerical aberrations.

- Experiment 2 (Continuous treatment and confirmation test):
Cytotoxicity test: Marked growth inhibitions where the growth rate showed 50% or less were seen both in 24-hour and 48-hour treatment. No precipitation of the test substance was observed at the beginning or at the end of treatment.
Chromosomal Aberration Test:
No precipitation of the test substance was observed at the beginning or at the end of treatment both in the 24 h and 48h exposure.
Metaphases were observable in all test substance-treated groups, and no conspicuous cell-cycle delay was observed in any test substance-treated group. No increase found in the incidence of cells with structural or numerical aberrations were observed.

- Experiment 3 (Confirmation test):
Chromosomal aberration test:
Metaphases were observable in all test substance-treated groups and no conspicuous cell-cycle delay was observed in any test substance-treated group. There was no increase found in the incidence of cells with structural or numerical aberrations.

- Control group:
The incidences of cells with chromosomal aberrations (structural aberration and polyploidy) in the solvent control groups were within the range expected from the background data throughout the test.
An apparent increase in the incidence of cells with structural aberration was observed in the positive control substance-treated groups (treated with MMC in the absence of S9 mix, and treated with CP in the presence of S9 mix).

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
see attached backgroud material.
Conclusions:
The test item has no potential to induce chromosomal aberrations in CHL/IU cells under the conditions of this test.
Executive summary:

An in vitro chromosomal aberration test of test item was conducted using cultured cells derived from Chinese hamster lung (CHL/IU) according to OECD 473. Treatment of the test substance was conducted in the presence or absence of a rat liver drug metabolizing enzyme system (S9 mix).

In Experiment 1, cells were treated with the test substance for 6 hours in the presence or absence of S9 mix and then cultured for 18 hours in fresh medium (24 hours harvest; shot- term treatment). Chromosome preparations from the test item-treated cultures of 87.5, 175 and 350 μg/mL in the presence of S9 mix and 60.0, 70.0 and 80.0 μg/mL in the absence of S9 mix were analyzed. There was no increase in the incidence of cells with structural aberrations or polyploid cells in any test-substance-treated group.

In Experiment 2, cells were treated with the test substance for 24 hours or 48 hours continuously without S9 mix (continuous treatment, confirmation test). Chromosome preparations from 17.5, 20.0 and 22.5 μg/mL without S9 mix for 24 hours treatment and 1.68, 3.36 and 6.72 μg/mL without S9 mix for 48 hours treatment were analyzed. There was no increase in the incidence of cells with chromosomal aberration (cells with structural aberrations or polyploid cell) in any test-substance treated group.

In Experiment 3, cells were treated with the same procedure as in Experiment 1 in the presence of S9 mix in order to confirm reproducibility (short-term treatment, confirmation test). Chromosome preparations from the test item-treated cultures of 87.5, 175 and 350 μg/mL in the presence of S9 mix were analyzed. There was no increase in the incidence of cells with chromosomal aberration (cells with structural aberrations or polyploid cell) in any test-substance-treated group.

Based on these findings, it was concluded that test item has no potential to induce chromosomal aberration in CHL/IU cells under the test conditions.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Feom 2018-07-03 to 2018-08-08
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 using the Hprt and xprt genes)
Version / remarks:
adopted 29 July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
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:
Batch No.: C170806
Purity: 100%
Target gene:
HPRT (hypoxanthine-guanine phosphoribosyl transferase)
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: V79 cell line, Laboratory for Mutagenicity Testing; Technical University, 64287 Darmstadt, Germany
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years. Especially the high proliferation rate and a good cloning efficiency of untreated cells (as a rule more than 50%) both necessary for the appropriate performance of the study, recommend the use of this cell line.

For cell lines:
- Methods for maintenance in cell culture: Thawed stock cultures were propagated at 37 °C in 75 cm2 plastic flasks. About 2-3E+06 cells were seeded into each flask with 15 mL of MEM (minimal essential medium) containing Hank’s salts supplemented with 10% fetal bovine serum (FBS), neomycin (5 μg/mL) and amphotericin B (1%). The cells were sub-cultured once or twice weekly. All incubations were done at 37°C with 1.5% carbon dioxide (CO2) in humidified air.
- Doubling time: 12 - 16 h
- Modal number of chromosomes: 22
- Periodically checked for karyotype stability: yes
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
MEM containing Hank’s salts, neomycin (5 μg/mL), 10% FBS, and amphotericin B (1 %).
During treatment no FBS was added to the medium. For the selection of mutant cells the complete medium was supplemented with 11 μg/mL 6-thioguanine. All cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : prepared and stored according to the currently valid version of the Lab SOP for rat liver S9 preparation.
- method of preparation of S9 mix: An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. S9 mix contained MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4). The protein concentration of the S9 preparation was 34.3 mg/mL (Lot. No. 161117) in the pre-experiment and in the main experiment.
- concentration or volume of S9 mix and S9 in the final culture medium: 50 μL/mL S9 mix
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.
Test concentrations with justification for top dose:
53.5, 107.1, 214.1, , 428.3, 856.5, 1713.0 μg/mL
In the pre-experiment there was no relevant shift of pH-values and osmolarity of the medium at the maximum concentration (1713.0 μg/mL) of the test item.
The dose range of the main experiment was set according to the data generated in the pre-experiment. The individual concentrations were spaced by a factor of 2.0.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Sterile deionized water, final concentration of sterile deionized water in the culture medium was 10% (v/v).

- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures on request of the sponsor.
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:
- Pre-Test on Toxicity:
A pre-test was performed in order to determine the toxicity of the test item.
The pH-value and the osmolarity were measured. Precipitation was checked at the beginning and at the end of treatment by the unaided eye and with a microscope. In this pre-test approximately 1.5 million cells were seeded in 25 cm² flasks 24 hours prior to treatment. After approximately 24 hours the test item was added and the treatment proceeded for 4 hours (duplicate cultures per concentration level). Immediately after treatment the test item was removed by rinsing with PBS. Subsequently, the cells were trypsinized and suspended in complete culture medium. After an appropriate dilution the cell density was determined with a cell counter.
Toxicity of the test item is evident as a reduction of the cell density compared to a corresponding solvent control. A cell density of approximately 1.5 million cells in 25 cm² flasks is about the same as approximately 10 million cells seeded in 175 cm² bottles 24 hours prior to treatment with the main experiment.
- Main Experiment:
- Seeding:
Two to four days after sub-cultivation stock cultures were trypsinized at 37 °C for 5-10 minutes. Then the enzymatic digestion was stopped by adding complete culture medium with 10% FBS and a single cell suspension was prepared. The trypsin concentration for all sub-culturing steps was 0.2% in phosphate buffered saline (PBS).
Prior to the trypsin treatment the cells were rinsed with PBS. Approximately 0.7 to 1.2E+07 cells were seeded in plastic flasks. The cells were grown for 24 hours prior to treatment.
- Treatment:
After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 μL/mL S9 mix. Concurrent negative, solvent, and positive controls were treated in parallel. Four hours after treatment, this medium was replaced with complete medium following two washing steps with "saline G".
Immediately after the end of treatment the cells were trypsinised as described above and sub-cultivated. At least 2.0E+06 cells per experimental point (concentration series plus controls) were subcultivated in 175 cm² flasks containing 30 mL medium.
Two additional 25 cm² flasks were seeded per experimental point with approx. 500 cells each to determine the relative survival (RS) as measure of test item induced cytotoxicity. The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2.
The colonies used to determine the relative survival (RS) were fixed and stained approximately 6 to 8 days after treatment as described below.
Three or four days after the first sub-cultivation, at least 2.0E+06 cells per experimental point were again, sub-cultivated in 175 cm² flasks containing 30 mL medium.
Following the expression time of approximately 7 days ten 75 cm² cell culture flasks were seeded with about 4 – 5E+05 cells each in medium containing 6-TG (11 μg/mL). Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability. The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2.
After 8 – 11 days the colonies were stained with 10 % methylene blue in 0.01 % KOH solution. Colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.
Subculturing of a log-phase the cultures showed an initial spontaneous mutation rate at the beginning of the experiment of 17.1 mutant colonies per 1E+06 cells.
Evaluation criteria:
A test item is classified as clearly mutagenic if, in any of the experimental conditions examined, all of the following criteria are met:
a. at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent solvent control,
b. the increase is dose-related when evaluated with an appropriate trend test,
c. any of the results are outside the distribution of the historical solvent control data (e.g. Poisson-based 95% control limits).
A test item is classified as clearly non-mutagenic if, in all experimental conditions examined, all of the following criteria are met:
a. none of the test concentrations exhibits a statistically significant increase compared with the concurrent solvent control,
b. there is no concentration-related increase when evaluated with an appropriate trend test,
c. all results are inside the distribution of the historical solvent control data (e.g. Poisson-based 95% control limits).
There is no requirement for verification of a clearly positive or negative response. In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and further investigations.
A test item producing a reproducible concentration-related increase of the mutant frequency above the historical solvent control range is considered to be mutagenic in this system.
When a test item cannot be classified as mutagenic, a test item is considered to be non-mutagenic in this system.
Statistics:
A linear regression was performed using a validated test script of "R", a language and environment for statistical computing and graphics (p < 0.05), to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
However, both, biological and statistical significance were considered together.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
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
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: 7.32 for test item at 1713.0 μg/mL, 7.31 for Solvent control
- Data on osmolality: 300 mOsm for test item at 1713.0 μg/mL, 276 mOsm for Solvent control
- Stability in Solvent: Stable in water (5% solution) at room temperature for 4 hours
- Precipitation and time of the determination: No precipitation was noted neither in the absence nor in the presence of metabolic activation at the beginning and at the end of treatment (4 hours) prior to the removal of the test item

RANGE-FINDING/SCREENING STUDIES:
The pre-experiment was performed in the presence and absence (4 h treatment) of metabolic activation. Test item concentrations between 13.4 μg/mL and 1713.0 μg/mL were used. The highest concentration of the pre-experiment was chosen with respect to the current guideline OECD 476 (10 mM).
No cytotoxic effects were noted up to the highest concentration of 1713.0 μg/mL after 4 hours treatment.
No precipitation or phase separation was observed macroscopically and microscopically at the beginning and at the end of treatment (4 hours) prior to the removal of the test item in the absence and presence of metabolic activation.
In the pre-experiment there was no relevant shift of pH-values and osmolarity of the medium at the maximum concentration of the test item.

STUDY RESULTS
- Concurrent vehicle negative and positive control data
The mean mutant frequency obtained in the solvent controls was 8.7 without S9 mix and 12.4 with S9 mix. In the negative control the mean mutant frequency was 19.5 without S9 mix and 17.6 with S9 mix. These values were well within the 95% confidence interval of the laboratory’s historical solvent control data.
EMS (300 μg/mL) and DMBA (2.3 μg/mL) were used as positive controls. They showed a statistically significant increase in induced mutant colonies compared with the concurrent solvent control and remain within the historical control range of positive controls.
In addition, the cloning efficiency II (absolute value) of the solvent controls all exceeded 50%.

For all test methods and criteria for data analysis and interpretation:
No precipitation was noted neither in the absence nor in the presence of metabolic activation.
No relevant cytotoxic effect indicated by a relative adjusted cloning efficiency I below 50% was observed neither in the absence nor in the presence of metabolic activation.
No biologically relevant increase in mutation frequency as the mean of both parallel cultures was observed in the main experiment up to the maximum concentrations scored for gene mutations.
The range of the mean mutant frequencies of the groups treated with the test item was from 7.5 up to 16.3 mutants per 1E+06 cells. The values of the treatment groups were all within the 95% confidence interval of the laboratory’s historical solvent control data.
The linear regression analysis based on the mean values of both cultures, showed no significant dose dependent trend of the mutation frequency at any of the experimental groups.
A t-test was not performed since the 95% confidence interval was not exceeded at any experimental point.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: EMS 150 and 300 μg/mL without metabolic activation: range: 53.9-872.3, mean 221 per 1E+06 cells, SD: 102.6, number of studies: 168; DMBA 1.1 and 2.3 μg/mL with metabolic activation: range: 55.6 – 739.9, mean 191.4 per 1E+06 cells, SD: 100.0, number of studies: 162
- Negative (solvent/vehicle) historical control data: without metabolic activation: range: 3.4 – 41.0, mean 16.9 per 1E+06 cells, SD: 7.1, number of studies: 168; with metabolic activation: range: 2.4 – 40.4, mean 16.9 per 1E+06 cells, SD: 7.0, number of studies: 162
Conclusions:
Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.
Therefore, the test item is considered to be non-mutagenic in this HPRT assay.
Executive summary:

The study was performed to investigate the potential of test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster in compliance with OECD 476.

The experiment was performed with a treatment time of 4 hours with and without metabolic activation.

The maximum test item concentration of the pre-experiment was 1713.0 μg/mL (10 mM) with respect to the current guideline.

In the pre-experiment, neither cytotoxicity nor precipitation were observed up to the maximum concentration under both conditions of with and without metabolic activation. Therefore, the maximum concentration of the main experiment was set to 1713.0 μg/mL.

No substantial dose dependent increase of the mutation frequency was observed in the main experiments.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test item is considered to be non-mutagenic in this HPRT assay.

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

Data:

Ames study: OECD 471, negative

In Vitro Mammalian Chromosome Aberration Test: OECD 473, negative

In vitro mammalian cell gene mutation test: OECD 476, negative

Therefore in accordance with Regulation (EC) No. 1272/2008 Table 3.5.1, the test substance should not be classified as germ cell mutagens.